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USOO951 2236B2

(12) United States Patent (10) Patent No.: US 9,512,236 B2 Adams et al. (45) Date of Patent: Dec. 6, 2016

(54) AMINO ACID SEQUENCES DIRECTED (52) U.S. Cl. AGAINST GPCRS AND POLYPEPTIDES CPC ...... C07K 16/468 (2013.01); C07K 16/2866 COMPRISING THE SAME FOR THE (2013.01); C07K 16/2869 (2013.01); C07K TREATMENT OF GPCR-RELATED 231 7/22 (2013.01); C07K 23.17/31 (2013.01); DISEASES AND DISORDERS C07K 23.17/34 (2013.01); C07K 23.17/35 (2013.01); C07K 2317/92 (2013.01) (71) Applicant: Ablynx N.V., Zwijnaarde (BE) (58) Field of Classification Search None (72) Inventors: Hendrik Adams, Rotterdam (NL); See application file for complete search history. Michael John Scott Saunders, Brussels (BE); Johannes Joseph Wilhelmus De Haard, Oudelande (56) References Cited (NL); Christophe Blanchetot, Destelbergen (BE); Martine Smit, U.S. PATENT DOCUMENTS Amsterdam (NL); Regorius Leurs, 8,937,164 B2 1/2015 Descamps et al. Amsterdam (NL); Sven Jahnichen, 9,212,226 B2 12/2015 Blanchetot et al. Schwarme (DE); Peter Vanlandschoot, 2002/0106.739 A1 8/2002 Oakley et al. 2004/0170634 A1 9, 2004 Burns et al. Bellem (BE); Francis Descamps, 2007/0167443 A1 7/2007 Melikian et al. Roeselare (BE); Maria Gonzalez 2010.0062004 A1 3/2010 Adams et al. Pajuelo, Porto (PT); Pascal Gerard 2011/0206660 A1 8/2011 Blanchetot et al. Merchiers, Tielen (BE); Catelijne 2011/0262438 A1 10/2011 Descamps et al. Stortelers, Ghent (BE); Philippe Van 2014/0178390 A1 6/2014 Descamps et al. Rompaey, Melle (BE); David Andre 2014/0227 270 A1 8/2014 Descamps et al. Baptiste Maussang-Detaille, Rotterdam (NL); Maarten Van Roy, Zwijnaarde FOREIGN PATENT DOCUMENTS (BE) EP O 368 684 A1 5, 1990 EP O 542 810 5, 1993 (73) Assignee: Ablynx N.V., Zwijnaarde (BE) EP 1316 801 A1 9, 2002 JP 2008-539772 A 11/2008 (*) Notice: Subject to any disclaimer, the term of this JP 2008-539775 A 11/2008 patent is extended or adjusted under 35 (Continued) U.S.C. 154(b) by 0 days. OTHER PUBLICATIONS (21) Appl. No.: 13/650,600 No Author Listed Nature Reviews Drug Discovery GPCR Ques (22) Filed: Oct. 12, 2012 tionnaire Participants. The state of GPCR research in 2004. Nat Rev Drug Discov. Jul. 2004:3(7):575, 577-626. (65) Prior Publication Data (Continued) US 2013/O 130379 A1 May 23, 2013 Primary Examiner — Ruixiang Li Related U.S. Application Data (74) Attorney, Agent, or Firm — Wolf, Greenfield & (63) Continuation-in-part of application No. 12/520,299. Sacks, P.C. filed as application No. PCT/EP2007/064243 on Dec. 19, 2007, now abandoned, application No. (57) ABSTRACT 13/650,600, which is a continuation-in-part of application No. 12/992,982, filed as application No. The present invention relates to amino acid sequences that PCT/EP2009/056026 on May 18, 2009, now Pat. No. are directed against G- coupled receptors (GPCRs), 9,212.226, application No. 13/650,600, which is a as well as to compounds or constructs, and in particular continuation-in-part of application No. and polypeptides, that comprise or essentially con PCT/EP2012/055499, filed on Mar. 28, 2012. sist of one or more such amino acid sequences. The inven tion also relates to nucleic acids encoding Such amino acid (60) Provisional application No. 60/875,860, filed on Dec. sequences and; to methods for preparing such amino acid 19, 2006, provisional application No. 61/053,847, sequences and polypeptides; to host cells expressing or filed on May 16, 2008, provisional application No. capable of expressing Such amino acid sequences or poly 61/102,142, filed on Oct. 2, 2008, provisional ; to compositions, and in particular to pharmaceu application No. 61/468,250, filed on Mar. 28, 2011, tical compositions, that comprise Such amino acid provisional application No. 61/540.272, filed on Sep. sequences, polypeptides, nucleic acids and/or host cells; and 28, 2011, provisional application No. 61/600,263, to uses of Such amino acid sequences or polypeptides, filed on Feb. 17, 2012. nucleic acids, host cells and/or compositions, in particular for prophylactic, therapeutic or diagnostic purposes. (51) Int. C. C07K I6/46 (2006.01) C07K 6/28 (2006.01) 4 Claims, 3 Drawing Sheets US 9,512.236 B2 Page 2

(56) References Cited Halaby et al., The immunoglobulin fold family: Sequence analysis and 3D structure comparisons. Protein Eng. Jul. 1999; 12(7):563-71. Hassaine et al., Semliki Forest virus vectors for overexpression of FOREIGN PATENT DOCUMENTS 101 -coupled receptors in mammalian host cells. Protein JP 2010-500876 A 1, 2010 Expr Purif. Feb. 2006:45(2):343-51. Epub Jul. 11, 2005. JP 2010-5069 12 A 3, 2010 Hoffman et al., A biosensor assay for studying -membrane WO WO 91/O1743 A1 2, 1991 interactions: binding of and HIV-1 Env to WO WO99/50461 A1 10, 1999 chemokine receptors. Proc Natl Acad Sci U S A. Oct. 10, WO WO O1/45746 A2 6, 2001 WO WO O2/O76489 A1 10, 2002 2000:97(21): 11215-20. WO WO 03/050531 A2 6, 2003 Holliger et al., Engineered fragments and the rise of single WO WO 03/0668.30 A2 8, 2003 domains. Nat Biotechnol. Sep. 2005:23(9): 1126-36. WO WO 2004/041867 A2 5, 2004 Holt et al., Domain antibodies: proteins for therapy. Trends WO WO 2004/051268 A1 6, 2004 Biotechnol. 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Jul. 8, 2002:448(4):410-22. comparison of more than 100 GPCRs in 3 expression systems. J Dahmen et al., Expression of olfactory receptors in Xenopus Struct Funct Genomics. Jun. 2006;7(2):77-91. Epub Nov. 22, 2006. oocytes. J Neurochem. Mar. 1992:58(3): 1176-9. Marinissen et al., G-protein-coupled receptors and signaling net Dimitrov et al. A mechanism of resistance to HIV-1 entry: ineffi works: emerging paradigms. Trends Pharmacol Sci. Jul. cient interactions of CXCR4 with CD4 and gp120 in macrophages. 2001:22(7):368-76. Virology. Jun. 20, 1999:259(1):1-6. McIntosh et al., CB1 : cellular regulation and Dove Pettit et al., Immunohistochemical localization of the neural distribution in N18TG2 neuroblastoma cells. Mol Brain Res. Jan. cannabinoid receptor in rat brain. J Neurosci Res. Feb. 1. 1998:53(1-2):163–73. 1998:51(3):391-402. Milligan, Constitutive activity and inverse of G protein Gensure et al., Parathyroid hormone and parathyroid hormone coupled receptors: a current perspective. Mol Pharmacol. Dec. related , and their receptors. Biochem Biophys Res Com 2003:64(6): 1271-6. mun. Mar. 18, 2005:328(3):666-78. Misumi et al. A novel cyclic peptide immunization strategy for George et al., G-protein-coupled receptor oligomerization and its preventing HIV-1/AIDS infection and progression. J Biol Chem. potential for drug discovery. Nat Rev Drug Discov. Oct. Aug. 22, 2003:278(34):32335-43. Epub May 27, 2003. 2002:1(10):808-20. Mitrirattanakul et al., Expression of cannabinoid 1 receptors in rat Getting, Targeting melanocortin receptors as potential novel thera dorsal root ganglia remains unchanged after spinal nerve ligation. peutics. Pharmacol Ther. Jul. 2006:111(1): 1-15. Epub Feb. 20, 33 Annual Meeting Soc Neurosci. Nov. 10, 2003;Program No. 2006. 483.9. Abstract. US 9,512.236 B2 Page 3

(56) References Cited Genbank Submission; NCBI; Accession No. NM 000609.5; Bracci et al.; Mar. 21, 2010. 4 pages. OTHER PUBLICATIONS Giusti et al., Somatic diversification of S107 from an antiphosphocholine to an anti-DNA autoantibody is due to a single Nicholson et al., Peripheral administration of a melanocortin 4-re ceptor inverse prevents loss of lean body mass in tumor base change in its heavy chain variable region. Proc Natl Acad Sci bearing mice. J Pharmacol Exp Ther. May 2006:317(2):771-7. Epub U S A. May 1987:84(9):2926-30. Jan. 25, 2006. Gissow et al., Humanization of monoclonal antibodies. Methods Pacher et al., The endocannabinoid system as an emerging target of Enzymol. 1991:203:99-121. pharmacotherapy. Pharmacol Rev. Sep. 2006:58(3):389-462. Hachet-Haas et al. Small Neutralizing Molecules to Inhibit Actions Pierce et al., Seven-transmembrane receptors. Nat Rev Mol Cell of the Chemokine CXCL12. Journal of Biological Chemistry, 2008; Biol. 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J Mol essential for ligand contact to engineer a less immunogenic human Biol. Nov. 19, 1999:294(1): 151-62. ized monoclonal antibody. J Immunol. Sep. 15, 2002; 169(6):3076 Zabel et al., Elucidation of CXCR7-mediated signaling events and 84. inhibition of CXCR4-mediated tumor cell transendothelial migra Desmyter et al., Three camelid VHH domains in complex with tion by CXCR7 ligands. J Immunol. Sep. 1, 2009:183(5):3204-11. porcine pancreatic alpha-amylase. Inhibition and versatility of bind Chen et al., Selection and analysis of an optimized anti-VEGF ing topology. J Biol Chem. Jun. 28, 2002:277(26):23645-50. Epub antibody: crystal structure of an affinity-matured Fab in complex Apr. 17, 2002. with antigen. J Mol Biol. Nov. 5, 1999:293(4):865-81. US 9,512.236 B2 Page 4

(56) References Cited Hattermann et al., The chemokine receptor CXCR7 is highly expressed in human glioma cells and mediates antiapoptotic effects. OTHER PUBLICATIONS Cancer Res. Apr. 15, 2010:70(8):3299-308. doi: 10.1158,0008 5472. CAN-09-3642. Epub Apr. 13, 2010. Lamminmäki et al., Crystal structure of a recombinant anti-estradiol Maussang et al., Llama-derived single variable domains (nanobod Fab fragment in complex with 17beta-estradiol. J. Biol Chem. Sep. ies) directed against chemokine receptor CXCR7 reduce head and 28, 2001:276(39):36687-94. Epub Jul. 12, 2001. neck cancer cell growth in vivo. J. Biol Chem. Oct. 11, Muyldermans, Single domain camel antibodies: current status. J 2013:288(41):29562-72. doi: 10.1074/bc.M113,498436. Epub Biotechnol. Jun. 2001:74(4):277-302. Aug. 26, 2013. Padlan et al., Structure of an antibody-antigen complex: crystal Xia et al., Expressions of CXCR7/ligands may be involved in oral structure of the HyHEL-10 Fab-lysozyme complex. Proc Natl Acad carcinogenesis. J Mol Histol. Apr. 2011:42(2): 175-80. doi:10.1007/ Sci U S A. Aug. 1989;86(15):5938-42. s10735-011-9322-X. Epub Mar. 26, 2011. Brown et al., Tolerance of single, but not multiple, amino acid Zheng et al., Chemokine receptor CXCR7 regulates the invasion, replacements in antibody VHCDR 2: a means of minimizing B cell angiogenesis and tumor growth of human hepatocellular carcinoma wastage from Somatic hypermutation? J Immunol. May 1, cells. J Exp Clin Cancer Res. Apr. 11, 2010:29:31, doi:10.1186/ 1996;156(9):3285-91. 1756-9966-29-31. U.S. Patent Dec. 6, 2016 Sheet 1 of 3 US 9,512,236 B2

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U.S. Patent Dec. 6, 2016 Sheet 3 of 3 US 9,512,236 B2

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& 3e 3 & { 8 f s :'s Ssys Ss & &s's' & & iss XC2 30 st US 9,512,236 B2 1. 2 AMINO ACID SEQUENCES DIRECTED and in the periphery. GPCRs are AGAINST GPCRS AND POLYPEPTIDES proteins with 7 transmembrane domains with highly con COMPRISING THE SAME FOR THE served domains. It was estimated that in the year 2000 half TREATMENT OF GPCR-RELATED of all modern drugs and almost one-quarter of the top 200 DISEASES AND DISORDERS best-selling drugs are directed against or modulate GPCR targets (approximately 30 in total). However, due to their RELATED APPLICATIONS architecture of 7 membrane-spanning helices and their strong tendency to aggregate, its a very challenging target This application is a continuation-in-part of international class. application PCT/EP2012/055499, filed Mar. 28, 2012, 10 GPCRs are a well-known class of receptors. Reference is which was published under PCT Article 21(2) in English, for example made to the following reviews: Surgand et al., and claims the benefit under 35 U.S.C. S 119(e) of U.S. provisional application Ser. No. 61/600,263 filed Feb. 17, Proteins 62:509-538 (2006); Vassilatis et al., Proc Natl Acad 2012, U.S. provisional application Ser. No. 61/540.272 filed Sci USA 100:4903-4908 (2003) and Pierce et al., Nat Rev Sep. 28, 2011, and U.S. provisional application Ser. No. 15 Mol Cell Biol 3:639-650 (2002); as well as to for example: 61/468,250 filed Mar. 28, 2011; and of U.S. application Ser. George et al., Nat Rev Drug Discov 1:808-820 (2002); No. 12/992,982, filed May 18, 2009, which is a national Kenakin, Trends Pharmacol Sci 25:186-192 (2002); Rios et stage filing under 35 U.S.C. S371 of international applica al., Pharmacol Ther 92:71-87 (2001); Jacoby et al., tion PCT/EP2009/056026, filed May 18, 2009, which was ChemMedChem 2006, 1,760-782; and Schlyer and Horuk, published under PCT Article 21(2) in English, and claims Drug Discovery Today, 11, 11/12. June 2006, 481; and also the benefit under 35 U.S.C. S 119(e) of U.S. provisional for example to Rosenkilde. Oncogene (2001), 20, 1582 application Ser. No. 61/102,142, filed Oct. 2, 2008, and U.S. 1593 and Sadee et al., AAPS PharmSci 2001; 3: 1-16; as provisional application Ser. No. 61/053,847, filed May 16, well as to the further references cited therein. 2008; and of U.S. application Ser. No. 12/520,299, filed Dec. G-protein-coupled receptors (GPCRs) are the largest class 19, 2007, which is a national stage filing under 35 U.S.C. 25 of cell-surface receptors (more than 1000 are present S371 of international application PCT/EP2007/064243, filed in the human ). They can be activated by a diverse Dec. 19, 2007, which was published under PCT Article array of stimuli, e.g. hormones, peptides, amino acids, 21(2) in English, and claims the benefit under 35 U.S.C. photons of light, and these receptors play a large role in the S119(e) of U.S. provisional application Ser. No. 60/875,860, central nervous system and in the periphery. GPCRs are filed Dec. 19, 2006; the disclosures of each of which are 30 proteins with 7 transmembrane domains with highly con incorporated by reference herein in their entireties. served domains. As half of all known drugs work through G-protein FIELD OF THE INVENTION coupled receptors, it is commercially very attractive to select Nanobodies against this protein family. It was estimated that The present invention relates to amino acid sequences that 35 in the year 2000 half of all modern drugs and almost are directed against (as defined herein) G-protein coupled one-quarter of the top 200 best-selling drugs are directed receptors (GPCRs), as well as to compounds or constructs, against or modulate GPCR targets (approximately 30 in and in particular proteins and polypeptides, that comprise or total). However, due to their architecture of 7 membrane essentially consist of one or more such amino acid spanning helices and their strong tendency to aggregate, it’s sequences (also referred to herein as "amino acid sequences 40 a very challenging target class. of the invention”, “compounds of the invention', and “poly GPCRs can be grouped on the basis of sequence homol peptides of the invention', respectively). ogy into several distinct families. Although all GPCRs have The invention also relates to nucleic acids encoding Such a similar architecture of seven membrane-spanning C-heli amino acid sequences and polypeptides (also referred to ces, the different families within this receptor class show no herein as “nucleic acids of the invention' or “nucleotide 45 to one another, thus Suggesting that the sequences of the invention'); to methods for preparing Such similarity of their transmembrane domain structure might amino acid sequences and polypeptides; to host cells define common functional requirements. Depending on the expressing or capable of expressing Such amino acid size of the extracellular domain three families are discrimi sequences or polypeptides; to compositions, and in particu nated (FIG. 1). lar to pharmaceutical compositions, that comprise Such 50 Members of Family 1 (also called family A or amino acid sequences, polypeptides, nucleic acids and/or like family; panela of FIG. 1) only have small extra host cells; and to uses of Such amino acid sequences or cellular loops and the interaction of the ligands occurs polypeptides, nucleic acids, host cells and/or compositions, with residues within the transmembrane cleft. This is in particular for prophylactic, therapeutic or diagnostic pur by far the largest group (>90% of the GPCRs) and poses. Such as the prophylactic, therapeutic or diagnostic 55 contains receptors for odorants, Small molecules Such purposes mentioned herein. as catecholamines and amines, (neuro)peptides and Other aspects, embodiments, advantages and applications glycoprotein hormones. Rhodopsin, which belongs to of the invention will become clear from the further descrip this family, is the only GPCR for which the structure tion herein. has been solved. 60 Family 2 or family B GPCRs (FIG. 1, panel b) are BACKGROUND characterized by a relatively long amino terminal extra cellular domain involved in ligand-binding. Little is G-protein-coupled receptors (GPCRs) are the largest class known about the orientation of the transmembrane of cell-surface receptors (more than 1000 genes are present domains, but it is probably quite different from that of in the ). They can be activated by a diverse 65 rhodopsin. Ligands for these GPCRs are hormones, array of stimuli, e.g. hormones, peptides, amino acids, Such as glucagon, gonadotropin-releasing hormone and photons of light, and these receptors play a large role in the parathyroid hormone. US 9,512,236 B2 3 4 Family 3 members (FIG. 1, panel c) also have a large and/or treated, respectively, by Suitably administering to a extracellular domain, which functions like a “Venus fly subject in need thereof (i.e. having the disease or disorder or trap' since it can open and close with the agonist bound at least one symptom thereof and/or at risk of attracting or inside. Family members are the metabotropic gluta developing the disease or disorder) of either a polypeptide or mate, the Ca2+-sensing and the y-aminobutyric acid 5 composition of the invention (and in particular, of a phar (GABA) B receptors. maceutically active amount thereof) and/or of a known active principle active against GPCRs or a biological path SUMMARY way or mechanism in which GPCRs is involved (and in particular, of a pharmaceutically active amount thereof). Traditionally small molecules are used for development 10 Examples of such GPCR-related diseases and disorders will of drugs directed against GPCRs, not only because pharma be clear to the skilled person based on the disclosure herein. ceutical companies have historical reasons to work with Thus, without being limited thereto, the amino acid these, but more importantly because of the structural con sequences and polypeptides of the invention can for example straints of Family 1 GPCRs, which have the ligand binding be used to prevent and/or to treat all diseases and disorders site within the transmembrane cleft (Nat Rev Drug Discov. 15 that are currently being prevented or treated with active (2004) The state of GPCR research in 2004. Nature Reviews principles that can modulate GPCRs-mediated signalling, Drug Discovery GPCR Questionnaire Participants 3(7):575, Such as those mentioned in the prior art cited above. It is also 577-626). For this reason it proved to be difficult or impos envisaged that the polypeptides of the invention can be used sible to generate monoclonal antibodies against this target to prevent and/or to treat all diseases and disorders for which class. The amino acid sequences of the invention (and in treatment with Such active principles is currently being particular Nanobodies of the invention) can solve this par developed, has been proposed, or will be proposed or ticular problem by means of their intrinsic property of developed in future. In addition, it is envisaged that, because binding via extended CDR loops into cavities (as further of their favourable properties as further described herein, the described herein). polypeptides of the present invention may be used for the Some non-limiting examples of therapeutically relevant 25 prevention and treatment of other diseases and disorders GPCRs are for example the following, which are all targets than those for which these known active principles are being of known drugs that have either been approved or are in used or will be proposed or developed; and/or that the clinical development. The text between brackets indicates polypeptides of the present invention may provide new the desired action of an amino acid sequence, a NANO methods and regimens for treating the diseases and disorders BODYR (V) or a polypeptide of the invention (i.e. as 30 described herein. agonist or antagonist): Other applications and uses of the amino acid sequences Class A GPCRS and polypeptides of the invention will become clear to the Muscarinic M1 receptor (agonist) skilled person from the further disclosure herein. Adrenoceptor (agonist); Generally, it is an object of the invention to provide receptor (agonist); 35 pharmacologically active agents, as well as compositions 5-HT GPCR (agonist); comprising the same, that can be used in the diagnosis, Cannabinoid receptor (agonist); prevention and/or treatment of GPCR-related diseases and Class A hormone protein GPCR (agonist); disorders and of the further diseases and disorders men Chemokine (agonist); tioned herein; and to provide methods for the diagnosis, Galanin (agonist); 40 prevention and/or treatment of Such diseases and disorders Melanocortin (agonist); that involve the administration and/or use of Such agents and (agonist); compositions. (agonist); In particular, it is an object of the invention to provide Opioid (agonist); Such pharmacologically active agents, compositions and/or Somatostatin (agonist); 45 methods that have certain advantages compared to the Vasopressin like receptor (agonist); agents, compositions and/or methods that are currently used Prostanoid receptor (agonist) and/or known in the art. These advantages will become clear Class B GPCRS from the further description below. ACTH releasing factor receptor (modulator); More in particular, it is an object of the invention to Class C GPCRS 50 provide therapeutic proteins that can be used as pharmaco GABAB receptor (agonist); logically active agents, as well as compositions comprising Metabotropic glutamate receptor (agonist); the same, for the diagnosis, prevention and/or treatment of Some other non-limiting examples of therapeutically rel GPCR-related diseases and disorders and of the further evant GPCRs are mentioned in Table C. A more extensive diseases and disorders mentioned herein; and to provide list of human GPCRs is given in Table D. 55 methods for the diagnosis, prevention and/or treatment of The polypeptides and compositions of the present inven Such diseases and disorders that involve the administration tion can generally be used to modulate (as defined herein), and/or the use of Such therapeutic proteins and composi and in particular inhibit and/or prevent, GPCRs mediated tions. signalling and/or to modulate the biological pathways in Accordingly, it is a specific object of the present invention which GPCRs are involved, and/or to modulate the biologi 60 to provide amino acid sequences that are directed against (as cal mechanisms, responses and effects associated with Such defined herein) GPCRs, in particular against GPCRs from a signalling or these pathways. warm-blooded animal, more in particular against GPCRs As such, the polypeptides and compositions of the present from a mammal, and especially against human GPCRs; and invention can be used for the prevention and treatment (as to provide proteins and polypeptides comprising or essen defined herein) of GPCR-related diseases and disorders. 65 tially consisting of at least one such amino acid sequence. Generally, “GPCR-related diseases and disorders' can be In particular, it is a specific object of the present invention defined as diseases and disorders that can be prevented to provide Such amino acid sequences and Such proteins US 9,512,236 B2 5 6 and/or polypeptides that are Suitable for prophylactic, thera Some preferred IC50 values for binding of the amino acid peutic and/or diagnostic use in a warm-blooded animal, and sequences or polypeptides of the invention to GPCRs will in particular in a mammal, and more in particular in a human become clear from the further description and examples being. herein. More in particular, it is a specific object of the present 5 For binding to GPCRs, an amino acid sequence of the invention to provide Such amino acid sequences and Such invention will usually contain within its amino acid proteins and/or polypeptides that can be used for the pre sequence one or more amino acid residues or one or more vention, treatment, alleviation and/or diagnosis of one or stretches of amino acid residues (i.e. with each “stretch' more diseases, disorders or conditions associated with comprising two or amino acid residues that are adjacent to GPCRs and/or mediated by GPCRs (such as the diseases, 10 each other or in close proximity to each other, i.e. in the disorders and conditions mentioned herein) in a warm primary or tertiary structure of the amino acid sequence) via blooded animal, in particular in a mammal, and more in which the amino acid sequence of the invention can bind to particular in a human being. GPCRs, which amino acid residues or stretches of amino It is also a specific object of the invention to provide such acid residues thus form the “site' for binding to GPCRs (also amino acid sequences and Such proteins and/or polypeptides 15 referred to herein as the “antigen binding site'). that can be used in the preparation of pharmaceutical or The amino acid sequences provided by the invention are veterinary compositions for the prevention and/or treatment preferably in essentially isolated form (as defined herein), or of one or more diseases, disorders or conditions associated form part of a protein or polypeptide of the invention (as with and/or mediated by GPCRs (such as the diseases, defined herein), which may comprise or essentially consist disorders and conditions mentioned herein) in a warm of one or more amino acid sequences of the invention and blooded animal, in particular in a mammal, and more in which may optionally further comprise one or more further particular in a human being. amino acid sequences (all optionally linked via one or more In the invention, generally, these objects are achieved by suitable linkers). For example, and without limitation, the the use of the amino acid sequences, proteins, polypeptides one or more amino acid sequences of the invention may be and compositions that are described herein. 25 used as a binding unit in Such a protein or polypeptide, In general, the invention provides amino acid sequences which may optionally contain one or more further amino that are directed against (as defined herein) and/or can acid sequences that can serve as a binding unit (i.e. against specifically bind (as defined herein) to GPCRs; as well as one or more other targets than GPCRs), so as to provide a compounds and constructs, and in particular proteins and monovalent, multivalent or multispecific polypeptide of the polypeptides, that comprise at least one such amino acid 30 invention, respectively, all as described herein. Preferably, Sequence. two or more amino acid sequences of the invention may be More in particular, the invention provides amino acid used as a binding unit against the same GPCR, so as to sequences that can bind to GPCRs with an affinity (suitably provide a biparatopic or multiparatopic protein or polypep measured and/or expressed as a K-value (actual or appar tide. Such a protein or polypeptide may also be in essentially ent), a K-value (actual or apparent), a k-rate and/or a 35 isolated form (as defined herein). For example a “bivalent” k-rate, or alternatively as an ICso value, as further polypeptide of the invention comprises two amino acid described herein) that is as defined herein; as well as sequences which may be the same or different (i.e. regard compounds and constructs, and in particular proteins and less of their antigen binding specificity), optionally linked polypeptides, that comprise at least one such amino acid via a Suitable linker sequence. Polypeptides that comprise Sequence. 40 two the same amino acid sequences will be referred herein In particular, amino acid sequences and polypeptides of to mono-specific bivalent polypeptides, whereas polypep the invention are preferably such that they: tides comprising two different amino acid sequences will be bind to GPCRs with a dissociation constant (Ki) of 10 referred to “bispecific' or “biparatopic' polypeptides as to 10° moles/liter or less, and preferably 107 to 10° further described herein. In particular, a polypeptide of the moles/liter or less and more preferably 10 to 10° 45 invention which comprises two amino acid sequences which moles/liter (i.e. with an association constant (K) of are directed against different targets (e.g. have a different 10 to 10' liter/moles or more, and preferably 107 to antigen specificity) will be referred to as a “bispecific' 10' liter/moles or more and more preferably 10 to polypeptide of the invention. Thus, for example, a “bispe 10' liter/moles): cific' polypeptide of the invention is a polypeptide that and/or such that they: 50 comprises at least one amino acid sequence directed against bind to GPCRs with a k-rate of between 10 M's to a first antigen (i.e. GPCR), and at least one further amino about 107 M's', preferably between 10 M's and acid sequence directed against a different antigen (i.e. dif 107 M's', more preferably between 10 M's and ferent from said GPCR), and optionally linked via a suitable 107 M's, such as between 10 M' s and 107M linker sequence. In a most preferred embodiment the poly s'; 55 peptide of the invention is a “biparatopic' polypeptide. As and/or such that they: used herein the term “biparatopic' means that a polypeptide bind to GPCRs with a krate between 1 s' (t=0.69s) comprises two amino acid sequences which are directed and 10 s' (providing a near irreversible complex against different antigenic determinants, epitopes, parts, with a ta of multiple days), preferably between 10° domains, Subunits or confirmations of a same antigen (i.e. s' and 10's", more preferably between 10s and 60 have a different specificity with respect to the epitopes of an 10 s, such as between 10 s and 10 s. antigen). Thus, for example a “biparatopic polypeptide of Preferably, a monovalent amino acid sequence of the the invention is a polypeptide that comprises at least one invention (or a polypeptide that contains only one amino amino acid sequence directed against a first antigenic deter acid sequence of the invention) is preferably such that it will minant, epitope, part, domain, Subunit or confirmation of a bind to GPCRs with an affinity less than 500 nM, preferably 65 GPCR, and at least one further amino acid sequence directed less than 200 nM, more preferably less than 10 nM, such as against a second antigenic determinant, epitope, part, less than 500 pM. domain, subunit or confirmation of said GPCR different US 9,512,236 B2 7 8 from the first, in which said first and second amino acid system (Dahmen et al., Supra), which can detect either a sequence may optionally be linked via a linker sequence (as depolarization or hyperpolarization of the membrane poten defined herein). Accordingly, in its simplest form, a bispe tial. cific or biparatopic polypeptide of the invention is a bivalent Other techniques for screening GPCRs will be clear to the polypeptide of the invention comprising two amino acid 5 skilled person, for example from the handbooks, reviews sequences, optionally linked via a suitable linker sequence. and prior art cited herein. These include for example the The amino acid sequences and polypeptides of the inven radioligand binding assays, as for example used in Lund tion as such preferably essentially consist of a single amino strom et al., J Struct Funct Genomics. 2006 Nov. 22; Epub acid chain that is not linked via disulphide bridges to any ahead of print and as described in Andre et al., Protein Sci 10 5:1115 (2006); Hassaine et al., (2006) Prot Purif Expr other amino acid sequence or chain (but that may or may not 45:343; Nicholson et al. J Pharmacol Exp Ther. 2006 May; contain one or more intramolecular disulphide bridges. For 317(2):771-7. Epub 2006 Jan. 25. and Vilardaga et al., J example, it is known that Nanobodies—as described Biol Chem. 2001 Sep. 7:276(36):33435-43. Epub 2001 May herein—may sometimes contain a disulphide bridge 31, for example using membrane preparations that can be between CDR3 and CDR1 or FR2). However, it should be 15 made as described in Hovius et al., (1998) J Neurochem noted that one or more amino acid sequences of the inven 70:824. Some HTS techniques for screening GPCRs are tion may be linked to each other and/or to other amino acid mentioned in Table 4 of the review by Jacoby et al., sequences (e.g. via disulphide bridges) to provide peptide Also, according to the invention, amino acid sequences constructs that may also be useful in the invention (for and polypeptides that are directed against GPCRs from a example Fab' fragments, F(ab')2 fragments, ScPv constructs, first species of warm-blooded animal may or may not show "diabodies' and other multispecific constructs. Reference is cross-reactivity with GPCRs from one or more other species for example made to the review by Holliger and Hudson, of warm-blooded animal. For example, amino acid Nat Biotechnol. 2005 September; 23(9): 1126-36), which is sequences and polypeptides directed against human GPCRs incorporated herein by reference. may or may not show cross reactivity with GPCRs from one Generally, when an amino acid sequence of the invention 25 or more other species of primates (such as, without limita (or a compound, construct or polypeptide comprising the tion, monkeys from the genus Macaca (Such as, and in same) is intended for administration to a Subject (for particular, cynomolgus monkeys (Macaca fascicularis) and/ example for therapeutic and/or diagnostic purposes as or rhesus monkeys (Macaca mulata)) and baboon (Papio described herein), it is preferably either an amino acid ursinus)) and/or with GPCRs from one or more species of 30 animals that are often used in animal models for diseases sequence that does not occur naturally in said Subject; or, (for example mouse, rat, rabbit, pig or dog), and in particular when it does occur naturally in said subject, in essentially in animal models for diseases and disorders associated with isolated form (as defined herein). GPCRs (such as the species and animal models mentioned It will also be clear to the skilled person that for phar herein). In this respect, it will be clear to the skilled person maceutical use, the amino acid sequences of the invention 35 that such cross-reactivity, when present, may have advan (as well as compounds, constructs and polypeptides com tages from a drug development point of view, since it allows prising the same) are preferably directed against human the amino acid sequences and polypeptides against human GPCRs; whereas for veterinary purposes, the amino acid GPCRs to be tested in such disease models. sequences and polypeptides of the invention are preferably More generally, amino acid sequences and polypeptides directed against GPCRs from the species to be treated, or at 40 of the invention that are cross-reactive with GPCRs from least cross-reactive with GPCRs from the species to be multiple species of mammal will usually be advantageous treated. for use in veterinary applications, since it will allow the Furthermore, an amino acid sequence of the invention same amino acid sequence or polypeptide to be used across may optionally, and in addition to the at least one binding multiple species. Thus, it is also encompassed within the site for binding against GPCRs, contain one or more further 45 Scope of the invention that amino acid sequences and binding sites for binding against other antigens, proteins or polypeptides directed against GPCRs from one species of targets. animal (such as amino acid sequences and polypeptides The efficacy of the amino acid sequences and polypep against human GPCRs) can be used in the treatment of tides of the invention, and of compositions comprising the another species of animal, as long as the use of the amino same, can be tested using any suitable in vitro assay, 50 acid sequences and/or polypeptides provide the desired cell-based assay, in vivo assay and/or animal model known effects in the species to be treated. perse, or any combination thereof, depending on the specific The present invention is in its broadest sense also not disease or disorder involved. Suitable assays and animal particularly limited to or defined by a specific antigenic models will be clear to the skilled person, and for example determinant, epitope, part, domain, Subunit or conformation include the expression of the receptors in Xenopus oocytes, 55 (where applicable) of GPCRs against which the amino acid after which the coupling of many GPCRs to ion channels sequences and polypeptides of the invention are directed. allows the activation or inhibition of these GPCRs to be For example, the amino acid sequences and polypeptides monitored in oocytes via Voltage clamping techniques. Het may or may not be directed against an “interaction site' (as erologous GPCRs can be functionally expressed in the defined herein). For example, amino acid sequences of the oocyte by injecting exogenous, GPCR-encoding mRNA into 60 invention may be raised by Suitably immunizing a mammal the oocyte and then allowing 20 the oocyte's endogenous (such as a Camelid) with GPCR that has been expressed in cellular machinery to translate and insert the receptors into a suitable expression system or that has been isolated from the plasma membrane. (See, e.g., Houamed et al., Science a Suitable cell or cell fraction. In particular, amino acid 252:1318-21, 1991; Dahmen et al., J. Neurochem. 58:1176 sequences of the invention may be raised against GPCRs (or 79, 1992.) Following functional expression of receptors, the 65 suitable parts or fragments thereof) that have been refolded ability of ligands to induce transmembrane conductance (for example using the techniques described in the review by changes can be observed via a two-electrode Voltage clamp Kiefer, Biochim. Biophys. Acta, 1610 (2003), 57-62), and US 9,512,236 B2 9 10 amino acid sequences, Nanobodies and polypeptides that are more, and preferably 107 to 10" liter/moles or more directed against and/or that have been raised against a and more preferably 10 to 10' liter/moles): refolded GPCR form a further aspect of the invention. and/or such that they: The amino acid sequences and polypeptides of the inven bind to an extracellular part, region, domain, loop or other tion may generally be directed against any desired GPCR, extracellular epitope of a GPCR (as described herein) and may in particular be directed against a GPCR that has at or against a peptide derived therefrom with a k-rate of least one extracellular loop or domain. Examples of Such between 10 M's to about 107 M's', preferably GPCRs will be clear to the skilled person based on the prior between 10 M's and 107M s', more preferably art cited herein between 10" M'S' and 107 M's', such as between According to a specific aspect of the invention, an amino 10 10 M's and 107 M' s: acid sequence or polypeptide of the invention may be and/or such that they: directed against (as defined herein) a GPCR that is expressed bind to an extracellular part, region, domain, loop or other on the Surface of a cell and/or against at least one extracel extracellular epitope of a GPCR (as described herein) lular region, domain, loop or other extracellular epitope of a or against a peptide derived therefrom with a k rate GPCR. For example, Such amino acid sequences may be 15 between 1 s' (t=0.69s) and 10 s' (providing a raised by Suitably immunizing a mammal (such as a Cam near irreversible complex with at of multiple days), elid) with a cell or cell fraction that has a GPCR on its preferably between 10° s' and 10 s', more pref Surface. For example, the mammal (and in particular, Cam erably between 10 s' and 10's", such as between elid) may be suitably immunized with whole cells that 10 s. and 10 s. express (and preferably overexpress) the desired GPCR, Preferably, a monovalent amino acid sequence of the such as with cells of a cell line that (over)expresses the invention (or a polypeptide that contains only one amino desired GPCR. acid sequence of the invention) is preferably such that it will In particular, according to this aspect, an amino acid bind to an extracellular part, region, domain, loop or other sequence or polypeptide of the invention is directed against extracellular epitope of a GPCR (as described herein) with (as defined herein) at least one extracellular region, domain, 25 an affinity less than 500 nM, preferably less than 200 nM, loop or other extracellular epitope of a GPCR; and is more preferably less than 10 nM, such as less than 500 pM. preferably further Such that said amino acid sequence or Also, according to this aspect, any multivalent or multi polypeptide of the invention is capable of modulating (as specific (as defined herein) polypeptides of the invention defined herein) said GPCR. More in particular, according to may also be suitably directed against two or more different this aspect, an amino acid sequence or polypeptide of the 30 extracellular parts, regions, domains, loops or other extra invention is directed against (as defined herein) at least one cellular epitopes on the same antigen, for example against extracellular region, domain, loop or other extracellular two different extracellular loops, against two different extra epitope of a GPCR; and is preferably further such that said cellular parts of the transmembrane domains or against one amino acid sequence or polypeptide of the invention is extracellular loops and one extracellular loop. More particu capable of (fully or partially) blocking said GPCR. 35 larly, such multivalent or multispecific polypeptides may for According to this aspect of the invention, the amino acid example be suitably directed against two or more different sequence or polypeptide of the invention may be directed extracellular parts, regions, domains, loops or other extra against any suitable extracellular part, region, domain, loop cellular epitopes on the same GPCR; or such a polypeptide or other extracellular epitope of a GPCR, but is preferably may be biparatopic? and or multiparatopic (as defined directed against one of the extracellular parts of the trans 40 herein). Such multivalent or multispecific polypeptides of membrane domains or more preferably against one of the the invention may also have (or be engineered and/or extracellular loops that link the transmembrane domains. selected for) increased avidity and/or improved selectivity The amino acid sequence of Such Suitable extracellular for the desired GPCR, and/or for any other desired property parts, regions, domains, loops or epitopes may be derived by or combination of desired properties that may be obtained by Kyte-Doolittle analysis of the amino acid sequence of the 45 the use of Such multivalent or multispecific polypeptides. pertinent GPCR; by aligning GPCRs belonging to the same Generally, it is expected that amino acid sequences and (sub)families and identifying the various transmembrane polypeptides of the invention that are directed against an domains and extracellular parts, regions, domain or loops; extracellular loop or domain of a GPCR (or against a small by TMAP-analysis; or by any suitable combination thereof. peptide derived therefrom or based thereon), and/or that The invention also relates to amino acid sequences and (as 50 have been screened against, selected using and/or raised further defined herein) that are directed against and/or have against an extracellular loop or domain of a GPCR (or been raised against Such extracellular parts, regions, against a small peptide derived therefrom or based thereon) domains, loops or epitopes (and/or that are directed against will also be able to bind (and in particular, to specifically and/or have been raised against Suitable parts or fragments bind, as defined herein) to such an extracellular loop or of Such extracellular parts, regions, domains, loops or 55 domain (or peptide derived therefrom) that forms part of a epitopes and/or against Synthetic or semi-synthetic peptides GPCR (or at least one subunit thereof) that is present on the that are derived from or based on such extracellular parts, surface of a cell. Thus, such (peptides derived from) an regions, domains, loops or epitopes). extracellular loop or domain may find particular use in In particular, amino acid sequences and polypeptides of methods for generating amino acid sequences and polypep the invention are preferably such that they: 60 tides of the invention (as defined herein); and such methods bind to an extracellular part, region, domain, loop or other and uses form further aspects of the invention; as do amino extracellular epitope of a GPCR (as described herein) acid sequences, Nanobodies and polypeptides of the inven or against a peptide derived therefrom with a dissocia tion that are directed against or raised against Such an tion constant (Ki) of 10 to 10' moles/liter or less, extracellular loop, domain or peptide derived therefrom. and preferably 107 to 10° moles/liter or less and 65 For example, such a method may comprise one of the more preferably 10 to 10' moles/liter (i.e. with an following steps or a suitable combination of both of the association constant (K) of 10 to 10' liter/moles or following steps: US 9,512,236 B2 11 12 a) a step of Suitably immunizing a Camelid with a suitable sequences, Nanobodies and polypeptides of the invention antigen that comprises the desired extracellular part, may mediate modulation of the receptor function by binding region, domain, loop or other extracellular epitope(s), or to different regions in the receptor (e.g. at allosteric sites). with a suitable peptide derived therefrom or based Reference is for example made to George et al., Nat Rev thereon, such that an immune response against the desired Drug Discov 1:808-820 (2002); Kenakin, Trends Pharmacol extracellular part, region, domain, loop or other extracel Sci 25:186-192 (2002) and Rios et al., Pharmacol Ther lular epitope(s) is raised. The antigen may be any suitable 92:71-87 (2001)). antigen that is capable of raising an immune response The amino acid sequences, Nanobodies and polypeptides against the desired extracellular part, region, domain, loop of the invention may also bind to the GPCR in such a way or other extracellular epitope(s); Such as, for example and 10 that they inhibit or enhance the assembly of GPCR func without limitation, whole cells that have the desired tional homodimers or heterodimers. extracellular part, region, domain, loop or other extracel The amino acid sequences, Nanobodies and polypeptides lular epitope(s) on their surface, cell wall fragments of the invention may also bind to the GPCR in such a way thereof or any other suitable preparation derived from that they prolong the duration of the GPCR-mediated sig such cells, vesicles that have the desired extracellular part, 15 nalling. region, domain, loop or other extracellular epitope(s) on The amino acid sequences, Nanobodies and polypeptides their Surface, a subunit or fragment of a subunit of a of the invention may also enhance receptor signalling by GPCR that comprises the desired extracellular part, increasing receptor-ligand affinity. region, domain, loop or other extracellular epitope(s), or Polypeptides of the invention that are directed against a a synthetic or semi-synthetic peptide that comprises and/ GPCR and its ligand may also provide for enhanced binding or is based on (the amino acid sequence of) the desired of the ligand to the GPCR by cross-linking the ligand to the extracellular part, region, domain, loop or other extracel orthosteric site; and/or stabilize the binding of the ligand to lular epitope(s): the orthosteric site. Thus, a further aspect of the invention and/or relates to a multispecific polypeptide of the invention (as b) a step of screening for affinity and/or binding for the 25 defined herein) that comprises at least one amino acid desired extracellular part, region, domain, loop or other sequence of the invention (such as a NANOBODYR (V)) extracellular epitope(s). This may for example be per against a GPCR receptor and at least one binding unit formed by screening a set, collection or library of cells directed against its natural ligand. Such multispecific pro that express heavy chain antibodies on their Surface (e.g. teins may further be as described herein. B-cells obtained from a suitably immunized Camelid), by 30 Also, as will be clear from the further disclosure herein, screening of a (naive or immune) library of VHH and depending on the GPCR against which they are directed sequences or NANOBODYR (V) sequences, or by and their desired (therapeutic) effect, the amino acid Screening of a (naive or immune) library of nucleic acid sequences, Nanobodies and polypeptides of the invention sequences that encode VHH sequences or NANO may act as (full or partial) agonists, (full or partial, and BODYR (V) sequences; which may all be performed 35 competitive or non-competitive) antagonists or as inverse in a manner known per se, for which reference is made to agonists of the GPCR (and/or of the ligand of the GPCR) the further disclosure and prior art mentioned herein; and/or of the biological function, pathway, mechanism, and which method may optionally further comprise one or effect, signalling or response associated therewith. They more other Suitable steps known perse, Such as, for example may do so in an irreversible but preferably reversible and without limitation, a step of affinity maturation, a step of 40 a. expressing the desired amino acid sequence, a step of In one embodiment, the amino acid sequence or polypep screening for binding and/or for activity against the desired tide of the invention is a "monoclonal amino acid sequence antigen (in this case, the GPCR), a step of determining the or polypeptide, by which is meant that at least each of the desired amino acid sequence or nucleotide sequence, a step one or more amino acid sequences directed against the of introducing one or more humanizing Substitutions (e.g. as 45 GPCR that are present in said protein or polypeptide (and further described herein), a step of formatting in a suitable preferably all of the immunoglobulin sequences that are multivalent and/or multispecific format, a step of screening present in said protein or polypeptide) are "monoclonal as for the desired biological and/or physiological properties commonly understood by the skilled person. In this respect, (i.e. using a suitable assay, Such as those described herein); it should however be noted that, as further described herein, and/or any Suitable combination of one or more of Such 50 the present invention explicitly covers multivalent or mul steps, in any Suitable order. tispecific proteins that comprise two or more immunoglobu Such methods and the amino acid sequences obtained via lin sequences (and in particular monoclonal immunoglobu Such methods, as well as proteins and polypeptides com lin sequences) that are directed against different parts, prising or essentially consisting of the same, form further regions, domains, loops or epitopes of the same GPCR, and aspects of this invention. 55 in particular against different extracellular parts, regions, The amino acid sequences, Nanobodies and polypeptides domains, loops or epitopes of the same GPCR. of the invention may also bind to the GPCR at the same site As further described herein, a polypeptide of the inven as the endogenous agonist (i.e. at an orthosteric site), so as tion may contain two or more amino acid sequences of the to activate or increase receptor signalling; or alternatively so invention that are directed against a GPCR. Generally, such as to decrease or inhibit receptor signalling. 60 polypeptides will bind to a GPCR with increased avidity The amino acid sequences, Nanobodies and polypeptides compared to a single amino acid sequence of the invention. of the invention may also bind to the GPCR in such a way Such a polypeptide may for example comprise two amino that they block the constitutive activity of the GPCR. acid sequences of the invention that are directed against the The amino acid sequences, Nanobodies and polypeptides same antigenic determinant, epitope, part, domain, Subunit of the invention may also bind to the GPCR in such a way 65 or conformation (where applicable) of a GPCR (which may that they mediate allosteric modulation (e.g. bind to the or may not be an interaction site); or comprise at least one GPCR at an allosteric site). In this way, the amino acid “first amino acid sequence of the invention that is directed US 9,512,236 B2 13 14 against a first same antigenic determinant, epitope, part, ments with an affinity and/or specificity that are the same as, domain, Subunit or conformation (where applicable) of a or that are different from (i.e. higher than or lower than), the GPCR (which may or may not be an interaction site); and at affinity and specificity with which the amino acid sequences least one 'second amino acid sequence of the invention that of the invention bind to (wild-type) GPCRs. It is also is directed against a second antigenic determinant, epitope, included within the scope of the invention that the amino part, domain, Subunit or conformation (where applicable) acid sequences and polypeptides of the invention bind to different from the first (and which again may or may not be Some analogs, variants, mutants, alleles, parts and fragments an interaction site). Preferably, in such “biparatopic poly of GPCRs, but not to others. peptides of the invention, at least one amino acid sequence When GPCRs exists in a monomeric form and in one or of the invention is directed against an interaction site (as 10 more multimeric forms, it is within the scope of the inven defined herein), although the invention in its broadest sense tion that the amino acid sequences and polypeptides of the is not limited thereto. invention only bind to GPCRs in monomeric form, only bind For example, when a polypeptide of the invention is a to GPCRs in multimeric form, or bind to both the mono biparatopic polypeptide of the invention, it may contain at meric and the multimeric form. Again, in Such a case, the least one amino acid sequence of the invention (such as a 15 amino acid sequences and polypeptides of the invention may NANOBODYR (V)) that is directed against a first extra bind to the monomeric form with an affinity and/or speci cellular part, region, domain, loop or other extracellular ficity that are the same as, or that are different from (i.e. epitope of a GPCR and at least one amino acid sequence of higher than or lower than), the affinity and specificity with the invention (such as a NANOBODYR (V)) that is which the amino acid sequences of the invention bind to the directed against a second extracellular part, region, domain, multimeric form. loop or other extracellular epitope of a GPCR different from Also, when GPCRs can associate with other proteins or said first extracellular part, region, domain, loop or other polypeptides to form protein complexes (e.g. with multiple extracellular epitope. subunits), it is within the scope of the invention that the Also, when the target is part of a binding pair (for amino acid sequences and polypeptides of the invention bind example, a receptor-ligand binding pair), the amino acid 25 to GPCRs in its non-associated state, bind to GPCRs in its sequences and polypeptides may be such that they compete associated State, or bind to both. In all these cases, the amino with the cognate binding partner (e.g. the ligand, receptor or acid sequences and polypeptides of the invention may bind other binding partner, as applicable) for binding to the target, to Such multimers or associated protein complexes with an and/or such that they (fully or partially) neutralize binding of affinity and/or specificity that may be the same as or different the binding partner to the target. 30 from (i.e. higher than or lower than) the affinity and/or It is also within the scope of the invention that, where specificity with which the amino acid sequences and poly applicable, an amino acid sequence of the invention can bind peptides of the invention bind to GPCRs in its monomeric to two or more antigenic determinants, epitopes, parts, and non-associated State. domains, subunits or conformations of GPCRs. In such a Also, as will be clear to the skilled person, proteins or case, the antigenic determinants, epitopes, parts, domains or 35 polypeptides that contain two or more amino acid sequences subunits of GPCRs to which the amino acid sequences directed against GPCRs may bind with higher avidity to and/or polypeptides of the invention bind may be essentially GPCRs than the corresponding monomeric amino acid the same (for example, if GPCRs contains repeated struc sequence(s). For example, and without limitation, proteins tural motifs or occurs in a multimeric form) or may be or polypeptides that contain two or more amino acid different (and in the latter case, the amino acid sequences 40 sequences directed against different epitopes of GPCRs (e.g. and polypeptides of the invention may bind to such different biparatopic polypeptides) may (and usually will) bind with antigenic determinants, epitopes, parts, domains, subunits of higher avidity than each of the different monomers. Proteins GPCRs with an affinity and/or specificity which may be the or polypeptides that contain two or more amino acid same or different). Also, for example, when GPCRs exists in sequences directed against GPCRS may (and usually will) an activated conformation and in an inactive conformation, 45 bind also with higher avidity to a multimer of GPCRs. The the amino acid sequences and polypeptides of the invention inventors have prepared Such polypeptides in accordance may bind to either one of these conformations, or may bind with the invention. The amino acid sequences of monovalent to both these conformations (i.e. with an affinity and/or and bivalent polypeptides directed against different repre specificity which may be the same or different). Also, for sentative GPCRs are shown in Tables B-4, J and M as example, the amino acid sequences and polypeptides of the 50 described herein. For example, polypeptides of the invention invention may bind to a conformation of GPCRs in which it may be formatted e.g., in a biparatopic way Such as to is bound to a pertinent ligand, may bind to a conformation combine two monomeric amino acid sequences against of GPCRs in which it is not bound to a pertinent ligand, or different epitopes as characterized in the experimental part may bind to both Such conformations (again with an affinity (see Examples 5A, 6 and 7). Of these, particularly preferred and/or specificity which may be the same or different). 55 polypeptides in accordance with the invention are It is also expected that the amino acid sequences and biparatopic polypeptides directed against GPCRs. Preferred polypeptides of the invention will generally bind to all examples hereof are the biparatopic constructs against naturally occurring or synthetic analogs, variants, mutants, human CXCR4 as previously described in WO09/138519. alleles, parts and fragments of GPCRs; or at least to those Particularly preferred polypeptides are the biparatopic con analogs, variants, mutants, alleles, parts and fragments of 60 structs that comprise 238D2 and/or 238D4 (see for example GPCRs that contain one or more antigenic determinants or SEQID NO:s 261 to 266 in WO 09/138519 or SEQID NO:s epitopes that are essentially the same as the antigenic 528 to 531 herein, and more particularly 238D2-20GS determinant(s) or epitope(s) to which the amino acid 238D4). As will be clear to the skilled person, polypeptides sequences and polypeptides of the invention bind in GPCRs that are bivalent may (and usually will) bind with higher (e.g. in wild-type GPCRs). Again, in Such a case, the amino 65 avidity to their target than a corresponding monovalent acid sequences and polypeptides of the invention may bind binder. However present inventors have demonstrated for to Such analogs, variants, mutants, alleles, parts and frag the first time that the biparatopic polypeptides of the inven US 9,512,236 B2 15 16 tion (e.g. polypeptides comprising two amino acid they comprise an immunoglobulin fold or are capable for sequences which are directed against different epitopes of forming, under Suitable conditions, an immunoglobulin fold. the same target as defined herein) completely unexpectedly In particular, but without limitation, the amino acid resulted in a significantly increased affinity. This affinity was sequences of the invention may be amino acid sequences much higher to that obtained in the mere bivalent constructs that essentially consist of 4 framework regions (FR1 to FR4 which mono-specifically bind to their target, i.e. to the same respectively) and 3 complementarity determining regions epitope. Thus, by constructing biparatopic constructs (CDR1 to CDR3 respectively); or any suitable fragment of directed against GPCRs the inventors were able to signifi Such an amino acid sequence (which will then usually cantly increase the potency compared to corresponding contain at least Some of the amino acid residues that form at monovalent and mono-specific bivalent binders. These spe 10 least one of the CDR's, as further described herein). cial technical features will become clear from the experi The amino acid sequences of the invention may in par mental data provided herein. ticular be an immunoglobulin sequence or a Suitable frag Generally, amino acid sequences and polypeptides of the ment thereof, and more in particular be an immunoglobulin invention will at least bind to those forms of GPCRs variable domain sequence or a Suitable fragment thereof, (including monomeric, multimeric and associated forms) 15 Such as light chain variable domain sequence (e.g. a V that are the most relevant from a biological and/or thera sequence) or a Suitable fragment thereof, or a heavy chain peutic point of view, as will be clear to the skilled person. variable domain sequence (e.g. a V-sequence) or a Suitable It is also within the scope of the invention to use parts, fragment thereof. When the amino acid sequence of the fragments, analogs, mutants, variants, alleles and/or deriva invention is a heavy chain variable domain sequence, it may tives of the amino acid sequences and polypeptides of the be a heavy chain variable domain sequence that is derived invention, and/or to use proteins or polypeptides comprising from a conventional four-chain antibody (such as, without or essentially consisting of one or more of Such parts, limitation, a V sequence that is derived from a human fragments, analogs, mutants, variants, alleles and/or deriva antibody) or be a so-called V-sequence (as defined tives, as long as these are Suitable for the uses envisaged herein) that is derived from a so-called “heavy chain anti herein. Such parts, fragments, analogs, mutants, variants, 25 body” (as defined herein). alleles and/or derivatives will usually contain (at least part However, it should be noted that the invention is not of) a functional antigen-binding site for binding against limited as to the origin of the amino acid sequence of the GPCRs; and more preferably will be capable of specific invention (or of the nucleotide sequence of the invention binding to GPCRs, and even more preferably capable of used to express it), nor as to the way that the amino acid binding to GPCRs with an affinity (suitably measured and/or 30 sequence or nucleotide sequence of the invention is (or has expressed as a K-value (actual or apparent), a K-value been) generated or obtained. Thus, the amino acid sequences (actual or apparent), a k-rate and/or a k-rate, or alterna of the invention may be naturally occurring amino acid tively as an ICso value, as further described herein) that is as sequences (from any Suitable species) or synthetic or semi defined herein. Some non-limiting examples of Such parts, synthetic amino acid sequences. In a specific but non fragments, analogs, mutants, variants, alleles, derivatives, 35 limiting aspect of the invention, the amino acid sequence is proteins and/or polypeptides will become clear from the a naturally occurring immunoglobulin sequence (from any further description herein. Additional fragments or polypep Suitable species) or a synthetic or semi-synthetic immuno tides of the invention may also be provided by suitably globulin sequence, including but not limited to "humanized' combining (i.e. by linking or genetic fusion) one or more (as defined herein) immunoglobulin sequences (such as (Smaller) parts or fragments as described herein. 40 partially or fully humanized mouse or rabbit immunoglobu In one specific, but non-limiting aspect of the invention, lin sequences, and in particular partially or fully humanized which will be further described herein, such analogs, V. sequences or Nanobodies), "camelized” (as defined mutants, variants, alleles, derivatives have an increased herein) immunoglobulin sequences, as well as immuno half-life in serum (as further described herein) compared to globulin sequences that have been obtained by techniques the amino acid sequence from which they have been derived. 45 Such as affinity maturation (for example, starting from For example, an amino acid sequence of the invention may synthetic, random or naturally occurring immunoglobulin be linked (chemically or otherwise) to one or more groups sequences), CDR grafting, Veneering, combining fragments or moieties that extend the half-life (such as PEG), so as to derived from different immunoglobulin sequences, PCR provide a derivative of an amino acid sequence of the assembly using overlapping primers, and similar techniques invention with increased half-life. 50 for engineering immunoglobulin sequences well known to In one specific, but non-limiting aspect, the amino acid the skilled person; or any suitable combination of any of the sequence of the invention may be an amino acid sequence foregoing. Reference is for example made to the standard that comprises an immunoglobulin fold or may be an amino handbooks, as well as to the further description and prior art acid sequence that, under Suitable conditions (such as physi mentioned herein. ological conditions) is capable of forming an immunoglobu 55 Similarly, the nucleotide sequences of the invention may lin fold (i.e. by folding). Reference is interalia made to the be naturally occurring nucleotide sequences or synthetic or review by Halaby et al., J. (1999) Protein Eng. 12, 563-71. semi-synthetic sequences, and may for example be Preferably, when properly folded so as to form an immu sequences that are isolated by PCR from a suitable naturally noglobulin fold, Such an amino acid sequence is capable of occurring template (e.g. DNA or RNA isolated from a cell), specific binding (as defined herein) to GPCRs; and more 60 nucleotide sequences that have been isolated from a library preferably capable of binding to GPCRs with an affinity (and in particular, an expression library), nucleotide (Suitably measured and/or expressed as a K-value (actual sequences that have been prepared by introducing mutations or apparent), a K-value (actual or apparent), a k-rate into a naturally occurring nucleotide sequence (using any and/or a k-rate, or alternatively as an ICso value, as further Suitable technique known per se, Such as mismatch PCR). described herein) that is as defined herein. Also, parts, 65 nucleotide sequence that have been prepared by PCR using fragments, analogs, mutants, variants, alleles and/or deriva overlapping primers, or nucleotide sequences that have been tives of Such amino acid sequences are preferably such that prepared using techniques for DNA synthesis known perse. US 9,512,236 B2 17 18 The amino acid sequence of the invention may in par In particular, a NANOBODYR (V) can be an amino ticular be a domain antibody (or an amino acid sequence that acid sequence with the (general) structure is suitable for use as a domain antibody), a single domain FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 antibody (or an amino acid sequence that is Suitable for use in which FR1 to FR4 refer to framework regions 1 to 4, as a single domain antibody), a “dAb’ (or an amino acid respectively, and in which CDR1 to CDR3 refer to the sequence that is suitable for use as a dAb) or a NANO complementarity determining regions 1 to 3, respectively, BODYR (V) (as defined herein, and including but not and in which the framework sequences are as further defined limited to a V sequence); other single variable domains, herein. or any suitable fragment of any one thereof. Note: domain More in particular, a NANOBODYR (V) can be an 10 amino acid sequence with the (general) structure antibody, domain antibodies, dAb and dAbs are trademarks FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 of the GlaxoSmithKline group of companies. in which FR1 to FR4 refer to framework regions 1 to 4, For a general description of (single) domain antibodies, respectively, and in which CDR1 to CDR3 refer to the reference is also made to the prior art cited above, as well as complementarity determining regions 1 to 3, respectively, to EP 0 368 684. For the term “dAbs’, reference is for and in which: example made to Ward et al. (Nature 1989 Oct. 12: 341 i) preferably one or more of the amino acid residues at (6242): 544-6), to Holt et al., Trends Biotechnol., 2003, positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 21 (11):484-490; as well as to for example WO 06/030220, according to the Kabat numbering are chosen from the WO 06/003388 and other published patent applications of Hallmark residues mentioned in Table A-3 below: Domantis Ltd. It should also be noted that, although less and in which: preferred in the context of the present invention because ii) said amino acid sequence has at least 80% amino acid they are not of mammalian origin, single domain antibodies identity with at least one of the amino acid sequences of or single variable domains can be derived from certain SEQ ID NO’s: 1 to 22, in which for the purposes of species of shark (for example, the so-called “IgNAR determining the degree of amino acid identity, the amino domains”, see for example WO 05/18629). 25 acid residues that form the CDR sequences (indicated In particular, the amino acid sequence of the invention with X in the sequences of SEQ ID NO’s: 1 to 22) are may be a NANOBODYR (V) (as defined herein) or a disregarded. suitable fragment thereof. Note: Nanobody(R), Nanobod In these Nanobodies, the CDR sequences are generally as ies(R) and Nanoclone(R) are registered trademarks of Ablynx further defined herein. N.V. Such Nanobodies directed against GPCRs will also be 30 Thus, the invention also relates to such Nanobodies that referred to herein as “Nanobodies of the invention'. The can bind to (as defined herein) and/or are directed against terms “amino acid sequence” and “Nanobody' are used GPCRs, to suitable fragments thereof, as well as to poly interchangeably herein, unless the context indicates other peptides that comprise or essentially consist of one or more wise. of such Nanobodies and/or suitable fragments. 35 SEQID NO’s 526 to 527, 538 to 541, 413 to 453 and 517 For a general description of Nanobodies, reference is to 525 give the amino acid sequences of a number of V. made to the further description below, as well as to the prior sequences that have been raised against five representative art cited herein. In this respect, it should however be noted GPCRs, and in particular against the Chemokine Receptors that this description and the prior art mainly described CXCR4 and CXCR7, the Cannabinoid Receptor 1 (CB1R, Nanobodies of the so-called “V3 class” (i.e. Nanobodies 40 see for example Pacher et al. Pharmacol Rev 58:389-462); with a high degree of sequence homology to human germ the parathyroid hormone receptor (PTHR1, see for example line sequences of the V3 class such as DP-47, DP-51 or Gensure et al. Biochem Biophys Res Commun 328:666 DP-29), which Nanobodies form a preferred aspect of this 678); and (MC4R), which are given invention. It should however be noted that the invention in in SEQID NO’s 526 to 527; SEQID NO's 538 to 541; 413 its broadest sense generally covers any type of NANO 45 to 430, 517 and 518; SEQID NO’s 431 to 453; and SEQID BODYR (V) directed against GPCRs, and for example NO’s: 519 to 525, respectively. also covers the Nanobodies belonging to the so-called “V4 In one embodiment, the amino acid sequences and pep class” (i.e. Nanobodies with a high degree of sequence tides of the invention (which may be as further described homology to human germline sequences of the V4 class herein) are directed against the chemokine receptor CXCR4, such as DP-78), as for example described in the U.S. 50 preferably human CXCR4 (Table N, SEQ ID NO: 584). provisional application 60/792.279 by Ablynx N.V. entitled It is expected that amino acid sequences, Nanobodies and “DP-78-like Nanobodies' filed on Apr. 14, 2006 (see also polypeptides of the invention directed against CXCR4, as WO07/118670). well as compositions comprising the same, may find par Generally, Nanobodies (in particular V. sequences and ticular use in the prevention and treatment of diseases 55 involving CXCR4 mediated signaling, Such as the various partially humanized Nanobodies) can in particular be char diseases in the group of cancer Such as hematopoietic acterized by the presence of one or more “Hallmark resi like CLL, AML. ALL, MM, Non-Hodgkin lym dues” (as described herein) in one or more of the framework phoma, Solid tumors such as breast cancer, lung cancer, sequences (again as further described herein). brain tumors, ovarian cancer, Stromal chemoresistance of Thus, generally, a NANOBODYR (V) can be defined 60 tumors, leukemia and other cancers, disrupting adhesive as an amino acid sequence with the (general) structure stromal interactions that confer tumor cell Survival and drug FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 resistance, mobilizing tumour cells from tissue sites and in which FR1 to FR4 refer to framework regions 1 to 4, making them better accessible to conventional therapy, respectively, and in which CDR1 to CDR3 refer to the inhibiting of migration and dissemination of tumor cells complementarity determining regions 1 to 3, respectively, 65 (metastasis), inhibiting or paracrine growth and Survival and in which one or more of the Hallmark residues are as signals, inhibiting pro-angiogenesis effects of SDF-1, further defined herein. and inflammatory disorders such as bowel US 9,512,236 B2 19 20 diseases (colitis, Crohn disease, IBD), infectious diseases, tion directed against Such receptors, and in particular against psioriasis, autoimmune diseases (such as MS), Sarcoidosis, MC4R, as well as compositions comprising the same, may transplant rejection, cystic fibrosis, , chronic obstruc find particular use in the prevention and treatment of dis tive pulmonary disease, , viral infection, eases and disorders associated with melanocortin receptors HIV. West Nile Virus encephalitis, common variable immu (e.g. with insufficient, undesired or abnormal signalling nodeficiency. Furthermore, the amino acid sequences of the mediated by melanocortin receptors). Such as diseases and invention can be used for stem cell mobilization in various disorders that are caused by and/or associated with aberra patients in need of stem cells after X-ray radiation Such as tions in physiological processes such as energy homeostasis e.g. cancer patients after radiation treatment to replenish the (for example, cachexia and/or obesity), immunity, inflam stem cell pool after radiation in cancer patients, or in patients 10 mation, sexual function, pigmentation and neurite out in need of more stem cells, e.g. in patients with ischemic growth. Reference is inter alia made to the review by diseases such as myocardial infarction (MI), stroke and/or Bednarek and Fong, Expert Opin. Ther. Patents (2004) diabetes (i.e. patients in need of tissue repair) wherein more 14(3): 327-336, which also lists a number of synthetic stem cell would be re-transfused (after mobilization, screen ligands for receptors belonging to the family of melanocor ing, selection for lineage in need (e.g. cardiac, Vascular 15 tin receptors, and it is expected that the amino acid lineages) and ex-Vivo expansion of patient’s own stem sequences and polypeptides of the invention may be used for cells). all diseases and disorders that arefcan be treated with such In one embodiment, the amino acid sequences and pep ligands and/or for which said ligands are being developed. tides of the invention (which may be as further described Reference is also made to the review by Getting, S.J. 2006, herein) are directed against the chemokine receptor CXCR7. Pharmacol Ther 111:1-15. preferably human CXCR7 (Table N, SEQ ID NO: 585). Accordingly, some particularly preferred Nanobodies of It is expected that amino acid sequences, Nanobodies and the invention are Nanobodies which can bind (as further polypeptides of the invention directed against CXCR7, as defined herein) to and/or are directed against to GPCRs and well as compositions comprising the same, may find par which: ticular use in the prevention and treatment of diseases 25 i) have at least 80% amino acid identity with at least one of involving CXCR7 mediated metastasis, chemotaxis, cell the amino acid sequences of SEQ ID NO’s: 526 to 527, adhesion, trans endothelial migration, cell proliferation and/ 538 to 541, 413 to 453 and 517 to 525, in which for the or survival. purposes of determining the degree of amino acid identity, In one embodiment, the amino acid sequences and pep the amino acid residues that form the CDR sequences are tides of the invention (which may be as further described 30 disregarded. In this respect, reference is also made to herein) are directed against a receptor (i.e. a GPCR) from the Table A-1, which lists the framework 1 sequences (SEQ family of cannabinoid receptor family ID NO’s: 542 to 547: 126 to 166 and 454 to 462), It is expected that amino acid sequences, Nanobodies and framework 2 sequences (SEQ ID NO’s: 554 to 559; 208 polypeptides of the invention of the invention directed to 248 and 472 to 480), framework 3 sequences (SEQ ID against CB1R (and in particular antagonists), as well as 35 NO’s: 566 to 571; 290 to 330 and 490 to 498) and compositions comprising the same, may find particular use framework 4 sequences (SEQ ID NO’s: 578 to 583; 372 in the prevention and treatment of for example cachexia and to 412 and 508 to 516) of the Nanobodies of SEQ ID anorexia; pain and inflammation; mental disorders such as NO’s: 526 to 527, 538 to 541, 413 to 453 and 517 to 525 Schizophrenia; anxiety and depression; drug abuse disorders (with respect to the amino acid residues at positions 1 to and insomnia: Syndrome X: nicotine dependence; obesity; 40 4 and 27 to 30 of the framework 1 sequences, reference psychosis; atherosclerosis; hypotension; lipid metabolism is also made to the comments made below. Thus, for disorders; metabolic disorders; non-insulin dependent dia determining the degree of amino acid identity, these betes; as an appetite Suppressant. Antagonists of CB1R may residues are preferably disregarded); also find use as antiarteriosclerotic agents; antipsychotic and in which: agents; hypoglycemic agents; and antihyperlipidemic 45 ii) preferably one or more of the amino acid residues at agents. positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 In one embodiment, the amino acid sequences and pep according to the Kabat numbering are chosen from the tides of the invention (which may be as further described Hallmark residues mentioned in Table A-3 below. herein) are directed against a receptor (i.e. a GPCR) from the In these Nanobodies, the CDR sequences are generally as family of parathyroid hormone receptors (for examples, 50 further defined herein. receptors for parathyroid hormone (PTH) and/or parathyroid Again, Such Nanobodies may be derived in any Suitable hormone-like hormone (PTHLH)). manner and from any suitable source, and may for example It is expected that amino acid sequences, Nanobodies and be naturally occurring V. sequences (i.e. from a Suitable polypeptides of the invention of the invention directed species of Camelid) or synthetic or semi-synthetic amino against PTHR1, as well as compositions comprising the 55 acid sequences, including but not limited to "humanized” (as same, may find particular use in the prevention and treat defined herein) Nanobodies, “camelized' (as defined herein) ment of osteoporosis and disorders of bone metabolism immunoglobulin sequences (and in particular camelized (agonist); hyperparathyroidism (antagonist) and Jansen’s heavy chain variable domain sequences), as well as Nano chondrodysplasia (). Antagonists of PTHR1 bodies that have been obtained by techniques such as affinity may also find use as agents for the prevention or treatment 60 maturation (for example, starting from Synthetic, random or of hypercalcemia; bone metastases and cachexia. naturally occurring immunoglobulin sequences), CDR graft In one embodiment, the amino acid sequences and poly ing, Veneering, combining fragments derived from different peptides of the invention (which may be as further described immunoglobulin sequences, PCR assembly using overlap herein) are directed against a receptor from the family of ping primers, and similar techniques for engineering immu melanocortin receptors, such as MC1R, MC2R, MC3R, 65 noglobulin sequences well known to the skilled person; or MC4R and MC4R. It is expected that amino acid sequences, any suitable combination of any of the foregoing as further Nanobodies and polypeptides of the invention of the inven described herein. Also, when a NANOBODYR (V) com US 9,512,236 B2 21 22 prises a V sequence, said NANOBODYR (V) may be such CDR sequences, as further described herein), they will suitably humanized, as further described herein, so as to also generally be referred to herein as “CDR sequences” (i.e. provide one or more further (partially or fully) humanized as CDR1 sequences, CDR2 sequences and CDR3 sequences, Nanobodies of the invention. Similarly, when a NANO respectively). It should however be noted that the invention BODYR (V) comprises a synthetic or semi-synthetic in its broadest sense is not limited to a specific structural role sequence (such as a partially humanized sequence), said or function that these stretches of amino acid residues may NANOBODYR (V) may optionally be further suitably have in an amino acid sequence of the invention, as long as humanized, again as described herein, again so as to provide these stretches of amino acid residues allow the amino acid one or more further (partially or fully) humanized Nano sequence of the invention to bind to GPCRs. Thus, gener bodies of the invention. 10 ally, the invention in its broadest sense comprises any amino In particular, humanized Nanobodies may be amino acid acid sequence that is capable of binding to GPCRs and that sequences that are as generally defined for Nanobodies in comprises one or more CDR sequences as described herein, the previous paragraphs, but in which at least one amino acid and in particular a suitable combination of two or more Such residue is present (and in particular, in at least one of the CDR sequences, that are suitably linked to each other via framework residues) that is and/or that corresponds to a 15 one or more further amino acid sequences, such that the humanizing Substitution (as defined herein). Some preferred, entire amino acid sequence forms a binding domain and/or but non-limiting humanizing Substitutions (and Suitable binding unit that is capable of binding to GPCRs. It should combinations thereof) will become clear to the skilled however also be noted that the presence of only one such person based on the disclosure herein. In addition, or alter CDR sequence in an amino acid sequence of the invention natively, other potentially useful humanizing Substitutions may by itself already be sufficient to provide an amino acid can be ascertained by comparing the sequence of the frame sequence of the invention that is capable of binding to work regions of a naturally occurring V. sequence with the GPCRs; reference is for example again made to the so-called corresponding framework sequence of one or more closely “Expedite fragments' described in WO 03/050531. related human V. sequences, after which one or more of the Thus, in another specific, but non-limiting aspect, the potentially useful humanizing Substitutions (or combina 25 amino acid sequence of the invention may be an amino acid tions thereof) thus determined can be introduced into said sequence that comprises at least one amino acid sequence V. sequence (in any manner known per se, as further that is chosen from the group consisting of the CDR1 target, for stability, for ease and level of expression, and/or sequences, CDR2 sequences and CDR3 sequences that are for other desired properties. In this way, by means of a described herein (or any suitable combination thereof). In limited degree of trial and error, other suitable humanizing 30 particular, an amino acid sequence of the invention may be substitutions (or suitable combinations thereof) can be deter an amino acid sequence that comprises at least one antigen mined by the skilled person based on the disclosure herein. binding site, wherein said antigen binding site comprises at Also, based on the foregoing, (the framework regions of) a least one amino acid sequence that is chosen from the group NANOBODYR (V) may be partially humanized or fully consisting of the CDR1 sequences, CDR2 sequences and humanized. 35 CDR3 sequences that are described herein (or any suitable Some particularly preferred humanized Nanobodies of the combination thereof). invention are humanized variants of the Nanobodies of SEQ Generally, in this aspect of the invention, the amino acid ID NO’s: 526 to 527, 538 to 541, 413 to 453 and/or 517 to sequence of the invention may be any amino acid sequence 525. that comprises at least one stretch of amino acid residues, in Thus, some other preferred Nanobodies of the invention 40 which said stretch of amino acid residues has an amino acid are Nanobodies which can bind (as further defined herein) to sequence that corresponds to the sequence of at least one of GPCRs and which: the CDR sequences described herein. Such an amino acid i) are a humanized variant of one of the amino acid sequence may or may not comprise an immunoglobulin fold. sequences of SEQID NO’s: 526 to 527, 538 to 541, 413 For example, and without limitation, Such an amino acid to 453 and/or 517 to 525; and/or 45 sequence may be a suitable fragment of an immunoglobulin ii) have at least 80% amino acid identity with at least one of sequence that comprises at least one such CDR sequence, the amino acid sequences of SEQ ID NO’s: 526 to 527, but that is not large enough to form a (complete) immuno 538 to 541, 413 to 453 and/or 517 to 525, in which for the globulin fold (reference is for example again made to the purposes of determining the degree of amino acid identity, “Expedite fragments' described in WO 03/050531). Alter the amino acid residues that form the CDR sequences are 50 natively, such an amino acid sequence may be a Suitable disregarded; “protein scaffold' that comprises least one stretch of amino and in which: acid residues that corresponds to Such a CDR sequence (i.e. i) preferably one or more of the amino acid residues at as part of its antigen binding site). Suitable scaffolds for positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 presenting amino acid sequences will be clear to the skilled according to the Kabat numbering are chosen from the 55 person, and for example comprise, without limitation, to Hallmark residues mentioned in Table A-3 below. binding scaffolds based on or derived from immunoglobu According to another specific aspect of the invention, the lins (i.e. other than the immunoglobulin sequences already invention provides a number of stretches of amino acid described herein), protein scaffolds derived from protein A residues (i.e. Small peptides) that are particularly Suited for domains (such as AffibodiesTM), tendamistat, fibronectin, binding to GPCRs. These stretches of amino acid residues 60 lipocalin, CTLA-4, T-cell receptors, designed ankyrin may be present in, and/or may be corporated into, an amino repeats, avimers and PDZ domains (Binz et al., Nat. Biotech acid sequence of the invention, in particular in Such a way 2005, Vol 23:1257), and binding moieties based on DNA or that they form (part of) the antigen binding site of an amino RNA including but not limited to DNA or RNA aptamers acid sequence of the invention. As these stretches of amino (Ulrich et al., Comb Chem High Throughput Screen 2006 acid residues were first generated as CDR sequences of 65 9(8):619-32). heavy chain antibodies or V. sequences that were raised Again, any amino acid sequence of the invention that against GPCRs (or may be based on and/or derived from comprises one or more of these CDR sequences is preferably US 9,512,236 B2 23 24 such that it can specifically bind (as defined herein) to g) the amino acid sequences of SEQ ID NO’s: 572 to 577; GPCRs, and more in particular such that it can bind to 331 to 371 and/or 499 to 507: GPCRs with an affinity (suitably measured and/or expressed h) amino acid sequences that have at least 80% amino acid as a K-value (actual or apparent), a K-value (actual or identity with at least one of the amino acid sequences of apparent), a k-rate and/or a k-rate, or alternatively as an SEQ ID NO’s: 572 to 577; 331 to 371 and/or 499 to 507: ICso value, as further described herein), that is as defined i) amino acid sequences that have 3, 2, or 1 amino acid herein. difference with at least one of the amino acid sequences More in particular, the amino acid sequences according to of SEQ ID NO’s: 572 to 577; 331 to 371 and/or 499 to this aspect of the invention may be any amino acid sequence 507; that comprises at least one antigen binding site, wherein said 10 or any suitable combination thereof. antigen binding site comprises at least two amino acid When an amino acid sequence of the invention contains sequences that are chosen from the group consisting of the one or more amino acid sequences according to b) and/or c): CDR1 sequences described herein, the CDR2 sequences 1) any amino acid substitution in Such an amino acid described herein and the CDR3 sequences described herein, 15 sequence according to b) and/or c) is preferably, and Such that (i) when the first amino acid sequence is chosen compared to the corresponding amino acid sequence from the CDR1 sequences described herein, the second according to a), a conservative amino acid Substitution, amino acid sequence is chosen from the CDR2 sequences (as defined herein); described herein or the CDR3 sequences described herein; and/or (ii) when the first amino acid sequence is chosen from the 2) the amino acid sequence according to b) and/or c) CDR2 sequences described herein, the second amino acid preferably only contains amino acid substitutions, and no sequence is chosen from the CDR1 sequences described amino acid deletions or insertions, compared to the cor herein or the CDR3 sequences described herein; or (iii) responding amino acid sequence according to a); when the first amino acid sequence is chosen from the CDR3 and/or sequences described herein, the second amino acid sequence 25 3) the amino acid sequence according to b) and/or c) may be is chosen from the CDR1 sequences described herein or the an amino acid sequence that is derived from an amino acid CDR3 sequences described herein. sequence according to a) by means of affinity maturation Even more in particular, the amino acid sequences of the using one or more techniques of affinity maturation invention may be amino acid sequences that comprise at known per se. least one antigen binding site, wherein said antigen binding 30 Similarly, when an amino acid sequence of the invention site comprises at least three amino acid sequences that are contains one or more amino acid sequences according to e) chosen from the group consisting of the CDR1 sequences and/or f): described herein, the CDR2 sequences described herein and 1) any amino acid substitution in Such an amino acid the CDR3 sequences described herein, such that the first sequence according to e) and/or f) is preferably, and amino acid sequence is chosen from the CDR1 sequences 35 compared to the corresponding amino acid sequence described herein, the second amino acid sequence is chosen according to d), a conservative amino acid Substitution, from the CDR2 sequences described herein, and the third (as defined herein); amino acid sequence is chosen from the CDR3 sequences and/or described herein. Preferred combinations of CDR1, CDR2 2) the amino acid sequence according to e) and/or f) pref and CDR3 sequences will become clear from the further 40 erably only contains amino acid Substitutions, and no description herein. As will be clear to the skilled person, amino acid deletions or insertions, compared to the cor Such an amino acid sequence is preferably an immunoglobu responding amino acid sequence according to d); lin sequence (as further described herein), but it may for and/or example also be any other amino acid sequence that com 3) the amino acid sequence according to e) and/or f) may be prises a Suitable scaffold for presenting said CDR sequences. 45 an amino acid sequence that is derived from an amino acid Thus, in one specific, but non-limiting aspect, the inven sequence according to d) by means of affinity maturation tion relates to an amino acid sequence directed against using one or more techniques of affinity maturation GPCRs, that comprises one or more stretches of amino acid known per se. residues chosen from the group consisting of: Also, similarly, when an amino acid sequence of the a) the amino acid sequences of SEQ ID NO’s: 548 to 553; 50 invention contains one or more amino acid sequences 167 to 207 and/or 463 to 471; according to h) and/or i): b) amino acid sequences that have at least 80% amino acid 1) any amino acid substitution in Such an amino acid identity with at least one of the amino acid sequences of sequence according to h) and/or i) is preferably, and SEQID NO’s: 548 to 553; 167 to 207 and/or 463 to 471; compared to the corresponding amino acid sequence c) amino acid sequences that have 3, 2, or 1 amino acid 55 according to g), a conservative amino acid Substitution, difference with at least one of the amino acid sequences (as defined herein); of SEQ ID NO’s: 548 to 553; 167 to 207 and/or 463 to and/or 471; 2) the amino acid sequence according to h) and/or i) pref d) the amino acid sequences of SEQ ID NO’s: 560 to 565: erably only contains amino acid Substitutions, and no 249 to 289 and/or 481 to 489: 60 amino acid deletions or insertions, compared to the cor e) amino acid sequences that have at least 80% amino acid responding amino acid sequence according to g); identity with at least one of the amino acid sequences of and/or SEQID NO’s: 560 to 565; 249 to 289 and/or 481 to 489: 3) the amino acid sequence according to h) and/or i) may be f) amino acid sequences that have 3, 2, or 1 amino acid an amino acid sequence that is derived from an amino acid difference with at least one of the amino acid sequences 65 sequence according to g) by means of affinity maturation of SEQ ID NO’s: 560 to 565; 249 to 289 and/or 481 to using one or more techniques of affinity maturation 489: known per se. US 9,512,236 B2 25 26 It should be understood that the last preceding paragraphs In one specific aspect, such a fragment comprises a single also generally apply to any amino acid sequences of the CDR sequence as described herein (and in particular a invention that comprise one or more amino acid sequences CDR3 sequence), that is flanked on each side by (part of) a according to b), c), e), f), h) or i), respectively. framework sequence (and in particular, part of the frame In this specific aspect, the amino acid sequence preferably work sequence(s) that, in the immunoglobulin sequence comprises one or more stretches of amino acid residues from which the fragment is derived, are adjacent to said chosen from the group consisting of: CDR sequence. For example, a CDR3 sequence may be i) the amino acid sequences of SEQ ID NO’s: 548 to 553; preceded by (part of) a FR3 sequence and followed by (part 167 to 207 and/or 463 to 471; of) a FR4 sequence). Such a fragment may also contain a ii) the amino acid sequences of SEQ ID NO’s: 560 to 565: 10 disulphide bridge, and in particular a disulphide bridge that 249 to 289 and/or 481 to 489; and links the two framework regions that precede and follow the iii) the amino acid sequences of SEQID NO’s: 572 to 577; CDR sequence, respectively (for the purpose of forming 331 to 371 and/or 499 to 507: Such a disulphide bridge, cysteine residues that naturally or any suitable combination thereof. occur in said framework regions may be used, or alterna Also, preferably, in Such an amino acid sequence, at least 15 tively cysteine residues may be synthetically added to or one of said stretches of amino acid residues forms part of the introduced into said framework regions). For a further antigen binding site for binding against GPCRs. description of these "Expedite fragments', reference is again In all of the above amino acid sequences of the invention, made to WO 03/050531, as well as to as well as to the US the framework sequences may be any suitable framework provisional application of Ablynx N.V. entitled “Peptides sequences, and examples of Suitable framework sequences capable of binding to serum proteins” of Ablynx N.V. will be clear to the skilled person, for example on the basis (inventors: Revets, Hilde Adi Pierrette; Kolkman, Joost the standard handbooks and the further disclosure and prior Alexander; and Hoogenboom, Hendricus Renerus Jacobus art mentioned herein. Mattheus) filed on Dec. 5, 2006 (see also WO08/068280). The framework sequences are preferably (a Suitable com In another aspect, the invention relates to a compound or bination of) immunoglobulin framework sequences or 25 construct, and in particular a protein or polypeptide (also framework sequences that have been derived from immu referred to herein as a “compound of the invention” or noglobulin framework sequences (for example, by human "polypeptide of the invention', respectively) that comprises ization or camelization). For example, the framework or essentially consists of one or more amino acid sequences sequences may be framework sequences derived from a light of the invention (or Suitable fragments thereof), and option chain variable domain (e.g. a V-sequence) and/or from a 30 ally further comprises one or more other groups, residues, heavy chain variable domain (e.g. a V-sequence). In one moieties or binding units. As will become clear to the skilled particularly preferred aspect, the framework sequences are person from the further disclosure herein, such further either framework sequences that have been derived from a groups, residues, moieties, binding units or amino acid V-sequence (in which said framework sequences may sequences may or may not provide further functionality to optionally have been partially or fully humanized) or are 35 the amino acid sequence of the invention (and/or to the conventional V sequences that have been camelized (as compound or construct in which it is present) and may or defined herein). may not modify the properties of the amino acid sequence of The framework sequences are preferably such that the the invention. amino acid sequence of the invention is a domain antibody For example, such further groups, residues, moieties or (or an amino acid sequence that is suitable for use as a 40 binding units may be one or more additional amino acid domain antibody); is a single domain antibody (or an amino sequences. Such that the compound or construct is a (fusion) acid sequence that is suitable for use as a single domain protein or (fusion) polypeptide. In a preferred but non antibody); is a "dAb’ (or an amino acid sequence that is limiting aspect, said one or more other groups, residues, suitable for use as a dAb); or is a NANOBODYR (V) moieties or binding units are immunoglobulin sequences. (including but not limited to V. sequence). Again, Suitable 45 Even more preferably, said one or more other groups, framework sequences will be clear to the skilled person, for residues, moieties or binding units are chosen from the example on the basis the standard handbooks and the further group consisting of domain antibodies, amino acid disclosure and prior art mentioned herein. sequences that are suitable for use as a domain antibody, In particular, the framework sequences present in the single domain antibodies, amino acid sequences that are amino acid sequences of the invention may contain one or 50 Suitable for use as a single domain antibody, “dAb's, amino more of Hallmark residues (as defined herein), such that the acid sequences that are Suitable for use as a dAb, or amino acid sequence of the invention is a NANOBODYR Nanobodies. (V. Some preferred, but non-limiting examples of (Suit Alternatively, Such groups, residues, moieties or binding able combinations of) Such framework sequences will units may for example be chemical groups, residues, moi become clear from the further disclosure herein. 55 eties, which may or may not by themselves be biologically Again, as generally described herein for the amino acid and/or pharmacologically active. For example, and without sequences of the invention, it is also possible to use Suitable limitation, such groups may be linked to the one or more fragments (or combinations of fragments) of any of the amino acid sequences of the invention so as to provide a foregoing, Such as fragments that contain one or more CDR "derivative of an amino acid sequence or polypeptide of the sequences, Suitably flanked by and/or linked via one or more 60 invention, as further described herein. framework sequences (for example, in the same order as Also within the scope of the present invention are com these CDR's and framework sequences may occur in the pounds or constructs, that comprises or essentially consists full-sized immunoglobulin sequence from which the frag of one or more derivatives as described herein, and option ment has been derived). Such fragments may also again be ally further comprises one or more other groups, residues, Such that they comprise or can form an immunoglobulin 65 moieties or binding units, optionally linked via one or more fold, or alternatively be such that they do not comprise or linkers. Preferably, said one or more other groups, residues, cannot form an immunoglobulin fold. moieties or binding units are amino acid sequences. US 9,512,236 B2 27 28 In the compounds or constructs described above, the one based on the further disclosure herein; and for example or more amino acid sequences of the invention and the one include, without limitation, polypeptides in which the one or or more groups, residues, moieties or binding units may be more amino acid sequences of the invention are Suitable linked directly to each other and/or via one or more suitable linked to one or more serum proteins or fragments thereof linkers or spacers. For example, when the one or more (such as (human) serum albumin or Suitable fragments groups, residues, moieties or binding units are amino acid thereof) or to one or more binding units that can bind to sequences, the linkers may also be amino acid sequences, so serum proteins (such as, for example, domain antibodies, that the resulting compound or construct is a fusion (protein) amino acid sequences that are Suitable for use as a domain or fusion (polypeptide). antibody, single domain antibodies, amino acid sequences As will be clear from the further description above and 10 that are suitable for use as a single domain antibody, herein, this means that the amino acid sequences of the "dAb’s, amino acid sequences that are Suitable for use as a invention can be used as “building blocks” to form poly dAb, or Nanobodies that can bind to serum proteins such as peptides of the invention, i.e. by Suitably combining them serum albumin (such as human serum albumin), serum with other groups, residues, moieties or binding units, in immunoglobulins such as IgG, or transferrin; reference is order to form compounds or constructs as described herein 15 made to the further description and references mentioned (such as, without limitations, the biparatopic, bi/multivalent herein); polypeptides in which an amino acid sequence of and bi/multispecific polypeptides of the invention described the invention is linked to an Fc portion (such as a human Fc) herein) which combine within one molecule one or more or a suitable part or fragment thereof, or polypeptides in desired properties or biological functions. Particularly pre which the one or more amino acid sequences of the inven ferred polypeptides of the invention are biparatopic poly tion are Suitable linked to one or more Small proteins or peptides as will be clear from the further description herein. peptides that can bind to serum proteins (such as, without The compounds or polypeptides of the invention can limitation, the proteins and peptides described in WO generally be prepared by a method which comprises at least 91/01743, WO 01/45746, WO 02/076489 and to the US one step of Suitably linking the one or more amino acid provisional application of Ablynx N.V. entitled “Peptides sequences of the invention to the one or more further groups, 25 capable of binding to serum proteins of Ablynx N.V. filed residues, moieties or binding units, optionally via the one or on Dec. 5, 2006. (see also WO08/068280). more Suitable linkers, so as to provide the compound or Generally, the compounds or polypeptides of the inven polypeptide of the invention. Polypeptides of the invention tion with increased half-life preferably have a half-life that can also be prepared by a method which generally comprises is at least 1.5 times, preferably at least 2 times, such as at at least the steps of providing a nucleic acid that encodes a 30 least 5 times, for example at least 10 times or more than 20 polypeptide of the invention, expressing said nucleic acid in times, greater than the half-life of the corresponding amino a suitable manner, and recovering the expressed polypeptide acid sequence of the invention per se. For example, the of the invention. Such methods can be performed in a compounds or polypeptides of the invention with increased manner known per se, which will be clear to the skilled half-life may have a half-life that is increased with more than person, for example on the basis of the methods and tech 35 1 hours, preferably more than 2 hours, more preferably more niques further described herein. than 6 hours, such as more than 12 hours, or even more than The process of designing/selecting and/or preparing a 24, 48 or 72 hours, compared to the corresponding amino compound or polypeptide of the invention, starting from an acid sequence of the invention per se. amino acid sequence of the invention, is also referred to In a preferred, but non-limiting aspect of the invention, herein as “formatting” said amino acid sequence of the 40 Such compounds or polypeptides of the invention have a invention; and an amino acid of the invention that is made serum half-life that is increased with more than 1 hours, part of a compound or polypeptide of the invention is said preferably more than 2 hours, more preferably more than 6 to be “formatted' or to be “in the format of said compound hours, such as more than 12 hours, or even more than 24, 48 or polypeptide of the invention. Examples of ways in which or 72 hours, compared to the corresponding amino acid an amino acid sequence of the invention can be formatted 45 sequence of the invention per se. and examples of such formats will be clear to the skilled In another preferred, but non-limiting aspect of the inven person based on the disclosure herein; and Such formatted tion, Such compounds or polypeptides of the invention amino acid sequences form a further aspect of the invention. exhibit a serum half-life in human of at least about 12 hours, In one specific aspect of the invention, a compound of the preferably at least 24 hours, more preferably at least 48 invention or a polypeptide of the invention may have an 50 hours, even more preferably at least 72 hours or more. For increased half-life, compared to the corresponding amino example, compounds or polypeptides of the invention may acid sequence of the invention. Some preferred, but non have a half-life of at least 5 days (such as about 5 to 10 days), limiting examples of Such compounds and polypeptides will preferably at least 9 days (such as about 9 to 14 days), more become clear to the skilled person based on the further preferably at least about 10 days (such as about 10 to 15 disclosure herein, and for example comprise amino acid 55 days), or at least about 11 days (Such as about 11 to 16 days), sequences or polypeptides of the invention that have been more preferably at least about 12 days (such as about 12 to chemically modified to increase the half-life thereof (for 18 days or more), or more than 14 days (such as about 14 to example, by means of pegylation); amino acid sequences of 19 days). the invention that comprise at least one additional binding In another aspect, the invention relates to a nucleic acid site for binding to a serum protein (Such as serum albumin); 60 that encodes an amino acid sequence of the invention or a or polypeptides of the invention that comprise at least one polypeptide of the invention (or a suitable fragment thereof). amino acid sequence of the invention that is linked to at least Such a nucleic acid will also be referred to herein as a one moiety (and in particular at least one amino acid “nucleic acid of the invention' and may for example be in sequence) that increases the half-life of the amino acid the form of a genetic construct, as further described herein. sequence of the invention. Examples of polypeptides of the 65 In another aspect, the invention relates to a host or host invention that comprise Such half-life extending moieties or cell that expresses (or that under Suitable circumstances is amino acid sequences will become clear to the skilled person capable of expressing) an amino acid sequence of the US 9,512,236 B2 29 30 invention and/or a polypeptide of the invention; and/or that herein. Some preferred but non-limiting examples of Such contains a nucleic acid of the invention. Some preferred but methods will become clear from the further description non-limiting examples of Such hosts or host cells will herein. become clear from the further description herein. Generally, these methods may comprise the steps of: The invention further relates to a product or composition a) providing a set, collection or library of amino acid containing or comprising at least one amino acid sequence sequences; and of the invention, at least one polypeptide of the invention (or b) screening said set, collection or library of amino acid a suitable fragment thereof) and/or at least one nucleic acid sequences for amino acid sequences that can bind to of the invention, and optionally one or more further com and/or have affinity for GPCRs: 10 and ponents of Such compositions known per se, i.e. depending c) isolating the amino acid sequence(s) that can bind to on the intended use of the composition. Such a product or and/or have affinity for GPCRs. composition may for example be a pharmaceutical compo In particular, in step b) of Such a method, the set, sition (as described herein), a veterinary composition or a collection or library may be screened for amino acid product or composition for diagnostic use (as also described 15 sequences that can bind to and/or have affinity for GPCRs herein). Some preferred but non-limiting examples of Such that are expressed on the surface of a suitable cell; for amino products or compositions will become clear from the further acid sequences that can bind to and/or have affinity for an description herein. extracellular part, region, domain, loop or other extracellular The invention also relates to the use of an amino acid epitope of a GPCR (as described herein); and/or for amino sequence, NANOBODYR (V) or polypeptide of the acid sequences that can bind to and/or have affinity for a invention, or of a composition comprising the same, in peptide that has been derived from or based on the amino (methods or compositions for) modulating a GPCR, either in acid sequence of an extracellular part, region, domain, loop vitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in or other extracellular epitope of a GPCR. This can be an a single cell or in a multicellular organism, and in performed using methods and techniques known per se, for particular in a mammal, and more in particular in a human 25 example those mentioned herein. being, such as in a human being that is at risk of or Suffers In such a method, the set, collection or library of amino from a GPCR related disease or disorder). acid sequences may be any Suitable set, collection or library The invention also relates to methods for modulating a of amino acid sequences. For example, the set, collection or GPCR, either in vitro (e.g. in an in vitro or cellular assay) or library of amino acid sequences may be a set, collection or 30 library of immunoglobulin sequences (as described herein), in vivo (e.g. in an a single cell or multicellular organism, and Such as a naive set, collection or library of immunoglobulin in particular in a mammal, and more in particular in a human sequences; a synthetic or semi-synthetic set, collection or being, such as in a human being that is at risk of or Suffers library of immunoglobulin sequences; and/or a set, collec from a GPCR related disease or disorder), which method tion or library of immunoglobulin sequences that have been comprises at least the step of contacting a GPCR with at 35 Subjected to affinity maturation. least one amino acid sequence, NANOBODYR (V) or Also, in such a method, the set, collection or library of polypeptide of the invention, or with a composition com amino acid sequences may be a set, collection or library of prising the same, in a manner and in an amount Suitable to heavy chain variable domains (such as V. domains or V. modulate a GPCR, with at least one amino acid sequence, domains) or of light chain variable domains. For example, NANOBODYR (V) or polypeptide of the invention. 40 the set, collection or library of amino acid sequences may be The invention also relates to the use of an amino acid a set, collection or library of domain antibodies or single sequence, NANOBODYR (V) or polypeptide of the domain antibodies, or may be a set, collection or library of invention in the preparation of a composition (Such as, amino acid sequences that are capable of functioning as a without limitation, a pharmaceutical composition or prepa domain antibody or single domain antibody. ration as further described herein) for modulating (as defined 45 In a preferred aspect of this method, the set, collection or herein) a GPCR, either in vitro (e.g. in an in vitro or cellular library of amino acid sequences may be an immune set, assay) or in vivo (e.g. in an a single cell or multicellular collection or library of immunoglobulin sequences, for organism, and in particular in a mammal, and more in example derived from a mammal that has been suitably particular in a human being, Such as in a human being that immunized with GPCRs or with a suitable antigenic deter is at risk of or suffers from a GPCR related disease or 50 minant based thereon or derived therefrom, Such as an disorder). antigenic part, fragment, region, domain, loop or other In the context of the present specification, “modulating epitope thereof. In one particular aspect, said antigenic may be as generally defined herein, but may for also involve determinant may be an extracellular part, region, domain, allosteric modulation (see for example George et al., Nat loop or other extracellular epitope(s), or a Suitable peptide Rev Drug Discov 1:808-820 (2002); Kenakin, Trends Phar 55 derived therefrom. Alternatively, as mentioned herein, the macol Sci 25:186-192 (2002) and Rios et al., Pharmacol set, collection or library of amino acid sequences may be an Ther 92:71-87 (2001)) and/or reducing or inhibiting the immune set, collection or library of immunoglobulin binding of a GPCR to one of its substrates or ligands and/or sequences, for example derived from a mammal that has competing with a natural ligand, Substrate for binding to a been suitably immunized with a refolded GPCR or with a GPCR. Modulating may also involve activating a GPCR or 60 cell, or cell fraction or preparation derived from a cell that the mechanism or pathway in which it is involved. Modu has a GPCR on its surface. lating may be reversible or irreversible, but for pharmaceu In the above methods, the set, collection or library of tical and pharmacological purposes will usually be in a amino acid sequences may be displayed on a phage, reversible manner. phagemid, ribosome or Suitable micro-organism (such as The invention further relates to methods for preparing or 65 yeast). Such as to facilitate screening. Suitable methods, generating the amino acid sequences, polypeptides, nucleic techniques and host organisms for displaying and screening acids, host cells, products and compositions described (a set, collection or library of) amino acid sequences will be US 9,512,236 B2 31 32 clear to the person skilled in the art, for example on the basis sequences that encode amino acid sequences that can bind to of the further disclosure herein. Reference is also made to and/or have affinity for GPCRs that are expressed on the the review by Hoogenboom in Nature Biotechnology, 23, 9, Surface of a Suitable cell; for nucleotide sequences that 1105-1116 (2005). encode amino acid sequences that can bind to and/or have In another aspect, the method for generating amino acid affinity for an extracellular part, region, domain, loop or sequences comprises at least the steps of: other extracellular epitope of a GPCR (as described herein); a) providing a collection or sample of cells expressing amino and/or for nucleotide sequences that encode amino acid acid sequences: sequences that can bind to and/or have affinity for a peptide b) screening said collection or sample of cells for cells that that has been derived from or based on the amino acid express an amino acid sequence that can bind to and/or 10 have affinity for GPCRs: sequence of an extracellular part, region, domain, loop or and other extracellular epitope of a GPCR. This can be per c) either (i) isolating said amino acid sequence; or (ii) formed using methods and techniques known per se, for isolating from said cell a nucleic acid sequence that example those mentioned herein. encodes said amino acid sequence, followed by express 15 In such a method, the set, collection or library of nucleic ing said amino acid sequence. acid sequences encoding amino acid sequences may for In particular, in step b) of Such a method, the set, example be a set, collection or library of nucleic acid collection or library may be screened for cells that express sequences encoding a naive set, collection or library of amino acid sequences that can bind to and/or have affinity immunoglobulin sequences; a set, collection or library of for GPCRs that are expressed on the surface of a suitable nucleic acid sequences encoding a synthetic or semi-syn cell; for cells that express amino acid sequences that can thetic set, collection or library of immunoglobulin bind to and/or have affinity for an extracellular part, region, sequences; and/or a set, collection or library of nucleic acid domain, loop or other extracellular epitope of a GPCR (as sequences encoding a set, collection or library of immuno described herein); and/or for cells that express amino acid globulin sequences that have been Subjected to affinity sequences that can bind to and/or have affinity for a peptide 25 maturation. that has been derived from or based on the amino acid Also, in such a method, the set, collection or library of sequence of an extracellular part, region, domain, loop or nucleic acid sequences may encode a set, collection or other extracellular epitope of a GPCR. This can be per library of heavy chain variable domains (such as V, formed using methods and techniques known per se, for domains or V. domains) or of light chain variable example those mentioned herein. 30 domains. For example, the set, collection or library of For example, when the desired amino acid sequence is an nucleic acid sequences may encode a set, collection or immunoglobulin sequence, the collection or sample of cells library of domain antibodies or single domain antibodies, or may for example be a collection or sample of B-cells. Also, a set, collection or library of amino acid sequences that are in this method, the sample of cells may be derived from a capable of functioning as a domain antibody or single mammal that has been suitably immunized with GPCRs or 35 domain antibody. with a suitable antigenic determinant based thereon or In a preferred aspect of this method, the set, collection or derived therefrom, Such as an antigenic part, fragment, library of amino acid sequences may be an immune set, region, domain, loop or other epitope thereof. In one par collection or library of nucleic acid sequences, for example ticular aspect, said antigenic determinant may be an extra derived from a mammal that has been suitably immunized cellular part, region, domain, loop or other extracellular 40 with GPCRs or with a suitable antigenic determinant based epitope(s), or a suitable peptide derived therefrom. Alterna thereon or derived therefrom, such as an antigenic part, tively, as mentioned herein, the sample of cells may be fragment, region, domain, loop or other epitope thereof. In derived from a mammal that has been suitably immunized one particular aspect, said antigenic determinant may be an with a refolded GPCR or with a cell, or cell fraction or extracellular part, region, domain, loop or other extracellular preparation derived from a cell that has a GPCR on its 45 epitope(s), or a suitable peptide derived therefrom. Alterna Surface. tively, as mentioned herein, the set, collection or library of The above method may be performed in any suitable nucleic acid sequences may be an immune set, collection or manner, as will be clear to the skilled person. Reference is library derived from a mammal that has been suitably for example made to EP 0 542 810, WO 05/19824, WO immunized with a refolded GPCR or with a cell, or cell 04/051268 and WO 04/106377. The screening of step b) is 50 fraction or preparation derived from a cell that has a GPCR preferably performed using a flow cytometry technique Such on its Surface. as FACS. For this, reference is for example made to Lieby The set, collection or library of nucleic acid sequences et al., Blood, Vol. 97, No. 12, 3820 (2001). may for example encode an immune set, collection or library In another aspect, the method for generating an amino of heavy chain variable domains or of light chain variable acid sequence directed against GPCRS may comprise at least 55 domains. In one specific aspect, the set, collection or library the steps of: of nucleotide sequences may encode a set, collection or a) providing a set, collection or library of nucleic acid library of V, Sequences. sequences encoding amino acid sequences; In the above methods, the set, collection or library of b) screening said set, collection or library of nucleic acid nucleotide sequences may be displayed on a phage, sequences for nucleic acid sequences that encode an 60 phagemid, ribosome or Suitable micro-organism (such as amino acid sequence that can bind to and/or has affinity yeast). Such as to facilitate screening. Suitable methods, for GPCRs: techniques and host organisms for displaying and screening and (a set, collection or library of) nucleotide sequences encod c) isolating said nucleic acid sequence, followed by express ing amino acid sequences will be clear to the person skilled ing said amino acid sequence. 65 in the art, for example on the basis of the further disclosure In particular, in step b) of Such a method, the set, herein. Reference is also made to the review by Hoogen collection or library may be screened for nucleotide boom in Nature Biotechnology, 23, 9, 1105-1116 (2005). US 9,512,236 B2 33 34 The invention also relates to amino acid sequences that FIG. 2: GPCR binding specificity determined in a phage are obtained by the above methods, or alternatively by a ELISA binding assay (as described in Example 3). method that comprises the one of the above methods and in FIG. 3: Snake plot representation of human CXCR4 with addition at least the steps of determining the nucleotide highlighted amino acid residues involved in the binding of sequence or amino acid sequence of said immunoglobulin 5 238D2 (green filled circles) and 238D4 (red filled circles) sequence; and of expressing or synthesizing said amino acid FIG. 4: Bivalent nanobodies inhibit CXCR4-mediated sequence in a manner known per se, such as by expression signalings. CXCL12-induced inositol phosphate accumula in a Suitable host cell or host organism or by chemical tion in cells transfected with CXCR4 and the chimeric synthesis. GO.gi5, protein is inhibited by monovalent and bivalent Also, following the steps above, one or more amino acid 10 nanobodies. sequences of the invention may be suitably humanized (or alternatively camelized); and/or the amino acid sequence(s) DETAILED DESCRIPTION OF THE thus obtained may be linked to each other or to one or more INVENTION other Suitable amino acid sequences (optionally via one or more suitable linkers) so as to provide a polypeptide of the 15 G protein-coupled receptors (GPCRs), also known as invention. Also, a nucleic acid sequence encoding an amino seven transmembrane receptors, 7TM receptors, heptaheli acid sequence of the invention may be suitably humanized cal receptors, and G protein linked receptors (GPLR), are a (or alternatively camelized) and Suitably expressed; and/or protein family of transmembrane receptors that transduce an one or more nucleic acid sequences encoding an amino acid extracellular signal (ligand binding) into an intracellular sequence of the invention may be linked to each other or to signal (G protein activation). GPCRs are integral membrane one or more nucleic acid sequences that encode other proteins that possess seven membrane-spanning domains or Suitable amino acid sequences (optionally via nucleotide transmembrane helices. The extracellular parts of the recep sequences that encode one or more Suitable linkers), after tor can be glycosylated. These extracellular loops also which the nucleotide sequence thus obtained may be suit contain two highly conserved cysteine residues which build ably expressed so as to provide a polypeptide of the inven 25 disulfide bonds to stabilize the receptor structure. tion. The GPCRs form the largest and most diverse group of The invention further relates to applications and uses of transmembrane proteins involved in signal transduction the amino acid sequences, polypeptides, nucleic acids, host (Howard et al., Trends Pharmacol. Sci. 22:132-40, 2001). cells, products and compositions described herein, as well as GPCRs are involved in various cellular and biological to methods for the prevention and/or treatment for diseases 30 functions, such as stimulus-response pathways (from inter and disorders associated with GPCRs. Some preferred but cellular communication to physiological senses), including, non-limiting applications and uses will become clear from for example, embryogenesis, release, neu the further description herein. For example, as mentioned rosensation (e.g., 15 chemosensory functions such as taste herein, it is expected that amino acid sequences, Nanobodies and smell) (Mombaerts, Science 286:707-711, 1999), neu and polypeptides of the invention that are directed against 35 ronal axon pathfinding (Mombaerts et al., Cell 87:675, 1996; olfactory GPCRs can find use as artificial flavourings or Mombaerts et al., Cold Spring Harbor Symp. Quant. Biol. even perfumes. The amino acid sequences, Nanobodies and 56:135, 1996), leukocyte targeting to sites of inflammation polypeptides of the invention may also find use as markers (Tager et al., J. Exp. Med., 192:439-46, 2000), and cell for detecting cells that express the GPCRs against which survival, proliferation, and differentiation. (Ryan et al., J. they are directed, for example in vitro (e.g. using Western 40 Biol Chem. 273: 13613-24, 1998). blot, immunoprecipitation or immunofluorescence tech The complexity of the GPCR repertoire surpasses that of niques) or in Vivo (e.g. using suitable imaging techniques). the immunoglobulin and receptor genes combined, The amino acid sequences, Nanobodies and polypeptides of with members of the GPCR superfamily estimated at as the invention may also find use in affinity purification many as 2,000, or more than 1.5% of the human genome. techniques for (cells expressing) the GPCRs against which 45 Further, members of the GPCR superfamily are the director they are directed. indirect target of more than 50% of the current pharmaceu Other aspects, embodiments, advantages and applications tical drugs used clinically in . of the invention will also become clear from the further The diversity of functions is matched by the wide range description herein, in which the invention will be described of ligands recognized by members of the family, from and discussed in more detail with reference to the Nano 50 photons (rhodopsin, the archetypal GPCR) to small mol bodies of the invention and polypeptides of the invention ecules (in the case of the histamine receptors) to proteins (for comprising the same, which form some of the preferred example, chemokine receptors). For an overview of the aspects of the invention. human GPCR family and ligands of human GPCRs refer As will become clear from the further description herein, ence is made to FIG. 1 in the US application 2002/0106739. Nanobodies generally offer certain advantages (outlined 55 GPCRs can be grouped into 4 classes based on structural herein) compared to “dAbs' or similar (single) domain homology and functional similarity: Class A (rhodopsin antibodies or immunoglobulin sequences, which advantages like), Class B (secretin-like), Class C (metabotropic/phero are also provided by the Nanobodies of the invention. mone), and Class D (Fungal ), of which Class A However, it will be clear to the skilled person that the more receptors, Class B receptors, and receptors with virtually general aspects of the teaching below can also be applied 60 non-existent carboxyl-terminal tails form the major classes. (either directly or analogously) to other amino acid GPCRs can be classified accordingly based on their inter sequences of the invention. actions with an affinity for rat beta-arrestin-2 in HEK-293 cells and may be predicted based on the amino acid residues BRIEF DESCRIPTION OF THE DRAWINGS in their carboxyl-terminal tail and the length of their car 65 boxyl-terminal tail. A Class B receptor is a GPCR that has FIG. 1: Schematic representation of the structure of the one or more sites of phosphorylation (e.g., clusters of three different classes of GPCRs. phosphorylation sites) properly positioned in its carboxyl US 9,512,236 B2 35 36 terminal tail such that it does recruit rat beta-arrestin-2 to 3 and 5 subtypes), MI muscarinic, adenosine 1, CC endosomes in HEK-293 cells under conditions as described adrenergic (alA, alB and a I C Subtypes), in U.S. Pat. No. 5,891,646, Oakley, et al., Journal of Bio (ATIA subtype), bombesin (BBI and B132 subtypes), logical Chemistry, Vol 275, No. 22, pp 17201-17210, Jun. 2, bradykinin (132 subtype), C5a, cholycystokinin (CCKa 2000, and Oakley et al., and CCKb subtypes), endothelin (Eta and Etb Sub Journal of Biological Chemistry, Vol. 276, No. 22, pp types), glutamate (mGlul, 5 subtypes), 5HT (2A, B and 19452-19460, 2001. A Class A receptor is a GPCR that does C Subtypes), histamine (H I subtype), neurotensin, not have one or more sites of phosphorylation (e.g., clusters neurokinin (NK2, 3 subtypes), oxytocin, thyrotropin of phosphorylation sites) properly positioned in its carboxyl releasing hormone (TRIJ), thyroid stimulating hor terminal tail such that it does not recruit rat p-arrestin-2 to 10 mone (TSH), thromoboxane A2 and vasopressin (VI a endosomes in HEK-293 cells under conditions as described Subtypes); above for Class B receptors. Receptors with virtually non GPCRs that couple to a GS G-protein, such as the fol existent carboxyl-terminal tails include, for example, olfac lowing receptors: P2-adrenergic, cardiac P-adrenergic, tory and taste receptors. histamine (H2 Subtype), thyrotropin, growth hormone Some examples of the biological and physiological roles 15 releasing factor, adrenocorticotropic hormone (ACTH), of GPCRs include: 5HT4, follicle stimulating hormone (FSH), thyroid the visual sense: the use a photoisomerization stimulating hormone (TSH), GLP-1, glucagon, dop reaction to translate electromagnetic radiation into cel amine5 (D5), doparninel (DI), calcitonin, adenosine2p lular signals. Rhodopsin, for example, uses the conver (A2p), vasopressin2, vasoactive intestinal polypeptide sion of 11-cis-retinal to all-trans-retinal for this pur and parathyroid hormone; pose. GPCRs that couple to a GiG-protein, such as the follow the sense of smell: receptors of the olfactory epithelium ing receptors: 5HT (IA, IB, I D and I F subtypes), bind odorants (olfactory receptors) and mGlutamineR (2, 3 subtypes), dopamine4 (D4), dop (vomeronasal receptors) amine-2 (D2) cannabinoid, adenosine3 (A3), Soma behavioral and mood regulation: receptors in the mam 25 to statin (4, 3 subtypes), t-opioid, 6-opioid, K-Opioid, malian brain bind several different , neuropeptide Y (1, 2 subtypes): including serotonin, dopamine, GABA and glutamate. The GPCRs mentioned in US 2002/0106739: regulation of immune system activity and inflammation: The GPCRs listed in Table 1 of Lundstrom et al., J. Struct. chemokine receptors bind ligands that mediate inter Funct. Genomics, 2006 Nov. 22; Epub ahead of print cellular communication between cells of the immune 30 GPCRs that are so-called “orphan’ receptors, i.e. a GPCR system; receptors such as histamine receptors bind that is structurally similar to other GPCRs but for inflammatory mediators and engage target cell types in which the natural ligand is not yet known; the inflammatory response The GPCRs mentioned in Table C: autonomic nervous system transmission: both the sympa The GPCRS mentioned in Table D. thetic and parasympathetic nervous systems are regu 35 Other GPCRs will be clear to the skilled person, for lated by GPCR pathways. These systems are respon example from the standard handbooks, such as the G Protein sible for control of many automatic functions of the Coupled Receptors Handbook, L. Devi (Ed.), Humana body such as blood pressure, rate and digestive Press, 2005; as well as from the standard databases, such as processes. GPCRDB (see for example http://www.gper.org/7tm/htmls/ Generally, for GPCRs reference is made to the standard 40 entries.html). handbooks, such as the G Protein Coupled Receptors Hand In the present description, examples and claims: book, L. Devi (Ed.), Humana Press, 2005, as well as to the a) Unless indicated or defined otherwise, all terms used have available databases, such as GPCRDB (see for example their usual meaning in the art, which will be clear to the http://www.gper.org/7tm/htmls/entries.html). skilled person. Reference is for example made to the Thus, generally, as used herein, the term "G-protein 45 standard handbooks, such as Sambrook et al., “Molecular coupled receptor' (or “GPCR) refers to a receptor that, Cloning: A Laboratory Manual' (2nd.Ed.), Vols. 1-3, Cold when expressed by a cell, associates with a G-protein (e.g., Spring Harbor Laboratory Press (1989); F. Ausubel et al. a protein composed of cc, P and y Subunits and which eds. “Current protocols in molecular biology'. Green hydrolyzes GTP). Preferably, the GPCR is a “seven trans Publishing and Wiley Interscience, New York (1987); membrane segment receptor' (or "7 TMS receptor), which 50 Lewin, “Genes II”, John Wiley & Sons, New York, N.Y., refers to a protein that structurally comprises seven hydro (1985); Old et al., “Principles of Gene Manipulation: An phobic transmembrane spanning regions. Introduction to Genetic Engineering, 2nd edition, Uni Some non-limiting examples of GPCRs include, but are versity of California Press, Berkeley, Calif. (1981); Roitt not limited to: et al., “Immunology” (6th. Ed.), Mosby/Elsevier, Edin GPCRs that are known targets for pharmaceuticals (either 55 burgh (2001); Roitt et al., Roitt's Essential Immunology, Small molecules or biologicals) that are currently on the 10' Ed. Blackwell Publishing, UK (2001); and Janeway market or in clinical development (for example, those et al., “Immunobiology” (6th Ed.), Garland Science Pub mentioned herein); lishing/Churchill Livingstone, N.Y. (2005), as well as to the luteinizing hormone releasing hormone (LHRH) (also the general background art cited herein; known as gonadotropin releasing hormone, GnRH) 60 b) Unless indicated otherwise, the term “immunoglobulin receptor, the MI muscarinic receptor and the D2-adren sequence' whether used herein to refer to a heavy chain ergic receptor, antibody or to a conventional 4-chain antibody—is used opioid receptors, endothelin receptors, angiotensin recep as a general term to include both the full-size antibody, the tors, neuropeptide Y receptors and serotonin K recep individual chains thereof, as well as all parts, domains or tors; 65 fragments thereof (including but not limited to antigen GPCRs that couple to (i.e., associates with) a Gq/1 I binding domains or fragments such as V. domains or G-protein, such as LHRH (=GnRH), acetylcholine (ml, V/V, domains, respectively). In addition, the term US 9,512,236 B2 37 38 'sequence as used herein (for example in terms like the second nucleotide sequence by the total number of “immunoglobulin sequence', 'antibody sequence', 'vari nucleotides in the first nucleotide sequence and multi able domain sequence”, “VHH sequence' or “protein plying by 100%, in which each deletion, insertion, sequence'), should generally be understood to include substitution or addition of a nucleotide in the second both the relevant amino acid sequence as well as nucleic nucleotide sequence—compared to the first nucleotide acids or nucleotide sequences encoding the same, unless sequence—is considered as a difference at a single the context requires a more limited interpretation. Also, nucleotide (position). the term “nucleotide sequence' as used herein also Alternatively, the degree of sequence identity between encompasses a nucleic acid molecule with said nucleotide two or more nucleotide sequences may be calculated sequence, so that the terms “nucleotide sequence' and 10 using a known computer algorithm for sequence align “nucleic acid should be considered equivalent and are ment such as NCBI Blast v2.0, using standard settings. used interchangeably herein; Some other techniques, computer algorithms and settings c) Unless indicated otherwise, all methods, steps, techniques for determining the degree of sequence identity are for and manipulations that are not specifically described in example described in WO 04/037999, EP 0967 284, detail can be performed and have been performed in a 15 EP 1 085 089, WO 00/55318, WO 00/78972, WO manner known per se, as will be clear to the skilled 98/491.85 and GB 2 357 768-A. person. Reference is for example again made to the Usually, for the purpose of determining the percentage of standard handbooks and the general background art men 'sequence identity” between two nucleotide sequences tioned herein and to the further references cited therein; as in accordance with the calculation method outlined well as to for example the following reviews Presta, Adv. hereinabove, the nucleotide sequence with the greatest Drug Deliv. Rev. 2006, 58 (5-6): 640-56; Levin and number of nucleotides will be taken as the “first Weiss, Mol. Biosyst. 2006, 201): 49-57; Irving et al., J. nucleotide sequence, and the other nucleotide sequence Immunol. Methods, 2001, 248(1-2), 31-45; Schmitz et al., will be taken as the “second nucleotide sequence: Placenta, 2000, 21 Suppl. A, S106-12, Gonzales et al., f) For the purposes of comparing two or more amino acid Tumour Biol., 2005, 26(1), 31-43, which describe tech 25 sequences, the percentage of "sequence identity” between niques for protein engineering, such as affinity maturation a first amino acid sequence and a second amino acid and other techniques for improving the specificity and sequence (also referred to herein as "amino acid identity”) other desired properties of proteins such as immunoglobu may be calculated by dividing the number of amino acid lins. residues in the first amino acid sequence that are identical d) Amino acid residues will be indicated according to the 30 to the amino acid residues at the corresponding positions standard three-letter or one-letter amino acid code, as in the second amino acid sequence by the total number mentioned in Table A-2: of amino acid residues in the first amino acid sequence and multiplying by 100%, in which each deletion, TABLE A-2 insertion, Substitution or addition of an amino acid residue 35 in the second amino acid sequence—compared to the first one-letter and three-letter amino acid code amino acid sequence—is considered as a difference at a Nonpolar, Alanine Ala A. single amino acid residue (position), i.e. as an "amino acid uncharged Valine Wall V difference” as defined herein. (at pH 6.0–7.0)(3) Leucine Leu L Alternatively, the degree of sequence identity between Isoleucine Ile I Phenylalanine Phe F 40 two amino acid sequences may be calculated using a Methionine Met M known computer algorithm, such as those mentioned Tryptophan Trp W above for determining the degree of sequence identity Proline Pro P for nucleotide sequences, again using standard settings. Polar, Glycine’’’ Gly G uncharged Serine Ser S Usually, for the purpose of determining the percentage of (at pH 6.0–7.0) Threonine Thr T 45 'sequence identity” between two amino acid sequences Cysteine Cys C in accordance with the calculation method outlined Asparagine ASn N Glutamine Gln Q hereinabove, the amino acid sequence with the greatest Tyrosine Tyr Y number of amino acid residues will be taken as the Polar, Lysine Lys K “first amino acid sequence, and the other amino acid charged Arginine Arg R 50 sequence will be taken as the 'second amino acid (at pH 6.0–7.0) Histidine His H Sequence. Aspartate Asp D Also, in determining the degree of sequence identity Glutamate Glu E between two amino acid sequences, the skilled person Notes: may take into account so-called “conservative' amino '''Sometimes also considered to be a polar uncharged amino acid. 55 'Sometimes also considered to be a nonpolar uncharged amino acid, acid Substitutions, which can generally be described as As will be clear to the skilled person, the fact that an amino acid residue is referred to amino acid Substitutions in which an amino acid resi in this Table as being either charged or uncharged at pH 6.0 to 7.0 does not reflect in any way on the charge said amino acid residue may have at a pH lower than 6.0 and or at a due is replaced with another amino acid residue of pH higher than 7.0; the amino acid residues mentioned in the Table can be either charged similar chemical structure and which has little or essen and or uncharged at such a higher or lower pH, as will be clear to the skilled person, As is known in the art, the charge of a His residue is greatly dependant upon even small tially no influence on the function, activity or other shifts in pH, but a His residue can generally be considered essentially uncharged at a pH 60 biological properties of the polypeptide. Such conser of about 6.5. vative amino acid substitutions are well known in the e) For the purposes of comparing two or more nucleotide art, for example from WO 04/037999, GB-A-3357 sequences, the percentage of “sequence identity” between 768, WO 98/49185, WO 00/46383 and WO 01/09300; a first nucleotide sequence and a second nucleotide and (preferred) types and/or combinations of Such sequence may be calculated by dividing the number of 65 substitutions may be selected on the basis of the nucleotides in the first nucleotide sequence that are iden pertinent teachings from WO 04/037999 as well as WO tical to the nucleotides at the corresponding positions in 98/49185 and from the further references cited therein. US 9,512,236 B2 39 40 Such conservative substitutions preferably are substitu respectively, as the latter sequence, irrespective of how tions in which one amino acid within the following the firstmentioned sequence has actually been generated groups (a)-(e) is substituted by another amino acid or obtained (which may for example be by any suitable residue within the same group: (a) Small aliphatic, method described herein). By means of a non-limiting nonpolar or slightly polar residues: Ala, Ser. Thr, Pro example, when a NANOBODYR (V) of the invention and Gly; (b) polar, negatively charged residues and is said to comprise a CDR sequence, this may mean that their (uncharged) amides: Asp, ASn, Glu and Gln; (c) said CDR sequence has been incorporated into the polar, positively charged residues: His, Arg and Lys; (d) NANOBODYR (V) of the invention, but more usually large aliphatic, nonpolar residues: Met, Leu, Ile, Val this generally means that the NANOBODYR (V) of and Cys; and (e) aromatic residues: Phe, Tyr and Trp. 10 the invention contains within its sequence a stretch of Particularly preferred conservative substitutions are as amino acid residues with the same amino acid sequence as follows: Ala into Gly or into Ser; Arg into Lys; ASn into said CDR sequence, irrespective of how said NANO Gln or into His; Asp into Glu: Cys into Ser; Gln into BODYR (V) of the invention has been generated or ASn; Glu into Asp: Gly into Ala or into Pro: His into obtained. It should also be noted that when the latter Asin or into Gln: Ile into Leu or into Val; Leu into Ile 15 amino acid sequence has a specific biological or structural or into Val; Lys into Arg, into Gln or into Glu; Met into function, it preferably has essentially the same, a similar Leu, into Tyr or into Ile; Phe into Met, into Leu or into or an equivalent biological or structural function in the Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into firstmentioned amino acid sequence (in other words, the Trp; and/or Phe into Val, into Ile or into Leu. firstmentioned amino acid sequence is preferably Such Any amino acid substitutions applied to the polypeptides that the latter sequence is capable of performing essen described herein may also be based on the analysis of tially the same, a similar or an equivalent biological or the frequencies of amino acid variations between structural function). For example, when a NANO homologous proteins of different species developed by BODYR (V) of the invention is said to comprise a Schulz et al., Principles of Protein Structure, Springer CDR sequence or framework sequence, respectively, the Verlag, 1978, on the analyses of structure forming 25 CDR sequence and framework are preferably capable, in potentials developed by Chou and Fasman, Biochem said NANOBODYR (V), of functioning as a CDR istry 13: 211, 1974 and Adv. Enzymol., 47: 45-149, sequence or framework sequence, respectively. Also, 1978, and on the analysis of hydrophobicity patterns in when a nucleotide sequence is said to comprise another proteins developed by Eisenberg et al., Proc. Nad. Acad nucleotide sequence, the firstmentioned nucleotide Sci. USA 81: 140-144, 1984; Kyte & Doolittle: J 30 sequence is preferably such that, when it is expressed into Molec. Biol. 157: 105-132, 1981, and Goldman et al., an expression product (e.g. a polypeptide), the amino acid Ann. Rev. Biophys. Chem. 15: 321-353, 1986, all sequence encoded by the latter nucleotide sequence forms incorporated herein in their entirety by reference. Infor part of said expression product (in other words, that the mation on the primary, secondary and tertiary structure latter nucleotide sequence is in the same reading frame as of Nanobodies is given in the description herein and in 35 the firstmentioned, larger nucleotide sequence). the general background art cited above. Also, for this j) A nucleic acid sequence or amino acid sequence is purpose, the crystal structure of a V. domain from a considered to be “(in) essentially isolated (form) —for llama is for example given by Desmyter et al., Nature example, compared to its native biological Source and/or Structural Biology, Vol. 3, 9,803 (1996); Spinelli et al., the reaction medium or cultivation medium from which it Natural Structural Biology (1996): 3, 752-757; and 40 has been obtained when it has been separated from at Decanniere et al., Structure, Vol. 7, 4, 361 (1999). least one other component with which it is usually asso Further information about some of the amino acid ciated in said source or medium, Such as another nucleic residues that in conventional V. domains form the acid, another protein/polypeptide, another biological V/V, interface and potential camelizing Substitutions component or macromolecule or at least one contaminant, on these positions can be found in the prior art cited 45 impurity or minor component. In particular, a nucleic acid above. sequence or amino acid sequence is considered “essen g) Amino acid sequences and nucleic acid sequences are said tially isolated when it has been purified at least 2-fold, in to be “exactly the same' if they have 100% sequence particular at least 10-fold, more in particular at least identity (as defined herein) over their entire length; 100-fold, and up to 1000-fold or more. A nucleic acid h) When comparing two amino acid sequences, the term 50 sequence or amino acid sequence that is “in essentially “amino acid difference” refers to an insertion, deletion or isolated form' is preferably essentially homogeneous, as Substitution of a single amino acid residue on a position determined using a suitable technique, such as a Suitable of the first sequence, compared to the second sequence; it chromatographical technique. Such as polyacrylamide-gel being understood that two amino acid sequences can electrophoresis; contain one, two or more such amino acid differences; 55 k) The term “domain as used herein generally refers to a i) When a nucleotide sequence or amino acid sequence is globular region of an amino acid sequence (such as an said to "comprise another nucleotide sequence or amino antibody chain, and in particular to a globular region of a acid sequence, respectively, or to “essentially consist of heavy chain antibody), or to a polypeptide that essentially another nucleotide sequence or amino acid sequence, this consists of Such a globular region. Usually, Such a domain may mean that the latter nucleotide sequence or amino 60 will comprise peptide loops (for example 3 or 4 peptide acid sequence has been incorporated into the firstmen loops) stabilized, for example, as a sheet or by disulfide tioned nucleotide sequence or amino acid sequence, bonds. The term “binding domain refers to such a respectively, but more usually this generally means that domain that is directed against an antigenic determinant the firstmentioned nucleotide sequence or amino acid (as defined herein); sequence comprises within its sequence a stretch of 65 l) The term “antigenic determinant” refers to the epitope on nucleotides or amino acid residues, respectively, that has the antigen recognized by the antigen-binding molecule the same nucleotide sequence or amino acid sequence, (such as a NANOBODYR (V) or a polypeptide of the US 9,512,236 B2 41 42 invention) and more in particular by the antigen-binding to measure dissociation constants of more then 10 site of said molecule. The terms “antigenic determinant moles/liter or 10 moles/liter (e.g., of 10° moles/ and "epitope' may also be used interchangeably herein. liter). Optionally, as will also be clear to the skilled m) An amino acid sequence (such as a NANOBODYR person, the (actual or apparent) dissociation constant (V), an antibody, a polypeptide of the invention, or may be calculated on the basis of the (actual or appar generally an antigen binding protein or polypeptide or a ent) association constant (K), by means of the rela fragment thereof) that can (specifically) bind to, that has tionship K-1/K. affinity for and/or that has specificity for a specific anti The affinity denotes the strength or stability of a molecular genic determinant, epitope, antigen or protein (or for at interaction. The affinity is commonly given as by the least one part, fragment or epitope thereof) is said to be 10 "against” or “directed against” said antigenic determinant, K or dissociation constant, which has units of mol/ epitope, antigen or protein. liter (or M). The affinity can also be expressed as an n) The term “specificity” refers to the number of different association constant, K, which equals 1/K and has types of antigens or antigenic determinants to which a units of (mol/liter)'' (or M'). In the present specifi particular antigen-binding molecule or antigen-binding 15 cation, the stability of the interaction between two protein (such as a NANOBODYR (V) or a polypeptide molecules (such as an amino acid sequence, NANO of the invention) molecule can bind. The specificity of an BODYR (V) or polypeptide of the invention and its antigen-binding protein can be determined based on affin intended target) will mainly be expressed in terms of ity and/or avidity. The affinity, represented by the equi the K value of their interaction; it being clear to the librium constant for the dissociation of an antigen with an skilled person that in view of the relation K=1/K, antigen-binding protein (K), is a measure for the binding specifying the strength of molecular interaction by its strength between an antigenic determinant and an antigen K. Value can also be used to calculate the correspond binding site on the antigen-binding protein: the lesser the ing K value. The K-value characterizes the strength value of the K, the stronger the binding strength between of a molecular interaction also in a thermodynamic an antigenic determinant and the antigen-binding mol 25 sense as it is related to the free energy (DG) of binding ecule (alternatively, the affinity can also be expressed as by the well known relation DG=RT ln(K) (equiva the affinity constant (K), which is 1/K). As will be clear lently DG=-RT. ln(K)), where R equals the gas con to the skilled person (for example on the basis of the stant, T equals the absolute temperature and ln denotes further disclosure herein), affinity can be determined in a the natural logarithm. The K for biological interac manner known per se, depending on the specific antigen 30 tions which are considered meaningful (e.g. specific) of interest. Avidity is the measure of the strength of are typically in the range of 10'M (0.1 nM) to 10M binding between an antigen-binding molecule (such as a (10000 nM). The stronger an interaction is, the lower is NANOBODYR (V) or polypeptide of the invention) its K. and the pertinent antigen. Avidity is related to both the The K can also be expressed as the ratio of the disso affinity between an antigenic determinant and its antigen 35 ciation rate constant of a complex, denoted as k, to binding site on the antigen-binding molecule and the the rate of its association, denoted k, (so that Kok? number of pertinent binding sites present on the antigen k, and Ki-k/km). The off-rate k has units s binding molecule. Typically, antigen-binding proteins (where s is the SI unit notation of second). The on-rate (such as the amino acid sequences, Nanobodies and/or k, has units M' s'. The on-rate may vary between polypeptides of the invention) will bind to their antigen 40 10 M' s to about 107 M' s', approaching the with a dissociation constant (Ki) of 10 to 10' moles/ diffusion-limited association rate constant for bimo liter or less, and preferably 107 to 10° moles/liter or lecular interactions. The off-rate is related to the half less and more preferably 10 to 10° moles/liter (i.e. life of a given molecular interaction by the relation with an association constant (K) of 10 to 10' liter/ to ln(2)/k. The off-rate may vary between 106 Sl moles or more, and preferably 107 to 10' liter/moles or 45 (near irreversible complex with at of multiple days) more and more preferably 10 to 10" liter/moles). Any to 1 s' (t=0.69s). K, value greater than 10 mol/liter (or any K value lower The affinity of a molecular interaction between two mol than 10 M') liters/mol is generally considered to indi ecules can be measured via different techniques known cate non-specific binding. Preferably, a monovalent per se, such as the well known Surface plasmon reso immunoglobulin sequence of the invention will bind to 50 nance (SPR) biosensor technique (see for example the desired antigen with an affinity less than 500 nM, Ober et al., Intern. Immunology, 13, 1551-1559, 2001) preferably less than 200 nM, more preferably less than 10 where one molecule is immobilized on the biosensor nM, such as less than 500 pM. Specific binding of an chip and the other molecule is passed over the immo antigen-binding protein to an antigen or antigenic deter bilized molecule under flow conditions yielding k, minant can be determined in any Suitable manner known 55 k, measurements and hence K, (or K) values. This per se, including, for example, Scatchard analysis and/or can for example be performed using the well-known competitive binding assays, such as radioimmunoassays BIACORE instruments. (RIA), enzyme immunoassays (EIA) and sandwich com It will also be clear to the skilled person that the measured petition assays, and the different variants thereof known K may correspond to the apparent K if the measuring perse in the art; as well as the other techniques mentioned 60 process somehow influences the intrinsic binding affin herein. ity of the implied molecules for example by artefacts The dissociation constant may be the actual or apparent related to the coating on the biosensor of one molecule. dissociation constant, as will be clear to the skilled Also, an apparent K, may be measured if one molecule person. Methods for determining the dissociation con contains more than one recognition sites for the other stant will be clear to the skilled person, and for example 65 molecule. In Such situation the measured affinity may include the techniques mentioned herein. In this be affected by the avidity of the interaction by the two respect, it will also be clear that it may not be possible molecules. US 9,512,236 B2 43 44 Another approach that may be used to assess affinity is the mining the level or concentration of the amino acid 2-step ELISA (Enzyme-LinkedImmunosorbent Assay) sequence, compound or polypeptide of the invention in procedure of Friguet et al. (J. Immunol. Methods, 77, said blood sample; and calculating, from (a plot of) the 305-19, 1985). This method establishes a solution data thus obtained, the time until the level or concentra phase binding equilibrium measurement and avoids tion of the amino acid sequence, compound or polypep possible artefacts relating to adsorption of one of the tide of the invention has been reduced by 50% compared molecules on a Support Such as plastic. to the initial level upon dosing. Reference is for example However, the accurate measurement of K may be quite made to the Experimental Part below, as well as to the labor-intensive and as consequence, often apparent K, standard handbooks, such as Kenneth, A et al: Chemical values are determined to assess the binding strength of 10 Stability of Pharmaceuticals: A Handbook for Pharma two molecules. It should be noted that as long all cists and Peters et al., Pharmacokinete analysis: A Practi measurements are made in a consistent way (e.g. keep cal Approach (1996). Reference is also made to “Phar ing the assay conditions unchanged) apparent K, mea macokinetics', M Gibaldi & D Perron, published by Surements can be used as an approximation of the true Marcel Dekker, 2nd Rev. edition (1982). K. and hence in the present document K, and apparent 15 As will also be clear to the skilled person (see for example K, should be treated with equal importance or rel pages 6 and 7 of WO 04/003019 and in the further VaC. references cited therein), the half-life can be expressed Finally, it should be noted that in many situations the using parameters such as the t1/2-alpha, t1/2-beta and experienced scientist may judge it to be convenient to the area under the curve (AUC). In the present speci determine the binding affinity relative to some refer fication, an “increase in half-life” refers to an increase ence molecule. For example, to assess the binding in any one of these parameters. Such as any two of these strength between molecules A and B, one may e.g. use parameters, or essentially all three these parameters. As a reference molecule C that is known to bind to B and used herein “increase in half-life' or “increased half that is suitably labelled with a fluorophore or chro life' in particular refers to an increase in the t1/2-beta, mophore group or other chemical moiety, Such as biotin 25 either with or without an increase in the t1/2-alpha for easy detection in an ELISA or FACS (Fluorescent and/or the AUC or both. activated cell sorting) or other format (the fluorophore p) In the context of the present invention, “modulating or for fluorescence detection, the chromophore for light “to modulate” generally means either reducing or inhib absorption detection, the biotin for streptavidin-medi iting the activity of, or alternatively increasing the activity ated ELISA detection). Typically, the reference mol 30 of a target or antigen, as measured using a suitable in ecule C is kept at a fixed concentration and the con vitro, cellular or in vivo assay. In particular, "modulating centration of A is varied for a given concentration or or “to modulate may mean either reducing or inhibiting amount of B. As a result an ICso value is obtained the activity of, or alternatively increasing a (relevant or corresponding to the concentration of A at which the intended) biological activity of a target or antigen, as signal measured for C in absence of A is halved. 35 measured using a suitable in vitro, cellular or in vivo Provided K, a the K, of the reference molecule, is assay (which will usually depend on the target or antigen known, as well as the total concentration c, of the involved), by at least 1%, preferably at least 5%, such as reference molecule, the apparent K, for the interaction at least 10% or at least 25%, for example by at least 50%, A-B can be obtained from following formula: at least 60%, at least 70%, at least 80%, or 90% or more, KICso/(1+c/K, ). Note that if c, isK, a 40 compared to activity of the target or antigen in the same KasiCo. Provided the measurement of the ICso is assay under the same conditions but without the presence performed in a consistent way (e.g. keeping c, fixed) of the construct of the invention. for the binders that are compared, the strength or As will be clear to the skilled person, “modulating may stability of a molecular interaction can be assessed by also involve effecting a change (which may either be an the ICso and this measurement is judged as equivalent 45 increase or a decrease) in affinity, avidity, specificity to K, or to apparent K, throughout this text. and/or selectivity of a target or antigen for one or more o) The half-life of an amino acid sequence, compound or of its ligands, binding partners, partners for association polypeptide of the invention can generally be defined as into a homomultimeric or heteromultimeric form, or the time taken for the serum concentration of the amino Substrates; and/or effecting a change (which may either acid sequence, compound or polypeptide to be reduced by 50 be an increase or a decrease) in the sensitivity of the 50%, in vivo, for example due to degradation of the target or antigen for one or more conditions in the sequence or compound and/or clearance or sequestration medium or Surroundings in which the target or antigen of the sequence or compound by natural mechanisms. The is present (Such as pH, ion strength, the presence of in vivo half-life of an amino acid sequence, compound or co-factors, etc.), compared to the same conditions but polypeptide of the invention can be determined in any 55 without the presence of the construct of the invention. manner known per se, such as by pharmacokinetic analy As will be clear to the skilled person, this may again be sis. Suitable techniques will be clear to the person skilled determined in any suitable manner and/or using any in the art, and may for example generally involve the steps Suitable assay known per se, depending on the target or of Suitably administering to a warm-blooded animal (i.e. antigen involved. to a human or to another Suitable mammal, such as a 60 "Modulating may also mean effecting a change (i.e. an mouse, rabbit, rat, pig, dog or a primate, for example activity as an agonist, as an antagonist or as a reverse monkeys from the genus Macaca (such as, and in par agonist, respectively, depending on the target or antigen ticular, cynomolgus monkeys (Macaca fascicularis) and/ and the desired biological or physiological effect) with or rhesus monkeys (Macaca mulata)) and baboon (Papio respect to one or more biological or physiological ursinus)) a suitable dose of the amino acid sequence, 65 mechanisms, effects, responses, functions, pathways or compound or polypeptide of the invention; collecting activities in which the target or antigen (or in which its blood samples or other samples from said animal; deter Substrate(s), ligand(s) or pathway(s) are involved. Such US 9,512,236 B2 45 46 as its signalling pathway or metabolic pathway and at least 1000 times less, such as 10.000 times less or even their associated biological or physiological effects) is less than that, than the K, with which said amino acid involved. Again, as will be clear to the skilled person, sequence or polypeptide binds to the second target or Such an action as an agonist or an antagonist may be polypeptide. Preferably, when an amino acid sequence or determined in any Suitable manner and/or using any polypeptide is 'specific for a first target or antigen Suitable (in vitro and usually cellular or in assay) assay compared to a second target or antigen, it is directed known per se, depending on the target or antigen against (as defined herein) said first target or antigen, but involved. In particular, an action as an agonist or not directed against said second target or antigen. antagonist may be such that an intended biological or s) The terms “cross-block”. “cross-blocked' and “cross physiological activity is increased or decreased, respec 10 blocking are used interchangeably herein to mean the tively, by at least 1%, preferably at least 5%, such as at ability of an amino acid sequence or other binding agents least 10% or at least 25%, for example by at least 50%, (such as a polypeptide of the invention) to interfere with at least 60%, at least 70%, at least 80%, or 90% or the binding of other amino acid sequences or binding more, compared to the biological or physiological agents of the invention to a given target. The extend to activity in the same assay under the same conditions 15 which an amino acid sequence or other binding agents of but without the presence of the construct of the inven the invention is able to interfere with the binding of tion. Modulating may for example also involve allos another to target, and therefore whether it can be said to teric modulation of the target or antigen; and/or reduc cross-block according to the invention, can be determined ing or inhibiting the binding of the target or antigen to using competition binding assays. One particularly Suit one of its Substrates or ligands and/or competing with able quantitative assay uses a Biacore machine which can a natural ligand, Substrate for binding to the target or measure the extent of interactions using Surface plasmon antigen. Modulating may also involve activating the resonance technology. Another Suitable quantitative target or antigen or the mechanism or pathway in which cross-blocking assay uses an ELISA-based approach to it is involved. Modulating may for example also measure competition between amino acid sequence or involve effecting a change in respect of the folding or 25 another binding agents in terms of their binding to the conformation of the target or antigen, or in respect of target. the ability of the target or antigen to fold, to change its The following generally describes a suitable Biacore conformation (for example, upon binding of a ligand), assay for determining whether an amino acid sequence to associate with other (sub)units, or to disassociate. or other binding agent cross-blocks or is capable of Modulating may for example also involve effecting a 30 cross-blocking according to the invention. It will be change in the ability of the target or antigen to transport appreciated that the assay can be used with any of the other compounds or to serve as a channel for other amino acid sequence or other binding agents described compounds (such as ions). Modulating may be revers herein. The Biacore machine (for example the Biacore ible or irreversible, but for pharmaceutical and phar 3000) is operated in line with the manufacturer's rec macological purposes will usually be in a reversible 35 ommendations. Thus in one cross-blocking assay, the a. target protein is coupled to a CM5 Biacore chip using q) In respect of a target or antigen, the term “interaction site' standard amine coupling chemistry to generate a Sur on the target or antigen means a site, epitope, antigenic face that is coated with the target. Typically 200-800 determinant, part, domain or stretch of amino acid resi resonance units of the target would be coupled to the dues on the target or antigen that is a site for binding to 40 chip (an amount that gives easily measurable levels of a ligand, receptor or other binding partner, a catalytic site, binding but that is readily saturable by the concentra a cleavage site, a site for allosteric interaction, a site tions of test reagent being used). Two test amino acid involved in multimerisation (Such as homomerization or sequences (termed A* and B) to be assessed for their heterodimerization) of the target or antigen; or any other ability to cross-block each other are mixed at a one to site, epitope, antigenic determinant, part, domain or 45 one molar ratio of binding sites in a suitable buffer to stretch of amino acid residues on the target or antigen that create the test mixture. When calculating the concen is involved in a biological action or mechanism of the trations on a binding site basis the molecular weight of target or antigen. More generally, an “interaction site' can an amino acid sequence is assumed to be the total be any site, epitope, antigenic determinant, part, domain molecular weight of the amino acid sequence divided or stretch of amino acid residues on the target or antigen 50 by the number of target binding sites on that amino acid to which an amino acid sequence or polypeptide of the sequence. The concentration of each amino acid invention can bind Such that the target or antigen (and/or sequence in the test mix should be high enough to any pathway, interaction, signalling, biological mecha readily Saturate the binding sites for that amino acid nism or biological effect in which the target or antigen is sequence on the target molecules captured on the involved) is modulated (as defined herein). 55 Biacore chip. The amino acid sequences in the mixture r) An amino acid sequence or polypeptide is said to be are at the same molar concentration (on a binding basis) 'specific for a first target or antigen compared to a and that concentration would typically be between 1.00 second target or antigen when is binds to the first antigen and 1.5 micromolar (on a binding site basis). Separate with an affinity (as described above, and suitably solutions containing A* alone and B* alone are also expressed as a K value, K value, K rate and/or K, 60 prepared. A* and B* in these solutions should be in the rate) that is at least 10 times, such as at least 100 times, same buffer and at the same concentration as in the test and preferably at least 1000 times, and up to 10.000 times mix. The test mixture is passed over the target-coated or more better than the affinity with which said amino acid Biacore chip and the total amount of binding recorded. sequence or polypeptide binds to the second target or The chip is then treated in such a way as to remove the polypeptide. For example, the first antigen may bind to 65 bound amino acid sequences without damaging the the target or antigen with a K value that is at least 10 chip-bound target. Typically this is done by treating the times less, such as at least 100 times less, and preferably chip with 30 mM HCl for 60 seconds. The solution of US 9,512,236 B2 47 48 A* alone is then passed over the target-coated Surface amino acid sequence and the amino acid sequence in and the amount of binding recorded. The chip is again solution compete for binding of the limited number of treated to remove all of the bound amino acid target molecules. The plate is washed to remove excess sequences without damaging the chip-bound target. target that has not been bound by the coated amino acid The solution of B* alone is then passed over the sequence and to also remove the second, Solution phase target-coated Surface and the amount of binding amino acid sequence as well as any complexes formed recorded. The maximum theoretical binding of the between the second, Solution phase amino acid mixture of A* and B is next calculated, and is the sum sequence and target. The amount of bound target is then of the binding of each amino acid sequence when measured using a reagent that is appropriate to detect passed over the target Surface alone. If the actual 10 the target. An amino acid sequence in Solution that is recorded binding of the mixture is less than this theo able to cross-block the coated amino acid sequence will retical maximum then the two amino acid sequences be able to cause a decrease in the number of target are cross-blocking each other. Thus, in general, a molecules that the coated amino acid sequence can bind cross-blocking amino acid sequence or other binding relative to the number of target molecules that the agent according to the invention is one which will bind 15 coated amino acid sequence can bind in the absence of to the target in the above Biacore cross-blocking assay the second, Solution phase, amino acid sequence. In the Such that during the assay and in the presence of a instance where the first amino acid sequence, e.g. an second amino acid sequence or other binding agent of Ab-X, is chosen to be the immobilized amino acid the invention the recorded binding is between 80% and sequence, it is coated onto the wells of the ELISA plate, 0.1% (e.g. 80% to 4%) of the maximum theoretical after which the plates are blocked with a suitable binding, specifically between 75% and 0.1% (e.g. 75% blocking Solution to minimize non-specific binding of to 4%) of the maximum theoretical binding, and more reagents that are Subsequently added. An excess specifically between 70% and 0.1% (e.g. 70% to 4%) of amount of the second amino acid sequence, i.e. Ab-Y. maximum theoretical binding (as just defined above) of is then added to the ELISA plate such that the moles of the two amino acid sequences or binding agents in 25 Ab-Y target binding sites per well are at least 10 fold combination. The Biacore assay described above is a higher than the moles of Ab-X target binding sites that primary assay used to determine if amino acid were used, per well, during the coating of the ELISA sequences or other binding agents cross-block each plate. target is then added such that the moles of other according to the invention. On rare occasions target added per well are at least 25-fold lower than particular amino acid sequences or other binding agents 30 the moles of Ab-X target binding sites that were used may not bind to target coupled via amine chemistry to for coating each well. Following a suitable incubation a CM5 Biacore chip (this usually occurs when the period the ELISA plate is washed and a reagent for relevant binding site on target is masked or destroyed detecting the target is added to measure the amount of by the coupling to the chip). In Such cases cross target specifically bound by the coated anti-target blocking can be determined using a tagged version of 35 amino acid sequence (in this case Ab-X). The back the target, for example a N-terminal His-tagged version ground signal for the assay is defined as the signal (R & D Systems, Minneapolis, Minn., USA; 2005 obtained in wells with the coated amino acid sequence cati 1406-ST-025). In this particular format, an anti-His (in this case Ab-X), second solution phase amino acid amino acid sequence would be coupled to the Biacore sequence (in this case Ab-Y), target buffer only (i.e. chip and then the His-tagged target would be passed 40 no target) and target detection reagents. The positive over the surface of the chip and captured by the anti-His control signal for the assay is defined as the signal amino acid sequence. The cross blocking analysis obtained in wells with the coated amino acid sequence would be carried out essentially as described above, (in this case Ab-X), second solution phase amino acid except that after each chip regeneration cycle, new sequence buffer only (i.e. no second solution phase His-tagged target would be loaded back onto the anti 45 amino acid sequence), target and target detection His amino acid sequence coated Surface. In addition to reagents. The ELISA assay may be run in Such a the example given using N-terminal His-tagged tar manner so as to have the positive control signal be at get, C-terminal His-tagged target could alternatively least 6 times the background signal. To avoid any be used. Furthermore, various other tags and tag bind artefacts (e.g. significantly different affinities between ing protein combinations that are known in the art 50 Ab-X and Ab-Y for target) resulting from the choice could be used for Such a cross-blocking analysis (e.g. of which amino acid sequence to use as the coating HA tag with anti-HA antibodies; FLAG tag with anti amino acid sequence and which to use as the second FLAG antibodies; biotin tag with streptavidin). (competitor) amino acid sequence, the cross-blocking The following generally describes an ELISA assay for assay may to be run in two formats: 1) format 1 is determining whether an amino acid sequence or other 55 where Ab-X is the amino acid sequence that is coated binding agent directed against a target cross-blocks or onto the ELISA plate and Ab-Y is the competitor amino is capable of cross-blocking as defined herein. It will be acid sequence that is in Solution and 2) format 2 is appreciated that the assay can be used with any of the where Ab-Y is the amino acid sequence that is coated amino acid sequences (or other binding agents such as onto the ELISA plate and Ab-X is the competitor amino polypeptides of the invention) described herein. The 60 acid sequence that is in solution. Ab-X and Ab-Y are general principal of the assay is to have an amino acid defined as cross-blocking if, either in format 1 or in sequence or binding agent that is directed against the format 2, the solution phase anti-target amino acid target coated onto the wells of an ELISA plate. An sequence is able to cause a reduction of between 60% excess amount of a second, potentially cross-blocking, and 100%, specifically between 70% and 100%, and anti-target amino acid sequence is added in Solution 65 more specifically between 80% and 100%, of the target (i.e. not bound to the ELISA plate). A limited amount detection signal {i.e. the amount of target bound by the of the target is then added to the wells. The coated coated amino acid sequence) as compared to the target US 9,512,236 B2 49 50 detection signal obtained in the absence of the Solution limiting the scope of the invention and/or of the appended phase anti-target amino acid sequence (i.e. the positive claims in any way, unless explicitly indicated otherwise control wells). herein. t) As further described herein, the total number of amino For a general description of heavy chain antibodies and acid residues in a NANOBODYR (V) can be in the 5 the variable domains thereof, reference is interalia made to region of 110-120, is preferably 112-115, and is most the prior art cited herein, to the review article by Muylder preferably 113. It should however be noted that parts, mans in Reviews in Molecular Biotechnology 74(2001), fragments, analogs or derivatives (as further described 277-302; as well as to the following patent applications, herein) of a NANOBODYR (V) are not particularly which are mentioned as general background art: WO limited as to their length and/or size, as long as such parts, 10 fragments, analogs or derivatives meet the further require 94/04678, WO95/04079 and WO 96/34103 of the Vrije ments outlined herein and are also preferably suitable for Universiteit Brussel; WO 94/25591, WO 99/37681, WO the purposes described herein; 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, u) The amino acid residues of a NANOBODYR (V) are WO 01/44301, EP 1134231 and WO 02/48.193 of Unilever, numbered according to the general numbering for V. 15 WO 97/49805, WO 01/21817, WO 03/035694, WO domains given by Kabat et al. (“Sequence of proteins of O3/O54016 and WO 03/055527 of the Vlaams Instituut voor immunological interest', US Public Health Services, NIH Biotechnologie (VIB); WO 03/050531 of Algonomics N.V. Bethesda, Md., Publication No. 91), as applied to V and Ablynx N.V., WO 01/90190 by the National Research domains from Camelids in the article of Riechmann and Council of Canada; WO 03/025020 (=EP 1433 793) by the Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 Institute of Antibodies; as well as WO 04/041867, WO (1-2): 185-195 (see for example FIG. 2 of this publica 04/041862, WO 04/041865, WO 04/041863, WO tion); or referred to herein. According to this numbering, 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, FR1 of a NANOBODYR (V) comprises the amino WO 06/122786, WO 06/122787 and WO 06/122825, by acid residues at positions 1-30, CDR1 of a NANO Ablynx N.V. and the further published patent applications by BODY (R) (V) comprises the amino acid residues at 25 Ablynx N.V. Reference is also made to the further prior art positions 31-35, FR2 of a NANOBODYR (V) com mentioned in these applications, and in particular to the list prises the amino acids at positions 36-49, CDR2 of a of references mentioned on pages 41-43 of the International NANOBODYR (V) comprises the amino acid residues application WO 06/04.0153, which list and references are at positions 50-65, FR3 of a NANOBODYR (V) incorporated herein by reference. comprises the amino acid residues at positions 66-94, 30 In accordance with the terminology used in the art (see the CDR3 of a NANOBODYR (V) comprises the amino above references), the variable domains present in naturally acid residues at positions 95-102, and FR4 of a NANO occurring heavy chain antibodies will also be referred to as BODYR (V) comprises the amino acid residues at “V. domains, in order to distinguish them from the heavy positions 103-113. In this respect, it should be noted chain variable domains that are present in conventional that—as is well known in the art for V, domains and for 35 4-chain antibodies (which will be referred to hereinbelow as V. domains—the total number of amino acid residues in “V, domains”) and from the light chain variable domains each of the CDR's may vary and may not correspond to that are present in conventional 4-chain antibodies (which the total number of amino acid residues indicated by the will be referred to hereinbelow as “V, domains'). Kabat numbering (that is, one or more positions according As mentioned in the prior art referred to above, V. to the Kabat numbering may not be occupied in the actual 40 domains have a number of unique structural characteristics sequence, or the actual sequence may contain more amino and functional properties which make isolated V. domains acid residues than the number allowed for by the Kabat (as well as Nanobodies based thereon, which share these numbering). This means that, generally, the numbering structural characteristics and functional properties with the according to Kabat may or may not correspond to the naturally occurring V, domains) and proteins containing actual numbering of the amino acid residues in the actual 45 the same highly advantageous for use as functional antigen sequence. Generally, however, it can be said that, accord binding domains or proteins. In particular, and without being ing to the numbering of Kabat and irrespective of the limited thereto, V, domains (which have been “designed number of amino acid residues in the CDR's, position 1 by nature to functionally bind to an antigen without the according to the Kabat numbering corresponds to the start presence of, and without any interaction with, a light chain of FR1 and vice versa, position 36 according to the Kabat 50 variable domain) and Nanobodies can function as a single, numbering corresponds to the start of FR2 and vice versa, relatively small, functional antigen-binding structural unit, position 66 according to the Kabat numbering corre domain or protein. This distinguishes the V. domains from sponds to the start of FR3 and vice versa, and position 103 the V and V, domains of conventional 4-chain antibodies, according to the Kabat numbering corresponds to the start which by themselves are generally not suited for practical of FR4 and vice versa.. Alternative methods for num 55 application as single antigen-binding proteins or domains, bering the amino acid residues of V. domains, which but need to be combined in some form or another to provide methods can also be applied in an analogous manner to a functional antigen-binding unit (as in for example con V. domains from Camelids and to Nanobodies, are the ventional antibody fragments such as Fab fragments; in method described by Chothia et al. (Nature 342, 877-883 ScFv's fragments, which consist of a V. domain covalently (1989)), the so-called “AbM definition' and the so-called 60 linked to a V, domain). “contact definition’. However, in the present description, Because of these unique properties, the use of V. claims and figures, the numbering according to Kabat as domains and Nanobodies as single antigen-binding proteins applied to V. domains by Riechmann and Muyldermans or as antigen-binding domains (i.e. as part of a larger protein will be followed, unless indicated otherwise; and or polypeptide) offers a number of significant advantages V) The Figures, Sequence Listing and the Experimental 65 over the use of conventional V and V, domains, scEv’s or Part/Examples are only given to further illustrate the conventional antibody fragments (such as Fab- or F(ab')- invention and should not be interpreted or construed as fragments): US 9,512,236 B2 51 52 only a single domain is required to bind an antigen with (single) domain antibodies that may be derived from vari high affinity and with high selectivity, so that there is no able domains of conventional antibodies. These improved need to have two separate domains present, nor to and advantageous properties will become clear from the assure that these two domains are present in the right further description herein, and for example include, without spatial conformation and configuration (i.e. through the 5 limitation, one or more of: use of especially designed linkers, as with ScFv's); increased affinity and/or avidity for GPCRs, either in a V. domains and Nanobodies can be expressed from a monovalent format, in a multivalent format (for single gene and require no post-translational folding or example in a bivalent format) and/or in a multispecific modifications; format (for example one of the multispecific formats V. domains and Nanobodies can easily be engineered 10 described hereinbelow); into multivalent and multispecific formats (as further better suitability for formatting in a multivalent format discussed herein); (for example in a bivalent format): V. domains and Nanobodies are highly soluble and do better suitability for formatting in a multispecific format not have a tendency to aggregate (as with the mouse 15 (for example one of the multispecific formats described derived “dAb’s described by Ward et al., Nature, Vol. hereinbelow); 341, 1989, p. 544); improved suitability or susceptibility for “humanizing V. domains and Nanobodies are highly stable to heat, substitutions (as defined herein); pH, proteases and other denaturing agents or conditions less immunogenicity, either in a monovalent format, in a (see for example Ewert et al. Supra); multivalent format (for example in a bivalent format) V. domains and Nanobodies are easy and relatively and/or in a multispecific format (for example one of the cheap to prepare, even on a scale required for produc multispecific formats described hereinbelow); tion. For example, V. domains, Nanobodies and pro increased Stability, either in a monovalent format, in a teins/polypeptides containing the same can be pro multivalent format (for example in a bivalent format) duced using microbial fermentation (e.g. as further 25 and/or in a multispecific format (for example one of the described below) and do not require the use of mam multispecific formats described hereinbelow); malian expression systems, as with for example con increased specificity towards GPCRs, either in a monova ventional antibody fragments; lent format, in a multivalent format (for example in a V. domains and Nanobodies are relatively small (ap bivalent format) and/or in a multispecific format (for proximately 15 kDa, or 10 times smaller than a con 30 example one of the multispecific formats described ventional IgG) compared to conventional 4-chain anti hereinbelow); bodies and antigen-binding fragments thereof, and decreased or where desired increased cross-reactivity therefore show high(er) penetration into tissues (in with GPCRs from different species: cluding but not limited to solid tumors and other dense 35 and/or tissues) than Such conventional 4-chain antibodies and one or more other improved properties desirable for antigen-binding fragments thereof; pharmaceutical use (including prophylactic use and/or V. domains and Nanobodies can show so-called cavity therapeutic use) and/or for diagnostic use (including binding properties (inter alia due to their extended but not limited to use for imaging purposes), either in CDR3 loop, compared to conventional V. domains) 40 a monovalent format, in a multivalent format (for and can therefore also access targets and epitopes not example in a bivalent format) and/or in a multispecific accessible to conventional 4-chain antibodies and anti format (for example one of the multispecific formats gen-binding fragments thereof. For example, it has described hereinbelow). been shown that V. domains and Nanobodies can As generally described herein for the amino acid inhibit enzymes (see for example WO 97/498.05: Tran 45 sequences of the invention, the Nanobodies of the invention sue et al., Proteins 1998 Sep. 1; 32(4): 515-22: Lauw are preferably in essentially isolated form (as defined ereys et al., EMBO J. 1998 Jul. 1; 17(13): 3512-20). herein), or form part of a protein or polypeptide of the In a specific and preferred aspect, the invention provides invention (as defined herein), which may comprise or essen Nanobodies against GPCRs, and in particular Nanobodies tially consist of one or more Nanobodies of the invention against GPCRs from a warm-blooded animal, and more in 50 and which may optionally further comprise one or more particular Nanobodies against GPCRs from a mammal, and further amino acid sequences (all optionally linked via one especially Nanobodies against human GPCRs; as well as or more suitable linkers). For example, and without limita proteins and/or polypeptides comprising at least one Such tion, the one or more amino acid sequences of the invention NANOBODYR (V). may be used as a binding unit in Such a protein or polypep In particular, the invention provides Nanobodies against 55 GPCRs, and proteins and/or polypeptides comprising the tide, which may optionally contain one or more further same, that have improved therapeutic and/or pharmacologi amino acid sequences that can serve as a binding unit (i.e. cal properties and/or other advantageous properties (such as, against one or more other targets than GPCRs), so as to for example, improved ease of preparation and/or reduced provide a monovalent, multivalent or multispecific polypep costs of goods), compared to conventional antibodies 60 tide of the invention, respectively, all as described herein. In against GPCRs or fragments thereof, compared to constructs particular, Such a protein or polypeptide may comprise or that could be based on such conventional antibodies or essentially consist of one or more Nanobodies of the inven antibody fragments (such as Fab' fragments. F(ab') frag tion and optionally one or more (other) Nanobodies (i.e. ments, ScFv constructs, “diabodies' and other multispecific directed against other targets than GPCRs), all optionally constructs (see for example the review by Holliger and 65 linked via one or more Suitable linkers, so as to provide a Hudson, Nat Biotechnol. 2005 September; 23(9): 1126-36)), monovalent, multivalent or multispecific NANOBODYR and also compared to the so-called “dAbs' or similar (V) construct, respectively, as further described herein. US 9,512,236 B2 53 54 More particularly, the protein or polypeptide of the invention the Nanobodies can bind to GPCRs with a dissociation comprises or essentially consists of two or more Nanobodies constant (K) of 10 to 10° moles/liter or less, and wherein at least and “first NANOBODYR (V) is preferably 107 to 10' moles/liter or less and more directed against a first antigenic determinant, epitope, part, preferably 10 to 10' moles/liter (i.e. with an asso domain, subunit or confirmation of a GPCR; and wherein at ciation constant(K) of 10 to 10' liter/moles or more, least one “second NANOBODYR (V) is directed and preferably 10 to 10' liter/moles or more and more against a second antigenic determinant, epitope, part, preferably 10 to 10' liter/moles): domain, subunit or confirmation of said GPCR different and/or such that: from the first, so as to provide biparatopic or multiparatopic the Nanobodies can bind to GPCRs with a k-rate of NANOBODYR (V) constructs as further described 10 herein. Such proteins or polypeptides may also be in essen between 10 M's to about 107M s', preferably tially isolated form (as defined herein). between 10 M's and 107 M's', more preferably In a NANOBODYR (V) of the invention, the binding between 10 M-'s and 107M s', such as between site for binding against GPCRs is preferably formed by the 10 M's and 107 Ms; CDR sequences. Optionally, a NANOBODYR (V) of the 15 and/or such that they: invention may also, and in addition to the at least one the Nanobodies can bind to GPCRs with a k, rate binding site for binding against GPCRs, contain one or more between 1 s' (t=0.69s) and 10 s' (providing a further binding sites for binding against other antigens, near irreversible complex with at of multiple days), proteins or targets. For methods and positions for introduc preferably between 10° s' and 10 s', more pref ing Such second binding sites, reference is for example made erably between 10 s' and 10's", such as between to Keck and Huston, Biophysical Journal, 71, October 1996, 10 s. and 10 s. 2002-2011; EP 0 640 130 and WO 06/07260. Preferably, (the CDR sequences present in) the Nanobod As generally described herein for the amino acid ies of the invention are such that: a monovalent NANO sequences of the invention, when a NANOBODYR (V) BODYR (V) of the invention (or a polypeptide that of the invention (or a polypeptide of the invention compris 25 contains only one NANOBODYR (V) of the invention) ing the same) is intended for administration to a Subject (for is preferably such that it will bind to GPCRs with an affinity example for therapeutic and/or diagnostic purposes as less than 500 nM, preferably less than 200 nM, more described herein), it is preferably directed against human preferably less than 10 nM, such as less than 500 pM. GPCRs; whereas for veterinary purposes, it is preferably The affinity of the NANOBODYR (V) of the invention directed against GPCRs from the species to be treated. Also, 30 against GPCRs can be determined in a manner known perse, as with the amino acid sequences of the invention, a NANO for example using the general techniques for measuring K. BODYR (V) of the invention may or may not be cross reactive (i.e. directed against GPCRs from two or more K., k, or k, mentioned herein, as well as some of the species of mammal. Such as against human GPCRs and specific assays described herein. GPCRs from at least one of the species of mammal men 35 Some preferred IC50 values for binding of the Nanobod tioned herein). ies of the invention (and of polypeptides comprising the Also, again as generally described herein for the amino same) to GPCRs will become clear from the further descrip acid sequences of the invention, the Nanobodies of the tion and examples herein. invention may generally be directed against any antigenic Some particularly preferred, but non-limiting combina determinant, epitope, part, domain, Subunit or conformation 40 tions of CDR sequences, as well as preferred combinations (where applicable) of GPCRs. However, it is generally of CDR sequences and framework sequences, are mentioned assumed and preferred that the Nanobodies of the invention in Table A-1 below, which lists the CDR sequences and (and polypeptides comprising the same) are directed against framework sequences that are present in a number of pre and/or have been raised against at least one extracellular ferred (but non-limiting) Nanobodies of the invention. As region, domain, loop or other extracellular epitope of a 45 will be clear to the skilled person, a combination of CDR1. GPCR (or a suitable peptide derived therefrom). CDR2 and CDR3 sequences that occur in the same clone As already described herein, the amino acid sequence and (i.e. CDR1, CDR2 and CDR3 sequences that are mentioned structure of a NANOBODYR (V) can be considered— on the same line in Table A-1) will usually be preferred without however being limited thereto—to be comprised of (although the invention in its broadest sense is not limited four framework regions or “FR’s” (or sometimes also 50 thereto, and also comprises other suitable combinations of referred to as “FW’s”), which are referred to in the art and the CDR sequences mentioned in Table A-1). Also, a com herein as “Framework region 1 or “FR1'; as “Framework bination of CDR sequences and framework sequences that region 2' or “FR2'; as “Framework region 3” or “FR3”; and occur in the same clone (i.e. CDR sequences and framework as “Framework region 4” or “FR4', respectively; which sequences that are mentioned on the same line in Table A-1) framework regions are interrupted by three complementary 55 will usually be preferred (although the invention in its determining regions or “CDR's', which are referred to in the broadest sense is not limited thereto, and also comprises art as “Complementarity Determining Region 1 or other suitable combinations of the CDR sequences and “CDR1; as “Complementarity Determining Region 2' or framework sequences mentioned in Table A-1, as well as “CDR2; and as “Complementarity Determining Region 3 combinations of such CDR sequences and other suitable or “CDR3, respectively. Some preferred framework 60 framework sequences, e.g. as further described herein). sequences and CDR's (and combinations thereof) that are Also, in the Nanobodies of the invention that comprise the present in the Nanobodies of the invention are as described combinations of CDR's mentioned in Table A-1, each CDR herein. Other suitable CDR sequences can be obtained by can be replaced by a CDR chosen from the group consisting the methods described herein. of amino acid sequences that have at least 80%, preferably According to a non-limiting but preferred aspect of the 65 at least 90%, more preferably at least 95%, even more invention, (the CDR sequences present in) the Nanobodies preferably at least 99% sequence identity (as defined herein) of the invention are such that: with the mentioned CDR's; in which:

US 9,512,236 B2 61 62 In another preferred, but non-limiting aspect, the inven The polypeptides of the invention comprise or essentially tion relates to a NANOBODYR (V) in which the CDR consist of at least one NANOBODYR (V) of the inven sequences (as defined herein) have more than 80%, prefer tion. ably more than 90%, more preferably more than 95%, such It will be clear to the skilled person that the Nanobodies as 99% or more sequence identity (as defined herein) with 5 that are mentioned herein as “preferred (or “more pre the CDR sequences of at least one of the amino acid ferred”, “even more preferred, etc.) are also preferred (or sequences of SEQID NO’s: 526 to 527; SEQ ID NO's 538 more preferred, or even more preferred, etc.) for use in the to 541; 413 to 453 and 517 to 525. polypeptides described herein. Thus, polypeptides that com Generally, Nanobodies with the above CDR sequences prise or essentially consist of one or more “preferred 10 Nanobodies of the invention will generally be preferred, and may be as further described herein, and preferably have polypeptides that comprise or essentially consist of one or framework sequences that are also as further described more “more preferred Nanobodies of the invention will herein. Thus, for example and as mentioned herein, Such generally be more preferred, etc. Nanobodies may be naturally occurring Nanobodies (from Generally, proteins or polypeptides that comprise or any suitable species), naturally occurring V, Sequences 15 essentially consist of a single NANOBODYR (V) (such (i.e. from a suitable species of Camelid) or synthetic or as a single NANOBODYR (V) of the invention) will be semi-synthetic amino acid sequences or Nanobodies, includ referred to herein as "monovalent proteins or polypeptides ing but not limited to partially humanized Nanobodies or or as "monovalent constructs”. Proteins and polypeptides V. sequences, fully humanized Nanobodies or V. that comprise or essentially consist of two or more Nano sequences, camelized heavy chain variable domain 20 bodies (such as at least two Nanobodies of the invention or sequences, as well as Nanobodies that have been obtained by at least one NANOBODYR (V) of the invention and at the techniques mentioned herein. least one other NANOBODYR (V)) will be referred to Thus, in one specific, but non-limiting aspect, the inven herein as “multivalent proteins or polypeptides or as “mul tion relates to a humanized NANOBODYR (V), which tivalent constructs', and these may provide certain advan consists of 4 framework regions (FR1 to FR4 respectively) 25 tages compared to the corresponding monovalent Nanobod and 3 complementarity determining regions (CDR1 to ies of the invention. Some non-limiting examples of Such CDR3 respectively), in which CDR1 to CDR3 are as defined multivalent constructs will become clear from the further herein and in which said humanized NANOBODYR (V) description herein. comprises at least one humanizing Substitution (as defined According to one specific, but non-limiting aspect, a herein), and in particular at least one humanizing Substitu 30 polypeptide of the invention comprises or essentially con sists of at least two Nanobodies of the invention, such as two tion in at least one of its framework sequences (as defined or three Nanobodies of the invention. As further described herein). herein, Such multivalent constructs can provide certain In another preferred, but non-limiting aspect, the inven advantages compared to a protein or polypeptide comprising tion relates to a NANOBODYR (V) in which the CDR 35 or essentially consisting of a single NANOBODYR (V) sequences have at least 70% amino acid identity, preferably of the invention, Such as a much improved avidity for at least 80% amino acid identity, more preferably at least GPCRs. Such multivalent constructs will be clear to the 90% amino acid identity, such as 95% amino acid identity or skilled person based on the disclosure herein. According to more or even essentially 100% amino acid identity with the a particular preferred aspect, a polypeptide of the invention CDR sequences of at least one of the amino acid sequences 40 comprises or essentially consists of two or more Nanobodies of SEQID NO’s: 526 to 527; SEQID NO's 538 to 541; 413 of the invention, which are directed against GPCRs, wherein to 453 and 517 to 525. This degree of amino acid identity at least one “first NANOBODYR (V) is directed against can for example be determined by determining the degree of a first antigenic determinant, epitope, part, domain, Subunit amino acid identity (in a manner described herein) between or confirmation of a GPCR; and wherein at least one said NANOBODYR (V) and one or more of the 45 “second NANOBODYR (V) is directed against a second sequences of SEQID NO’s: 526 to 527; SEQ ID NO's 538 antigenic determinant, epitope, part, domain, Subunit or to 541; 413 to 453 and 517 to 525, in which the amino acid confirmation of said GPCR different from the first, so as to residues that form the framework regions are disregarded. provide biparatopic or multiparatopic constructs as Such Nanobodies can be as further described herein. described herein. Although it will be clear to the skilled In another preferred, but non-limiting aspect, the inven- 50 person that Such constructs may provide favourable proper tion relates to a NANOBODYR (V) with an amino acid ties over the monovalent constructs, present inventors how sequence that is chosen from the group consisting of SEQID ever demonstrated completely unexpectedly that the affinity NO’s: 526 to 527; SEQID NO’s 538 to 541; 413 to 453 and of the resulting “biparatopic polypeptides of the invention 517 to 525 or from the group consisting of from amino acid was significantly improved compared to the corresponding sequences that have more than 80%, preferably more than 55 monovalent and mono-specific bivalent constructs. 90%, more preferably more than 95%, such as 99% or more Examples of Such biparatopic polypeptides of the invention sequence identity (as defined herein) with at least one of the will become clear from the further description herein. amino acid sequences of SEQID NO’s: 526 to 527; SEQ ID According to another specific, but non-limiting aspect, a NO’s 538 to 541; 413 to 453 and 517 to 525. polypeptide of the invention comprises or essentially con Another preferred, but non-limiting aspect of the inven- 60 sists of at least one NANOBODYR (V) of the invention tion relates to humanized variants of the Nanobodies of SEQ and at least one other binding unit (i.e. directed against ID NO’s: 526 to 527; SEQID NO's 538 to 541; 413 to 453 another epitope, antigen, target, protein or polypeptide), and 517 to 525, that comprise, compared to the correspond which is preferably also a NANOBODYR (V). Such ing native V. sequence, at least one humanizing Substitu proteins or polypeptides are also referred to herein as tion (as defined herein), and in particular at least one 65 “multispecific' proteins or polypeptides or as “multispecific humanizing Substitution in at least one of its framework constructs', and these may provide certain advantages com sequences (as defined herein). pared to the corresponding monovalent Nanobodies of the US 9,512,236 B2 63 64 invention (as will become clear from the further discussion more Small proteins or peptides that can bind to serum herein of Some preferred, but-nonlimiting multispecific con proteins (such as, without limitation, the proteins and pep structs). Such multispecific constructs will be clear to the tides described in WO 91/01743, WO 01/45746, WO skilled person based on the disclosure herein. 02/076489 and to the US provisional application of Ablynx According to yet another specific, but non-limiting aspect, 5 N.V. entitled “Peptides capable of binding to serum pro a polypeptide of the invention comprises or essentially teins” of Ablynx N.V. filed on Dec. 5, 2006 (see also consists of at least one NANOBODYR (V) of the inven WO08/068280). tion, optionally one or more further Nanobodies, and at least Again, as will be clear to the skilled person, such Nano one other amino acid sequence (such as a protein or poly bodies, compounds, constructs or polypeptides may contain peptide) that confers at least one desired property to the 10 NANOBODYR (V) of the invention and/or to the result one or more additional groups, residues, moieties or binding ing fusion protein. Again, such fusion proteins may provide units, such as one or more further amino acid sequences and certain advantages compared to the corresponding monova in particular one or more additional Nanobodies (i.e. not lent Nanobodies of the invention. Some non-limiting directed against GPCRs), so as to provide a tri- of multi examples of Such amino acid sequences and of Such fusion 15 specific NANOBODYR (V) construct. constructs will become clear from the further description Generally, the Nanobodies of the invention (or com herein. pounds, constructs or polypeptides comprising the same) It is also possible to combine two or more of the above with increased half-life preferably have a half-life that is at aspects, for example to provide a trivalent bispecific con least 1.5 times, preferably at least 2 times, such as at least 5 struct comprising two Nanobodies of the invention and one 20 times, for example at least 10 times or more than 20 times, other NANOBODYR (V), and optionally one or more greater than the half-life of the corresponding amino acid other amino acid sequences. Further non-limiting examples sequence of the invention per se. For example, the Nano of Such constructs, as well as some constructs that are bodies, compounds, constructs or polypeptides of the inven particularly preferred within the context of the present tion with increased half-life may have a half-life that is invention, will become clear from the further description 25 increased with more than 1 hours, preferably more than 2 herein. hours, more preferably more than 6 hours, such as more than In the above constructs, the one or more Nanobodies 12 hours, or even more than 24, 48 or 72 hours, compared and/or other amino acid sequences may be directly linked to to the corresponding amino acid sequence of the invention each other and/or suitably linked to each other via one or per se. more linker sequences. Some Suitable but non-limiting 30 In a preferred, but non-limiting aspect of the invention, examples of such linkers will become clear from the further Such Nanobodies, compound, constructs or polypeptides of description herein. the invention exhibit a serum half-life in human of at least In one specific aspect of the invention, a NANOBODYR about 12 hours, preferably at least 24 hours, more preferably (V) of the invention or a compound, construct or poly at least 48 hours, even more preferably at least 72 hours or peptide of the invention comprising at least one NANO- 35 more. For example, compounds or polypeptides of the BODYR (V) of the invention may have an increased invention may have a half-life of at least 5 days (such as half-life, compared to the corresponding amino acid about 5 to 10 days), preferably at least 9 days (such as about sequence of the invention. Some preferred, but non-limiting 9 to 14 days), more preferably at least about 10 days (such examples of Such Nanobodies, compounds and polypeptides as about 10 to 15 days), or at least about 11 days (such as will become clear to the skilled person based on the further 40 about 11 to 16 days), more preferably at least about 12 days disclosure herein, and for example comprise Nanobodies (such as about 12 to 18 days or more), or more than 14 days sequences or polypeptides of the invention that have been (such as about 14 to 19 days). chemically modified to increase the half-life thereof (for In another one aspect of the invention, a polypeptide of example, by means of pegylation); amino acid sequences of the invention comprises one or more (such as two or the invention that comprise at least one additional binding 45 preferably one) Nanobodies of the invention linked (option site for binding to a serum protein (such as serum albumin, ally via one or more Suitable linker sequences) to one or see for example EP 0368 684 B1, page 4); or polypeptides more (such as two and preferably one) amino acid sequences of the invention that comprise at least one NANOBODYR that allow the resulting polypeptide of the invention to cross (V) of the invention that is linked to at least one moiety the blood brain barrier. In particular, said one or more amino (and in particular at least one amino acid sequence) that 50 acid sequences that allow the resulting polypeptides of the increases the half-life of the NANOBODYR (V) of the invention to cross the blood brain barrier may be one or invention. Examples of polypeptides of the invention that more (such as two and preferably one) Nanobodies, such as comprise Such half-life extending moieties or amino acid the Nanobodies described in WO 02/057445, of which FC44 sequences will become clear to the skilled person based on (SEQID NO: 189 of WO 06/04.0153) and FC5 (SEQID NO: the further disclosure herein; and for example include, 55 190 of WO 06/040154) are preferred examples. without limitation, polypeptides in which the one or more In particular, polypeptides comprising one or more Nano Nanobodies of the invention are suitable linked to one or bodies of the invention are preferably such that they: more serum proteins or fragments thereof (such as serum bind to GPCRs with a dissociation constant (Ki) of 10 albumin or suitable fragments thereof) or to one or more to 10' moles/liter or less, and preferably 107 to 10° binding units that can bind to serum proteins (such as, for 60 moles/liter or less and more preferably 10 to 10° example, Nanobodies or (single) domain antibodies that can moles/liter (i.e. with an association constant (K) of bind to serum proteins such as serum albumin, serum 10 to 10' liter/moles or more, and preferably 107 to immunoglobulins such as IgG, or transferrin); polypeptides 10' liter/moles or more and more preferably 10 to in which a NANOBODYR (V) of the invention is linked 10' liter/moles): to an Fc portion (Such as a human Fc) or a Suitable part or 65 and/or such that they: fragment thereof, or polypeptides in which the one or more bind to GPCRs with a k-rate of between 10 M's to Nanobodies of the invention are suitable linked to one or about 107M s', preferably between 10 M's and US 9,512,236 B2 65 66 107 M's', more preferably between 10 M's and limited to a specific biological source or to a specific method 107 M's, such as between 10 M' s and 107M of preparation. For example, as will be discussed in more s'; detail below, the Nanobodies of the invention can generally and/or such that they: be obtained: (1) by isolating the V. domain of a naturally bind to GPCRs with a krate between 1 s' (t=0.69s) occurring heavy chain antibody; (2) by expression of a and 10 s' (providing a near irreversible complex nucleotide sequence encoding a naturally occurring V with at of multiple days), preferably between 10° domain; (3) by “humanization' (as described herein) of a s' and 10's", more preferably between 10s and naturally occurring V domain or by expression of a 10 s, such as between 10's and 10 s. nucleic acid encoding a such humanized V. domain; (4) by Preferably, a polypeptide that contains only one amino 10 “camelization' (as described herein) of a naturally occurring acid sequence of the invention is preferably such that it will V domain from any animal species, and in particular a from bind to GPCRs with an affinity less than 500 nM, preferably species of mammal. Such as from a human being, or by less than 200 nM, more preferably less than 10 nM, such as expression of a nucleic acid encoding such a camelized V less than 500 pM. In this respect, it will be clear to the skilled domain; (5) by “camelisation of a “domain antibody” or person that a polypeptide that contains two or more Nano 15 “dAb” as described by Ward et al (supra), or by expression bodies of the invention may bind to GPCRs with an of a nucleic acid encoding such a camelized V. domain; (6) increased avidity, compared to a polypeptide that contains by using synthetic or semi-synthetic techniques for prepar only one amino acid sequence of the invention. ing proteins, polypeptides or other amino acid sequences Some preferred ICs values for binding of the amino acid known per se; (7) by preparing a nucleic acid encoding a sequences or polypeptides of the invention to GPCRs will NANOBODYR (V) using techniques for nucleic acid become clear from the further description and examples synthesis known per se, followed by expression of the herein. nucleic acid thus obtained; and/or (8) by any combination of Another aspect of this invention relates to a nucleic acid one or more of the foregoing. Suitable methods and tech that encodes an amino acid sequence of the invention (Such niques for performing the foregoing will be clear to the as a NANOBODYR (V) of the invention) or a polypep 25 skilled person based on the disclosure herein and for tide of the invention comprising the same. Again, as gener example include the methods and techniques described in ally described herein for the nucleic acids of the invention, more detail herein. Such a nucleic acid may be in the form of a genetic construct, One preferred class of Nanobodies corresponds to the as defined herein. V. domains of naturally occurring heavy chain antibodies In another aspect, the invention relates to host or host cell 30 directed against GPCRs. As further described herein, such that expresses or that is capable of expressing an amino acid V. sequences can generally be generated or obtained by sequence (such as a NANOBODYR (V)) of the invention suitably immunizing a species of Camelid with GPCRs (i.e. and/or a polypeptide of the invention comprising the same; So as to raise an immune response and/or heavy chain and/or that contains a nucleic acid of the invention. Some antibodies directed against GPCRs), by obtaining a suitable preferred but non-limiting examples of Such hosts or host 35 biological sample from said Camelid (such as a blood cells will become clear from the further description herein. sample, serum sample or sample of B-cells), and by gener Another aspect of the invention relates to a product or ating V. sequences directed against GPCRs, starting from composition containing or comprising at least one amino said sample, using any suitable technique known per se. acid sequence of the invention, at least one polypeptide of Such techniques will be clear to the skilled person and/or are the invention and/or at least one nucleic acid of the inven 40 further described herein. tion, and optionally one or more further components of Such Alternatively, such naturally occurring V domains compositions known per se, i.e. depending on the intended against GPCRs, can be obtained from naive libraries of use of the composition. Such a product or composition may Camelid V. sequences, for example by Screening Such a for example be a pharmaceutical composition (as described library using GPCRs, or at least one part, fragment, anti herein), a veterinary composition or a product or composi 45 genic determinant or epitope thereof using one or more tion for diagnostic use (as also described herein). Some screening techniques known per se. Such libraries and preferred but non-limiting examples of Such products or techniques are for example described in WO99/37681, WO compositions will become clear from the further description 01/90190, WO 03/025020 and WO 03/035694. Alterna herein. tively, improved synthetic or semi-synthetic libraries The invention further relates to methods for preparing or 50 derived from naive V. libraries may be used, such as V. generating the amino acid sequences, compounds, con libraries obtained from naive V. libraries by techniques structs, polypeptides, nucleic acids, host cells, products and Such as random mutagenesis and/or CDR shuffling, as for compositions described herein. Some preferred but non example described in WO 00/43507. limiting examples of Such methods will become clear from Thus, in another aspect, the invention relates to a method the further description herein. 55 for generating Nanobodies, that are directed against GPCRs. The invention further relates to applications and uses of In one aspect, said method at least comprises the steps of: the amino acid sequences, compounds, constructs, polypep a) providing a set, collection or library of NANOBODYR tides, nucleic acids, host cells, products and compositions (Vit) sequences; and described herein, as well as to methods for the prevention b) screening said set, collection or library of NANOBODYR and/or treatment for diseases and disorders associated with 60 (V) sequences for NANOBODYR (V) sequences GPCRs. Some preferred but non-limiting applications and that can bind to and/or have affinity for GPCRs: uses will become clear from the further description herein. and Other aspects, embodiments, advantages and applications c) isolating the amino acid sequence(s) that can bind to of the invention will also become clear from the further and/or have affinity for GPCRs. description hereinbelow. 65 In such a method, the set, collection or library of NANO Generally, it should be noted that the term NANO BODYR (V) sequences may be a naive set, collection or BODYR (V) as used herein in its broadest sense is not library of NANOBODYR (V) sequences; a synthetic or US 9,512,236 B2 67 68 semi-synthetic set, collection or library of NANOBODYR In another aspect, the method for generating an amino (V) sequences; and/or a set, collection or library of acid sequence directed against GPCRS may comprise at least NANOBODYR (V) sequences that have been subjected the steps of: to affinity maturation. a) providing a set, collection or library of nucleic acid In a preferred aspect of this method, the set, collection or sequences encoding heavy chain antibodies or NANO library of NANOBODYR (V) sequences may be an BODYR (V) sequences; immune set, collection or library of NANOBODYR (V) b) screening said set, collection or library of nucleic acid sequences, and in particular an immune set, collection or sequences for nucleic acid sequences that encode a heavy library of V. sequences, that have been derived from a chain antibody or a NANOBODYR (V) sequence that 10 can bind to and/or has affinity for GPCRs: species of Camelid that has been suitably immunized with and GPCRs or with a suitable antigenic determinant based c) isolating said nucleic acid sequence, followed by express thereon or derived therefrom, Such as an antigenic part, ing the V. sequence present in said heavy chain anti fragment, region, domain, loop or other epitope thereof. In body or by expressing said NANOBODYR (V) one particular aspect, said antigenic determinant may be an 15 sequence, respectively. extracellular part, region, domain, loop or other extracellular In such a method, the set, collection or library of nucleic epitope(s). acid sequences encoding heavy chain antibodies or NANO In the above methods, the set, collection or library of BODYR (V) sequences may for example be a set, col NANOBODYR (V) or V. sequences may be displayed lection or library of nucleic acid sequences encoding a naive on a phage, phagemid, ribosome or Suitable micro-organism set, collection or library of heavy chain antibodies or V. (such as yeast). Such as to facilitate Screening. Suitable sequences; a set, collection or library of nucleic acid methods, techniques and host organisms for displaying and sequences encoding a synthetic or semi-synthetic set, col screening (a set, collection or library of) NANOBODYR lection or library of NANOBODYR (V) sequences; and/ (V) sequences will be clear to the person skilled in the art, or a set, collection or library of nucleic acid sequences for example on the basis of the further disclosure herein. 25 encoding a set, collection or library of NANOBODYR Reference is also made to WO 03/054016 and to the review (V) sequences that have been subjected to affinity matu by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 ration. (2005). In a preferred aspect of this method, the set, collection or In another aspect, the method for generating NANO library of amino acid sequences may be an immune set, BODYR (V) sequences comprises at least the steps of: 30 collection or library of nucleic acid sequences encoding heavy chain antibodies or V. sequences derived from a a) providing a collection or sample of cells derived from a Camelid that has been suitably immunized with GPCRs or species of Camelid that express immunoglobulin with a suitable antigenic determinant based thereon or Sequences; derived therefrom, such as an antigenic part, fragment, b) screening said collection or sample of cells for (i) cells 35 region, domain, loop or other epitope thereof. In one par that express an immunoglobulin sequence that can bind to ticular aspect, said antigenic determinant may be an extra and/or have affinity for GPCRs; and (ii) cells that express cellular part, region, domain, loop or other extracellular heavy chain antibodies, in which Substeps (i) and (ii) can epitope(s). be performed essentially as a single Screening step or in In the above methods, the set, collection or library of any Suitable order as two separate Screening steps, so as 40 nucleotide sequences may be displayed on a phage, to provide at least one cell that expresses a heavy chain phagemid, ribosome or Suitable micro-organism (such as antibody that can bind to and/or has affinity for GPCRs: yeast). Such as to facilitate screening. Suitable methods, and techniques and host organisms for displaying and screening c) either (i) isolating from said cell the V. sequence present (a set, collection or library of) nucleotide sequences encod in said heavy chain antibody; 45 ing amino acid sequences will be clear to the person skilled or (ii) isolating from said cell a nucleic acid sequence that in the art, for example on the basis of the further disclosure encodes the V. sequence present in said heavy chain herein. Reference is also made to WO 03/054016 and to the antibody, followed by expressing said V. domain. review by Hoogenboom in Nature Biotechnology, 23, 9, In the method according to this aspect, the collection or 1105-1116 (2005). sample of cells may for example be a collection or sample 50 As will be clear to the skilled person, the screening step of B-cells. Also, in this method, the sample of cells may be of the methods described herein can also be performed as a derived from a Camelid that has been suitably immunized selection step. Accordingly the term "screening as used in with GPCRs or a suitable antigenic determinant based the present description can comprise selection, screening or thereon or derived therefrom, Such as an antigenic part, any Suitable combination of selection and/or screening tech fragment, region, domain, loop or other epitope thereof. In 55 niques. Also, when a set, collection or library of sequences one particular aspect, said antigenic determinant may be an is used, it may contain any suitable number of sequences, extracellular part, region, domain, loop or other extracellular such as 1, 2, 3 or about 5, 10, 50, 100, 500, 1000, 5000, 10, epitope(s). 10, 10, 107, 10 or more Sequences. The above method may be performed in any suitable Also, one or more or all of the sequences in the above set, manner, as will be clear to the skilled person. Reference is 60 collection or library of amino acid sequences may be for example made to EP 0 542 810, WO 05/19824, WO obtained or defined by rational, or semi-empirical 04/051268 and WO 04/106377. The screening of step b) is approaches such as computer modelling techniques or bio preferably performed using a flow cytometry technique Such statics or datamining techniques. as FACS. For this, reference is for example made to Lieby Furthermore, such a set, collection or library can comprise et al., Blood, Vol. 97, No. 12, 3820. Particular reference is 65 one, two or more sequences that are variants from one made to the so-called “NanocloneTM technique described in another (e.g. with designed point mutations or with random International application WO 06/079372 by Ablynx N.V. ized positions), comprise multiple sequences derived from a US 9,512,236 B2 69 70 diverse set of naturally diversified sequences (e.g. an on humanization referred to herein. Again, it should be noted immune library)), or any other source of diverse sequences that such humanized Nanobodies of the invention can be (as described for example in Hoogenboom et al. Nat Bio obtained in any suitable manner known per se (i.e. as technol 23:1105, 2005 and BinZ. et al, Nat Biotechnol 2005, indicated under points (1)–(8) above) and thus are not strictly 23:1247). Such set, collection or library of sequences can be limited to polypeptides that have been obtained using a displayed on the Surface of a phage particle, a ribosome, a polypeptide that comprises a naturally occurring V bacterium, a yeast cell, a mammalian cell, and linked to the domain as a starting material. nucleotide sequence encoding the amino acid sequence Another particularly preferred class of Nanobodies of the within these carriers. This makes Such set, collection or invention comprises Nanobodies with an amino acid library amenable to selection procedures to isolate the 10 sequence that corresponds to the amino acid sequence of a desired amino acid sequences of the invention. More gen naturally occurring V domain, but that has been "camel erally, when a sequence is displayed on a Suitable host or ized', i.e. by replacing one or more amino acid residues in host cell, it is also possible (and customary) to first isolate the amino acid sequence of a naturally occurring V domain from said host or host cell a nucleotide sequence that from a conventional 4-chain antibody by one or more of encodes the desired sequence, and then to obtain the desired 15 the amino acid residues that occur at the corresponding sequence by Suitably expressing said nucleotide sequence in position(s) in a V. domain of a heavy chain antibody. This a suitable host organism. Again, this can be performed in any can be performed in a manner known per se, which will be suitable manner known per se, as will be clear to the skilled clear to the skilled person, for example on the basis of the person. further description herein. Such "camelizing substitutions Yet another technique for obtaining V. sequences or are preferably inserted at amino acid positions that form NANOBODYR (V) sequences directed against GPCRs and/or are present at the V-V, interface, and/or at the involves Suitably immunizing a transgenic mammal that is so-called Camelidae hallmark residues, as defined herein capable of expressing heavy chain antibodies (i.e. So as to (see for example WO94/04678 and Davies and Riechmann raise an immune response and/or heavy chain antibodies (1994 and 1996), supra). Preferably, the V sequence that is directed against GPCRs), obtaining a suitable biological 25 used as a starting material or starting point for generating or sample from said transgenic mammal that contains (nucleic designing the camelized NANOBODYR (V) is prefer acid sequences encoding) said V. sequences or NANO ably a V sequence from a mammal, more preferably the V BODYR (V) sequences (such as a blood sample, serum sequence of a human being, such as a V-3 sequence. sample or sample of B-cells), and then generating V However, it should be noted that such camelized Nanobod sequences directed against GPCRs, starting from said 30 ies of the invention can be obtained in any suitable manner sample, using any suitable technique known per se (such as known per se (i.e. as indicated under points (1)–(8) above) any of the methods described herein or a hybridoma tech and thus are not strictly limited to polypeptides that have nique). For example, for this purpose, the heavy chain been obtained using a polypeptide that comprises a naturally antibody-expressing mice and the further methods and tech occurring V domain as a starting material. niques described in WO 02/085945, WO 04/049794 and 35 For example, again as further described herein, both WO 06/008548 and Janssens et al., Proc. Natl. Acad. Sci. “humanization' and “camelization can be performed by USA. 2006 Oct. 10; 103(41): 15130-5 can be used. For providing a nucleotide sequence that encodes a naturally example, Such heavy chain antibody expressing mice can occurring V domain or V. domain, respectively, and then express heavy chain antibodies with any Suitable (single) changing, in a manner known perse, one or more codons in variable domain, such as (single) variable domains from 40 said nucleotide sequence in Such a way that the new nucleo natural sources (e.g. human (single) variable domains, Cam tide sequence encodes a “humanized' or “camelized elid (single) variable domains or shark (single) variable NANOBODYR (V) of the invention, respectively. This domains), as well as for example synthetic or semi-synthetic nucleic acid can then be expressed in a manner known per (single) variable domains. se, so as to provide the desired NANOBODYR (V) of the The invention also relates to the V. sequences or 45 invention. Alternatively, based on the amino acid sequence NANOBODYR (V) sequences that are obtained by the of a naturally occurring V, domain or V. domain, respec above methods, or alternatively by a method that comprises tively, the amino acid sequence of the desired humanized or the one of the above methods and in addition at least the camelized NANOBODYR (V) of the invention, respec steps of determining the nucleotide sequence or amino acid tively, can be designed and then synthesized de novo using sequence of said V. sequence or NANOBODYR (V) 50 techniques for peptide synthesis known per se. Also, based sequence; and of expressing or synthesizing said V. on the amino acid sequence or nucleotide sequence of a sequence or NANOBODYR (V) sequence in a manner naturally occurring V domain or V. domain, respectively, known per se, such as by expression in a suitable host cell a nucleotide sequence encoding the desired humanized or or host organism or by chemical synthesis. camelized NANOBODYR (V) of the invention, respec As mentioned herein, a particularly preferred class of 55 tively, can be designed and then synthesized de novo using Nanobodies of the invention comprises Nanobodies with an techniques for nucleic acid synthesis known per se, after amino acid sequence that corresponds to the amino acid which the nucleic acid thus obtained can be expressed in a sequence of a naturally occurring V domain, but that has manner known per se, so as to provide the desired NANO been “humanized', i.e. by replacing one or more amino acid BODYR (V) of the invention. residues in the amino acid sequence of said naturally occur 60 Other suitable methods and techniques for obtaining the ring V. sequence (and in particular in the framework Nanobodies of the invention and/or nucleic acids encoding sequences) by one or more of the amino acid residues that the same, starting from naturally occurring V. sequences or occur at the corresponding position(s) in a V. domain from preferably V. sequences, will be clear from the skilled a conventional 4-chain antibody from a human being (e.g. person, and may for example comprise combining one or indicated above). This can be performed in a manner known 65 more parts of one or more naturally occurring V. sequences per se, which will be clear to the skilled person, for example (such as one or more FR sequences and/or CDR sequences), on the basis of the further description herein and the prior art one or more parts of one or more naturally occurring V US 9,512,236 B2 71 72 sequences (such as one or more FR sequences or CDR In particular, a NANOBODYR (V) in its broadest sequences), and/or one or more synthetic or semi-synthetic sense can be generally defined as a polypeptide comprising: sequences, in a Suitable manner, so as to provide a NANO a) an amino acid sequence that is comprised of four frame BODYR (V) of the invention or a nucleotide sequence or work regions/sequences interrupted by three complemen nucleic acid encoding the same (which may then be suitably tarity determining regions/sequences, in which the amino expressed). Nucleotide sequences encoding framework acid residue at position 108 according to the Kabat sequences of V. sequences or Nanobodies will be clear to numbering is Q; the skilled person based on the disclosure herein and/or the and/or: further prior art cited herein (and/or may alternatively be b) an amino acid sequence that is comprised of four frame obtained by PCR starting from the nucleotide sequences 10 work regions/sequences interrupted by three complemen obtained using the methods described herein) and may be tarity determining regions/sequences, in which the amino Suitably combined with nucleotide sequences that encode acid residue at position 44 according to the Kabat num the desired CDR's (for example, by PCR assembly using bering is E and in which the amino acid residue at position overlapping primers), so as to provide a nucleic acid encod 15 45 according to the Kabat numbering is an R; ing a NANOBODYR (V) of the invention. and/or: As mentioned herein, Nanobodies may in particular be c) an amino acid sequence that is comprised of four frame characterized by the presence of one or more “Hallmark work regions/sequences interrupted by three complemen residues” (as described herein) in one or more of the tarity determining regions/sequences, in which the amino framework sequences. acid residue at position 103 according to the Kabat Thus, according to one preferred, but non-limiting aspect numbering is chosen from the group consisting of P. Rand of the invention, a NANOBODYR (V) in its broadest S, and is in particular chosen from the group consisting of sense can be generally defined as a polypeptide comprising: R and S. a) an amino acid sequence that is comprised of four frame Thus, according to a preferred, but non-limiting aspect, a work regions/sequences interrupted by three complemen 25 NANOBODYR (V) of the invention may have the struc tarity determining regions/sequences, in which the amino ture acid residue at position 108 according to the Kabat FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 numbering is Q; in which FR1 to FR4 refer to framework regions 1 to 4, and/or: respectively, and in which CDR1 to CDR3 refer to the b) an amino acid sequence that is comprised of four frame 30 complementarity determining regions 1 to 3, respectively, work regions/sequences interrupted by three complemen and in which tarity determining regions/sequences, in which the amino a) the amino acid residue at position 108 according to the acid residue at position 45 according to the Kabat num Kabat numbering is Q; and/or in which: bering is a charged amino acid (as defined herein) or a b) the amino acid residue at position 44 according to the cysteine residue, and position 44 is preferably an E: 35 Kabat numbering is E and in which the amino acid residue and/or: at position 45 according to the Kabat numbering is an R; c) an amino acid sequence that is comprised of four frame and/or in which: work regions/sequences interrupted by three complemen c) the amino acid residue at position 103 according to the tarity determining regions/sequences, in which the amino Kabat numbering is chosen from the group consisting of acid residue at position 103 according to the Kabat 40 P. R and S, and is in particular chosen from the group numbering is chosen from the group consisting of P. Rand consisting of R and S. S, and is in particular chosen from the group consisting of and in which: R and S. d) CDR1, CDR2 and CDR3 are as defined herein, and are Thus, in a first preferred, but non-limiting aspect, a preferably as defined according to one of the preferred NANOBODYR (V) of the invention may have the struc 45 aspects herein, and are more preferably as defined accord ture ing to one of the more preferred aspects herein. FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 In particular, a NANOBODYR (V) against GPCRs in which FR1 to FR4 refer to framework regions 1 to 4, according to the invention may have the structure: respectively, and in which CDR1 to CDR3 refer to the FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 complementarity determining regions 1 to 3, respectively, 50 in which FR1 to FR4 refer to framework regions 1 to 4, and in which respectively, and in which CDR1 to CDR3 refer to the a) the amino acid residue at position 108 according to the complementarity determining regions 1 to 3, respectively, Kabat numbering is Q; and/or in which: and in which b) the amino acid residue at position 45 according to the a) the amino acid residue at position 108 according to the Kabat numbering is a charged amino acid or a cysteine 55 Kabat numbering is Q; and the amino acid residue at position 44 according to the and/or in which: Kabat numbering is preferably E: b) the amino acid residue at position 44 according to the and/or in which: Kabat numbering is E and in which the amino acid residue c) the amino acid residue at position 103 according to the at position 45 according to the Kabat numbering is an R; Kabat numbering is chosen from the group consisting of 60 and/or in which: P. R and S, and is in particular chosen from the group c) the amino acid residue at position 103 according to the consisting of R and S. Kabat numbering is chosen from the group consisting of and in which: P. R and S, and is in particular chosen from the group d) CDR1, CDR2 and CDR3 are as defined herein, and are consisting of R and S. preferably as defined according to one of the preferred 65 and in which: aspects herein, and are more preferably as defined accord d) CDR1, CDR2 and CDR3 are as defined herein, and are ing to one of the more preferred aspects herein. preferably as defined according to one of the preferred US 9,512,236 B2 73 74 aspects herein, and are more preferably as defined accord a-1) the amino acid residue at position 44 according to the ing to one of the more preferred aspects herein. Kabat numbering is chosen from the group consisting of In particular, according to one preferred, but non-limiting A, G, E, D, G, Q, R. S. L.; and is preferably chosen from aspect of the invention, a NANOBODYR (V) can gen the group consisting of G, E or Q; erally be defined as a polypeptide comprising an amino acid and in which: sequence that is comprised of four framework regions/ a-2) the amino acid residue at position 45 according to the sequences interrupted by three complementarity determin Kabat numbering is chosen from the group consisting of ing regions/sequences, in which: L., R or C; and is preferably chosen from the group a-1) the amino acid residue at position 44 according to the consisting of L or R; Kabat numbering is chosen from the group consisting of 10 and in which: A, G, E, D, G, Q, R. S. L.; and is preferably chosen from a-3) the amino acid residue at position 103 according to the the group consisting of G, E or Q, and Kabat numbering is chosen from the group consisting of a-2) the amino acid residue at position 45 according to the W. R or S; and is preferably W or R, and is most Kabat numbering is chosen from the group consisting of 15 preferably W: L., R or C; and is preferably chosen from the group and in which consisting of L or R; and a-4) the amino acid residue at position 108 according to the a-3) the amino acid residue at position 103 according to the Kabat numbering is Q; Kabat numbering is chosen from the group consisting of and in which: W. R or S; and is preferably W or R, and is most preferably W: d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred a-4) the amino acid residue at position 108 according to the aspects herein, and are more preferably as defined accord Kabat numbering is Q; ing to one of the more preferred aspects herein. or in which: In another preferred, but non-limiting aspect, a NANO b-1) the amino acid residue at position 44 according to the 25 BODYR (V) of the invention may have the structure Kabat numbering is chosen from the group consisting of E and Q; and in which FR1 to FR4 refer to framework regions 1 to 4, b-2) the amino acid residue at position 45 according to the respectively, and in which CDR1 to CDR3 refer to the Kabat numbering is R; and 30 complementarity determining regions 1 to 3, respectively, b-3) the amino acid residue at position 103 according to the and in which: Kabat numbering is chosen from the group consisting of b-1) the amino acid residue at position 44 according to the W. R and S; and is preferably W: Kabat numbering is chosen from the group consisting of b-4) the amino acid residue at position 108 according to the E and Q; Kabat numbering is chosen from the group consisting of 35 and in which: Q and L; and is preferably Q; b-2) the amino acid residue at position 45 according to the or in which: Kabat numbering is R; c-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of and in which: A. G. E. D. Q. R. S. and L; and is preferably chosen from 40 b-3) the amino acid residue at position 103 according to the the group consisting of G, E and Q, and Kabat numbering is chosen from the group consisting of c-2) the amino acid residue at position 45 according to the W. R and S; and is preferably W: Kabat numbering is chosen from the group consisting of and in which: L. R and C.; and is preferably chosen from the group b-4) the amino acid residue at position 108 according to the consisting of L and R; and 45 Kabat numbering is chosen from the group consisting of c-3) the amino acid residue at position 103 according to the Q and L; and is preferably Q; Kabat numbering is chosen from the group consisting of and in which: P. R and S; and is in particular chosen from the group d) CDR1, CDR2 and CDR3 are as defined herein, and are consisting of R and S; and 50 preferably as defined according to one of the preferred c-4) the amino acid residue at position 108 according to the aspects herein, and are more preferably as defined accord Kabat numbering is chosen from the group consisting of ing to one of the more preferred aspects herein. Q and L. is preferably Q; In another preferred, but non-limiting aspect, a NANO and in which BODYR (V) of the invention may have the structure d) CDR1, CDR2 and CDR3 are as defined herein, and are 55 preferably as defined according to one of the preferred in which FR1 to FR4 refer to framework regions 1 to 4, aspects herein, and are more preferably as defined accord respectively, and in which CDR1 to CDR3 refer to the ing to one of the more preferred aspects herein. complementarity determining regions 1 to 3, respectively, Thus, in another preferred, but non-limiting aspect, a and in which: NANOBODYR (V) of the invention may have the struc 60 c-1) the amino acid residue at position 44 according to the ture Kabat numbering is chosen from the group consisting of A. G. E. D. Q. R. S. and L; and is preferably chosen from in which FR1 to FR4 refer to framework regions 1 to 4, the group consisting of G, E and Q; respectively, and in which CDR1 to CDR3 refer to the 65 and in which: complementarity determining regions 1 to 3, respectively, c-2) the amino acid residue at position 45 according to the and in which: Kabat numbering is chosen from the group consisting of US 9,512,236 B2 75 76 L. R and C.; and is preferably chosen from the group Nanobodies of the invention in which the amino acid consisting of L and R; residues at positions 44-47 according to the Kabat number and in which: ing form the sequence GLEW, the amino acid residue at c-3) the amino acid residue at position 103 according to the position 37 is chosen from the group consisting of Y, H., I, Kabat numbering is chosen from the group consisting of 5 L, V or F, and is most preferably V. P. R and S; and is in particular chosen from the group Thus, without being limited hereto in any way, on the consisting of R and S. basis of the amino acid residues present on the positions and in which: mentioned above, the Nanobodies of the invention can c-4) the amino acid residue at position 108 according to the generally be classified on the basis of the following three Kabat numbering is chosen from the group consisting of 10 groups: Q and L. is preferably Q; i) The “GLEW-group': Nanobodies with the amino acid and in which: sequence GLEW at positions 44-47 according to the d) CDR1, CDR2 and CDR3 are as defined herein, and are Kabat numbering and Q at position 108 according to the preferably as defined according to one of the preferred Kabat numbering. As further described herein, Nanobod aspects herein, and are more preferably as defined accord- 15 ies within this group usually have a V at position 37, and ing to one of the more preferred aspects herein. can have a W. P. R or S at position 103, and preferably Two particularly preferred, but non-limiting groups of the have a W at position 103. The GLEW group also com Nanobodies of the invention are those according to a) above: prises some GLEW-like sequences such as those men according to (a-1) to (a-4) above; according to b) above; tioned in Table A-3 below More generally, and without according to (b-1) to (b-4) above; according to (c) above; 20 limitation, Nanobodies belonging to the GLEW-group can and/or according to (c-1) to (c-4) above, in which either: be defined as Nanobodies with a G at position 44 and/or i) the amino acid residues at positions 44-47 according to the with a Wat position 47, in which position 46 is usually E Kabat numbering form the sequence GLEW (or a GLEW and in which preferably position 45 is not a charged like sequence as described herein) and the amino acid amino acid residue and not cysteine; residue at position 108 is Q; 25 ii) The “KERE-group': Nanobodies with the amino acid or in which: sequence KERE or KQRE (or another KERE-like ii) the amino acid residues at positions 43-46 according to sequence) at positions 43-46 according to the Kabat the Kabat numbering form the sequence KERE or KQRE numbering and Q or L at position 108 according to the (or a KERE-like sequence as described) and the amino Kabat numbering. As further described herein, Nanobod acid residue at position 108 is Q or L, and is preferably Q. 30 ies within this group usually have a F at position 37, an Thus, in another preferred, but non-limiting aspect, a L or F at position 47; and can have a W. P. R or S at NANOBODYR (V) of the invention may have the struc position 103, and preferably have a W at position 103. ture More generally, and without limitation, Nanobodies FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 belonging to the KERE-group can be defined as Nano in which FR1 to FR4 refer to framework regions 1 to 4, 35 bodies with a K, Q or R at position 44 (usually K) in respectively, and in which CDR1 to CDR3 refer to the which position 45 is a charged amino acid residue or complementarity determining regions 1 to 3, respectively, cysteine, and position 47 is as further defined herein; and in which: iii) The “103 P, R, S-group': Nanobodies with a P R or S at i) the amino acid residues at positions 44-47 according to the position 103. These Nanobodies can have either the amino Kabat numbering form the sequence GLEW (or a GLEW- 40 acid sequence GLEW at positions 44-47 according to the like sequence as defined herein) and the amino acid Kabat numbering or the amino acid sequence KERE or residue at position 108 is Q; KQRE at positions 43-46 according to the Kabat num and in which: bering, the latter most preferably in combination with an ii) CDR1, CDR2 and CDR3 are as defined herein, and are Fat position 37 and an L or an F at position 47 (as defined preferably as defined according to one of the preferred 45 for the KERE-group); and can have Q or L at position 108 aspects herein, and are more preferably as defined accord according to the Kabat numbering, and preferably have Q. ing to one of the more preferred aspects herein. Also, where appropriate, Nanobodies may belong to (i.e. In another preferred, but non-limiting aspect, a NANO have characteristics of) two or more of these classes. For BODYR (V) of the invention may have the structure example, one specifically preferred group of Nanobodies has FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 50 GLEW or a GLEW-like sequence at positions 44-47: P R or in which FR1 to FR4 refer to framework regions 1 to 4, S (and in particular R) at position 103; and Qat position 108 respectively, and in which CDR1 to CDR3 refer to the (which may be humanized to L). complementarity determining regions 1 to 3, respectively, More generally, it should be noted that the definitions and in which: referred to above describe and apply to Nanobodies in the i) the amino acid residues at positions 43-46 according to the 55 form of a native (i.e. non-humanized) V. sequence, and Kabat numbering form the sequence KERE or KQRE (or that humanized variants of these Nanobodies may contain a KERE-like sequence) and the amino acid residue at other amino acid residues than those indicated above (i.e. position 108 is Q or L, and is preferably Q; one or more humanizing Substitutions as defined herein). For and in which: example, and without limitation, in Some humanized Nano ii) CDR1, CDR2 and CDR3 are as defined herein, and are 60 bodies of the GLEW-group or the 103 P. R. S-group, Q at preferably as defined according to one of the preferred position 108 may be humanized to 108L. As already men aspects herein, and are more preferably as defined accord tioned herein, other humanizing Substitutions (and Suitable ing to one of the more preferred aspects herein. combinations thereof) will become clear to the skilled In the Nanobodies of the invention in which the amino person based on the disclosure herein. In addition, or alter acid residues at positions 43-46 according to the Kabat 65 natively, other potentially useful humanizing Substitutions numbering form the sequence KERE or KQRE, the amino can be ascertained by comparing the sequence of the frame acid residue at position 37 is most preferably F. In the work regions of a naturally occurring V. sequence with the US 9,512,236 B2 77 78 corresponding framework sequence of one or more closely Nanobodies corresponding to naturally occurring V related human V. sequences, after which one or more of the domains) or R (for “humanized Nanobodies, as described potentially useful humanizing Substitutions (or combina herein). tions thereof) thus determined can be introduced into said Furthermore, in one aspect of the Nanobodies of the V. sequence (in any manner known per se, as further invention, the amino acid residue at position 104 is chosen described herein) and the resulting humanized V from the group consisting of G and D; and is most preferably sequences can be tested for affinity for the target, for G. stability, for ease and level of expression, and/or for other Collectively, the amino acid residues at positions 11, 37, desired properties. In this way, by means of a limited degree 10 44, 45, 47, 83, 84, 103, 104 and 108, which in the Nano of trial and error, other Suitable humanizing Substitutions (or bodies are as mentioned above, will also be referred to suitable combinations thereof) can be determined by the herein as the “Hallmark Residues'. The Hallmark Residues skilled person based on the disclosure herein. Also, based on and the amino acid residues at the corresponding positions the foregoing, (the framework regions of) a NANOBODYR (V) may be partially humanized or fully humanized. of the most closely related human V, domain, V3, are Thus, in another preferred, but non-limiting aspect, a 15 summarized in Table A-3. NANOBODYR (V) of the invention may be a NANO Some especially preferred but non-limiting combinations BODYR (V) belonging to the GLEW-group (as defined of these Hallmark Residues as occur in naturally occurring herein), and in which CDR1, CDR2 and CDR3 are as V. domains are mentioned in Table A-4. For comparison, defined herein, and are preferably as defined according to the corresponding amino acid residues of the human V3 one of the preferred aspects herein, and are more preferably called DP-47 have been indicated in italics. as defined according to one of the more preferred aspects herein. TABLE A-3 In another preferred, but non-limiting aspect, a NANO Hallmark Residues in Nanobodies BODYR (V) of the invention may be a NANOBODYR 25 (V) belonging to the KERE-group (as defined herein), Position Human V3 Hallmark Residues and CDR1, CDR2 and CDR3 are as defined herein, and are 11 L. V. predominantly L. L., M. S. V. W: preferably L preferably as defined according to one of the preferred 37 V, I, F: usually V FC, Y, H, I, L or V. aspects herein, and are more preferably as defined according preferably For Y 30 44(8) G G(2), EG, A, D, Q, R, S, L; to one of the more preferred aspects herein. preferably G’, E' or Q: Thus, in another preferred, but non-limiting aspect, a most preferably G2) or E. 45 (8) L L(2, RG, C, I, L., P, Q, V: NANOBODYR (V) of the invention may be a NANO preferably L(2) or R3) BODYR (V) belonging to the 103 P. R. S-group (as 47(8) W, Y W(2), L() or FC, A, G, I, M, R, S, V defined herein), and in which CDR1, CDR2 and CDR3 are 35 or Y; preferably W2), L(1), F(i) or R as defined herein, and are preferably as defined according to 83 R or K; usually R R, KS, N, E, G, I, M, Q or T: preferably K or R; most preferably K one of the preferred aspects herein, and are more preferably 84 A, T, D; predominantly P. A., L, R, S, T, D, V, preferably P as defined according to one of the more preferred aspects A. herein. 103 W W(4), P(6), R(6), S: preferably W 104 G G or D; preferably G Also, more generally and in addition to the 108O, 43E/ 40 108 L, M or T: Q, L, or R: preferably Q or L7 44R and 103 P.R.S residues mentioned above, the Nano predominantly L. bodies of the invention can contain, at one or more positions Notes: that in a conventional V. domain would form (part of) the (In particular, but not exclusively, in combination with KERE or KQRE at positions V/V, interface, one or more amino acid residues that are 43-46. Usually as GLEW at positions 44-47. more highly charged than the amino acid residues that 45 Usually as KERE or KQRE at positions 43-46, e.g. as KEREL, KEREF, KQREL, naturally occur at the same position(s) in the corresponding KQREF or KEREG at positions 43-47. Alternatively, also sequences such as TERE (for example TEREL), KECE (for example KECEL or KECER), RERE (for example REREG), naturally occurring V. sequence, and in particular one or QERE (for example QEREG), KGRE (for example KGREG), KDRE (for example more charged amino acid residues (as mentioned in Table KDREV) are possible. Some other possible, but less preferred sequences include for A-2). Such substitutions include, but are not limited to, the exampleWith both DECKL GLEW and at NVCEL. positions 44-47 and KERE or KQRE at positions 43-46. GLEW-like sequences mentioned in Table A-3 below; as 50 Often as KP or EP at positions 83-84 of naturally occurring VHH domains, well as the substitutions that are described in the Interna (6)n particular, but not exclusively, in combination with GLEW at positions 44-47. (With the proviso that when positions 44-47 are GLEW, position 108 is always Q in tional Application WO 00/29004 for so-called “microbod non-humanized) VHH sequences that also contain a W at 103. The GLEW group also contains GLEW-like sequences at positions 44-47, such as for ies', e.g. so as to obtain a NANOBODYR (V) with Q at example GVEW, EPEW, GLER, DQEW, DLEW, GIEW, ELEW, GPEW, EWLP, GPER, position 108 in combination with KLEW at positions 44-47. GLER and ELEW. Other possible substitutions at these positions will be clear 55 to the skilled person based upon the disclosure herein. In one aspect of the Nanobodies of the invention, the TABLE A-4 amino acid residue at position 83 is chosen from the group Some preferred but non-limiting combinations of Hallmark Residues consisting of L. M. S. V and W. and is preferably L. in naturally occurring Nanobodies. For humanization of these Also, in one aspect of the Nanobodies of the invention, the 60 combinations, reference is made to the specification. amino acid residue at position 83 is chosen from the group 11 37 44 45 47 83 84 103 104 108 consisting of R. K. N. E. G. I. T and Q, and is most preferably either K or E (for Nanobodies corresponding to DP-47 (human) M V G L W R A W G L “KERE' group L. F E R L K P W G Q naturally occurring V domains) or R (for “humanized' L F E R F E P W G Q Nanobodies, as described herein). The amino acid residue at 65 L F E R F K P W G Q position 84 is chosen from the group consisting of P. A. R. L Y Q R L K P W G Q S, DT, and V in one aspect, and is most preferably P (for US 9,512,236 B2 79 80 TABLE A-4-continued is used). For a general explanation of sequence entropy, sequence variability and the methodology for determining Some preferred but non-limiting combinations of Hallmark Residues in naturally occurring Nanobodies. For humanization of these the same, see Oliveira et al., PROTEINS: Structure, Func combinations, reference is made to the Specification. tion and Genetics, 52: 544-552 (2003). 5 11 37 44 4S 47 83 84 103 104 108 TABLE A-5 L F L R V K P Q G Q L F Q R L K P W G Q Non-limiting examples of amino acid residues in FR1 (for the footnotes, L F E R F K P W G Q see the footnotes to Table A-3 “GLEW group L V G L W K S W G Q 10 M V G L W K P R G Q Amino acid residue(s): VHH VHH Pos. Human V3 Camelid V.'s Ent. War. In the Nanobodies, each amino acid residue at any other 1 E, Q Q, A, E position than the Hallmark Residues can be any amino acid 2 V V O.2 1 15 residue that naturally occurs at the corresponding position 3 Q Q, K O.3 2 (according to the Kabat numbering) of a naturally occurring 4 L L O.1 1 Vir domain. 5 V, L Q., E. L. V. O.8 3 Such amino acid residues will be clear to the skilled 6 E E. D., Q. A O.8 4 7 S, T S, F O.3 2 person. Tables A-5 to A-8 mention Some non-limiting resi 8 G, R G O.1 1 dues that can be present at each position (according to the 9 G G O 1 Kabat numbering) of the FR1, FR2, FR3 and FR4 of 10 G, V G, D, R O.3 2 naturally occurring V domains. For each position, the 11 Hallmark residue: L., M. S. V. W: preferably L O.8 2 12 V, V, A O.2 2 amino acid residue that most frequently occurs at each 13 Q, K, R Q, E, K, P, R 0.4 4 position of a naturally occurring V domain (and which is 25 14 P A, Q, A, G, P, S, T, V 1 5 the most preferred amino acid residue for said position in a 1S G G O 1 NANOBODYR (V)) is indicated in bold; and other 16 G, R G, A, E, D 0.4 3 preferred amino acid residues for each position have been 17 S S., F. O.S 2 18 L L, V O.1 1 underlined (note: the number of amino acid residues that are 19 R, K R, K, L, N, S, T O6 4 found at positions 26-30 of naturally occurring V, domains 30 20 L L., F., I, V O.S 4 Supports the hypothesis underlying the numbering by 21 S S, A, F, T O.2 3 Chothia (supra) that the residues at these positions already 22 C C O 1 form part of CDR1.) 23 A, T A, D, E, P, S, T, V 1.3 5 In Tables A-5-A-8, some of the non-limiting residues that 24 A A., I, L., S, T, V. 1 6 can be present at each position of a human V3 domain have 35 2S S S, A, F, P, T O.S 5 also been mentioned. Again, for each position, the amino 26 G G, A, D, E, R, S, T, V 0.7 7 27 F S, F, R, L, P. G., N, 2.3 13 acid residue that most frequently occurs at each position of 28 T N, T, E, D, S, I, R, A, G, R., F.Y 1.7 11 a naturally occurring human V3 domain is indicated in 29 F.V. F, L, D, S, I, G. V. A 1.9 11 bold; and other preferred amino acid residues have been 30 S, D, G N, S, E, G, A, D, M, T 1.8 11 underlined. 40 For reference only, Tables A-5-A-8 also contain data on the V entropy (V, Ent.) and V. Variability (V, Var.”) at each amino acid position for a representative TABLE A-6 sample of 1118 V sequences (data kindly provided by Non-limiting examples of amino acid residues in FR2 (for the footnotes, David Lutje Hulsing and Prof. Theo Verrips of Utrecht 45 see the footnotes to Table A-3 University). The values for the V entropy and the V variability provide a measure for the variability and degree Amino acid residue(s): VHH VHH of conservation of amino acid residues between the 1118 V. sequences analyzed: low values (i.e. <1, such as <0.5) Pos. Human V3 Camelid V's Ent. War. indicate that an amino acid residue is highly conserved 50 36 W W O.1 1 37 Hallmark residue: FG, H, I, L., Y or V, preferably 1.1 6 between the V. sequences (i.e. little variability). For F(1) or Y example, the G at position 8 and the G at position 9 have 38 R R O.2 1 values for the V entropy of 0.1 and 0 respectively, 39 Q Q, H, P, R O.3 2 indicating that these residues are highly conserved and have 40 A A, F, G, L, P, T, V O.9 7 little variability (and in case of position 9 is G in all 1118 55 41 P, S, T P, A, L, S 0.4 3 42 G G. E. O.2 2 sequences analysed), whereas for residues that form part of 43 K K, D, E, N, Q, R, T, V 0.7 6 the CDR's generally values of 1.5 or more are found (data 44 Hallmark residue: G’, E, A, D, Q, R, S, L: 1.3 5 not shown). Note that (1) the amino acid residues listed in preferably G’, E' or Q; most preferably G(2) or EC3). the second column of Tables A-5-A-8 are based on a bigger 45 Hallmark residue: L, R, C, I, L., P, Q, V: O6 4 sample than the 1118 V sequences that were analysed for 60 preferably L(2) or R3) determining the V entropy and V variability referred to 46 E, V E, D, K, Q, V 0.4 2 in the last two columns; and (2) the data represented below 47 Hallmark residue: W’, L' or F, A, G, I, 1.9 9 M, R, S, V or Y; preferably W2), L.), Support the hypothesis that the amino acid residues at F() or R positions 27-30 and maybe even also at positions 93 and 94 48 V V, I, L 0.4 3 already form part of the CDR's (although the invention is 65 49 SAG A, S, G, T, V O.8 3 not limited to any specific hypothesis or explanation, and as mentioned above, herein the numbering according to Kabat US 9,512,236 B2 81 82 TABLE A-7 and in which: Non-limiting examples of amino acid residues in FR3 ii) CDR1, CDR2 and CDR3 are as defined herein, and are for the footnotes, see the footnotes to Table A-3 preferably as defined according to one of the preferred aspects herein, and are more preferably as defined accord Amino acid residue(s): VHH VHH ing to one of the more preferred aspects herein. Pos. Human V3 Camelid VI's Ent. War. The above Nanobodies may for example be V. 66 R R O.1 1 sequences or may be humanized Nanobodies. When the 67 F F, L, V O.1 1 above NANOBODYR (V) sequences are V. sequences, 68 T T, A, N, S O.S 4 10 they may be suitably humanized, as further described herein. 69 I, L, M, V 0.4 4 70 S S, A, F, T O.3 4 When the Nanobodies are partially humanized Nanobodies, 71 R R, G, H, I, L., K, Q, S, T, W 1.2 8 they may optionally be further Suitably humanized, again as 72 D, E D, E, G, N, V O.S 4 described herein. 73 N, D. G. N, A, D, F, I, K, L, R, S, T, V, Y 1.2 9 74 A, S A, D, G, N, P, S, T, V 1 7 15 In particular, a NANOBODYR (V) of the invention 75 K K, A, E, K, L, N, Q, R O.9 6 can be an amino acid sequence with the (general) structure 76 N, S N, D, K, R, S, T, Y O.9 6 77 S, T, I T, A, E, I, M, P, S O.8 5 78 L., A V, L, A, F, G, I, M 1.2 5 79 Y, H. Y, A, D, F, H, N, S, T 1 7 in which FR1 to FR4 refer to framework regions 1 to 4, 8O L L., F, V O.1 1 respectively, and in which CDR1 to CDR3 refer to the 81 Q Q., E., I, L, R, T O6 5 complementarity determining regions 1 to 3, respectively, 82 M M, I, L, V O.2 2 and in which: 82a N, G N, D, G, H, S, T O.8 4 82b S S, N, D, G, R, T 1 6 i) (preferably) one or more of the amino acid residues at 82c L L., P. V O.1 2 83 Hallmark residue: R, KC, N, E, G, I, M, Q or T: 0.9 7 positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 preferably K or R; most preferably K 25 according to the Kabat numbering are chosen from the 84 Hallmark residue: P(, A, D, L, R, S, T, V: 0.7 6 Hallmark residues mentioned in Table A-3 (it being preferably P 85 E, G E, D, G, Q O.S 3 understood that V. sequences will contain one or more 86 D D O 1 Hallmark residues; and that partially humanized Nano 87 T, M T, A, S O.2 3 bodies will usually, and preferably, still contain one or 88 A A, G, S O.3 2 30 more Hallmark residues although it is also within the 89 V, L V, A, D, I, L, M, N, R, T 1.4 6 90 Y Y, F O 1 scope of the invention to provide where suitable in 91 Y, H. Y, D, F, H, L, S, T, V O6 4 accordance with the invention partially humanized 92 C C O 1 Nanobodies in which all Hallmark residues, but not one or 93 A, K, T A, N, G, H, K, N, R, S, T, V, Y 1.4 10 more of the other amino acid residues, have been human 94 K, R, T A, V, C, F, G, I, K, L, R, S or T 1.6 9 35 ized; and that in fully humanized Nanobodies, where Suitable in accordance with the invention, all amino acid residues at the positions of the Hallmark residues will be TABLE A-8 amino acid residues that occur in a human V-3 sequence. Non-limiting examples of amino acid residues in FR4 40 As will be clear to the skilled person based on the for the footnotes, see the footnotes to Table A-3 disclosure herein that Such V sequences, such partially humanized Nanobodies with at least one Hallmark resi Amino acid residue(s): VHH VHH due, such partially humanized Nanobodies without Hall Pos. Human V3 Camelid Vis Ent. War. mark residues and such fully humanized Nanobodies all 103 Hallmark residue: We', P, R, S: preferably W 0.4 2 45 form aspects of this invention); 104 Hallmark residue: G or D; preferably G O.1 1 and in which: 105 Q, R Q, E, K, P, R O6 4 106 G G O.1 1 ii) said amino acid sequence has at least 80% amino acid 107 T T, A, I O.3 2 identity with at least one of the amino acid sequences of 108 Hallmark residue: Q, L7 or R: preferably Q or L7 0.4 3 109 V V O.1 1 50 SEQ ID NO’s: 1 to 22, in which for the purposes of 110 T T, I, A O.2 1 determining the degree of amino acid identity, the amino 111 V V, A, I O.3 2 acid residues that form the CDR sequences (indicated 112 S S, F O.3 1 with X in the sequences of SEQ ID NO’s: 1 to 22) are 113 S S, A, L., P., T 0.4 3 disregarded; 55 Thus, in another preferred, but not limiting aspect, a and in which: NANOBODYR (V) of the invention can be defined as an iii) CDR1, CDR2 and CDR3 are as defined herein, and are amino acid sequence with the (general) structure preferably as defined according to one of the preferred FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 aspects herein, and are more preferably as defined accord in which FR1 to FR4 refer to framework regions 1 to 4, 60 ing to one of the more preferred aspects herein. respectively, and in which CDR1 to CDR3 refer to the The above Nanobodies may for example be V. complementarity determining regions 1 to 3, respectively, sequences or may be humanized Nanobodies. When the and in which: above NANOBODYR (V) sequences are V. sequences, i) one or more of the amino acid residues at positions 11, 37. they may be suitably humanized, as further described herein. 44, 45, 47, 83, 84, 103, 104 and 108 according to the 65 When the Nanobodies are partially humanized Nanobodies, Kabat numbering are chosen from the Hallmark residues they may optionally be further Suitably humanized, again as mentioned in Table A-3: described herein.

US 9,512,236 B2 85 86 In particular, a NANOBODYR (V) of the invention of TABLE A-11 - continued the KERE group can be an amino acid sequence with the (general) structure Representative FW2 sequences for Nanobodies of the FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 KERE-group. in which: i) the amino acid residue at position 45 according to the 5 KERE FW2 sequence no. 4 SEQ ID NO: 44 WYRQGPGKQRELVA Kabat numbering is a charged amino acid (as defined herein) or a cysteine residue, and position 44 is preferably KERE FW2 sequence no. 5 SEQ ID NO: 45 WIRQAPGKEREGVS an E: KERE FW2 sequence no. 6 SEQ ID NO: 46 WFREAPGKEREGIS and in which: 10 ii) FR1 is an amino acid sequence that has at least 80% KERE FW2 sequence no. 7 SEQ ID NO: 47 WYRQAPGKERDLVA amino acid identity with at least one of the following amino acid sequences: TABLE A-1 O Representative FW1 sequences for Nanobodies of the KERE- group. KERE FW1 sequence no. 1 SEQ ID NO: 23 QVQRVESGGGLVQAGGSLRLSCAASGRTSS KERE FW1 sequence no. 2 SEQ ID NO: 24 QVOLVESGGGLVQTGDSLSLSCSASGRTFS KERE FW1 sequence no. 3 SEQ ID NO: 25 QWKLEESGGGLVQAGDSLRLSCAATGRAFG KERE FW1 sequence no. 4 SEQ ID NO: 26 AVOLVESGGGLVQPGESLGLSCVASGRDFV KERE FW1 sequence no. 5 SEQ ID NO: 27 EVOLVESGGGLVQAGGSLRLSCEVLGRTAG KERE FW1 sequence no. 6 SEQ ID NO: 28 QVOLVESGGGWVQPGGSLRLSCAASETILS KERE FW1 sequence no. 7 SEQ ID NO : 29 QVOLVESGGGTVQPGGSLNLSCVASGNTFN KERE FW1 sequence no. 8 SEQ ID NO: 3O EVOLVESGGGLAQPGGSLQLSCSAPGFTLD

KERE FW1 sequence no. 9 SEQ ID NO : 31 AQELEESGGGLVQAGGSLRLSCAASGRTFN and in which: 35 TABLE A-11 - continued iii) FR2 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following Representative FW2 sequences for Nanobodies of the amino acid sequences: KERE-group.

40 TABLE A-11 KERE FW2 sequence no. 8 SEQ ID NO : 48 WFRQAPGKQREEVS Representative FW2 sequences for Nanobodies of the KERE-group. KERE FW2 sequence no. 9 SEQ ID NO: 49 WFRQPPGKVREFVG KERE FW2 sequence no. 1 SEQ ID No. 41 WFROAPGKEREFVA KERE FW2 sequence no. 2 SEQ ID NO: 42 WFROTPGREREFVA and in which: KERE FW2 sequence no. 3 SEQ ID NO : 43 WYRQAPGKQREMVA iv) FR3 is an amino acid sequence that has at least 80%O amino acid identity with at least one of the following amino acid sequences: TABLE A-12 Representative FW3 sequences for Nanobodies of the KERE-group. KE RE FW3 sequence no. 1 SEQ ID NO: 50 RFTISRDNAKNTVYLOMNSLKPEDTAVYRCYF KE RE FW3 sequence no. 2 SEQ ID NO: 51 RFAISRDNNKNTGYLQMNSLEPEDTAVYYCAA KE RE FW3 sequence no. 3 SEQ ID NO: 52 RFTVARNNAKNTVNLEMNSLKPEDTAVYYCAA

KE RE FW3 sequence no. 4 SEQ ID NO. 53 RFTISRDIAKNTVDLLMNNLEPEDTAVYYCAA

KE RE FW3 sequence no. 5 SEQ ID NO. 54 RLTISRDNAVDTMYLOMNSLKPEDTAVYYCAA

KE RE FW3 sequence no. 6 SEQ ID NO. 55 RFTISRDNAKNTVYLQMDNVKPEDTAIYYCAA

KE RE FW3 sequence no. 7 SEQ ID NO. 56 RFTISKDSGKNTVYLOMTSLKPEDTAVYYCAT

KE RE FW3 sequence no. 8 SEQ ID NO: 57 RFTISRDSAKNMMYLOMNNLKPODTAVYYCAA US 9,512,236 B2 87 88 TABLE A-12- Continued Representative FW3 sequences for Nanobodies of the KERE-group.

KERE FW3 sequence no. 9 SEQ ID NO: 58 RFTISRENDKSTWYLOLNSLKPEDTAVYYCAA KERE FW3 sequence no. 10 SEQ ID NO: 59 RFTISRDYAGNTAYLOMNSLKPEDTGVYYCAT and in which: 10 according to the Kabat numbering) may often be determined v) FR4 is an amino acid sequence that has at least 80% by the primer(s) that have been used to generate said nucleic amino acid identity with at least one of the following acid. Thus, for determining the degree of amino acid iden amino acid sequences: tity, the first four amino acid residues are preferably disre garded. TABLE A-13 15 Also, with regard to framework 1, and although amino acid positions 27 to 30 are according to the Kabat numbering Representative FW4 sequences for Nanobodies of the KERE-group. considered to be part of the framework regions (and not the CDR's), it has been found by analysis of a database of more KERE FW4 sequence no. 1 SEQ ID NO: 60 WGQGTQVTVSS than 1000 V sequences that the positions 27 to 30 have a KERE FW4 sequence no. 2 SEQ ID NO : 61 WGKGTLVTVSS variability (expressed in terms of V entropy and V. variability—see Tables A-5 to A-8) that is much greater than KERE FW4 sequence no. 3 SEQ ID NO: 62 RGQGTRVTVSS the variability on positions 1 to 26. Because of this, for determining the degree of amino acid identity, the amino KERE FW4 sequence no. 4 SEQ ID NO: 63 WGLGTQVTISS acid residues at positions 27 to 30 are preferably also 25 disregarded. and in which: In view of this, a NANOBODYR (V) of the KERE vi) CDR1, CDR2 and CDR3 are as defined herein, and are class may be an amino acid sequence that is comprised of preferably as defined according to one of the preferred four framework regions/sequences interrupted by three aspects herein, and are more preferably as defined accord 30 complementarity determining regions/sequences, in which: ing to one of the more preferred aspects herein. i) the amino acid residue at position 45 according to the In the above Nanobodies, one or more of the further Kabat numbering is a charged amino acid (as defined Hallmark residues are preferably as described herein (for herein) or a cysteine residue, and position 44 is preferably example, when they are V. sequences or partially human an E: ized Nanobodies). 35 and in which: Also, the above Nanobodies may for example be V. ii) FR1 is an amino acid sequence that, on positions 5 to 26 sequences or may be humanized Nanobodies. When the of the Kabat numbering, has at least 80% amino acid above NANOBODYR (V) sequences are V. sequences, identity with at least one of the following amino acid they may be suitably humanized, as further described herein. Sequences: TABLE A-14 Representative FW1 sequences (amino acid residues 5 to 26) for Nanobodies of the KERE- group. KERE FW1 sequence no. O SEQ ID NO: 32 WESGGGLVOPGGSLRLSCAASG KERE FW1 sequence no. 1 SEQ ID NO: 33 WDSGGGLVOAGDSLKLSCALTG KERE FW1 sequence no. 2 SEQ ID NO: 34WDSGGGLVOAGDSLRLSCAASG KERE FW1 sequence no. 3 SEQ ID NO: 35 WDSGGGLVEAGGSLRLSCQVSE KERE FW1 sequence no. 14 SEQ ID NO: 36 QDSGGGSVQAGGSLKLSCAASG KERE FW1 sequence no. 15 SEQ ID NO : 37 VQSGGRLVQAGDSLRLSCAASE KERE FW1 sequence no. 6 SEQ ID NO: 38WESGGTLVOSGDSLKLSCASST KERE FW1 sequence no. 7 SEQ ID NO: 39 MESGGDSWOSGGSLTLSCVASG KERE FW1 sequence no. 18 SEQ ID NO: 40 QASGGGLVQAGGSLRLSCSASV

60 When the Nanobodies are partially humanized Nanobodies, and in which: they may optionally be further Suitably humanized, again as iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 described herein. and FR4 of Nanobodies of the KERE-class; With regard to framework 1, it will be clear to the skilled and in which: person that, when an amino acid sequence as outlined above 65 is generated by expression of a nucleotide sequence, the first iv) CDR1, CDR2 and CDR3 are as defined herein, and are four amino acid sequences (i.e. amino acid residues 1-4 preferably as defined according to one of the preferred US 9,512,236 B2 89 90 aspects herein, and are more preferably as defined accord A NANOBODYR (V) of the GLEW class may be an ing to one of the more preferred aspects herein. amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity The above Nanobodies may for example be V. determining regions/sequences, in which sequences or may be humanized Nanobodies. When the 5 i) preferably, when the NANOBODYR (V) of the above NANOBODYR (V) sequences are V. sequences, GLEW-class is a non-humanized NANOBODYR (V), they may be suitably humanized, as further described herein. the amino acid residue in position 108 is Q; When the Nanobodies are partially humanized Nanobodies, ii) FR1 is an amino acid sequence that has at least 80% they may optionally be further Suitably humanized, again as amino acid identity with at least one of the following described herein. amino acid sequences: TABLE A-15

Representative FW1 sequences for Nanobodies of the GLEW-group.

GLEW FW1 sequence no. 1 SEQ ID NO: 64 QVOLVESGGGLVQPGGSLRLSCAASGFTFS

GLEW FW1 sequence no. 2 SEQ ID NO: 65 EVHLVESGGGLVRPGGSLRLSCAAFGFIFK

GLEW FW1 sequence no. 3 SEQ ID NO: 66 QWKLEESGGGLAQPGGSLRLSCVASGFTFS

GLEW FW1 sequence no. 4 SEQ ID NO: 67 EVOLVESGGGLVQPGGSLRLSCVCVSSGCT

GLEW FW1 sequence no. 5 SEQ ID NO: 68 EVOLVESGGGLALPGGSLTLSCVFSGSTFS

and in which: iii) FR2 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences: TABLE A-16 Representative FW2 sequences for Nanobodies of the GLEW-group. GLEW FW2 sequence no. 1 SEQ ID NO: 72 WVRQAPGKVLEWVS GLEW FW2 sequence no. 2 SEQ ID NO: 73 WVRRPPGKGLEWVS GLEW FW2 sequence no. 3 SEQ ID NO: 74 WVRQAPGMGLEWVS GLEW FW2 sequence no. 4 SEQ ID NO: 75 WVRQAPGKEPEWVS GLEW FW2 sequence no. 5 SEQ ID NO: 76 WVRQAPGKDQEWVS GLEW FW2 sequence no. 6 SEQ ID NO: 77 WVRQAPGKAEEWVS GLEW FW2 sequence no. 7 SEQ ID NO: 78 WVRQAPGKGLEWVA GLEW FW2 sequence no. 8 SEQ ID NO: 79 WVRQAPGRATEWVS

and in which: so iv) FR3 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences: TABLE A-17 Representative FW3 sequences for Nanobodies of the GLEW-group.

GLEW FW3 sequence no. 1 SEO ID NO: 80 RFTISRDNAKNTLYLOMNSLKPEDTAVYYCVK

GLEW FW3 sequence no. 2 SEO ID NO: 81 RFTISRDNARNTLYLOMDSLIPEDTALYYCAR

GLEW FW3 sequence no. 3 SEO ID NO: 82 RFTSSRDNAKSTLYLOMNDLKPEDTALYYCAR

GLEW FW3 sequence no. 4 SEO ID NO: 83 RFIISRDNAKNTLYLOMNSLGPEDTAMYYCOR

GLEW FW3 sequence no. 5 SEO ID NO: 84 RFTASRDNAKNTLYLOMNSLKSEDTARYYCAR

GLEW FW3 sequence no. 6 SEO ID NO: 85 RFTISRDNAKNTLYLOMDDLOSEDTAMYYCGR US 9,512,236 B2 91 92 and in which: aspects herein, and are more preferably as defined accord v) FR4 is an amino acid sequence that has at least 80% ing to one of the more preferred aspects herein. amino acid identity with at least one of the following In the above Nanobodies, one or more of the further amino acid sequences: Hallmark residues are preferably as described herein (for example, when they are V. sequences or partially human ized Nanobodies). TABLE A-18 With regard to framework 1, it will again be clear to the 10 skilled person that, for determining the degree of amino acid Representative FW4 sequences for Nanobodies of the GLEW-group. identity, the amino acid residues on positions 1 to 4 and 27 to 30 are preferably disregarded. GLEW FW4 sequence no. 1 SEQ ID NO: 86 GSQGTQVTVSS In view of this, a NANOBODYR (V) of the GLEW GLEW FW4 sequence no. 2 SEQ ID NO : 87 LRGGTQVTVSS 15 class may be an amino acid sequence that is comprised of GLEW FW4 sequence no. 3 SEQ ID NO: 88 RGQGTLVTVSS four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which: GLEW FW4 sequence no. 4 SEQ ID NO: 89 RSRGIQVTVSS i) preferably, when the NANOBODYR (V) of the GLEW FW4 sequence no. 5 SEQ ID NO: 90 WGKGTQVTVSS GLEW-class is a non-humanized NANOBODYR (V), GLEW FW4 sequence no. 6 SEQ ID NO: 91 WGQGTQVTVSS the amino acid residue in position 108 is Q; and in which: ii) FR1 is an amino acid sequence that, on positions 5 to 26 and in which: 25 of the Kabat numbering, has at least 80% amino acid vi) CDR1, CDR2 and CDR3 are as defined herein, and are identity with at least one of the following amino acid preferably as defined according to one of the preferred Sequences: TABLE A-19

Representative FW1 sequences (amino acid residues 5 to 26) for Nanobodies of the GLEW-group.

GLEW FW1 sequence no. 6 SEQ ID NO: 69 WESGGGLVOPGGSLRLSCAASG

GLEW FW1 sequence no. 7 SEQ ID NO: 70 EESGGGLAQPGGSLRLSCVASG

GLEW FW1 sequence no. 8 SEQ ID NO : 71 WESGGGLALPGGSLTLSCWFSG

and in which: iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4 of Nanobodies of the GLEW-class; 45 and in which: iv) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined accord ing to one of the more preferred aspects herein. 50 The above Nanobodies may for example be V. sequences or may be humanized Nanobodies. When the above NANOBODYR (V) sequences are V. sequences, they may be suitably humanized, as further described herein. 55 When the Nanobodies are partially humanized Nanobodies, they may optionally be further Suitably humanized, again as described herein. In the above Nanobodies, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V. sequences or 60 partially humanized Nanobodies). A NANOBODYR (V) of the P. R. S. 103 class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity 65 determining regions/sequences, in which i) the amino acid residue at position 103 according to the Kabat numbering is different from W: US 9,512,236 B2 93 94 and in which: and in which: ii) preferably the amino acid residue at position 103 accord- iii) FR1 is an amino acid sequence that has at least 80% ing to the Kabat numbering is P. R or S, and more amino acid identity with at least one of the following preferably R: amino acid sequences: TABLE A-2O Representative FW1 sequences for Nanobodies of the P, R, S 103 - group. P, R, S 103 FW1 sequence no. 1 SEQ ID NO: 92 AVOLVESGGGLVQAGGSLRLSCAASGRTFS P, R, S 103 FW1 sequence no. 2 SEQ ID NO: 93 QVQLQESGGGMVQPGGSLRLSCAASGFDFG P, R, S 103 FW1 sequence no. 3 SEQ ID NO: 94 EVHLVESGGGLVRPGGSLRLSCAAFGFIFK P, R, S 103 FW1 sequence no. 4 SEQ ID NO: 95 QVOLAESGGGLVQPGGSLKLSCAASRTIVS P, R, S 103 FW1 sequence no. 5 SEQ ID NO: 96 QEHLVESGGGLVDIGGSLRLSCAASERIFS P, R, S 103 FW1 sequence no. 6 SEQ ID NO: 97 QWKLEESGGGLAQPGGSLRLSCVASGFTFS P, R, S 103 FW1 sequence no. 7 SEQ ID NO: 98 EVOLVESGGGLVQPGGSLRLSCVCVSSGCT P, R, S 103 FW1 sequence no. 8 SEQ ID NO: 99 EVOLVESGGGLALPGGSLTLSCVFSGSTFS

and in which iv) FR2 is an amino acid sequence that has at least 80% 25 amino acid identity with at least one of the following amino acid sequences: TABLE A-21

Representative FW2 sequences for Nanobodies of the P, R, S 103-group.

P, R, S 103 FW2 sequence no. 1 SEQ ID NO: 102 WFRQAPGKEREFVA

P, R, S 103 FW2 sequence no. 2 SEQ ID NO: 103 WVRQAPGKVLEWVS

P, R, S 103 FW2 sequence no. 3 SEQ ID NO: 104 WVRRPPGKGLEWVS

P, R, S 103 FW2 sequence no. 4 SEQ ID NO: 105 WIRQAPGKEREGVS

P, R, S 103 FW2 sequence no. 5 SEQ ID NO: 106 WVRQYPGKEPEWVS

P, R, S 103 FW2 sequence no. 6 SEQ ID NO: 107 WFRQPPGKEHEFVA

P, R, S 103 FW2 sequence no. 7 SEQ ID NO: 108 WYRQAPGKRTELVA

P, R, S 103 FW2 sequence no. 8 SEQ ID NO: 109 WLRQAPGQGLEWVS

P, R, S 103 FW2 sequence no. 9 SEQ ID NO: 110 WLRQTPGKGLEWVG

P, R, S 103 FW2 sequence no. 10 SEQ ID NO: 111 WVRQAPGKAEEFWS

50 and in which: v) FR3 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences: TABLE A-22 Representative FW3 sequences for Nanobodies of the P, R, S 103-group. P, R, S 103 FW3 sequence no. 1 SEQ ID NO: 112 RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA P, R, S 103 FW3 sequence no. 2 SEQ ID NO: 113 RFTISRDNARNTLYLQMDSLIPEDTALYYCAR P, R, S 103 FW3 sequence no. 3 SEQ ID NO: 114 RFTISRDNAKNEMYLQMNNLKTEDTGVYWCGA P, R, S 103 FW3 sequence no. 4 SEQ ID NO: 115 RFTISSDSNRNMIYLOMNNLKPEDTAVYYCAA P, R, S 103 FW3 sequence no. 5 SEQ ID NO: 116 RFTISRDNAKNMLYLHLNNLKSEDTAVYYCRR US 9,512,236 B2 95 96 TABLE A-22 - continued Representative FW3 sequences for Nanobodies of the P, R, S 103 - group. P, R, S 103 FW3 sequence no. 6 SEQ ID NO: 117 RFTISRDNAKKTWYLRLNSLNPEDTAWYSCNL P, R, S 103 FW3 sequence no. 7 SEQ ID NO: 118 RFKISRDNAKKTLYLOMNSLGPEDTAMYYCOR

P, R, S 103 FW3 sequence no. 8 SEQ ID NO: 119 RFTWSRDNGKNTAYLRMNSLKIPEDTADYYCAW

10 and in which: and in which: vi) FR4 is an amino acid sequence that has at least 80% iv) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 amino acid identity with at least one of the following and FR4 of Nanobodies of the P, R, S 103 class; amino acid sequences: and in which: TABLE A-23 Representative FW4 sequences for Nanobodies of the P, R, S 103-group. P, R, S 103 FW4 sequence no. 1 SEO ID NO: 12O RGOGTOVTVSS P, R, S 103 FW4 sequence no. 2 SEO ID NO: 121 LRGGTOVTVSS P, R, S 103 FW4 sequence no. 3 SEO ID NO: 122 GNKGTLVTVSS P, R, S 103 FW4 sequence no. 4 SEO ID NO: 123 SSPGTOVTVSS P, R, S 103 FW4 sequence no. 5 SEO ID NO: 124 SSOGTLVTVSS P, R, S 103 FW4 sequence no. 6 SEO ID NO: 125 RSRGIOVTVSS and in which: 30 V) CDR1, CDR2 and CDR3 are as defined herein, and are vii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred preferably as defined according to one of the preferred aspects herein, and are more preferably as defined accord aspects herein, and are more preferably as defined accord ing to one of the more preferred aspects herein. ing to one of the more preferred aspects herein. The above Nanobodies may for example be V. In the above Nanobodies, one or more of the further 35 sequences or may be humanized Nanobodies. When the Hallmark residues are preferably as described herein (for above NANOBODYR (V) sequences are V. sequences, example, when they are V. sequences or partially human they may be suitably humanized, as further described herein. ized Nanobodies). When the Nanobodies are partially humanized Nanobodies, With regard to framework 1, it will again be clear to the they may optionally be further Suitably humanized, again as skilled person that, for determining the degree of amino acid 40 described herein. identity, the amino acid residues on positions 1 to 4 and 27 In the above Nanobodies, one or more of the further to 30 are preferably disregarded. Hallmark residues are preferably as described herein (for In view of this, a NANOBODYR (V) of the P.R.S 103 example, when they are V. sequences or partially human class may be an amino acid sequence that is comprised of ized Nanobodies). four framework regions/sequences interrupted by three *5 In another preferred, but non-limiting aspect, the inven complementarity determining regions/sequences, in which: tion relates to a NANOBODYR (V) as described above, i) the amino acid residue at position 103 according to the in which the CDR sequences have at least 70% amino acid Kabat numbering is different from W: identity, preferably at least 80% amino acid identity, more and in which: preferably at least 90% amino acid identity, such as 95% ii) preferably the amino acid residue at position 103 accord 50 amino acid identity or more or even essentially 100% amino ing to the Kabat numbering is P. R or S, and more acid identity with the CDR sequences of at least one of the preferably R: amino acid sequences of SEQ ID NO’s: 526 to 527, 538 to and in which: 541, 413 to 453 and 517 to 525. This degree of amino acid iii) FR1 is an amino acid sequence that, on positions 5 to 26 identity can for example be determined by determining the of the Kabat numbering, has at least 80% amino acid 55 degree of amino acid identity (in a manner described herein) identity with at least one of the following amino acid between said NANOBODYR (V) and one or more of the Sequences: sequences of SEQID NO’s: 526 to 527, 538 to 541, 413 to TABLE A-24

Representative FW1 sequences (amino acid residues 5 to 26 for Nanobodies of the P, R, S 103-group.

P, R, S 103 FW1 sequence no. 9 SEQ ID NO: 1OO WESGGGLVOAGGSLRLSCAASG

P, R, S 103 FW1 sequence no. 10 SEQ ID NO: 101. AESGGGLVOPGGSLKLSCAASR US 9,512,236 B2 97 98 453 and 517 to 525, in which the amino acid residues that the invention, a NANOBODYR (V) may be as defined form the framework regions are disregarded. Such Nano herein, but with the proviso that it has at least “one amino bodies can be as further described herein. acid difference' (as defined herein) at at least one of the As already mentioned herein, another preferred but non Hallmark residues (including those at positions 108, 103 limiting aspect of the invention relates to a NANOBODYR and/or 45) compared to the corresponding framework region (V) with an amino acid sequence that is chosen from the of a naturally occurring human V. domain, and in particular group consisting of SEQ ID NO’s: 526 to 527, 538 to 541, compared to the corresponding framework region of DP-47. 413 to 453 and 517 to 525 or from the group consisting of Usually, a NANOBODYR (V) will have at least one such from amino acid sequences that have more than 80%, amino acid difference with a naturally occurring V, domain preferably more than 90%, more preferably more than 95%, 10 in at least one of FR2 and/or FR4, and in particular at at least such as 99% or more sequence identity (as defined herein) one of the Hallmark residues in FR2 and/or FR4 (again, with at least one of the amino acid sequences of SEQ ID including those at positions 108, 103 and/or 45). NO’s: 526 to 527, 538 to 541, 413 to 453 and 517 to 525. Also, a humanized NANOBODYR (V) of the inven Also, in the above Nanobodies: tion may be as defined herein, but with the proviso that it has i) any amino acid Substitution (when it is not a humanizing 15 at least “one amino acid difference' (as defined herein) in at substitution as defined herein) is preferably, and com least one of the framework regions compared to the corre pared to the corresponding amino acid sequence of SEQ sponding framework region of a naturally occurring V ID NO’s: 526 to 527, 538 to 541, 413 to 453 and 517 to domain. More specifically, according to one non-limiting 525, a conservative amino acid substitution, (as defined aspect of the invention, a humanized NANOBODYR (V) herein); may be as defined herein, but with the proviso that it has at and/or: least “one amino acid difference' (as defined herein) at at ii) its amino acid sequence preferably contains either only least one of the Hallmark residues (including those at amino acid substitutions, or otherwise preferably no more positions 108, 103 and/or 45) compared to the correspond than 5, preferably no more than 3, and more preferably ing framework region of a naturally occurring V domain. only 1 or 2 amino acid deletions or insertions, compared 25 Usually, a humanized NANOBODYR (V) will have at to the corresponding amino acid sequence of SEQ ID least one Such amino acid difference with a naturally occur NO’s: 526 to 527, 538 to 541, 413 to 453 and 517 to 525; ring V domain in at least one of FR2 and/or FR4, and in and/or particular at at least one of the Hallmark residues in FR2 iii) the CDR's may be CDR's that are derived by means of and/or FR4 (again, including those at positions 108, 103 affinity maturation, for example starting from the CDR's 30 and/or 45). of to the corresponding amino acid sequence of SEQ ID As will be clear from the disclosure herein, it is also NO’s: 526 to 527, 538 to 541, 413 to 453 and 517 to 525. within the scope of the invention to use natural or synthetic Preferably, the CDR sequences and FR sequences in the analogs, mutants, variants, alleles, homologs and orthologs Nanobodies of the invention are such that the Nanobodies of (herein collectively referred to as “analogs’) of the Nano the invention (and polypeptides of the invention comprising 35 bodies of the invention as defined herein, and in particular the same): analogs of the Nanobodies of SEQID NO’s 526 to 527, 538 bind to GPCRs with a dissociation constant (Ki) of 10 to 541, 413 to 453 and 517 to 525. Thus, according to one to 10° moles/liter or less, and preferably 107 to 10° aspect of the invention, the term “NANOBODYR (V) of moles/liter or less and more preferably 10 to 10° the invention' in its broadest sense also covers such analogs. moles/liter (i.e. with an association constant (K) of 40 Generally, in Such analogs, one or more amino acid 10 to 10' liter/moles or more, and preferably 107 to residues may have been replaced, deleted and/or added, 10' liter/moles or more and more preferably 10 to compared to the Nanobodies of the invention as defined 10' liter/moles): herein. Such substitutions, insertions or deletions may be and/or such that they: made in one or more of the framework regions and/or in one bind to GPCRs with a k-rate of between 10 M's to 45 or more of the CDR's. When such substitutions, insertions about 107M s', preferably between 10 M's and or deletions are made in one or more of the framework 107 M's', more preferably between 10 M's and regions, they may be made at one or more of the Hallmark 107 M' such as between 10 M's and 107M s'; residues and/or at one or more of the other positions in the and/or such that they: framework residues, although Substitutions, insertions or bind to GPCRs with a krate between 1 s' (t=0.69s) 50 deletions at the Hallmark residues are generally less pre and 10 s' (providing a near irreversible complex ferred (unless these are suitable humanizing Substitutions as with a ta of multiple days), preferably between 10° described herein). s' and 10's", more preferably between 10s and By means of non-limiting examples, a Substitution may 10 s, such as between 10's and 10 s. for example be a conservative substitution (as described Preferably, CDR sequences and FR sequences present in 55 herein) and/or an amino acid residue may be replaced by the Nanobodies of the invention are such that the Nanobod another amino acid residue that naturally occurs at the same ies of the invention will bind to GPCRs with an affinity less position in another V. domain (see Tables A-5 to A-8 for than 500 nM, preferably less than 200 nM, more preferably Some non-limiting examples of such substitutions), although less than 10 nM, such as less than 500 pM. the invention is generally not limited thereto. Thus, any one According to one non-limiting aspect of the invention, a 60 or more substitutions, deletions or insertions, or any com NANOBODYR (V) may be as defined herein, but with bination thereof, that either improve the properties of the the proviso that it has at least “one amino acid difference' NANOBODYR (V) of the invention or that at least do not (as defined herein) in at least one of the framework regions detract too much from the desired properties or from the compared to the corresponding framework region of a balance or combination of desired properties of the NANO naturally occurring human V, domain, and in particular 65 BODYR (V) of the invention (i.e. to the extent that the compared to the corresponding framework region of DP-47. NANOBODYR (V) is no longer suited for its intended More specifically, according to one non-limiting aspect of use) are included within the scope of the invention. A skilled US 9,512,236 B2 99 100 person will generally be able to determine and select suitable The humanizing Substitutions should be chosen such that Substitutions, deletions or insertions, or Suitable combina the resulting humanized Nanobodies still retain the favour tions of thereof, based on the disclosure herein and option able properties of Nanobodies as defined herein, and more ally after a limited degree of routine experimentation, which preferably such that they are as described for analogs in the may for example involve introducing a limited number of 5 preceding paragraphs. A skilled person will generally be possible Substitutions and determining their influence on the able to determine and select Suitable humanizing Substitu properties of the Nanobodies thus obtained. tions or Suitable combinations of humanizing Substitutions, For example, and depending on the host organism used to based on the disclosure herein and optionally after a limited express the NANOBODYR (V) or polypeptide of the degree of routine experimentation, which may for example 10 involve introducing a limited number of possible humaniz invention, such deletions and/or Substitutions may be ing Substitutions and determining their influence on the designed in Such a way that one or more sites for post properties of the Nanobodies thus obtained. translational modification (such as one or more glycosy Generally, as a result of humanization, the Nanobodies of lation sites) are removed, as will be within the ability of the the invention may become more “human-like', while still person skilled in the art. Alternatively, substitutions or 15 retaining the favorable properties of the Nanobodies of the insertions may be designed so as to introduce one or more invention as described herein. As a result, such humanized sites for attachment of functional groups (as described Nanobodies may have several advantages. Such as a reduced herein), for example to allow site-specific pegylation (again immunogenicity, compared to the corresponding naturally as described herein). occurring V domains. Again, based on the disclosure As can be seen from the data on the V entropy and V. herein and optionally after a limited degree of routine variability given in Tables A-5 to A-8 above, some amino experimentation, the skilled person will be able to select acid residues in the framework regions are more conserved humanizing Substitutions or Suitable combinations of than others. Generally, although the invention in its broadest humanizing Substitutions which optimize or achieve a sense is not limited thereto, any Substitutions, deletions or desired or suitable balance between the favourable proper insertions are preferably made at positions that are less 25 ties provided by the humanizing Substitutions on the one conserved. Also, generally, amino acid substitutions are hand and the favourable properties of naturally occurring preferred over amino acid deletions or insertions. V. domains on the other hand. The analogs are preferably such that they can bind to The Nanobodies of the invention may be suitably human GPCRs with an affinity (suitably measured and/or expressed ized at any framework residue(s). Such as at one or more as a K-value (actual or apparent), a K-value (actual or 30 Hallmark residues (as defined herein) or at one or more other apparent), a k-rate and/or a k-rate, or alternatively as an framework residues (i.e. non-Hallmark residues) or any ICso value, as further described herein) that is as defined suitable combination thereof. One preferred humanizing herein for the Nanobodies of the invention. substitution for Nanobodies of the "P.R.S-103 group' or the The analogs are preferably also such that they retain the “KERE group” is Q108 into L108. Nanobodies of the favourable properties the Nanobodies, as described herein. 35 “GLEW class” may also be humanized by a Q108 into L108 Also, according to one preferred aspect, the analogs have substitution, provided at least one of the other Hallmark a degree of sequence identity of at least 70%, preferably at residues contains a camelid (camelizing) Substitution (as least 80%, more preferably at least 90%, such as at least 95% defined herein). For example, as mentioned above, one or 99% or more; and/or preferably have at most 20, prefer particularly preferred class of humanized Nanobodies has ably at most 10, even more preferably at most 5, such as 4, 40 GLEW or a GLEW-like sequence at positions 44-47: P R or 3, 2 or only 1 amino acid difference (as defined herein), with S (and in particular R) at position 103, and an L at position one of the Nanobodies of SEQ ID NOs: 526 to 527, 538 to 108. 541, 413 to 453 and 517 to 525. The humanized and other analogs, and nucleic acid Also, the framework sequences and CDR's of the analogs sequences encoding the same, can be provided in any are preferably such that they are in accordance with the 45 manner known per se. For example, the analogs can be preferred aspects defined herein. More generally, as obtained by providing a nucleic acid that encodes a naturally described herein, the analogs will have (a) a Q at position occurring V domain, changing the codons for the one or 108; and/or (b) a charged amino acid or a cysteine residue more amino acid residues that are to be substituted into the at position 45 and preferably an E at position 44, and more codons for the corresponding desired amino acid residues preferably Eat position 44 and R at position 45; and/or (c) 50 (e.g. by site-directed mutagenesis or by PCR using Suitable P, R or S at position 103. mismatch primers), expressing the nucleic acid/nucleotide One preferred class of analogs of the Nanobodies of the sequence thus obtained in a suitable host or expression invention comprise Nanobodies that have been humanized system; and optionally isolating and/or purifying the analog (i.e. compared to the sequence of a naturally occurring thus obtained to provide said analog in essentially isolated NANOBODYR (V) of the invention). As mentioned in 55 form (e.g. as further described herein). This can generally be the background art cited herein, such humanization gener performed using methods and techniques known per se, ally involves replacing one or more amino acid residues in which will be clear to the skilled person, for example from the sequence of a naturally occurring V with the amino the handbooks and references cited herein, the background acid residues that occur at the same position in a human V, art cited herein and/or from the further description herein. domain, such as a human V3 domain. Examples of possible 60 Alternatively, a nucleic acid encoding the desired analog can humanizing Substitutions or combinations of humanizing be synthesized in a manner known perse (for example using substitutions will be clear to the skilled person, for example an automated apparatus for synthesizing nucleic acid from the Tables herein, from the possible humanizing sub sequences with a predefined amino acid sequence) and can stitutions mentioned in the background art cited herein, then be expressed as described herein. Yet another technique and/or from a comparison between the sequence of a 65 may involve combining one or more naturally occurring NANOBODYR (V) and the sequence of a naturally and/or synthetic nucleic acid sequences each encoding a part occurring human V, domain. of the desired analog, and then expressing the combined US 9,512,236 B2 101 102 nucleic acid sequence as described herein. Also, the analogs residues at the N-terminal end, one or more amino acid can be provided using chemical synthesis of the pertinent residues at the C-terminal end, one or more contiguous amino acid sequence using techniques for peptide synthesis internal amino acid residues, or any combination thereof, known per se, Such as those mentioned herein. have been deleted and/or removed. In this respect, it will be also be clear to the skilled person 5 The parts or fragments are preferably Such that they can that the Nanobodies of the invention (including their ana bind to GPCRs with an affinity (suitably measured and/or logs) can be designed and/or prepared starting from human expressed as a K-value (actual or apparent), a K-value V. sequences (i.e. amino acid sequences or the correspond (actual or apparent), a k-rate and/or a k-rate, or alterna ing nucleotide sequences). Such as for example from human tively as an ICso value, as further described herein) that is as V3 sequences such as DP-47, DP-51 or DP-29, i.e. by 10 defined herein for the Nanobodies of the invention. introducing one or more camelizing Substitutions (i.e. Any part or fragment is preferably Such that it comprises changing one or more amino acid residues in the amino acid at least 10 contiguous amino acid residues, preferably at sequence of said human V. domain into the amino acid least 20 contiguous amino acid residues, more preferably at residues that occur at the corresponding position in a V least 30 contiguous amino acid residues, such as at least 40 domain), so as to provide the sequence of a NANOBODYR 15 contiguous amino acid residues, of the amino acid sequence (V) of the invention and/or so as to confer the favourable of the corresponding full length NANOBODYR (V) of properties of a NANOBODYR (V) to the sequence thus the invention. obtained. Again, this can generally be performed using the Also, any part or fragment is such preferably that it various methods and techniques referred to in the previous comprises at least one of CDR1, CDR2 and/or CDR3 or at paragraph, using an amino acid sequence and/or nucleotide least part thereof (and in particular at least CDR3 or at least sequence for a human V, domain as a starting point. part thereof). More preferably, any part or fragment is such Some preferred, but non-limiting camelizing Substitutions that it comprises at least one of the CDR's (and preferably can be derived from Tables A-5-A-8. It will also be clear that at least CDR3 or part thereof) and at least one other CDR camelizing Substitutions at one or more of the Hallmark (i.e. CDR1 or CDR2) or at least part thereof, preferably residues will generally have a greater influence on the 25 connected by Suitable framework sequence(s) or at least part desired properties than substitutions at one or more of the thereof. More preferably, any part or fragment is such that it other amino acid positions, although both and any Suitable comprises at least one of the CDR's (and preferably at least combination thereof are included within the scope of the CDR3 or part thereof) and at least part of the two remaining invention. For example, it is possible to introduce one or CDR's, again preferably connected by suitable framework more camelizing Substitutions that already confer at least 30 sequence(s) or at least part thereof. some the desired properties, and then to introduce further According to another particularly preferred, but non camelizing substitutions that either further improve said limiting aspect, such a part or fragment comprises at least properties and/or confer additional favourable properties. CDR3, such as FR3, CDR3 and FR4 of the corresponding Again, the skilled person will generally be able to determine full length NANOBODYR (V) of the invention, i.e. as for and select Suitable camelizing Substitutions or Suitable com 35 example described in the International application WO binations of camelizing Substitutions, based on the disclo 03/050531 (Lasters et al.). Sure herein and optionally after a limited degree of routine As already mentioned above, it is also possible to com experimentation, which may for example involve introduc bine two or more of Such parts or fragments (i.e. from the ing a limited number of possible camelizing Substitutions same or different Nanobodies of the invention), i.e. to and determining whether the favourable properties of Nano 40 provide an analog (as defined herein) and/or to provide bodies are obtained or improved (i.e. compared to the further parts or fragments (as defined herein) of a NANO original V. domain). Generally, however, such camelizing BODYR (V) of the invention. It is for example also Substitutions are preferably Such that the resulting an amino possible to combine one or more parts or fragments of a acid sequence at least contains (a) a Qat position 108; and/or NANOBODYR (V) of the invention with one or more (b) a charged amino acid or a cysteine residue at position 45 45 parts or fragments of a human V. domain. and preferably also an E at position 44, and more preferably According to one preferred aspect, the parts or fragments E at position 44 and Rat position 45; and/or (c) P R or S at have a degree of sequence identity of at least 50%, prefer position 103; and optionally one or more further camelizing ably at least 60%, more preferably at least 70%, even more substitutions. More preferably, the camelizing substitutions preferably at least 80%, such as at least 90%. 95% or 99% are such that they result in a NANOBODYR (V) of the 50 or more with one of the Nanobodies of SEQID NOS 526 to invention and/or in an analog thereof (as defined herein), 527; SEQID NO’s 538 to 541; 413 to 453 and 517 to 525. Such as in a humanized analog and/or preferably in an analog The parts and fragments, and nucleic acid sequences that is as defined in the preceding paragraphs. encoding the same, can be provided and optionally com As will also be clear from the disclosure herein, it is also bined in any manner known per se. For example, Such parts within the scope of the invention to use parts or fragments, 55 or fragments can be obtained by inserting a stop codon in a or combinations of two or more parts or fragments, of the nucleic acid that encodes a full-sized NANOBODYR (V) Nanobodies of the invention as defined herein, and in of the invention, and then expressing the nucleic acid thus particular parts or fragments of the Nanobodies of SEQ ID obtained in a manner known per se (e.g. as described NO’s: 526 to 527, 538 to 541, 413 to 453 and 517 to 525. herein). Alternatively, nucleic acids encoding such parts or Thus, according to one aspect of the invention, the term 60 fragments can be obtained by Suitably restricting a nucleic “NANOBODYR (V) of the invention” in its broadest acid that encodes a full-sized NANOBODYR (V) of the sense also covers such parts or fragments. invention or by synthesizing such a nucleic acid in a manner Generally, such parts or fragments of the Nanobodies of known perse. Parts or fragments may also be provided using the invention (including analogs thereof) have amino acid techniques for peptide synthesis known per se. sequences in which, compared to the amino acid sequence of 65 The invention in its broadest sense also comprises deriva the corresponding full length NANOBODYR (V) of the tives of the Nanobodies of the invention. Such derivatives invention (or analog thereof), one or more of the amino acid can generally be obtained by modification, and in particular US 9,512,236 B2 103 104 by chemical and/or biological (e.g. enzymatical) modifica modified so as to Suitably introduce one or more cysteine tion, of the Nanobodies of the invention and/or of one or residues for attachment of PEG, or an amino acid sequence more of the amino acid residues that form the Nanobodies of comprising one or more cysteine residues for attachment of the invention. PEG may be fused to the N- and/or C-terminus of a Examples of Such modifications, as well as examples of 5 NANOBODYR (V) of the invention, all using techniques amino acid residues within the NANOBODYR (V) of protein engineering known per se to the skilled person. sequence that can be modified in Such a manner (i.e. either Preferably, for the Nanobodies and proteins of the inven on the protein backbone but preferably on a side chain), tion, a PEG is used with a molecular weight of more than methods and techniques that can be used to introduce Such 5000, such as more than 10,000 and less than 200,000, such modifications and the potential uses and advantages of Such 10 as less than 100,000; for example in the range of 20,000 modifications will be clear to the skilled person. 80,000. For example, Such a modification may involve the intro Another, usually less preferred modification comprises duction (e.g. by covalent linking or in an other Suitable N-linked or O-linked , usually as part of co manner) of one or more functional groups, residues or translational and/or post-translational modification, depend moieties into or onto the NANOBODYR (V) of the 15 ing on the host cell used for expressing the NANOBODYR invention, and in particular of one or more functional (V) or polypeptide of the invention. groups, residues or moieties that confer one or more desired Yet another modification may comprise the introduction properties or functionalities to the NANOBODYR (V) of of one or more detectable labels or other signal-generating the invention. Example of such functional groups will be groups or moieties, depending on the intended use of the clear to the skilled person. labelled NANOBODYR (V). Suitable labels and tech For example, Such modification may comprise the intro niques for attaching, using and detecting them will be clear duction (e.g. by covalent binding or in any other Suitable to the skilled person, and for example include, but are not manner) of one or more functional groups that increase the limited to, fluorescent labels (such as fluorescein, isothio half-life, the solubility and/or the absorption of the NANO cyanate, rhodamine, phycoerythrin, phycocyanin, allophy BODYR (V) of the invention, that reduce the immuno 25 cocyanin, o-phthaldehyde, and fluorescamine and fluores genicity and/or the toxicity of the NANOBODYR (V) of cent metals such as ''Eu or others metals from the the invention, that eliminate or attenuate any undesirable lanthanide series), phosphorescent labels, chemiluminescent side effects of the NANOBODYR (V) of the invention, labels or bioluminescent labels (such as luminal, isoluminol, and/or that confer other advantageous properties to and/or theromatic acridinium ester, imidazole, acridinium salts, reduce the undesired properties of the Nanobodies and/or 30 oxalate ester, dioxetane or GFP and its analogs), radio polypeptides of the invention; or any combination of two or isotopes (such as H, I, P, S, 'C, Cr, C1, 7Co, more of the foregoing. Examples of such functional groups Co, Fe, and 'Se), metals, metal chelates or metallic and of techniques for introducing them will be clear to the cations (for example metallic cations such as "Tc, 'I, skilled person, and can generally comprise all functional ''' In, ''I, Ru, 'Cu, ''Ga, and Ga or other metals or groups and techniques mentioned in the general background 35 metallic cations that are particularly Suited for use in in vivo, art cited hereinabove as well as the functional groups and in vitro or in situ diagnosis and imaging, such as (''Gd, techniques known per se for the modification of pharma Mn, Dy, °Cr, and Fe), as well as chromophores and ceutical proteins, and in particular for the modification of enzymes (such as malate dehydrogenase, staphylococcal antibodies or antibody fragments (including Schv's and nuclease, delta-V-steroid isomerase, yeast alcohol dehydro single domain antibodies), for which reference is for 40 genase, alpha-glycerophosphate dehydrogenase, triose example made to Remington's Pharmaceutical Sciences, phosphate isomerase, biotinavidin peroxidase, horseradish 16th ed., Mack Publishing Co., Easton, Pa. (1980). Such peroxidase, alkaline phosphatase, asparaginase, glucose oxi functional groups may for example be linked directly (for dase, beta-galactosidase, ribonuclease, urease, catalase, glu example covalently) to a NANOBODYR (V) of the cose-VI-phosphate dehydrogenase, glucoamylase and ace invention, or optionally via a Suitable linker or spacer, as 45 tylcholine esterase). Other suitable labels will be clear to the will again be clear to the skilled person. skilled person, and for example include moieties that can be One of the most widely used techniques for increasing the detected using NMR or ESR spectroscopy. half-life and/or reducing the immunogenicity of pharmaceu Such labelled Nanobodies and polypeptides of the inven tical proteins comprises attachment of a suitable pharmaco tion may for example be used for in vitro, in vivo or in situ logically acceptable polymer, Such as poly(ethyleneglycol) 50 assays (including immunoassays known per se Such as (PEG) or derivatives thereof (such as methoxypoly(ethyl ELISA, RIA, EIA and other “sandwich assays, etc.) as well eneglycol) or mPEG). Generally, any suitable form of pegy as in Vivo diagnostic and imaging purposes, depending on lation can be used. Such as the pegylation used in the art for the choice of the specific label. antibodies and antibody fragments (including but not limited As will be clear to the skilled person, another modification to (single) domain antibodies and Schv's); reference is made 55 may involve the introduction of a chelating group, for to for example Chapman, Nat. Biotechnol. 54, 531-545 example to chelate one of the metals or metallic cations (2002); by Veronese and Harris, Adv. Drug Deliv. Rev. 54, referred to above. Suitable chelating groups for example 453-456 (2003), by Harris and Chess, Nat. Rev. Drug. include, without limitation, diethyl-enetriaminepentaacetic Discov. 2, (2003) and in WO 04/060965. Various reagents acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). for pegylation of proteins are also commercially available, 60 Yet another modification may comprise the introduction for example from Nektar Therapeutics, USA. of a functional group that is one part of a specific binding Preferably, site-directed pegylation is used, in particular pair, such as the biotin-(strept)avidin binding pair. Such a via a cysteine-residue (see for example Yang et al., Protein functional group may be used to link the NANOBODYR Engineering, 16, 10, 761-770 (2003). For example, for this (V) of the invention to another protein, polypeptide or purpose, PEG may be attached to a cysteine residue that 65 chemical compound that is bound to the other half of the naturally occurs in a NANOBODYR (V) of the inven binding pair, i.e. through formation of the binding pair. For tion, a NANOBODYR (V) of the invention may be example, a NANOBODYR (V) of the invention may be US 9,512,236 B2 105 106 conjugated to biotin, and linked to another protein, poly NANOBODYR (V), although the invention in its peptide, compound or carrier conjugated to avidin or strepta broadest sense is not limited thereto; vidin. For example, such a conjugated NANOBODYR may form a sequence or signal that allows the NANO (V) may be used as a reporter, for example in a diagnostic BODYR (V) to be directed towards and/or to pen system where a detectable signal-producing agent is conju etrate or enter into specific organs, tissues, cells, or gated to avidin or streptavidin. Such binding pairs may for parts or compartments of cells, and/or that allows the example also be used to bind the NANOBODYR (V) of NANOBODYR (V) to penetrate or cross a biologi the invention to a carrier, including carriers suitable for cal barrier Such as a cell membrane, a cell layer Such as pharmaceutical purposes. One non-limiting example are the a layer of epithelial cells, a tumor including Solid liposomal formulations described by Cao and Suresh, Jour 10 tumors, or the blood-brain-barrier. Examples of such nal of Drug Targetting, 8, 4, 257 (2000). Such binding pairs amino acid sequences will be clear to the skilled may also be used to link a therapeutically active agent to the person. Some non-limiting examples are the Small NANOBODYR (V) of the invention. peptide vectors (“Pep-trans vectors') described in WO For some applications, in particular for those applications 03/026700 and in Temsamani et al., Expert Opin. Biol. in which it is intended to kill a cell that expresses the target 15 Ther. 1, 773 (2001); Temsamani and Vidal, Drug against which the Nanobodies of the invention are directed Discov. Today, 9, 1012 (004) and Rousselle, J. Phar (e.g. in the treatment of cancer), or to reduce or slow the macol. Exp. Ther. 296, 124-131 (2001), and the mem growth and/or proliferation such a cell, the Nanobodies of brane translocator sequence described by Zhao et al., the invention may also be linked to a toxin or to a toxic , 8, 631-637 (2003). C-terminal and N-ter residue or moiety. Examples of toxic moieties, compounds minal amino acid sequences for intracellular targeting or residues which can be linked to a NANOBODYR (V) of antibody fragments are for example described by of the invention to provide—for example—a cytotoxic com Cardinale et al., Methods, 34, 171 (2004). Other suit pound will be clear to the skilled person and can for example able techniques for intracellular targeting involve the be found in the prior art cited above and/or in the further expression and/or use of so-called “intrabodies' com description herein. One example is the so-called ADEPTTM 25 prising a NANOBODYR (V) of the invention, as technology described in WO 03/055527. mentioned below: Other potential chemical and enzymatical modifications may form a “tag”, for example an amino acid sequence or will be clear to the skilled person. Such modifications may residue that allows or facilitates the purification of the also be introduced for research purposes (e.g. to study NANOBODYR (V), for example using affinity tech function-activity relationships). Reference is for example 30 niques directed against said sequence or residue. There made to Lundblad and Bradshaw, Biotechnol. Appl. Bio after, said sequence or residue may be removed (e.g. by chem., 26, 143-151 (1997). chemical or enzymatical cleavage) to provide the Preferably, the derivatives are such that they bind to NANOBODYR (V) sequence (for this purpose, the GPCRs with an affinity (suitably measured and/or expressed tag may optionally be linked to the NANOBODYR as a K-value (actual or apparent), a K-value (actual or 35 (V) sequence via a cleavable linker sequence or apparent), a k-rate and/or a k-rate, or alternatively as an contain a cleavable motif). Some preferred, but non ICso value, as further described herein) that is as defined limiting examples of Such residues are multiple histi herein for the Nanobodies of the invention. dine residues, glutathione residues and a myc-tag (see As mentioned above, the invention also relates to proteins for example SEQ ID NO:31 of WO 06/12282). or polypeptides that essentially consist of or comprise at 40 may be one or more amino acid residues that have been least one NANOBODYR (V) of the invention. By functionalized and/or that can serve as a site for attach “essentially consist of is meant that the amino acid ment of functional groups. Suitable amino acid residues sequence of the polypeptide of the invention either is exactly and functional groups will be clear to the skilled person the same as the amino acid sequence of a NANOBODYR and include, but are not limited to, the amino acid (V) of the invention or corresponds to the amino acid 45 residues and functional groups mentioned herein for the sequence of a NANOBODYR (V) of the invention which derivatives of the Nanobodies of the invention. has a limited number of amino acid residues, such as 1-20 According to another aspect, a polypeptide of the inven amino acid residues, for example 1-10 amino acid residues tion comprises a NANOBODYR (V) of the invention, and preferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 which is fused at its amino terminal end, at its carboxy or 6 amino acid residues, added at the amino terminal end, 50 terminal end, or both at its amino terminal end and at its at the carboxy terminal end, or at both the amino terminal carboxy terminal end to at least one further amino acid end and the carboxy terminal end of the amino acid sequence sequence, i.e. so as to provide a fusion protein comprising of the NANOBODYR (V). said NANOBODYR (V) of the invention and the one or Said amino acid residues may or may not change, alter or more further amino acid sequences. Such a fusion will also otherwise influence the (biological) properties of the 55 be referred to herein as a “NANOBODYR (V) fusion”. NANOBODYR (V) and may or may not add further The one or more further amino acid sequence may be any functionality to the NANOBODYR (V). For example, Suitable and/or desired amino acid sequences. The further Such amino acid residues: amino acid sequences may or may not change, alter or can comprise an N-terminal Met residue, for example as otherwise influence the (biological) properties of the result of expression in a heterologous host cell or host 60 NANOBODYR (V), and may or may not add further organism. functionality to the NANOBODYR (V) or the polypep may form a signal sequence or leader sequence that tide of the invention. Preferably, the further amino acid directs secretion of the NANOBODYR (V) from a sequence is such that it confers one or more desired prop host cell upon synthesis. Suitable secretory leader erties or functionalities to the NANOBODYR (V) or the peptides will be clear to the skilled person, and may be 65 polypeptide of the invention. as further described herein. Usually, such a leader For example, the further amino acid sequence may also sequence will be linked to the N-terminus of the provide a second binding site, which binding site may be US 9,512,236 B2 107 108 directed against any desired protein, polypeptide, antigen, serum albumin and amino acid sequences that can bind to antigenic determinant or epitope (including but not limited amino acid residues on (human) serum albumin that are not to the same protein, polypeptide, antigen, antigenic deter involved in binding of serum albumin to FcRn (see for minant or epitope against which the NANOBODYR (V) example WO 06/0122787) and/or amino acid sequences that of the invention is directed, or a different protein, polypep- 5 are capable of binding to amino acid residues on serum tide, antigen, antigenic determinant or epitope). albumin that do not form part of domain III of serum Example of Such amino acid sequences will be clear to the albumin (see again for example WO 06/0122787); amino skilled person, and may generally comprise all amino acid acid sequences that have or can provide an increased half sequences that are used in peptide fusions based on conven life (see for example the U.S. provisional application tional antibodies and fragments thereof (including but not 10 60/843,349 by Ablynx N.V. entitled “Serum albumin bind limited to Sclv’s and single domain antibodies). Reference ing proteins with long half-lives' filed on Sep. 8, 2006; see is for example made to the review by Holliger and Hudson, also WO08/028977); amino acid sequences against human Nature Biotechnology, 23, 9, 1126-1136 (2005), serum albumin that are cross-reactive with serum albumin For example, Such an amino acid sequence may be an from at least one species of mammal, and in particular with amino acid sequence that increases the half-life, the Solu 15 at least one species of primate (such as, without limitation, bility, or the absorption, reduces the immunogenicity or the monkeys from the genus Macaca (Such as, and in particular, toxicity, eliminates or attenuates undesirable side effects, cynomolgus monkeys (Macaca fascicularis) and/or rhesus and/or confers other advantageous properties to and/or monkeys (Macaca mulata)) and baboon (Papio ursinus), reduces the undesired properties of the polypeptides of the reference is again made to the U.S. provisional application invention, compared to the NANOBODYR (V) of the 60/843,349 and WO08/028977): amino acid sequences that invention perse. Some non-limiting examples of such amino can bind to serum albumin in a pH independent manner (see acid sequences are serum proteins, such as human serum for example the U.S. provisional application 60/850.774 by albumin (see for example WO 00/27435) or haptenic mol Ablynx N.V. entitled “Amino acid sequences that bind to ecules (for example haptens that are recognized by circu serum proteins in a manner that is essentially independent of lating antibodies, see for example WO 98/22141). 25 the pH, compounds comprising the same, and uses thereof. In particular, it has been described in the art that linking filed on Oct. 11, 2006; see also WO08/043821) and/or amino fragments of immunoglobulins (such as V. domains) to acid sequences that are conditional binders (see for example serum albumin or to fragments thereof can be used to the U.S. provisional application 60/850,775 by Ablynx N.V. increase the half-life. Reference is for made to WO entitled “Amino acid sequences that bind to a desired 00/27435 and WO 01/077137). According to the invention, 30 molecule in a conditional manner', filed on Oct. 11, 2006; the NANOBODYR (V) of the invention is preferably see also WO08/043822). either directly linked to serum albumin (or to a suitable According to another aspect, the one or more further fragment thereof) or via a suitable linker, and in particular amino acid sequences may comprise one or more parts, via a suitable peptide linked so that the polypeptide of the fragments or domains of conventional 4-chain antibodies invention can be expressed as a genetic fusion (protein). 35 (and in particular human antibodies) and/or of heavy chain According to one specific aspect, the NANOBODYR (V) antibodies. For example, although usually less preferred, a of the invention may be linked to a fragment of serum NANOBODYR (V) of the invention may be linked to a albumin that at least comprises the domain III of serum conventional (preferably human) V, or V, domain or to a albumin or part thereof. Reference is for example made to natural or synthetic analog of a V or V, domain, again the U.S. provisional application 60/788.256 of Ablynx N.V. 40 optionally via a linker sequence (including but not limited to entitled “Albumin derived amino acid sequence, use thereof other (single) domain antibodies, such as the dAb’s for increasing the half-life of therapeutic proteins and of described by Ward et al.). other therapeutic proteins and entities, and constructs com The at least one NANOBODYR (V) may also be prising the same filed on Mar. 31, 2006 (see also WO07/ linked to one or more (preferably human) C1, C2 and/or 112940). 45 C3 domains, optionally via a linker sequence. For instance, Alternatively, the further amino acid sequence may pro a NANOBODYR (V) linked to a suitable C1 domain vide a second binding site or binding unit that is directed could for example be used together with suitable light against a serum protein (Such as, for example, human serum chains—to generate antibody fragments/structures analo albumin or another serum protein such as IgG). So as to gous to conventional Fab fragments or F(ab') fragments, but provide increased half-life in serum. Such amino acid 50 in which one or (in case of an F(ab')2 fragment) one or both sequences for example include the Nanobodies described of the conventional V. domains have been replaced by a below, as well as the Small peptides and binding proteins NANOBODYR (V) of the invention. Also, two Nano described in WO 91/01743, WO 01/45746 and WO bodies could be linked to a C3 domain (optionally via a 02/076489 and the dAb’s described in WO 03/002609 and linker) to provide a construct with increased half-life in vivo. WO 04/003019. Reference is also made to Harmsen et al., 55 According to one specific aspect of a polypeptide of the Vaccine, 23 (41): 4926-42, 2005, as well as to EP 0368 684, invention, one or more Nanobodies of the invention may be as well as to the following the U.S. provisional applications linked (optionally via a suitable linker or hinge region) to 60/843,349 (see also WO08/028977), 60/850,774 (see also one or more constant domains (for example, 2 or 3 constant WO08/043821), 60/850,775 (see also WO08/043822) by domains that can be used as part of to form an Fc portion), Ablynx N.V. mentioned herein US provisional application of 60 to an Fc portion and/or to one or more antibody parts, Ablynx N.V. entitled “Peptides capable of binding to serum fragments or domains that confer one or more effector proteins' filed on Dec. 5, 2006 (see also WO08/068280). functions to the polypeptide of the invention and/or may Such amino acid sequences may in particular be directed confer the ability to bind to one or more Fc receptors. For against Serum albumin (and more in particular human serum example, for this purpose, and without being limited thereto, albumin) and/or against IgG (and more in particular human 65 the one or more further amino acid sequences may comprise IgG). For example, such amino acid sequences may be one or more C2 and/or C3 domains of an antibody, such amino acid sequences that are directed against (human) as from a heavy chain antibody (as described herein) and US 9,512,236 B2 109 110 more preferably from a conventional human 4-chain anti The further amino acid sequence may also form a body; and/or may form (part of) and Fc region, for example sequence or signal that allows the NANOBODYR (V) or from IgG (e.g. from IgG1, IgG2, IgG3 or IgG4), from IgE polypeptide of the invention to be directed towards and/or to or from another human Ig Such as IgA, Ig|D or IgM. For penetrate or enter into specific organs, tissues, cells, or parts example, WO 94/04678 describes heavy chain antibodies or compartments of cells, and/or that allows the NANO comprising a Camelid V. domain or a humanized deriva BODYR (V) or polypeptide of the invention to penetrate tive thereof (i.e. a NANOBODYR (V)), in which the or cross a biological barrier Such as a cell membrane, a cell Camelidae C2 and/or C3 domain have been replaced by layer Such as a layer of epithelial cells, a tumor including human C2 and C3 domains, so as to provide an immu solid tumors, or the blood-brain-barrier. Suitable examples 10 of such amino acid sequences will be clear to the skilled noglobulin that consists of 2 heavy chains each comprising person, and for example include, but are not limited to, the a NANOBODYR (V) and human C2 and C3 domains “Peptrans' vectors mentioned above, the sequences (but no C1 domain), which immunoglobulin has the effec described by Cardinale et al. and the amino acid sequences tor function provided by the C2 and C3 domains and and antibody fragments known per se that can be used to which immunoglobulin can function without the presence of 15 express or produce the Nanobodies and polypeptides of the any light chains. Other amino acid sequences that can be invention as so-called “intrabodies', for example as suitably linked to the Nanobodies of the invention so as to described in WO 94/02610, WO95/22618, U.S. Pat. No. provide an effector function will be clear to the skilled 7,004,940, WO 03/014960, WO 99/07414; WO 05/01690; person, and may be chosen on the basis of the desired EP 1 512 696; and in Cattaneo, A. & Biocca, S. (1997) effector function(s). Reference is for example made to WO Intracellular Antibodies: Development and Applications. 04/058820, WO 99/42077, WO 02/056910 and WO Landes and Springer-Verlag, and in Kontermann, Methods 05/017148, as well as the review by Holliger and Hudson, 34, (2004), 163-170, and the further references described supra; and WO09/068628. Coupling of a NANOBODYR therein. (V) of the invention to an Fc portion may also lead to an For some applications, in particular for those applications increased half-life, compared to the corresponding NANO 25 in which it is intended to kill a cell that expresses the target BODYR (V) of the invention. For some applications, the against which the Nanobodies of the invention are directed use of an Fc portion and/or of constant domains (i.e. C2 (e.g. in the treatment of cancer), or to reduce or slow the and/or C3 domains) that confer increased half-life without growth and/or proliferation of such a cell, the Nanobodies of any biologically significant effector function may also be the invention may also be linked to a (cyto)toxic protein or suitable or even preferred. Other suitable constructs com 30 polypeptide. Examples of Such toxic proteins and polypep prising one or more Nanobodies and one or more constant tides which can be linked to a NANOBODYR (V) of the domains with increased half-life in vivo will be clear to the invention to provide for example—a cytotoxic polypep skilled person, and may for example comprise two Nano tide of the invention will be clear to the skilled person and bodies linked to a C3 domain, optionally via a linker can for example be found in the prior art cited above and/or sequence. Generally, any fusion protein or derivatives with 35 in the further description herein. One example is the so increased half-life will preferably have a molecular weight called ADEPTTM technology described in WO 03/055527. of more than 50 kDa, the cut-off value for renal absorption. According to one preferred, but non-limiting aspect, said In another one specific, but non-limiting, aspect, in order one or more further amino acid sequences comprise at least to form a polypeptide of the invention, one or more amino one further NANOBODYR (V), so as to provide a acid sequences of the invention may be linked (optionally 40 polypeptide of the invention that comprises at least two, via a Suitable linker or hinge region) to naturally occurring, such as three, four, five or more Nanobodies, in which said synthetic or semisynthetic constant domains (or analogs, Nanobodies may optionally be linked via one or more linker variants, mutants, parts or fragments thereof) that have a sequences (as defined herein). Polypeptides of the invention reduced (or essentially no) tendency to self-associate into that comprise two or more Nanobodies, of which at least one dimers (i.e. compared to constant domains that naturally 45 is a NANOBODYR (V) of the invention, will also be occur in conventional 4-chain antibodies). Such monomeric referred to herein as “multivalent” polypeptides of the (i.e. not self-associating) Fc chain variants, or fragments invention, and the Nanobodies present in Such polypeptides thereof, will be clear to the skilled person. For example, will also be referred to herein as being in a “multivalent Helm et al., J Biol Chem 1996 271 7494, describe mono format'. For example a “bivalent” polypeptide of the inven meric Fce chain variants that can be used in the polypeptide 50 tion comprises two Nanobodies, optionally linked via a chains of the invention. linker sequence, whereas a “trivalent polypeptide of the Also, Such monomeric Fc chain variants are preferably invention comprises three Nanobodies, optionally linked via such that they are still capable of binding to the complement two linker sequences; etc.; in which at least one of the or the relevant Fc receptor(s) (depending on the Fc portion Nanobodies present in the polypeptide, and up to all of the from which they are derived), and/or such that they still have 55 Nanobodies present in the polypeptide, is/are a NANO some or all of the effector functions of the Fc portion from BODYR (V) of the invention. which they are derived (or at a reduced level still suitable for In a multivalent polypeptide of the invention, the two or the intended use). Alternatively, in Such a polypeptide chain more Nanobodies may be the same or different, and may be of the invention, the monomeric Fc chain may be used to directed against the same antigen or antigenic determinant confer increased half-life upon the polypeptide chain, in 60 (for example against the same part(s) or epitope(s) or against which case the monomeric Fc chain may also have no or different parts or epitopes) or may alternatively be directed essentially no effector functions. against different antigens or antigenic determinants; or any Bivalent/multivalent, bispecific/multispecific O suitable combination thereof. For example, a bivalent poly biparatopic/multiparatopic polypeptides of the invention peptide of the invention may comprise (a) two identical may also be linked to Fc portions, in order to provide 65 Nanobodies; (b) a first NANOBODYR (V) directed polypeptide constructs of the type that is described in against a first antigenic determinant of a protein or antigen WOO9/06863.O. and a second NANOBODYR (V) directed against the US 9,512,236 B2 111 112 same antigenic determinant of said protein or antigen which Completely unexpectedly, present inventors have con is different from the first NANOBODYR (V); (c) a first structed biparatopic Nanobodies, obtained by linkage of NANOBODYR (V) directed against a first antigenic 238D2 to 238D4 which resulted in a significantly increased determinant of a protein or antigen and a second NANO affinity for CXCR4 (27 and 17-fold increase over its respec BODYR (V) directed against another antigenic determi tive monovalent constructs, see Example 6 described nant of said protein or antigen (i.e. biparatopic NANO herein). BODYR (V) constructs); or (d) a first NANOBODYR WO2011/161266 describes a number of sequence-opti (V) directed against a first protein orantigen and a second mized/improved variants of the amino acid sequences NANOBODYR (V) directed against a second protein or 238D2 and 238D4, as well as biparatopic constructs that 10 comprise Such improved variants as building blocks. Also antigen (i.e. different from said first antigen). Similarly, a further exemplified herein are biparatopic polypeptides of trivalent polypeptide of the invention may, for example and the invention directed against another respresentative without being limited thereto. comprise (a) three identical GPCR, such as CXCR7; wherein the inventors repeatedly Nanobodies; (b) two identical NANOBODYR (V) demonstrate that a biparatopic polypeptide of the invention against a first antigenic determinant of an antigen and a third 15 is more effective in biological assays, e.g. B-arrestin assay, NANOBODYR (V) directed against a different antigenic than mono-specific bivalent polypeptides (Example 7 determinant of the same antigen; (c) two identical NANO described herein). Reference is for example made to BODYR (V) against a first antigenic determinant of an WO2012/130874. Thus, present inventors have demon antigen and a third NANOBODYR (V) directed against strated for the first time that the construction of biparatopic a second antigen different from said first antigen; (d) a first constructs directed against GPCRs completely unexpectedly NANOBODYR (V) directed against a first antigenic resulted in a significant increase in potency compared to its determinant of a first antigen, a second NANOBODYR corresponding monovalent and mono-specific bivalent bind (V) directed against a second antigenic determinant of ers. Particularly preferred biparatopic polypeptides in accor said first antigen and a third NANOBODYR (V) directed dance with the invention are those shown in Examples 5A, against a second antigen different from said first antigen; or 25 6 and 7 described herein. (e) a first NANOBODYR (V) directed against a first Polypeptides of the invention that contain at least two antigen, a second NANOBODYR (V) directed against a Nanobodies, in which at least one NANOBODYR (V) is second antigen different from said first antigen, and a third directed against a first antigen (i.e. against GPCRs) and at NANOBODYR (V) directed against a third antigen dif least one NANOBODYR (V) is directed against a second ferent from said first and second antigen. Particularly pre 30 antigen (i.e. different from GPCRs), will also be referred to ferred polypeptides in accordance with the invention are as “multispecific' polypeptides of the invention, and the biparatopic polypeptides directed against GPCRs. Such Nanobodies present in such polypeptides will also be polypeptides may comprise two or more Nanobodies which referred to herein as being in a “multispecific format. Thus, are directed against GPCRs, wherein at least one “first for example, a “bispecific polypeptide of the invention is a NANOBODYR (V) is directed against a first antigenic 35 polypeptide that comprises at least one NANOBODYR determinant, epitope, part, domain, Subunit or confirmation (V) directed against a first antigen (i.e. GPCRs) and at of a GPCR (e.g. CXCR4); and wherein at least one “second least one further NANOBODYR (V) directed against a NANOBODYR (V) is directed against a second antigenic second antigen (i.e. different from GPCRs), whereas a determinant, epitope, part, domain, Subunit or confirmation “trispecific' polypeptide of the invention is a polypeptide of said GPCR different from the first, in which said Nano 40 that comprises at least one NANOBODYR (V) directed bodies may be suitably linked, for example via a suitable against a first antigen (i.e. GPCRs), at least one further linker as further described herein. Such biparatopic NANO NANOBODYR (V) directed against a second antigen BODYR (V) constructs were prepared by the inventors (i.e. different from GPCRs) and at least one further NANO and are provided herein. For example, WO 09/138519 BODYR (V) directed against a third antigen (i.e. different describes a number of amino acid sequences and in particu 45 from both GPCRs, and the second antigen); etc. lar VHHs and constructs thereof that are directed against Accordingly, in its simplest form, a bispecific polypeptide human CXCR4 (see for example the amino acid sequences of the invention is a bivalent polypeptide of the invention (as mentioned such as SEQ ID NO. 238 and SEQ ID NO: 239 defined herein), comprising a first NANOBODYR (V) in Table B-1.1 of WO 09/138519). WO 09/138519 also directed against GPCRs, and a second NANOBODYR describes multivalent, multispecific and/or biparatopic con 50 (V) directed against a linked via a linker sequence (as structs (as defined in WO 09/138519) that are directed defined herein); whereas a trispecific polypeptide of the against human CXCR4. Reference is for example made to invention in its simplest form is a trivalent polypeptide of the the constructs referred to in Example 4 of WO 09/138519 invention (as defined herein), comprising a first NANO such as SEQ ID NO: 264 in Table B-5 of WO 09/138519). BODYR (V) directed against GPCRs, a second NANO One particularly preferred example of an amino acid 55 BODYR (V) directed against a second antigen and a third sequence against human CXCR4 from WO 09/138519 is the NANOBODYR (V) directed against a third antigen, in sequence designated 238D2 (see SEQ ID NO: 238 in WO which said first, second and third NANOBODYR (V) 09/1385.19 and SEQ ID NO: 526 herein). One other par may optionally be linked via one or more, and in particular ticularly preferred example of an amino acid sequence one and more, in particular two, linker sequences. against the human CXCR4 from WO 09/138519 is the 60 Polypeptides of the invention that contain at least two sequence designated 238D4 (see SEQ ID NO: 239 in WO Nanobodies which are directed against GPCRs, wherein at 09/1385.19 and SEQ ID NO. 527 herein). WO 09/138519 least one “first Nanbody is directed against a first antigenic further gives some non-limiting examples of multivalent, determinant, epitope, part, domain, Subunit or confirmation multispecific and/or biparatopic constructs that comprise of a GPCR (e.g. CXCR4); and wherein at least one “second 238D2 and/or 238D4 (see for example SEQ ID NO’s: 261 65 NANOBODYR (V) is directed against a second antigenic to 266 in WO 09/138519 and in particular the biparatopic determinant, epitope, part, domain, Subunit or confirmation construct 238D2-20CS-238D4. of said GPCR (e.g. CXCR4) different from the first, will also US 9,512,236 B2 113 114 be referred to as “multiparatopic polypeptides of the inven invention, it should be noted that this encompasses any order tion, and the Nanobodies present in such polypeptides will or arrangements of the relevant Nanobodies, unless explic also be referred to herein as being in a “multiparatopic itly indicated otherwise. format. Thus, for example, a “biparatopic' polypeptide of Finally, it is also within the scope of the invention that the the invention is a polypeptide that comprises at least one 5 polypeptides of the invention contain two or more Nano NANOBODYR (V) directed against a first antigenic bodies and one or more further amino acid sequences (as determinant, epitope, part, domain, Subunit or confirmation mentioned herein). of an antigen (i.e. GPCRs) and at least one further NANO For multivalent and multispecific polypeptides containing BODYR (V) directed against a second antigenic deter one or more V. domains and their preparation, reference is minant, epitope, part, domain, Subunit or confirmation of 10 said antigen (i.e. same GPCRs) different from the first, also made to Conrath et al., J. Biol. Chem., Vol. 276, 10. whereas a “triparatopic' polypeptide of the invention is a 7346-7350, 2001; Muyldermans, Reviews in Molecular Bio polypeptide that comprises at least one NANOBODYR technology 74 (2001), 277-302; as well as to for example (V) directed against a first antigenic determinant, epitope, WO 96/34103 and WO 99/23221. Some other examples of part, domain, Subunit or confirmation of an antigen (i.e. 15 Some specific multispecific and/or multivalent polypeptide GPCRs), at least one further NANOBODYR (V) directed of the invention can be found in the applications by Ablynx against a second antigenic determinant, epitope, part, N.V. referred to herein. domain, Subunit or confirmation of said antigen (i.e. same One preferred, but non-limiting example of a multispe GPCRs) different from the first, and at least one further cific polypeptide of the invention comprises at least one NANOBODYR (V) directed against a third antigenic NANOBODYR (V) of the invention and at least one determinant, epitope, part, domain, Subunit or confirmation NANOBODYR (V) that provides for an increased half of an antigen (i.e. same GPCRs) but different from said first life. Such Nanobodies may for example be Nanobodies that and said second antigenic determinant, epitope, part, are directed against a serum protein, and in particular a domain, Subunit or confirmation of said antigen, etc. human serum protein, Such as human serum albumin, thy Accordingly, in its simplest form, a biparatopic polypep 25 roxine-binding protein, (human) transferrin, fibrinogen, an tide of the invention is a bivalent polypeptide of the inven immunoglobulin Such as IgG, IgE or IgM, or against one of tion (as defined herein), comprising a first NANOBODYR the serum proteins listed in WO 04/003019. Of these, (V) directed against a first antigenic determinant, epitope, Nanobodies that can bind to serum albumin (and in particu part, domain, subunit or confirmation of a GPCR, and a lar human serum albumin) or to IgG (and in particular second NANOBODYR (V) directed against a second 30 human IgG, see for example NANOBODYR (V) VH-1 antigenic determinant, epitope, part, domain, Subunit or described in the review by Muyldermans, supra) are par confirmation of said GPCR different from the first, in which ticularly preferred (although for example, for experiments in said first and said second NANOBODYR (V) may optionally be linked via a linker sequence (as defined mice or primates, Nanobodies against or cross-reactive with herein); whereas a triparatopic polypeptide of the invention 35 mouse serum albumin (MSA) or serum albumin from said in its simplest form is a trivalent polypeptide of the invention primate, respectively, can be used. However, for pharma (as defined herein), comprising a first NANOBODYR ceutical use, Nanobodies against human serum albumin or (V) directed against a first antigenic determinant, epitope, human IgG will usually be preferred). Nanobodies that part, domain, subunit or confirmation of a GPCR, a second provide for increased half-life and that can be used in the NANOBODYR (V) directed against a second antigenic 40 polypeptides of the invention include the Nanobodies determinant, epitope, part, domain, Subunit or confirmation directed against serum albumin that are described in WO of said GPCR different from the first, and a third NANO 04/041865, in WO 06/122787 and in the further patent BODYR (V) directed against a third antigenic determi applications by Ablynx N.V., such as those mentioned nant, epitope, part, domain, Subunit or confirmation of the above. same GPCR but different from said first and said second 45 For example, the some preferred Nanobodies that provide antigenic determinant, epitope, part, domain, Subunit or for increased half-life for use in the present invention confirmation, in which said first, second and third NANO include Nanobodies that can bind to amino acid residues on BODYR (V) may optionally be linked via one or more, (human) serum albumin that are not involved in binding of and in particular one and more, in particular two, linker serum albumin to FcRn (see for example WO 06/0122787); Sequences. 50 Nanobodies that are capable of binding to amino acid However, as will be clear from the description herein residues on serum albumin that do not form part of domain above, the invention is not limited thereto, in the sense that III of serum albumin (see for example WO 06/0122787); a multispecific polypeptide of the invention may comprise at Nanobodies that have or can provide an increased half-life least one NANOBODYR (V) against GPCRs, and any (see for example the U.S. provisional application 60/843, number of Nanobodies directed against one or more antigens 55 349 by Ablynx N.V mentioned herein; see also WO08/ different from GPCRs. 028977); Nanobodies against human serum albumin that are Furthermore, although it is encompassed within the scope cross-reactive with serum albumin from at least one species of the invention that the specific order or arrangement of the of mammal, and in particular with at least one species of various Nanobodies in the polypeptides of the invention may primate (such as, without limitation, monkeys from the have some influence on the properties of the final polypep 60 genus Macaca (Such as, and in particular, cynomolgus tide of the invention (including but not limited to the affinity, monkeys (Macaca fascicularis) and/or rhesus monkeys specificity or avidity for GPCRs, or against the one or more (Macaca mulata)) and baboon (Papio ursinus)) (see for other antigens), said order or arrangement is usually not example the U.S. provisional application 60/843.349 by critical and may be suitably chosen by the skilled person, Ablynx N.V. see also WO08/028977); Nanobodies that can optionally after Some limited routine experiments based on 65 bind to serum albumin in a pH independent manner (see for the disclosure herein. Thus, when reference is made to a example the U.S. provisional application 60/850.774 by specific multivalent or multispecific polypeptide of the Ablynx N.V. mentioned herein) and/or Nanobodies that are US 9,512,236 B2 115 116 conditional binders (see for example the U.S. provisional In the polypeptides of the invention, the one or more application 60/850,775 by Ablynx N.V.: see also WO08/ Nanobodies and the one or more polypeptides may be 043822). directly linked to each other (as for example described in Some particularly preferred Nanobodies that provide for WO 99/23221) and/or may be linked to each other via one increased half-life and that can be used in the polypeptides 5 or more Suitable spacers or linkers, or any combination of the invention include the Nanobodies ALB-1 to ALB-10 thereof. disclosed in WO 06/122787 (see Tables II and III) of which Suitable spacers or linkers for use in multivalent and ALB-8 (SEQ ID NO: 62 in WO 06/122787) is particularly multispecific polypeptides will be clear to the skilled person, preferred. and may generally be any linker or spacer used in the art to According to a specific, but non-limiting aspect of the 10 link amino acid sequences. Preferably, said linker or spacer invention, the polypeptides of the invention contain, besides is suitable for use in constructing proteins or polypeptides the one or more Nanobodies of the invention, at least one that are intended for pharmaceutical use. NANOBODYR (V) against human serum albumin. Some particularly preferred spacers include the spacers Generally, any polypeptides of the invention with and linkers that are used in the art to link antibody fragments increased half-life that contain one or more Nanobodies of 15 or antibody domains. These include the linkers mentioned in the invention, and any derivatives of Nanobodies of the the general background art cited above, as well as for invention or of Such polypeptides that have an increased example linkers that are used in the art to construct diabodies half-life, preferably have a half-life that is at least 1.5 times, or ScFv fragments (in this respect, however, its should be preferably at least 2 times, such as at least 5 times, for noted that, whereas in diabodies and in ScFv fragments, the example at least 10 times or more than 20 times, greater than linker sequence used should have a length, a degree of the half-life of the corresponding NANOBODYR (V) of flexibility and other properties that allow the pertinent V the invention per se. For example, such a derivative or and V, domains to come together to form the complete polypeptides with increased half-life may have a half-life antigen-binding site, there is no particular limitation on the that is increased with more than 1 hours, preferably more length or the flexibility of the linker used in the polypeptide than 2 hours, more preferably more than 6 hours, such as 25 of the invention, since each NANOBODYR (V) by itself more than 12 hours, or even more than 24, 48 or 72 hours, forms a complete antigen-binding site). compared to the corresponding NANOBODYR (V) of For example, a linker may be a suitable amino acid the invention per se. sequence, and in particular amino acid sequences of between In a preferred, but non-limiting aspect of the invention, 1 and 50, preferably between 1 and 30, such as between 1 such derivatives or polypeptides may exhibit a serum half 30 and 10 amino acid residues. Some preferred examples of life in human of at least about 12 hours, preferably at least such amino acid sequences include GLY-SER linkers, for 24 hours, more preferably at least 48 hours, even more example of the type (GLYSER), such as (for example preferably at least 72 hours or more. For example, such (GLYSER), or (GLYSER), as described in WO derivatives or polypeptides may have a half-life of at least 5 99/42077 and the GS30, GS15, GS9 and GS7 linkers days (such as about 5 to 10 days), preferably at least 9 days 35 described in the applications by Ablynx mentioned herein (such as about 9 to 14 days), more preferably at least about (see for example WO 06/04.0153 and WO 06/122825), as 10 days (such as about 10 to 15 days), or at least about 11 well as hinge-like regions, such as the hinge regions of days (such as about 11 to 16 days), more preferably at least naturally occurring heavy chain antibodies or similar about 12 days (such as about 12 to 18 days or more), or more sequences (such as described in WO 94/04678). than 14 days (such as about 14 to 19 days). 40 Some other particularly preferred linkers are poly-alanine According to one aspect of the invention the polypeptides (such as AAA), as well as the linkers GS30 (SEQ ID NO: 85 are capable of binding to one or more molecules which can in WO 06/122825) and GS9 (SEQ ID NO: 84 in WO increase the half-life of the polypeptide in vivo. 06/122825). The polypeptides of the invention are stabilised in vivo Other Suitable linkers generally comprise organic com and their half-life increased by binding to molecules which 45 pounds or polymers, in particular those Suitable for use in resist degradation and/or clearance or sequestration. Typi proteins for pharmaceutical use. For instance, poly(ethyl cally, Such molecules are naturally occurring proteins which eneglycol) moieties have been used to link antibody themselves have a long half-life in vivo. domains, see for example WO 04/081026. Another preferred, but non-limiting example of a multi It is encompassed within the scope of the invention that specific polypeptide of the invention comprises at least one 50 the length, the degree of flexibility and/or other properties of NANOBODYR (V) of the invention and at least one the linker(s) used (although not critical, as it usually is for NANOBODYR (V) that directs the polypeptide of the linkers used in ScFv fragments) may have some influence on invention towards, and/or that allows the polypeptide of the the properties of the final polypeptide of the invention, invention to penetrate or to enter into specific organs, including but not limited to the affinity, specificity or avidity tissues, cells, or parts or compartments of cells, and/or that 55 for GPCRs, or for one or more of the other antigens. Based allows the NANOBODYR (V) to penetrate or cross a on the disclosure herein, the skilled person will be able to biological barrier Such as a cell membrane, a cell layer Such determine the optimal linker(s) for use in a specific poly as a layer of epithelial cells, a tumor including Solid tumors, peptide of the invention, optionally after some limited or the blood-brain-barrier. Examples of such Nanobodies routine experiments. include Nanobodies that are directed towards specific cell 60 For example, in multivalent polypeptides of the invention Surface proteins, markers or epitopes of the desired organ, that comprise Nanobodies directed against a multimeric tissue or cell (for example cell-Surface markers associated antigen (such as a multimeric receptor or other protein), the with tumor cells), and the single-domain brain targeting length and flexibility of the linker are preferably such that it antibody fragments described in WO 02/057445 and WO allows each NANOBODYR (V) of the invention present 06/04.0153, of which FC44 (SEQ ID NO: 189 of WO 65 in the polypeptide to bind to the antigenic determinant on 06/04.0153) and FC5 (SEQ ID NO: 190 of WO 06/040154) each of the subunits of the multimer. Similarly, in a multi are preferred examples. specific polypeptide of the invention that comprises Nano US 9,512,236 B2 117 118 bodies directed against two or more different antigenic NANOBODYR (V) and/or a polypeptide of the invention determinants on the same antigen (for example against generally comprises the steps of: different epitopes of an antigen and/or against different i) the expression, in a suitable host cell or host organism Subunits of a multimeric receptor, channel or protein), the (also referred to herein as a “host of the invention') or in length and flexibility of the linker are preferably such that it another Suitable expression system of a nucleic acid that allows each NANOBODYR (V) to bind to its intended encodes said amino acid sequence, NANOBODYR antigenic determinant. Again, based on the disclosure (V) or polypeptide of the invention (also referred to herein, the skilled person will be able to determine the herein as a “nucleic acid of the invention'), optionally optimal linker(s) for use in a specific polypeptide of the followed by: invention, optionally after some limited routine experi 10 ii) isolating and/or purifying the amino acid sequence, mentS. NANOBODYR (V) or polypeptide of the invention It is also within the scope of the invention that the thus obtained. linker(s) used confer one or more other favourable proper In particular, Such a method may comprise the steps of ties or functionality to the polypeptides of the invention, 15 i) cultivating and/or maintaining a host of the invention and/or provide one or more sites for the formation of under conditions that are such that said host of the derivatives and/or for the attachment of functional groups invention expresses and/or produces at least one amino (e.g. as described herein for the derivatives of the Nano acid sequence, NANOBODYR (V) and/or polypeptide bodies of the invention). For example, linkers containing of the invention; optionally followed by: one or more charged amino acid residues (see Table A-2 ii) isolating and/or purifying the amino acid sequence, above) can provide improved hydrophilic properties, NANOBODYR (V) or polypeptide of the invention whereas linkers that form or contain Small epitopes or tags thus obtained. can be used for the purposes of detection, identification A nucleic acid of the invention can be in the form of single and/or purification. Again, based on the disclosure herein, or double stranded DNA or RNA, and is preferably in the the skilled person will be able to determine the optimal 25 form of double stranded DNA. For example, the nucleotide linkers for use in a specific polypeptide of the invention, sequences of the invention may be genomic DNA, cDNA or optionally after some limited routine experiments. synthetic DNA (such as DNA with a codon usage that has Finally, when two or more linkers are used in the poly been specifically adapted for expression in the intended host peptides of the invention, these linkers may be the same or cell or host organism). different. Again, based on the disclosure herein, the skilled 30 According to one aspect of the invention, the nucleic acid person will be able to determine the optimal linkers for use of the invention is in essentially isolated from, as defined in a specific polypeptide of the invention, optionally after herein. Some limited routine experiments. The nucleic acid of the invention may also be in the form Usually, for easy of expression and production, a poly of be present in and/or be part of a vector, such as for peptide of the invention will be a linear polypeptide. How 35 example a plasmid, cosmid or YAC, which again may be in ever, the invention in its broadest sense is not limited essentially isolated form. thereto. For example, when a polypeptide of the invention The nucleic acids of the invention can be prepared or comprises three of more Nanobodies, it is possible to link obtained in a manner known perse, based on the information them by use of a linker with three or more “arms”, which on the amino acid sequences for the polypeptides of the each "arm' being linked to a NANOBODYR (V), so as 40 invention given herein, and/or can be isolated from a Suit to provide a “star-shaped construct. It is also possible, able natural source. To provide analogs, nucleotide although usually less preferred, to use circular constructs. sequences encoding naturally occurring V, domains can The invention also comprises derivatives of the polypep for example be subjected to site-directed mutagenesis, so at tides of the invention, which may be essentially analogous to provide a nucleic acid of the invention encoding said to the derivatives of the Nanobodies of the invention, i.e. as 45 analog. Also, as will be clear to the skilled person, to prepare described herein. a nucleic acid of the invention, also several nucleotide The invention also comprises proteins or polypeptides sequences. Such as at least one nucleotide sequence encod that “essentially consist of a polypeptide of the invention ing a NANOBODYR (V) and for example nucleic acids (in which the wording “essentially consist of has essentially encoding one or more linkers can be linked together in a the same meaning as indicated hereinabove). 50 Suitable manner. According to one aspect of the invention, the polypeptide Techniques for generating the nucleic acids of the inven of the invention is in essentially isolated from, as defined tion will be clear to the skilled person and may for instance herein. include, but are not limited to, automated DNA synthesis: The amino acid sequences, Nanobodies, polypeptides and site-directed mutagenesis; combining two or more naturally nucleic acids of the invention can be prepared in a manner 55 occurring and/or synthetic sequences (or two or more parts known per se, as will be clear to the skilled person from the thereof), introduction of mutations that lead to the expres further description herein. For example, the Nanobodies and sion of a truncated expression product; introduction of one polypeptides of the invention can be prepared in any manner or more restriction sites (e.g. to create cassettes and/or known per se for the preparation of antibodies and in regions that may easily be digested and/or ligated using particular for the preparation of antibody fragments (includ 60 suitable restriction enzymes), and/or the introduction of ing but not limited to (single) domain antibodies and ScHv mutations by means of a PCR reaction using one or more fragments). Some preferred, but non-limiting methods for “mismatched primers, using for example a sequence of a preparing the amino acid sequences, Nanobodies, polypep naturally occurring form of GPCRs as a template. These and tides and nucleic acids include the methods and techniques other techniques will be clear to the skilled person, and described herein. 65 reference is again made to the standard handbooks, such as As will be clear to the skilled person, one particularly Sambrook et al. and Ausubel et al., mentioned above, as well useful method for preparing an amino acid sequence, as the Examples below. US 9,512,236 B2 119 120 The nucleic acid of the invention may also be in the form orientation and usually also in the same reading frame. They of be present in and/or be part of a genetic construct, as will will usually also be essentially contiguous, although this be clear to the person skilled in the art. Such genetic may also not be required. constructs generally comprise at least one nucleic acid of the Preferably, the regulatory and further elements of the invention that is optionally linked to one or more elements genetic constructs of the invention are such that they are of genetic constructs known perse, Such as for example one capable of providing their intended biological function in the or more Suitable regulatory elements (such as a Suitable intended host cell or host organism. promoter(s), enhancer(s), terminator(s), etc.) and the further For instance, a promoter, enhancer or terminator should elements of genetic constructs referred to herein. Such be “operable' in the intended host cell or host organism, by 10 which is meant that (for example) said promoter should be genetic constructs comprising at least one nucleic acid of the capable of initiating or otherwise controlling/regulating the invention will also be referred to herein as “genetic con transcription and/or the expression of a nucleotide structs of the invention'. sequence—e.g. a coding sequence—to which it is operably The genetic constructs of the invention may be DNA or linked (as defined herein). RNA, and are preferably double-stranded DNA. The genetic 15 Some particularly preferred promoters include, but are not constructs of the invention may also be in a form suitable for limited to, promoters known perse for the expression in the transformation of the intended host cell or host organism, in host cells mentioned herein; and in particular promoters for a form suitable for integration into the genomic DNA of the the expression in the bacterial cells, such as those mentioned intended host cell or in a form suitable for independent herein and/or those used in the Examples. replication, maintenance and/or inheritance in the intended A selection marker should be such that it allows—i.e. host organism. For instance, the genetic constructs of the under appropriate selection conditions—host cells and/or invention may be in the form of a vector, Such as for example host organisms that have been (Successfully) transformed a plasmid, cosmid, YAC, a viral vector or transposon. In with the nucleotide sequence of the invention to be distin particular, the vector may be an expression vector, i.e. a guished from host cells/organisms that have not been (Suc vector that can provide for expression in vitro and/or in vivo 25 cessfully) transformed. Some preferred, but non-limiting (e.g. in a suitable host cell, host organism and/or expression examples of Such markers are genes that provide resistance system). against antibiotics (such as kanamycin or amplicillin), genes In a preferred but non-limiting aspect, a genetic construct that provide for temperature resistance, or genes that allow of the invention comprises the host cell or host organism to be maintained in the i) at least one nucleic acid of the invention; operably 30 absence of certain factors, compounds and/or (food) com connected to ponents in the medium that are essential for survival of the ii) one or more regulatory elements, such as a promoter and non-transformed cells or organisms. optionally a Suitable terminator; A leader sequence should be such that in the intended and optionally also host cell or host organism—it allows for the desired post iii) one or more further elements of genetic constructs 35 translational modifications and/or such that it directs the known per se; transcribed mRNA to a desired part or organelle of a cell. A in which the terms “regulatory element”, “promoter', “ter leader sequence may also allow for secretion of the expres minator' and “operably connected' have their usual mean sion product from said cell. As such, the leader sequence ing in the art (as further described herein); and in which said may be any pro-, pre-, or prepro-sequence operable in the “further elements' present in the genetic constructs may for 40 host cell or host organism. Leader sequences may not be example be 3'- or 5'-UTR sequences, leader sequences, required for expression in a bacterial cell. For example, selection markers, expression markers/reporter genes, and/ leader sequences known per se for the expression and or elements that may facilitate or increase (the efficiency of) production of antibodies and antibody fragments (including transformation or integration. These and other Suitable ele but not limited to single domain antibodies and ScFv frag ments for such genetic constructs will be clear to the skilled 45 ments) may be used in an essentially analogous manner. person, and may for instance depend upon the type of An expression marker or reporter gene should be such construct used, the intended host cell or host organism; the that in the host cell or host organism—it allows for detec manner in which the nucleotide sequences of the invention tion of the expression of (a gene or nucleotide sequence of interest are to be expressed (e.g. via constitutive, transient present on) the genetic construct. An expression marker may or inducible expression); and/or the transformation tech 50 optionally also allow for the localisation of the expressed nique to be used. For example, regulatory sequences, pro product, e.g. in a specific part or organelle of a cell and/or moters and terminators known perse for the expression and in (a) specific cell(s), tissue(s), organ(s) or part(s) of a production of antibodies and antibody fragments (including multicellular organism. Such reporter genes may also be but not limited to (single) domain antibodies and ScHv expressed as a protein fusion with the amino acid sequence fragments) may be used in an essentially analogous manner. 55 of the invention. Some preferred, but non-limiting examples Preferably, in the genetic constructs of the invention, said include fluorescent proteins such as GFP. at least one nucleic acid of the invention and said regulatory Some preferred, but non-limiting examples of suitable elements, and optionally said one or more further elements, promoters, terminator and further elements include those are “operably linked to each other, by which is generally that can be used for the expression in the host cells men meant that they are in a functional relationship with each 60 tioned herein; and in particular those that are suitable for other. For instance, a promoter is considered “operably expression in bacterial cells, such as those mentioned herein linked to a coding sequence if said promoter is able to and/or those used in the Examples below. For some (further) initiate or otherwise control/regulate the transcription and/or non-limiting examples of the promoters, selection markers, the expression of a coding sequence (in which said coding leader sequences, expression markers and further elements sequence should be understood as being “under the control 65 that may be present/used in the genetic constructs of the of said promoter). Generally, when two nucleotide invention—such as terminators, transcriptional and/or trans sequences are operably linked, they will be in the same lational enhancers and/or integration factors—reference is US 9,512,236 B2 121 122 made to the general handbooks Such as Sambrook et al. and BHK-cells (for example BHK-21 cells) and human Ausubel et al. mentioned above, as well as to the examples cells or cell lines such as HeLa, COS (for example that are given in WO95/07463, WO 96/23810, WO COS-7) and PER.C6 cells; 95/07463, WO95/21191, WO 97/11094, WO 97/42320, as well as all other hosts or host cells known perse for the WO 98/06737, WO 98/21355, U.S. Pat. No. 7,207,410, U.S. expression and production of antibodies and antibody frag Pat. No. 5,693,492 and EP 1 085089. Other examples will ments (including but not limited to (single) domain antibod be clear to the skilled person. Reference is also made to the ies and ScFv fragments), which will be clear to the skilled general background art cited above and the further refer person. Reference is also made to the general background art ences cited herein. cited hereinabove, as well as to for example WO94/29457: 10 WO 96/34103; WO99/42077; Frenken et al., (1998), supra: The genetic constructs of the invention may generally be Riechmann and Muyldermans, (1999), supra; Van der Lin provided by Suitably linking the nucleotide sequence(s) of den, (2000), supra; Thomassen et al., (2002), supra; Joosten the invention to the one or more further elements described et al., (2003), supra; Joosten et al., (2005), supra; and the above, for example using the techniques described in the further references cited herein. general handbooks such as Sambrook et al. and Ausubel et 15 The amino acid sequences, Nanobodies and polypeptides al., mentioned above. of the invention can also be introduced and expressed in one Often, the genetic constructs of the invention will be or more cells, tissues or organs of a multicellular organism, obtained by inserting a nucleotide sequence of the invention for example for prophylactic and/or therapeutic purposes in a Suitable (expression) vector known per se. Some pre (e.g. as a gene therapy). For this purpose, the nucleotide ferred, but non-limiting examples of Suitable expression sequences of the invention may be introduced into the cells vectors are those used in the Examples below, as well as or tissues in any Suitable way, for example as Such (e.g. those mentioned herein. using liposomes) or after they have been inserted into a The nucleic acids of the invention and/or the genetic suitable gene therapy vector (for example derived from constructs of the invention may be used to transform a host retroviruses Such as adenovirus, or parvoviruses such as cell or host organism, i.e. for expression and/or production 25 adeno-associated virus). As will also be clear to the skilled of the amino acid sequence, NANOBODYR (V) or person, such gene therapy may be performed in vivo and/or polypeptide of the invention. Suitable hosts or host cells will in situ in the body of a patient by administering a nucleic be clear to the skilled person, and may for example be any acid of the invention or a suitable gene therapy vector Suitable fungal, prokaryotic or eukaryotic cell or cell line or encoding the same to the patient or to specific cells or a any suitable fungal, prokaryotic or eukaryotic organism, for 30 specific tissue or organ of the patient; or Suitable cells (often example: taken from the body of the patient to be treated, such as a bacterial strain, including but not limited to gram explanted lymphocytes, bone marrow aspirates or tissue negative strains such as Strains of Escherichia coli, of biopsies) may be treated in vitro with a nucleotide sequence Proteus, for example of Proteus mirabilis; of of the invention and then be suitably (re-)introduced into the Pseudomonas, for example of Pseudomonas fluore 35 body of the patient. All this can be performed using gene scens; and gram-positive strains such as strains of therapy vectors, techniques and delivery systems which are Bacillus, for example of Bacillus subtilis or of Bacillus well known to the skilled person, and for example described brevis; of Streptomyces, for example of Streptomyces in Culver, K. W., “Gene Therapy”, 1994, p. xii, Mary Ann lividans; of Staphylococcus, for example of Staphyllo Liebert, Inc., Publishers, New York, N.Y); Giordano, Nature coccus carnosus; and of Lactococcus, for example of 40 F Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 Lactococcus lactis, (1996), 911-919; Anderson, Science 256 (1992),808–813; a fungal cell, including but not limited to cells from Verma, Nature 389 (1994).239; Isner, Lancet 348 (1996), species of Trichoderma, for example from Trichoderma 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086: reesei; of Neurospora, for example from Neurospora Onodera, Blood 91; (1998).30-36; Verma, Gene Ther. 5 crassa; of Sordaria, for example from Sordaria mac 45 (1998),692-699; Nabel, Ann. N.Y. Acad. Sci.: 811 (1997), rospora; of Aspergillus, for example from Aspergillus 289-292; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-51: niger or from Aspergillus sojae, or from other filamen Wang, Nature Medicine 2 (1996).714-716; WO 94/29469; tous fungi, WO 97/00957, U.S. Pat. No. 5,580,859; U.S. Pat. No. a yeast cell, including but not limited to cells from species 5,589.5466; or Schaper, Current Opinion in Biotechnology of Saccharomyces, for example of Saccharomyces cer 50 7 (1996), 635-640. For example, in situ expression of ScFv evisiae; of Schizosaccharomyces, for example of Schi fragments (Afanasieva et al., Gene Ther. 10, 1850-1859 zosaccharomyces pombe; of Pichia, for example of (2003)) and of diabodies (Blanco et al., J. Immunol. 171, Pichia pastoris or of Pichia methanolica; of Han 1070-1077 (2003)) has been described in the art. senula, for example of Hansenula polymorpha; of For expression of the Nanobodies in a cell, they may also Kluyveromyces, for example of Kluyveromyces lactis; 55 be expressed as so-called “intrabodies', as for example of ArXula, for example of ArXula adeninivorans; of described in WO94/02610, WO95/22618 and U.S. Pat. No. Yarrowia, for example of Yarrowia lipolytica, 7,004,940; WO 03/014960; in Cattaneo, A. & Biocca, S. an amphibian cell or cell line. Such as Xenopus oocytes; (1997) Intracellular Antibodies: Development and Applica an insect-derived cell or cell line, such as cells/cell lines tions. Landes and Springer-Verlag, and in Kontermann, derived from lepidoptera, including but not limited to 60 Methods 34, (2004), 163-170. Spodoptera SF9 and Sf21 cells or cells/cell lines The amino acid sequences, Nanobodies and polypeptides derived from Drosophila, such as Schneider and Kc of the invention can for example also be produced in the cells; milk of transgenic mammals, for example in the milk of a plant or plant cell, for example in tobacco plants; and/or rabbits, cows, goats or sheep (see for example U.S. Pat. No. a mammalian cell or cell line, for example a cell or cell 65 6,741,957, U.S. Pat. No. 6,304,489 and U.S. Pat. No. line derived from a human, a cell or a cell line from 6,849.992 for general techniques for introducing transgenes mammals including but not limited to CHO-cells, into mammals), in plants or parts of plants including but not US 9,512,236 B2 123 124 limited to their leaves, flowers, fruits, seed, roots or tubers Thus, according to one non-limiting aspect of the inven (for example in tobacco, maize, soybean or alfalfa) or in for tion, the amino acid sequence, NANOBODYR (V) or example pupae of the silkworm Bombix mori. polypeptide of the invention is glycosylated. According to Furthermore, the amino acid sequences, Nanobodies and another non-limiting aspect of the invention, the amino acid polypeptides of the invention can also be expressed and/or sequence, NANOBODYR (V) or polypeptide of the produced in cell-free expression systems, and Suitable invention is non-glycosylated. examples of such systems will be clear to the skilled person. According to one preferred, but non-limiting aspect of the Some preferred, but non-limiting examples include expres invention, the amino acid sequence, NANOBODYR (V) sion in the wheat germ system; in rabbit reticulocyte lysates; or polypeptide of the invention is produced in a bacterial or in the E. coli Zubay system. 10 As mentioned above, one of the advantages of the use of cell, in particular a bacterial cell suitable for large scale Nanobodies is that the polypeptides based thereon can be pharmaceutical production, such as cells of the strains prepared through expression in a suitable bacterial system, mentioned above. and Suitable bacterial expression systems, vectors, host cells, According to another preferred, but non-limiting aspect of regulatory elements, etc., will be clear to the skilled person, 15 the invention, the amino acid sequence, NANOBODYR for example from the references cited above. It should (V) or polypeptide of the invention is produced in a yeast however be noted that the invention in its broadest sense is cell, in particular a yeast cell Suitable for large scale phar not limited to expression in bacterial systems. maceutical production, such as cells of the species men Preferably, in the invention, an (in vivo or in vitro) tioned above. expression system, Such as a bacterial expression system, is According to yet another preferred, but non-limiting used that provides the polypeptides of the invention in a aspect of the invention, the amino acid sequence, NANO form that is suitable for pharmaceutical use, and Such BODYR (V) or polypeptide of the invention is produced expression systems will again be clear to the skilled person. in a mammalian cell, in particular in a human cell or in a cell As also will be clear to the skilled person, polypeptides of of a human cell line, and more in particular in a human cell the invention Suitable for pharmaceutical use can be pre 25 or in a cell of a human cell line that is suitable for large scale pared using techniques for peptide synthesis. pharmaceutical production, such as the cell lines mentioned For production on industrial scale, preferred heterologous hereinabove. hosts for the (industrial) production of Nanobodies or When expression in a host cell is used to produce the NANOBODYR (V)-containing protein therapeutics amino acid sequences, Nanobodies and the polypeptides of include strains of E. coli, Pichia pastoris, S. cerevisiae that 30 the invention, the amino acid sequences, Nanobodies and are Suitable for large scale expression/production/fermenta polypeptides of the invention can be produced either intra tion, and in particular for large scale pharmaceutical (i.e. cellularly (e.g. in the cytosol, in the periplasma or in GMP grade) expression/production/fermentation. Suitable inclusion bodies) and then isolated from the host cells and examples of such strains will be clear to the skilled person. optionally further purified; or can be produced extracellu Such strains and production/expression systems are also 35 larly (e.g. in the medium in which the host cells are cultured) made available by companies such as BioVitrum (Uppsala, and then isolated from the culture medium and optionally Sweden). further purified. When eukaryotic host cells are used, extra Alternatively, mammalian cell lines, in particular Chinese cellular production is usually preferred since this consider hamster ovary (CHO) cells, can be used for large scale ably facilitates the further isolation and downstream pro expression/production/fermentation, and in particular for 40 cessing of the Nanobodies and proteins obtained. Bacterial large scale pharmaceutical expression/production/fermenta cells such as the strains of E. coli mentioned above normally tion. Again, Such expression/production systems are also do not secrete proteins extracellularly, except for a few made available by Some of the companies mentioned above. classes of proteins such as toxins and hemolysin, and The choice of the specific expression system would secretory production in E. coli refers to the translocation of depend in part on the requirement for certain post-transla 45 proteins across the inner membrane to the periplasmic space. tional modifications, more specifically glycosylation. The Periplasmic production provides several advantages over production of a NANOBODYR (V)-containing recom cytosolic production. For example, the N-terminal amino binant protein for which glycosylation is desired or required acid sequence of the secreted product can be identical to the would necessitate the use of mammalian expression hosts natural gene product after cleavage of the secretion signal that have the ability to glycosylate the expressed protein. In 50 sequence by a specific signal peptidase. Also, there appears this respect, it will be clear to the skilled person that the to be much less protease activity in the periplasm than in the glycosylation pattern obtained (i.e. the kind, number and cytoplasm. In addition, protein purification is simpler due to position of residues attached) will depend on the cell or cell fewer contaminating proteins in the periplasm. Another line that is used for the expression. Preferably, either a advantage is that correct disulfide bonds may form because human cell or cell line is used (i.e. leading to a protein that 55 the periplasm provides a more oxidative environment than essentially has a human glycosylation pattern) or another the cytoplasm. Proteins overexpressed in E. coli are often mammalian cell line is used that can provide a glycosylation found in insoluble aggregates, so-called inclusion bodies. pattern that is essentially and/or functionally the same as These inclusion bodies may be located in the cytosol or in human glycosylation or at least mimics human glycosy the periplasm; the recovery of biologically active proteins lation. Generally, prokaryotic hosts Such as E. coli do not 60 from these inclusion bodies requires a denaturation/refold have the ability to glycosylate proteins, and the use of lower ing process. Many recombinant proteins, including thera eukaryotes such as yeast usually leads to a glycosylation peutic proteins, are recovered from inclusion bodies. Alter pattern that differs from human glycosylation. Nevertheless, natively, as will be clear to the skilled person, recombinant it should be understood that all the foregoing host cells and strains of bacteria that have been genetically modified so as expression systems can be used in the invention, depending 65 to secrete a desired protein, and in particular an amino acid on the desired amino acid sequence, NANOBODYR (V) sequence, NANOBODYR (V) or a polypeptide of the or polypeptide to be obtained. invention, can be used. US 9,512,236 B2 125 126 Thus, according to one non-limiting aspect of the inven vectors for expression in plants or plant cells: for example tion, the amino acid sequence, NANOBODYR (V) or vectors based on cauliflower mosaic virus or tobacco polypeptide of the invention is an amino acid sequence, mosaic virus, Suitable strains of Agrobacterium, or NANOBODYR (V) or polypeptide that has been pro Ti-plasmid based vectors. duced intracellularly and that has been isolated from the host 5 Some preferred, but non-limiting secretory sequences for cell, and in particular from a bacterial cell or from an use with these host cells include: inclusion body in a bacterial cell. According to another for use in bacterial cells such as E. coli: PelB, Bla, Omp.A, non-limiting aspect of the invention, the amino acid OmpC, OmpF, OmpT, StII, PhoA, PhoE, MalE, Lipp, sequence, NANOBODYR (V) or polypeptide of the LamB, and the like; TAT signal peptide, hemolysin 10 C-terminal secretion signal; invention is an amino acid sequence, NANOBODYR (V) for use in yeast: C.-mating factor prepro-sequence, phos or polypeptide that has been produced extracellularly, and phatase (phol), invertase (Suc), etc.; that has been isolated from the medium in which the host for use in mammalian cells: indigenous signal in case the cell is cultivated. target protein is of eukaryotic origin; murine Ig K-chain Some preferred, but non-limiting promoters for use with 15 V-J2-C signal peptide; etc. these host cells include, Suitable techniques for transforming a host or host cell of for expression in E. coli: lac promoter (and derivatives the invention will be clear to the skilled person and may thereof such as the lacUV5 promoter); arabinose pro depend on the intended host cell/host organism and the moter; left-(PL) and rightward (PR) promoter of phage genetic construct to be used. Reference is again made to the lambda; promoter of the trp operon; hybrid lac/trp handbooks and patent applications mentioned above. promoters (tac and trc). T7-promoter (more specifically After transformation, a step for detecting and selecting that of T7-phage gene 10) and other T-phage promot those host cells or host organisms that have been Success ers; promoter of the Tn 10 tetracycline resistance gene; fully transformed with the nucleotide sequence/genetic con engineered variants of the above promoters that include struct of the invention may be performed. This may for one or more copies of an extraneous regulatory opera 25 instance be a selection step based on a selectable marker tor Sequence; present in the genetic construct of the invention or a step for expression in S. cerevisiae: constitutive: ADH1 (alco involving the detection of the amino acid sequence of the hol dehydrogenase 1), ENO (enolase), CYC1 (cy invention, e.g. using specific antibodies. tochrome c iso-1), GAPDH (glyceraldehydes-3-phos The transformed host cell (which may be in the form or phate dehydrogenase), PGK1 (phosphoglycerate 30 a stable cell line) or host organisms (which may be in the form of a stable mutant line or strain) form further aspects kinase), PYK1 (pyruvate kinase); regulated: GAL1, of the present invention. 10.7 (galactose metabolic enzymes), ADH2 (alcohol Preferably, these host cells or host organisms are such that dehydrogenase 2), PHOS (acid phosphatase), CUP1 they express, or are (at least) capable of expressing (e.g. (copper metallothionein); heterologous: CaMV (cauli 35 under Suitable conditions), an amino acid sequence, NANO flower mosaic virus 35S promoter); BODYR (V) or polypeptide of the invention (and in case for expression in Pichia pastoris: the AOX1 promoter of a host organism: in at least one cell, part, tissue or organ (alcohol oxidase I); thereof). The invention also includes further generations, for expression in mammalian cells: human cytomegalo progeny and/or offspring of the host cell or host organism of virus (hCMV) immediate early enhancer/promoter; 40 the invention, that may for instance be obtained by cell human cytomegalovirus (hCMV) immediate early pro division or by sexual or asexual reproduction. moter variant that contains two tetracycline operator To produce? obtain expression of the amino acid sequences sequences such that the promoter can be regulated by of the invention, the transformed host cell or transformed the Tet repressor; Herpes Simplex Virus thymidine host organism may generally be kept, maintained and/or kinase (TK) promoter; Rous Sarcoma Virus long ter 45 cultured under conditions such that the (desired) amino acid minal repeat (RSV LTR) enhancer/promoter; elonga sequence, NANOBODYR (V) or polypeptide of the tion factor 1C.. (hEF-1C...) promoter from human, chim invention is expressed/produced. Suitable conditions will be panzee, mouse or rat; the SV40 early promoter: HIV-1 clear to the skilled person and will usually depend upon the long terminal repeat promoter, B-actin promoter, host cell/host organism used, as well as on the regulatory Some preferred, but non-limiting vectors for use with 50 elements that control the expression of the (relevant) nucleo these host cells include: tide sequence of the invention. Again, reference is made to vectors for expression in mammalian cells: pMAMneo the handbooks and patent applications mentioned above in (Clontech), pcDNA3 (Invitrogen), pMClneo (Strata the paragraphs on the genetic constructs of the invention. gene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC Generally, Suitable conditions may include the use of a 37593), pBPV-1 (8-2) (ATCC 37110), pdBPV-MMT 55 suitable medium, the presence of a suitable source of food neo (342-12) (ATCC 37224), pRSVgpt (ATCC37.199), and/or Suitable nutrients, the use of a suitable temperature, pRSVneo (ATCC37198), pSV2-dhfr (ATCC 37146), and optionally the presence of a suitable inducing factor or pUCTag (ATCC 37460) and 1 ZD35 (ATCC 37565), as compound (e.g. when the nucleotide sequences of the inven well as viral-based expression systems, such as those tion are under the control of an inducible promoter); all of based on adenovirus; 60 which may be selected by the skilled person. Again, under vectors for expression in bacterial cells: plT vectors Such conditions, the amino acid sequences of the invention (Novagen) and pCE vectors (Qiagen); may be expressed in a constitutive manner, in a transient vectors for expression in yeast or other fungal cells: manner, or only when Suitably induced. pYES2 (Invitrogen) and Pichia expression vectors (In It will also be clear to the skilled person that the amino vitrogen); 65 acid sequence, NANOBODYR (V) or polypeptide of the vectors for expression in insect cells: plBlueBacII (Invit invention may (first) be generated in an immature form (as rogen) and other baculovirus vectors mentioned above), which may then be subjected to post US 9,512,236 B2 127 128 translational modification, depending on the host cell/host and Solutions such as physiological phosphate-buffered organism used. Also, the amino acid sequence, NANO saline, Ringer's Solutions, dextrose solution, and Hank’s BODYR (V) or polypeptide of the invention may be Solution; water oils; glycerol; ethanol; glycols such as pro glycosylated, again depending on the host cell/host organ pylene glycol or as well as mineral oils, animal oils and ism used. 5 vegetable oils, for example peanut oil, soybean oil, as well The amino acid sequence, NANOBODYR (V) or as Suitable mixtures thereof. Usually, aqueous Solutions or polypeptide of the invention may then be isolated from the suspensions will be preferred. host cell/host organism and/or from the medium in which The amino acid sequences, Nanobodies and polypeptides said host cell or host organism was cultivated, using protein of the invention can also be administered using gene therapy isolation and/or purification techniques known per se, Such 10 methods of delivery. See, e.g., U.S. Pat. No. 5,399,346, as (preparative) chromatography and/or electrophoresis which is incorporated by reference in its entirety. Using a techniques, differential precipitation techniques, affinity gene therapy method of delivery, primary cells transfected techniques (e.g. using a specific, cleavable amino acid with the gene encoding an amino acid sequence, NANO sequence fused with the amino acid sequence, NANO BODYR (V) or polypeptide of the invention can addi BODYR (V) or polypeptide of the invention) and/or 15 tionally be transfected with tissue specific promoters to preparative immunological techniques (i.e. using antibodies target specific organs, tissue, grafts, tumors, or cells and can against the amino acid sequence to be isolated). additionally be transfected with signal and stabilization Generally, for pharmaceutical use, the polypeptides of the sequences for Subcellularly localized expression. invention may be formulated as a pharmaceutical prepara Thus, the amino acid sequences, Nanobodies and poly tion or compositions comprising at least one polypeptide of peptides of the invention may be systemically administered, the invention and at least one pharmaceutically acceptable e.g., orally, in combination with a pharmaceutically accept carrier, diluent or excipient and/or adjuvant, and optionally able vehicle such as an inert diluent or an assimilable edible one or more further pharmaceutically active polypeptides carrier. They may be enclosed in hard or soft shell gelatin and/or compounds. By means of non-limiting examples, capsules, may be compressed into tablets, or may be incor such a formulation may be in a form suitable for oral 25 porated directly with the food of the patient’s diet. For oral administration, for parenteral administration (such as by therapeutic administration, the amino acid sequences, Nano intravenous, intramuscular or Subcutaneous injection or bodies and polypeptides of the invention may be combined intravenous infusion), for topical administration, for admin with one or more excipients and used in the form of istration by inhalation, by a skin patch, by an implant, by a ingestible tablets, buccal tablets, troches, capsules, elixirs, Suppository, etc. Such suitable administration forms—which 30 Suspensions, syrups, wafers, and the like. Such compositions may be solid, semi-solid or liquid, depending on the manner and preparations should contain at least 0.1% of the amino of administration—as well as methods and carriers for use in acid sequence, NANOBODYR (V) or polypeptide of the the preparation thereof, will be clear to the skilled person, invention. Their percentage in the compositions and prepa and are further described herein. rations may, of course, be varied and may conveniently be Thus, in a further aspect, the invention relates to a 35 between about 2 to about 60% of the weight of a given unit pharmaceutical composition that contains at least one amino dosage form. The amount of the amino acid sequence, acid of the invention, at least one NANOBODY (R) (V) of NANOBODYR (V) or polypeptide of the invention in the invention or at least one polypeptide of the invention and Such therapeutically useful compositions is such that an at least one suitable carrier, diluent or excipient (i.e. suitable effective dosage level will be obtained. for pharmaceutical use), and optionally one or more further 40 The tablets, troches, pills, capsules, and the like may also active Substances. contain the following: binders such as gum tragacanth, Generally, the amino acid sequences, Nanobodies and acacia, corn starch or gelatin; excipients such as dicalcium polypeptides of the invention can be formulated and admin phosphate; a disintegrating agent Such as corn starch, potato istered in any Suitable manner known per se, for which starch, alginic acid and the like; a lubricant Such as magne reference is for example made to the general background art 45 sium Stearate; and a Sweetening agent such as Sucrose, cited above (and in particular to WO 04/041862, WO fructose, lactose or aspartame or a flavoring agent such as 04/041863, WO 04/041865 and WO 04/041867) as well as peppermint, oil of wintergreen, or cherry flavoring may be to the standard handbooks, such as Remington's Pharma added. When the unit dosage form is a capsule, it may ceutical Sciences, 18" Ed., Mack Publishing Company, USA contain, in addition to materials of the above type, a liquid (1990) or Remington, the Science and Practice of Pharmacy, 50 carrier, such as a vegetable oil or a polyethylene glycol. 21st Edition, Lippincott Williams and Wilkins (2005). Various other materials may be present as coatings or to For example, the amino acid sequences, Nanobodies and otherwise modify the physical form of the solid unit dosage polypeptides of the invention may be formulated and admin form. For instance, tablets, pills, or capsules may be coated istered in any manner known per se for conventional anti with gelatin, wax, shellac or Sugar and the like. A syrup or bodies and antibody fragments (including ScFv's and dia 55 elixir may contain the amino acid sequences, Nanobodies bodies) and other pharmaceutically active proteins. Such and polypeptides of the invention, Sucrose or fructose as a formulations and methods for preparing the same will be Sweetening agent, methyl and propylparabens as preserva clear to the skilled person, and for example include prepa tives, a dye and flavoring Such as cherry or orange flavor. Of rations Suitable for parenteral administration (for example course, any material used in preparing any unit dosage form intravenous, intraperitoneal, Subcutaneous, intramuscular, 60 should be pharmaceutically acceptable and Substantially intraluminal, intra-arterial or intrathecal administration) or non-toxic in the amounts employed. In addition, the amino for topical (i.e. transdermal or intradermal) administration. acid sequences, Nanobodies and polypeptides of the inven Preparations for parenteral administration may for tion may be incorporated into Sustained-release preparations example be sterile solutions, Suspensions, dispersions or and devices. emulsions that are suitable for infusion or injection. Suitable 65 Preparations and formulations for oral administration may carriers or diluents for Such preparations for example also be provided with an enteric coating that will allow the include, without limitation, sterile water and aqueous buffers constructs of the invention to resist the gastric environment US 9,512,236 B2 129 130 and pass into the intestines. More generally, preparations liquid compositions can be applied from absorbent pads, and formulations for oral administration may be suitably used to impregnate bandages and other dressings, or sprayed formulated for delivery into any desired part of the gastro onto the affected area using pump-type or aerosol sprayers. intestinal tract. In addition, Suitable Suppositories may be Thickeners such as Synthetic polymers, fatty acids, fatty used for delivery into the gastrointestinal tract. acid salts and esters, fatty alcohols, modified celluloses or The amino acid sequences, Nanobodies and polypeptides modified mineral materials can also be employed with liquid of the invention may also be administered intravenously or carriers to form spreadable pastes, gels, ointments, Soaps, intraperitoneally by infusion or injection. Solutions of the and the like, for application directly to the skin of the user. amino acid sequences, Nanobodies and polypeptides of the Examples of useful dermatological compositions which invention or their salts can be prepared in water, optionally 10 can be used to deliver the amino acid sequences, Nanobodies mixed with a nontoxic Surfactant. Dispersions can also be and polypeptides of the invention to the skin are known to prepared in glycerol, liquid polyethylene glycols, triacetin, the art; for example, see Jacquet et al. (U.S. Pat. No. and mixtures thereof and in oils. Under ordinary conditions 4,608.392), Geria (U.S. Pat. No. 4,992.478), Smith et al. of storage and use, these preparations contain a preservative (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. to prevent the growth of microorganisms. 15 4,820,508). The pharmaceutical dosage forms suitable for injection or Useful dosages of the amino acid sequences, Nanobodies infusion can include sterile aqueous solutions or dispersions and polypeptides of the invention can be determined by or sterile powders comprising the active ingredient which comparing their in vitro activity, and in vivo activity in are adapted for the extemporaneous preparation of sterile animal models. Methods for the extrapolation of effective injectable or infusible solutions or dispersions, optionally dosages in mice, and other animals, to humans are known to encapsulated in liposomes. In all cases, the ultimate dosage the art; for example, see U.S. Pat. No. 4,938,949. form must be sterile, fluid and stable under the conditions of Generally, the concentration of the amino acid sequences, manufacture and storage. The liquid carrier or vehicle can be Nanobodies and polypeptides of the invention in a liquid a solvent or liquid dispersion medium comprising, for composition, such as a lotion, will be from about 0.1-25 example, water, ethanol, a polyol (for example, glycerol, 25 wt-%, preferably from about 0.5-10 wt-%. The concentra propylene glycol, liquid polyethylene glycols, and the like), tion in a semi-solid or solid composition Such as a gel or a vegetable oils, nontoxic glyceryl esters, and Suitable mix powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 tures thereof. The proper fluidity can be maintained, for wt-%. example, by the formation of liposomes, by the maintenance The amount of the amino acid sequences, Nanobodies and of the required particle size in the case of dispersions or by 30 polypeptides of the invention required for use in treatment the use of surfactants. The prevention of the action of will vary not only with the particular amino acid sequence, microorganisms can be brought about by various antibacte NANOBODYR (V) or polypeptide selected but also with rial and antifungal agents, for example, parabens, chlorobu the route of administration, the nature of the condition being tanol, phenol, Sorbic acid, thimerosal, and the like. In many treated and the age and condition of the patient and will be cases, it will be preferable to include isotonic agents, for 35 ultimately at the discretion of the attendant physician or example, Sugars, buffers or sodium chloride. Prolonged clinician. Also the dosage of the amino acid sequences, absorption of the injectable compositions can be brought Nanobodies and polypeptides of the invention varies about by the use in the compositions of agents delaying depending on the target cell, tumor, tissue, graft, or organ. absorption, for example, aluminum monostearate and gela The desired dose may conveniently be presented in a tin. 40 single dose or as divided doses administered at appropriate Sterile injectable solutions are prepared by incorporating intervals, for example, as two, three, four or more Sub-doses the amino acid sequences, Nanobodies and polypeptides of per day. The sub-dose itself may be further divided, e.g., into the invention in the required amount in the appropriate a number of discrete loosely spaced administrations. Such as solvent with various of the other ingredients enumerated multiple inhalations from an insufflator or by application of above, as required, followed by filter sterilization. In the 45 a plurality of drops into the eye. case of sterile powders for the preparation of sterile inject An administration regimen could include long-term, daily able solutions, the preferred methods of preparation are treatment. By “long-term' is meant at least two weeks and vacuum drying and the freeze drying techniques, which preferably, several weeks, months, or years of duration. yield a powder of the active ingredient plus any additional Necessary modifications in this dosage range may be deter desired ingredient present in the previously sterile-filtered 50 mined by one of ordinary skill in the art using only routine Solutions. experimentation given the teachings herein. See Reming For topical administration, the amino acid sequences, ton's Pharmaceutical Sciences (Martin, E. W., ed. 4), Mack Nanobodies and polypeptides of the invention may be Publishing Co., Easton, Pa. The dosage can also be adjusted applied in pure form, i.e., when they are liquids. However, by the individual physician in the event of any complication. it will generally be desirable to administer them to the skin 55 In another aspect, the invention relates to a method for the as compositions or formulations, in combination with a prevention and/or treatment of at least one GPCR-related dermatologically acceptable carrier, which may be a solid or diseases and disorders, said method comprising administer a liquid. ing, to a Subject in need thereof, a pharmaceutically active Useful solid carriers include finely divided solids such as amount of an amino acid sequence of the invention, of a talc, clay, microcrystalline cellulose, silica, alumina and the 60 NANOBODYR (V) of the invention, of a polypeptide of like. Useful liquid carriers include water, hydroxyalkyls or the invention, and/or of a pharmaceutical composition com glycols or water-alcohol/glycol blends, in which the amino prising the same. acid sequences, Nanobodies and polypeptides of the inven In the context of the present invention, the term “preven tion can be dissolved or dispersed at effective levels, option tion and/or treatment not only comprises preventing and/or ally with the aid of non-toxic Surfactants. Adjuvants such as 65 treating the disease, but also generally comprises preventing fragrances and additional antimicrobial agents can be added the onset of the disease, slowing or reversing the progress of to optimize the properties for a given use. The resultant disease, preventing or slowing the onset of one or more US 9,512,236 B2 131 132 symptoms associated with the disease, reducing and/or suffering from or at risk of the diseases and disorders alleviating one or more symptoms associated with the dis mentioned herein, a pharmaceutically active amount of an ease, reducing the severity and/or the duration of the disease amino acid sequence of the invention, of a NANOBODYR and/or of any symptoms associated therewith and/or pre (V) of the invention, of a polypeptide of the invention, venting a further increase in the severity of the disease and/or of a pharmaceutical composition comprising the and/or of any symptoms associated therewith, preventing, SaC. reducing or reversing any physiological damage caused by In the above methods, the amino acid sequences, Nano the disease, and generally any pharmacological action that is bodies and/or polypeptides of the invention and/or the beneficial to the patient being treated. compositions comprising the same can be administered in The subject to be treated may be any warm-blooded 10 any suitable manner, depending on the specific pharmaceu animal, but is in particular a mammal, and more in particular tical formulation or composition to be used. Thus, the amino a human being. As will be clear to the skilled person, the acid sequences, Nanobodies and/or polypeptides of the subject to be treated will in particular be a person suffering invention and/or the compositions comprising the same can from, or at risk of the diseases and disorders mentioned for example be administered orally, intraperitoneally (e.g. herein. 15 intravenously, Subcutaneously, intramuscularly, or via any The invention relates to a method for the prevention other route of administration that circumvents the gastroin and/or treatment of at least one disease or disorder that is testinal tract), intranasally, transdermally, topically, by associated with GPCRs, with its biological or pharmaco means of a Suppository, by inhalation, again depending on logical activity, and/or with the biological pathways or the specific pharmaceutical formulation or composition to be signalling in which GPCRs is involved, said method com used. The clinician will be able to select a suitable route of prising administering, to a subject in need thereof, a phar administration and a suitable pharmaceutical formulation or maceutically active amount of an amino acid sequence of the composition to be used in Such administration, depending on invention, of a NANOBODYR (V) of the invention, of a the disease or disorder to be prevented or treated and other polypeptide of the invention, and/or of a pharmaceutical factors well known to the clinician. composition comprising the same. In particular, the inven 25 The amino acid sequences, Nanobodies and/or polypep tion relates to a method for the prevention and/or treatment tides of the invention and/or the compositions comprising of at least one disease or disorder that can be treated by the same are administered according to a regime of treat modulating GPCRs, its biological or pharmacological activ ment that is suitable for preventing and/or treating the ity, and/or the biological pathways or signalling in which disease or disorder to be prevented or treated. The clinician GPCRs is involved, said method comprising administering, 30 will generally be able to determine a suitable treatment to a Subject in need thereof, a pharmaceutically active regimen, depending on factors such as the disease or disor amount of an amino acid sequence of the invention, of a der to be prevented or treated, the severity of the disease to NANOBODYR (V) of the invention, of a polypeptide of be treated and/or the severity of the symptoms thereof, the the invention, and/or of a pharmaceutical composition com specific amino acid sequence, NANOBODYR (V) or prising the same. In particular, said pharmaceutically effec 35 polypeptide of the invention to be used, the specific route of tive amount may be an amount that is sufficient to modulate administration and pharmaceutical formulation or composi GPCRs, its biological or pharmacological activity, and/or tion to be used, the age, gender, weight, diet, general the biological pathways or signalling in which GPCRs is condition of the patient, and similar factors well known to involved; and/or an amount that provides a level of the the clinician. amino acid sequence of the invention, of a NANOBODYR 40 Generally, the treatment regimen will comprise the (V) of the invention, of a polypeptide of the invention in administration of one or more amino acid sequences, Nano the circulation that is sufficient to modulate GPCRs, its bodies and/or polypeptides of the invention, or of one or biological or pharmacological activity, and/or the biological more compositions comprising the same, in one or more pathways or signalling in which GPCRs is involved. pharmaceutically effective amounts or doses. The specific The invention furthermore relates to a method for the 45 amount(s) or doses to administer can be determined by the prevention and/or treatment of at least one disease or dis clinician, again based on the factors cited above. order that can be prevented and/or treated by administering Generally, for the prevention and/or treatment of the an amino acid sequence of the invention, a NANOBODYR diseases and disorders mentioned herein and depending on (V) of the invention or a polypeptide of the invention to the specific disease or disorder to be treated, the potency of a patient, said method comprising administering, to a subject 50 the specific amino acid sequence, NANOBODYR (V) in need thereof, a pharmaceutically active amount of an and polypeptide of the invention to be used, the specific amino acid sequence of the invention, of a NANOBODYR route of administration and the specific pharmaceutical (V) of the invention, of a polypeptide of the invention, formulation or composition used, the amino acid sequences, and/or of a pharmaceutical composition comprising the Nanobodies and polypeptides of the invention will generally SaC. 55 be administered in an amount between 1 gram and 0.01 More in particular, the invention relates to a method for microgram per kg body weight per day, preferably between the prevention and/or treatment of at least one disease or 0.1 gram and 0.1 microgram per kg body weight per day, disorder chosen from the group consisting of the diseases such as about 1, 10, 100 or 1000 microgram per kg body and disorders listed herein, said method comprising admin weight per day, either continuously (e.g. by infusion), as a istering, to a subject in need thereof, a pharmaceutically 60 single daily dose or as multiple divided doses during the day. active amount of an amino acid sequence of the invention, The clinician will generally be able to determine a suitable of a NANOBODYR (V) of the invention, of a polypep daily dose, depending on the factors mentioned herein. It tide of the invention, and/or of a pharmaceutical composi will also be clear that in specific cases, the clinician may tion comprising the same. choose to deviate from these amounts, for example on the In another aspect, the invention relates to a method for 65 basis of the factors cited above and his expert judgment. immunotherapy, and in particular for passive immuno Generally, some guidance on the amounts to be administered therapy, which method comprises administering, to a subject can be obtained from the amounts usually administered for US 9,512,236 B2 133 134 comparable conventional antibodies or antibody fragments Generally, the treatment regimen will be followed until against the same target administered via essentially the same the desired therapeutic effect is achieved and/or for as long route, taking into account however differences in affinity/ as the desired therapeutic effect is to be maintained. Again, avidity, efficacy, biodistribution, half-life and similar factors this can be determined by the clinician. well known to the skilled person. In another aspect, the invention relates to the use of an Usually, in the above method, a single amino acid amino acid sequence, NANOBODYR (V) or polypeptide sequence, NANOBODYR (V) or polypeptide of the of the invention in the preparation of a pharmaceutical invention will be used. It is however within the scope of the composition for prevention and/or treatment of at least one invention to use two or more amino acid sequences, Nano GPCR-related diseases and disorders; and/or for use in one 10 or more of the methods of treatment mentioned herein. bodies and/or polypeptides of the invention in combination. The subject to be treated may be any warm-blooded The Nanobodies, amino acid sequences and polypeptides animal, but is in particular a mammal, and more in particular of the invention may also be used in combination with one a human being. As will be clear to the skilled person, the or more further pharmaceutically active compounds or prin subject to be treated will in particular be a person suffering ciples, i.e. as a combined treatment regimen, which may or 15 from, or at risk of the diseases and disorders mentioned may not lead to a synergistic effect. Again, the clinician will herein. be able to select such further compounds or principles, as The invention also relates to the use of an amino acid well as a suitable combined treatment regimen, based on the sequence, NANOBODYR (V) or polypeptide of the factors cited above and his expert judgement. invention in the preparation of a pharmaceutical composi In particular, the amino acid sequences, Nanobodies and tion for the prevention and/or treatment of at least one polypeptides of the invention may be used in combination disease or disorder that can be prevented and/or treated by with other pharmaceutically active compounds or principles administering an amino acid sequence, NANOBODYR that are or can be used for the prevention and/or treatment (V) or polypeptide of the invention to a patient. of the diseases and disorders cited herein, as a result of More in particular, the invention relates to the use of an which a synergistic effect may or may not be obtained. 25 amino acid sequence, NANOBODYR (V) or polypeptide Examples of Such compounds and principles, as well as of the invention in the preparation of a pharmaceutical routes, methods and pharmaceutical formulations or com composition for the prevention and/or treatment of GPCR positions for administering them will be clear to the clini related diseases and disorders, and in particular for the cian. prevention and treatment of one or more of the diseases and When two or more substances or principles are to be used 30 disorders listed herein. as part of a combined treatment regimen, they can be Again, in Such a pharmaceutical composition, the one or administered via the same route of administration or via more amino acid sequences, Nanobodies or polypeptides of different routes of administration, at essentially the same the invention may also be suitably combined with one or time or at different times (e.g. essentially simultaneously, more other active principles, such as those mentioned consecutively, or according to an alternating regime). When 35 herein. the Substances or principles are to be administered simulta Finally, although the use of the Nanobodies of the inven neously via the same route of administration, they may be tion (as defined herein) and of the polypeptides of the administered as different pharmaceutical formulations or invention is much preferred, it will be clear that on the basis compositions or part of a combined pharmaceutical formu of the description herein, the skilled person will also be able lation or composition, as will be clear to the skilled person. 40 to design and/or generate, in an analogous manner, other Also, when two or more active Substances or principles amino acid sequences and in particular (single) domain are to be used as part of a combined treatment regimen, each antibodies against GPCRs, as well as polypeptides compris of the Substances or principles may be administered in the ing Such (single) domain antibodies. same amount and according to the same regimen as used For example, it will also be clear to the skilled person that when the compound or principle is used on its own, and Such 45 it may be possible to 'graft one or more of the CDR's combined use may or may not lead to a synergistic effect. mentioned above for the Nanobodies of the invention onto However, when the combined use of the two or more active Such (single) domain antibodies or other protein scaffolds, Substances or principles leads to a synergistic effect, it may including but not limited to human scaffolds or non-immu also be possible to reduce the amount of one, more or all of noglobulin scaffolds. Suitable scaffolds and techniques for the substances or principles to be administered, while still 50 such CDR grafting will be clear to the skilled person and are achieving the desired therapeutic action. This may for well known in the art, see for example U.S. Pat. No. example be useful for avoiding, limiting or reducing any 7,180.370, WO 01/27160, EPO 605522, EPO 460 167, U.S. unwanted side-effects that are associated with the use of one Pat. No. 7,054,297, Nicaise et al., Protein Science (2004), or more of the Substances or principles when they are used 13:1882-1891; Ewert et al., Methods, 2004 October; 34(2): in their usual amounts, while still obtaining the desired 55 184-199: Kettleborough et al., Protein Eng. 1991 October; pharmaceutical or therapeutic effect. 4(7): 773-783; O'Brien and Jones, Methods Mol. Biol. The effectiveness of the treatment regimen used according 2003: 207: 81-100: Skerra, J. Mol. Recognit. 2000: 13: to the invention may be determined and/or followed in any 167-187, and Saerens et al., J. Mol. Biol. 2005 Sep. 23; manner known perse for the disease or disorder involved, as 352(3):597-607, and the further references cited therein. For will be clear to the clinician. The clinician will also be able, 60 example, techniques known perse for grafting mouse or rat where appropriate and on a case-by-case basis, to change or CDR's onto human frameworks and scaffolds can be used in modify a particular treatment regimen, so as to achieve the an analogous manner to provide chimeric proteins compris desired therapeutic effect, to avoid, limit or reduce unwanted ing one or more of the CDR's of the Nanobodies of the side-effects, and/or to achieve an appropriate balance invention and one or more human framework regions or between achieving the desired therapeutic effect on the one 65 Sequences. hand and avoiding, limiting or reducing undesired side It should also be noted that, when the Nanobodies of the effects on the other hand. inventions contain one or more other CDR sequences than US 9,512,236 B2 135 136 the preferred CDR sequences mentioned above, these CDR nyl-KTSLHLWNRSSYRLHS (SP061354; lot. Nr. sequences can be obtained in any manner known per se, for CK01160). The peptide sequence is located in the extracel example from Nanobodies (preferred), V. domains from lular part of MC4R. conventional antibodies (and in particular from human anti Two llamas (086 and 087) were immunized with 6 boosts bodies), heavy chain antibodies, conventional 4-chain anti of refolded GPCRs (PTHR, CB1R) according to standard bodies (such as conventional human 4-chain antibodies) or protocols. The proteins were refolded using a standard other immunoglobulin sequences directed against GPCRs. protocol, based on the techniques described by Kiefer (Bio Such immunoglobulin sequences directed against GPCRs chim. Biophys. Acta, 1610 (2003), 57-62). Blood was col can be generated in any manner known per se, as will be lected from these animals after 7 and 10 days after boost 6 clear to the skilled person, i.e. by immunization with GPCRs 10 Two llamas (100 and 101) were immunized with 6 boosts or by Screening a suitable library of immunoglobulin of KLH-NT peptide (derived from MC4R) according to sequences with GPCRs, or any suitable combination thereof. Ablynx protocols. Blood was collected from these animals Optionally, this may be followed by techniques such as after 7 and 10 days after boost 6 random or site-directed mutagenesis and/or other techniques Phage display libraries were prepared according to a for affinity maturation known perse. Suitable techniques for 15 standard protocol. Peripheral blood mononuclear cells were generating Such immunoglobulin sequences will be clear to prepared from blood samples using Ficoll-Hypaque accord the skilled person, and for example include the screening ing to the manufacturers instructions. Total RNA was techniques reviewed by Hoogenboom, Nature Biotechnol extracted from these cells and used as starting material for ogy, 23, 9, 1105-1116 (2005) Other techniques for generat RT-PCR to amplify NANOBODYR (V) encoding gene ing immunoglobulins against a specified target include for fragments. These fragments were cloned into phagemid example the Nanoclone technology (as for example vector pAX50. Phage suspensions were prepared according described in the published US patent application 2006 to standard methods and stored after filter sterilization at 4° 0211088), so-called SLAM technology (as for example C. prior to selections. described in the European patent application 0.542 810), the use of transgenic mice expressing human immunoglobulins 25 Example 2 or the well-known hybridoma techniques (see for example Larricket al. Biotechnology, Vol. 7, 1989, p. 934). All these GPCR NANOBODYR (V) Selections techniques can be used to generate immunoglobulins against GPCRs, and the CDR's of such immunoglobulins can be Phage libraries were used for two rounds of selections on used in the Nanobodies of the invention, i.e. as outlined 30 either refolded CB1R or on refolded PTHR. A second round above. For example, the sequence of such a CDR can be on CB1R membranes was also performed for the CB1R determined, synthesized and/or isolated, and inserted into NANOBODYR (V) selection. All antigens were solid the sequence of a NANOBODYR (V) of the invention phase immobilized on Nunc Maxisorp ELISA plates at 10 (e.g. So as to replace the corresponding native CDR), all microg?well, 2 microg?well, 0.4 microg/well and 0 microg/ using techniques known per se Such as those described 35 well (background control) for the first round of selection. herein, or Nanobodies of the invention containing Such For the second round of selection either 2 microg?well, 0.4 CDR's (or nucleic acids encoding the same) can be synthe microg?well refolded GPCR was used or 5 microg?well and sized de novo, again using the techniques mentioned herein. 1 microg?well CB1R membranes were used in the MaxiSorp Further uses of the amino acid sequences, Nanobodies, plates. Interacting phages were retrieved from both first and polypeptides, nucleic acids, genetic constructs and hosts and 40 second round of selections using triethylamine elution. host cells of the invention will be clear to the skilled person Individual clones were isolated and were grown on 10 ml based on the disclosure herein. For example, and without or 50 ml scale, and induced by adding IPTG for NANO limitation, the amino acid sequences of the invention can be BODYR (V) expression. Periplasmic extracts (volume: 1 linked to a suitable carrier or solid Support so as to provide to 5 ml) were prepared according to standard methods. a medium than can be used in a manner known per se to 45 Two phage libraries were used for selections for two purify GPCRs from compositions and preparations compris rounds on biotinylated MC4R peptide. The peptide was ing the same. Derivatives of the amino acid sequences of the captured by neutravidin which was solid-phase immobilized invention that comprise a suitable detectable label can also on Nunc Maxisorp ELISA plates at 500 ng/well. The peptide be used as markers to determine (qualitatively or quantita was coated in the first round of selection at a concentration tively) the presence of GPCRs in a composition or prepa 50 ranging from 100 to 0.01 uM. For the second round of ration or as a marker to selectively detect the presence of selection the peptide was used at a concentration ranging GPCRs on the surface of a cell or tissue (for example, in from 5 to 0.05 uM. Interacting phages were retrieved from combination with Suitable cell sorting techniques). both first and second round of selections using triethylamine The invention will now be further described by means of elution. Individual clones were isolated and were grown 1 the following non-limiting examples and in FIG. 1 (which 55 ml deep well plates, and induced by adding IPTG for schematically shows the structure of the three different NANOBODYR (V) expression. Periplasmic extracts classes of GPCRs) and FIG. 2 (which is a graph showing the were prepared according to standard methods. screening results obtained in Example 3): Phage libraries 100 and 101 were used for selections for two rounds on biotinylated MC4R peptide. The peptide was Example 1 60 captured by neutravidin which was solid-phase immobilized on Nunc Maxisorp ELISA plates at 500 ng/well. The peptide Immunization and Library Construction was coated in the first round of selection at a concentration ranging from 100 to 0.01 uM. For the second round of Mouse CB1R Membranes were purchased from Bioxtal selection the peptide was used at a concentration ranging (Mundolshein, France). 65 from 5 to 0.05 uM. Interacting phages were retrieved from N-terminal biotinylated MC4R peptide was purchased both first and second round of selections using triethylamine from NeoMPS S.A. (Strasbourg, France). Sequence: bioti elution. US 9,512,236 B2 137 138 Individual clones were isolated and were grown 1 ml deep SEQ ID NO’s 413 to 430, 517 and 518: Nanobodies well plates, and induced by adding IPTG for NANO against CB1R BODYR (V) expression. Periplasmic extracts were pre SEQ ID NO’s 431 to 453: Nanobodies against PTHR1 pared according to standard methods. SEQ ID NO’s 519 to 525: Nanobodies against MCR4 Example 3 Example 5 Screening Highly Diverse Pool of Generated Nanobodies Underscores the Construction of Biparatopic For the determination of the binding specificity to the 10 Binders GPCRs, the clones were tested in a phage ELISA binding assay setup. 500 ng?well refolded GPCR or GPCR mem The CDR sequences of the different Nanobodies identi brane was solid-phase immobilized on Maxisorp microtiter fied for each of the GPCR targets (CB1R, PTHR1 and plates (Nunc), and free binding sites were blocked using 4% MCR4) as described herein were compared to each other to Marvel Milk in PBS. 10 ul of phage suspension of the 15 indicate the diversity of binding properties present within different clones in 100 ul 2% Marvel PBS was incubated the set of generated Nanobodies. The data for each of the with the immobilized antigen. After 1 h incubation, non GPCR targets are presented in Tables E, F and G respec bound phage was removed by washing with PBS-Tween, tively, and show a clear overview of the percentage of followed by PBS. Next mouse-anti-M13-HRPantibody was sequence identity (data presented below diagonal) and the incubated with the interacting phages. After 1 h incubation, number of different residues (data present above diagonal) non-bound phage was removed by washing with PBS amongst the generated Nanobodies previously provided in Tween, followed by PBS. Anti-M13 antibody presence was Table B-4 of US2O100062004. detected using TMB substrate (Pierce). Binding specificity The data recited in Tables E to F respectively, illustrate was determined based on OD values compared to a control 25 that a high sequence diversity is present within the previ GPCR. The results are schematically shown in FIG. 2. ously identified pool of Nanobodies (Table B-4). The amino acid sequences were tested for activity against Molecules with very different CDR sequences will bind to CB1R, PTHR1 and MC4R, respectively, using a standard divergent epitopes. Hence, it is self-explanatory that the high radioligand assay (see for example Andre et al., Protein Sci sequence diversity observed for the CDRS generated against 5:1115 (2006); Hassaine et al., (2006) Prot Purif Expr 30 all 3 GPCR targets, i.e. CB1R, PTHR1 and MCR4 is 45:343; Nicholson et al. J Pharmacol Exp Ther. 2006 May; correlated with a diversity of binding properties, and accord 317(2):771-7. Epub 2006 Jan. 25. and Villardaga et al., J ingly supports the production of biparatopic binders against Biol Chem. 2001 Sep. 7:276(36):33435-43. Epub 2001 May 31), for example using membrane preparations that can be these GPCR targets. made as described in Hovius et al., (1998) J Neurochem 35 Example 5A 70:824). Example 4 Biparatopic CB1R, PTHR1 and MCR4 Nanobodies Binding Data The monovalent CB1R, PTHR1 and MCR4 Nanobodies 40 which displayed a high CDR sequence diversity as demon Binding of 4 clones directed against CB1R and of 4 clones strated by Tables E, F and G respectively are now further against PTHR was measured on Biacore. The results are selected to engineer biparatopic binders. Different sets of given in Tables B-2 and B-3. CB1R, PTHR1 and MCR4 biparatopic Nanobodies are 45 prepared by recombinantly linking the diverse monovalent TABLE B-2 binders using amino linkers with repetitive GGGGS sequences of various sizes. The potency of these constructs Binding of clones against CB1R can be assessed by using the Path Hunter(R) eXpress B-Ar w6B7 w6H4 wSG11 wSD12 restin GPCR assay which provides a direct measure of 50 CB1R 12 nM 36 nM 12 nM non calculable B-Arrestin binding to the GPCR of interest. Example 6

TABLE B-3 Biparatopic CXCR4 Nanobodies 55 binding of clones on PTHR Another example of a therapeutic relevant GPCR is PTH (1-34) CXCR4, which was previously identified as such in Table C vHH22A3 v HH26F2 vHH23A3 v HH26A6 (ligand) and Table D of US20100062004. This representative GPCR has now been further investigated by the present inventors. PTHR- 9 nM 70 nM NT NT O2-16 nM CaCl2 60 Different pools of highly similar clones able to inhibit PTHR- 17 nM f 22 nM 17 nM 4-23 nM specific 'I-CXCL12 binding were identified in a CXCR4 CaCl2 competition binding assay. Nanobodies representing these PTHR NT 360 nM NT NT 260 M different pools were purified and further pharmacologically Fos12 analyzed as detailed in the Example section of WO09/ 65 138519. In particular two representative monovalent Nano Table B-4 gives the amino acid sequences of a number of bodies 238D2 and 238D4 and different mono- and hetero Nanobodies against some representative GPCRs. bivalent constructs thereof have been analyzed in depth: US 9,512,236 B2 139 140 6.1 Sequence Identity and Epitope Mapping nanobodies, 12G5 or 1 uM AMD3100 in Rosenkilde buffer Similar to Example 5, the CDR sequences of the two for 2 hours at 37°C. After this pre-stimulation, both 30 nM monovalent Nanobodies 238D2 and 238D4 were compared CXCL12 (or buffer for basal signaling) and 10 mM LiCl to each other to investigate their binding diversity. were added to each well (final concentrations), followed by Table Hindicates a CDR sequence similarity of only 37% a final 2 hour incubation at 37°C. After this final incubation, between the two CXCR4 Nanobodies 238D2 and 238D4 and the reaction was stopped by aspiration of stimulation are thus assumed to bind different epitopes within CXCR4 medium and addition of 10 mM formic acid (FIG. 4). as explained in Example 5. As seen in FIG. 4, the biparatopic 238D2-238D4 Nano Moreover this hypothesis was further corroborated using bodies (L3 and L8 constructs) clearly show a significantly the shotgun mutagenesis technology platform from Integral 10 Moleuclar, which mapped the epitopes of the anti-CXCR4 increased inhibitory potency compared to their monovalent Nanobodies at single amino acid resolution. This analysis counterparts and the monoclonal antibody 12G5. (depicted in FIG. 3) clearly indicates that the two Nanobod Moreover, the biparatopic constructs effectively inhibited ies bind distinct but partially overlapping sites in the extra CXCR4 function at a concentration of 60 nM, which is cellular loops. 15 approximately 16-fold lower than the concentration needed 6.2 Potency of Mono-Specific Bivalent CXCR4 Nanobodies for the clinically validated small molecule compound Versus Biparatopic CXCR4 Nanobodies plerixafor or AMD3100 (1 uM). Additionally, the inventors engineered a series of mono specific bivalent and biparatopic CXCR4 constructs. Example 7 Recombinantly linking of 238D2 to 238D2 and 238D4 to 238D4 (i.e. mono-specific bivalent constructs) using amino Biparatopic CXCR7 Nanobodies acid linkers with repetitive GGGGS sequences of different sizes resulted in a 14 and 4.4-fold increase in affinity to CXCR-7 is a member of the G-protein coupled receptor CXCR4, respectively (Table I). Overall, short peptide link family, which binds to the chemokines CXCL12/SDF-1 and ers were used favoring binding to CXCR4 monomers. 25 CXCL11. Ligand binding to CXCR7 activates MAP kinases Astonishingly, a more significant increase in affinity was through Beta-arrestins. observed when 238D2 was linked to 238D4 (i.e. biparatopic After immunizations of llamas, selections of phage dis constructs). In case of the obtained biparatopic NANO playing human CXCR7 binding Nanobodies were per BODYR (V)238D2-20GS-238D4, the affinity to CXCR4 formed. CXCR7 specific Nanobodies were identified by was increased by 27 and 17-fold over the respective mon 30 phage ELISA and FACS analysis. Nanobodies 08A05, ovalent counterparts 238D2 and 238D4. Alteration in the 08A10, 07C03, O1C10 and 14G03 were characterized in linker size between 15 amino acids and 20 amino acids does more detail. not show any influence in respect to the receptor affinity 7.1 Epitope Mapping (compare L3 and L8). The minimal epitope of Mab 11G8 (human CXCR7 6.3 Comparison Effectivity 12G5 vs Monovalent 238D2, 35 238D4 and Bivalent Nanobodies specific monoclonal antibody, R&D Systems) is known to be In membrane binding experiments mab 12G5, which is a F14SDISWP20 located at the CXCR7 N-terminus (see e.g., known neutralizing CXCR4 antibody, seems only to dis WO2008/048519). In order to map the epitope of the Nano place 50% of radiolabeled 125I-CXCL12 (CXCR4 ligand). bodies, cells were incubated simultaneously with 20 nM We were therefore interested to test whether 12G5 effec 40 Mab 11G8 APC (R&D) and with diluted test molecules for tively inhibits CXCR4 function. 2 h at 4°C. The level of competition with Mab 11G8 APC Inositol Phosphate Measurement ranges from about 20% to 100%, suggesting that the respec In an effort to functionally characterize the Nanobodies tive NANOBODYR (V) epitopes match to a high degree 238D2 and 238D4, the inventors measured their ability to (high% of competition) with the Mab 11G8 epitope or to a activate G-protein signaling or to inhibit the CXCL12 45 low degree (low % of competition) or induce allosteric induced G-protein signaling in HEK293T cells transiently changes affecting the Mab 11G8 binding. The results suggest co-transfected with cDNAs encoding CXCR4 and GO.gi5. that NANOBODYR (V) 01C10 hits an epitope distinct This assay is based upon the use of the chimeric Goldi5 from the epitope(s) hit by the other selected Nanobodies. protein that contains of a God backbone with the 5 C-ter Nanobodies 08A05, 08A10, 07C03, O1C10 and 14G03, minal amino acids replaced by those from Goli. The chimeric 50 Mab 8F 11 (Biolegend), Mab 11G8 (R&D) and Mab 9C4 G-protein is activated by CXCR4 like a Goli subunit but (MBL) were further tested for competition with Alexa647 transduces signals like Gold proteins (Coward, P. et al., labelled 14G03 in FACS analysis. Nanobodies 08A05, Chimeric G Proteins Allow a High-Throughput Signaling 08A10, 07C03, Mab 8F 11, Mab 11 G8 and Mab 9C4 com Assay of G1-Coupled Receptors, Analytical Biochemistry pete with 14G03 binding to CXCR7, while 01C10 does not, (1999) 270: 242-248). Thus, the activation of Goldi5 can be 55 quantified by the measurement of accumulated inositol Suggesting again that 01C10 hits an epitope distinct from the phosphates. epitope(s) hit by the other selected Nanobodies. 12G5 has been tested as a full curve (n=2) and single point In a third approach, Nanobodies were tested for their (10 nM) on hCXCR4/GO.gi5: cells were stimulated with 30 binding to a set of 10 point mutants of CXCR7 (S9A, F14Y. nM CXCL12. 60 117L, S18N, W 19A, S23G, D25A, V32A, M33Q, N36T), Methods: On day 1, HEK293T cells were plated out at a which yielded information on the individual NANO density of 2 million cells/dish. On day 2, these cells were BODYR (V) epitopes. For Nanobodies 08A05, 08A10, transfected with 2.5 lug CXCR4 DNA and 2.5 lug GO.gi5. 07C03 and 14G03, the epitope included residue M33, while using the PEI method. Cells were plated out the next day in that of 01C10 did not. The binding of 01C10 was affected by poly-L-lysine coated 24 well plates (500 ul/well) and labeled 65 the W19A , while this mutation did not affect the after 4-6 hours with 2 LLCi/ml 3H-inositol in inositol-free binding of 08A05, 08A10, 07C03 and 14G03. Once more, medium. On day 4, cells were pre-stimulated with either these data indicate that 01C10 hits a distinct epitope. US 9,512,236 B2 141 142 Based on the foregoing results, the Nanobodies 14G03, TABLE B-4 - continued 08A05,08A10 and 07C03 were grouped together (Group 2), Nanobodies against some representative GPCRs. while NANOBODYR (V) 01C10 was classified into SEQ ID NOS 413 to 430, 517 and 518 : Nanobodies another group (Group 1). against CB1R 7.2 Potency of Biparatopic Constructs Versus Bivalent and 5 SEQ ID NO's 431 to 453 : Nanobodies against PTHR1 Trivalent CXCR7 Nanobodies SEQ ID NO's 519 to 525 : Nanobodies against MCR4

The Path Hunter eXpress B-arrestin assay (DiscoverX) LGPMP5 B9 SEQ ID NO 416 was used to assess the potency of the CXCR7 NANO EVOLVESGGGLVOAGGSLRLSCAASGRTLNNHDMGWFROAPGKEREFW BODYR (V) constructs in inhibiting B-arrestin recruit 10 ATVRWGTSSTYYADSVKGRFTISSDNTKNTVALOMNRLTPEDTGVYYCAA ment. RSVYSYEYTYWGOGTOVTVSS The anti-CXCR7 constructs have an ALB8 building LGPMP5 D12 SEQ ID NO 417 block, which binds serum albumin (HSA). ALB8 is intended EVOLVESGGGLVOAGGSLRLSCAASRRTFSSFVMAWFROAPGKEREFV for half life extension (see for example WO08/028977 AAIYGTGGELVYYENSWKGRFTISRDNAKSTMYLOMNSLKPEDTGVYYCA described herein). The B-arrestin assay was run in the 15 WELTVRSIDLRRPLEYDYWGOGTOVTVSS presence of HSA. Longer linkers preceding the ALB8 build LGPMP5 F9 SEQ ID NO 418 ing block were evaluated to minimize sterical interference of EVOLVESGGGLVOAGGSLRLSCAASGRTLVNYDMGWFROAPGKEREFW HSA binding to the NANOBODYR (V) (Table K). ATIRWSTSETYYADSVKGRFTISSDNTKNTVDLOMNSLTPEDTAVYYCAA The biparatopic constructs 038,085 and 052 have an IC50 RSVYSYEYNYWGOGTOVTVSS value in the low nM range in the presence of HSA. LGPMP5 G11 SEQ ID NO 419 In contrast, the mono-specific bivalent constructs 055 and EVOLVKSGGGLVOAGGSLRLSCAASGRTLSNYDMGWFROAPGKEREFW 093 show no or weak antagonism. If only one CXCR7 ATIRWSTGETYYTDSVKGRFTISKDNTKNTVDLOMNSLTPEDTAVYYCAS building block is used in the construct, the concentration for RSVYSYEYNYWGOGTOVTVSS reaching IC50 is dramatically higher than with the 25 LGPMP6 B7 SEQ ID NO 420 biparatopic constructs. EVOLVESGGGLVOAGGSLRLSCAASGRTLRNYDMGWFROAPGKEREFW In conclusion, biparatopic constructs inhibit 3-arrestin ATIRWSTSETYYADSVKGRFTISSDNAKNTVDLOMNSLTPGDTGVYYCAA recruitment better than corresponding mono-specific biva RSVYSYDYNYWGOGTOVTVSS lent constructs. LGPMP6 C7 SEQ ID NO 421 7.3 Inhibition of B-Arrestin Recruitment of Trivalent 30 EVOLVESGGGLVOAGGSLRLSCAASGRTLSNYDWGWFROAPGKEREFW CXCR7 Nanobodies ATIRWSTSSTYYADPVKGRFTISSDNIKNTVDLOMNSLTPEDTAVYYCAA RSVYSYEYNYWGOGTOVTVSS The Path Hunter eXpress B-arrestin assay (DiscoverX) was also used to assess the antagonistic effect of trivalent LGPMP6 C9 SEQ ID NO 422 EVOLVESGGGLVOAGGSLRLSCAASGRTLSNYDMGWFROAPGKEREFW CXCR7 Nanobodies on recruitment of B-arrestin (see point 35 7.2 above). A panel of Nanobodies (clones) was screened at ATIRWSTSETYYADSVKGRFTISSSNTKNTVDLOMNSLTPEDTAVYYCAA a 100 nM concentration in the assay. Results are ranked in RSVYSYEYNYWGOGTOVTVSS Table L on the basis of efficiency of inhibition. The most LGPMP6 D2 SEQ ID NO 423 efficient molecules constitute combinations of Group 1 EVOLMESGGGLVOAGGSLRLSCAASGRTLTNYDMGWFROAPGKEREFW (O1C10) and Group 2 building blocks=biparatopic con 40 ATIRWSTSETYYADSVKGRFTISKDNTKNTVALOMNSLTPEDTALYYCAA structs, i.e. clones 038, 052, 021,023 and 049. RSVYSYEYNYWGOGTOVTVSS Although Nanobodies of Group 2 either monovalently or LGPMP6 D5 SEQ ID NO 424 bivalently demonstrate Superior binding and competition EVOLVESGGGLEOAGGSLRLSCAVSRSIFETNTMAWFROAPGKORELVA characteristics than the corresponding Nanobodies of Group RISSTGDTNYADSVKGRFTISRDNAKNTVYLOMNSLKPEDTAVYYCHVAG 1, Nanobodies of Group 1 combined with Nanobodies of 45 PYGSKFAWGOGTOVTVSS Group 2 gave wholly unexpectedly the best results in the LGPMP6 D12 SEQ ID NO 425 B-arrestin recruitment assay (see Table L). EVOLVESGGGLVOAGGSLRLSCAASGRTLSNHDWGWFROAPGKEREFW ATMRWSTGSTYYADSVKGRFTISSDNTKNTVDLOMNSLTPEDTAVYYCAA TABLE B-4 RSVYSYEYNYWGOGTOVTVSS 50 Nanobodies against some representative GPCRs. LGPMP6 E11 SEQ ID NO 426 SEQ ID NOS 413 to 43 O, 517 and 518 : Nanobodies EVOLVESGGGLVOAGGSLRLSCAASGRTLRNYDLGWFROAPGKEREFW against CB1R ATIRWSTSETYYADSVKGRFTISSDNAKNTVDLOMNSLTPEDTGVYYCAA SEQ ID NO's 431 to 453 : Nanobodies against PTHR1 RSVYSYDYNYWGOGTOVTVSS SEQ ID NO's 519 to 525: Nanobodies against MCR4 55 LGPMP6 F12SEQ ID NO 427 LGPMP5 A6 SEQ ID NO 413 EVOLVESGGGLVOAGGSLRLSCAASGRTLTNHDMGWFROAPGKEREFW EVOLVESGGGLVOAGGSLRLACAASGSIFSFNAMGWYROAPGKORELL ATIRWGTTDTYYADSVKGRFTISSDNTKNTVHLOMNSLTPEDTGVYYCAA AVITRDGITKYADSVKGRFTISRDNAKNTMYLOMNSLKPEDTAVYYCHTG RSVYSYEYTYWGOGTOVTVSS LPTGRGSHSDYWGOGTOVTVSS LGPMP6 H4 SEQ ID NO 428 LGPMP5 A7 SEQ ID NO 414 60 EVOLVESGGGLVOAGGSLRLSCAASGRTLSNYDMGWFROAPGKEREFW EVOLVESGGGLVOAGGSLRLACAASGSIFSFNAMGWYROAPGKORELL ATIRWSTSSTYYADSWKGRFTISSDNTKNTVDLRMNSLTPEDTAWYYCAA AVITRDGITKYADSVKGRFTISRDNAKNTMYLOMNSLKPEDTAVYYCHTG RSVYSYEYNYWGOGTOVTVSS LPTGRGSHSDYWGOGTOVTVSS LGPMP5 B6 SEQ ID NO 415 LGPMP6 H9 SEQ ID NO 429 EVOLVESGGGLVOAGGSLRLSCAASGRTLTNYDMGWFROAPGKEREFW EVOLVESGGGLVOAGGSLRLSCAASGRTLNDHDWGWFROAPGKEREFW ATIRWSTSETYYADSVKGRFTISKDNTKNTVALOMNSLTPEDTALYYCAA 65 ATIRWSSKSTYYADSWKGRFTISSDNTKNTVDLRMNSLTPEDTAWYYCAA RSVYSYEYNYWGOGTOVTVSS RSVYSYEYNYWGOGTOVTVSS

US 9,512,236 B2 145 146 TABLE B- 4 - continued TABLE C-continued Nanobodies against some representative GPCRs. non-limiting list of Some therapeutically relevant SEQ ID NOS 413 to 43 O, 517 and 518 : Nanobodies GPCRs (and desired action of an amino acid sequence, against CB1R a Nanobody or a polypeptide of the invention). SEQ ID NO's 431 to 453 : Nanobodies against PTHR1 5 SEQ ID NO's 519 to 525: Nanobodies against MCR4 5-HT2 (agonist), 5-HT 4 (agonist), 5-HT 5a (agonist), 58-4H SEQ ID NO 521 5-HT 5b (agonist) EVOLVESGGGLAOPGGSLRLSCWASGFSLADYAISWFROAPGKVREGWS 5-HT6 (agonist), CISSSDGSTYYADSVKGRFTISRDNAKNAVYLOMSTLKPEDTAVYYCAAE 10 5-HT7 (agonist), RSCLSSDYDSYDAWGOGTOVTVSS Trace amine-associated receptor (agonist), Trace amine-associated receptor-1 (agonist), 60-5E SEQ ID NO 522 Trace amine-associated receptor-2 (agonist) EVOLVESGGGLVOPGGSLRLSCAASGFTLDDYAIGWFROAPGKEREGWS Trace amine-associated receptor-3 (agonist) CISSSDGSTYYADSVKGRFTISRDNAKNTVYLOMNSLKPEDTAVYYCAAD Trace amine-associated receptor-4 (agonist) LGYGSNCLEYDFWGOGTOVTVSS 15 Trace amine-associated receptor-5 (agonist) Trace amine-associated receptor-6 (agonist) 60 - 4A SEQ ID NO 523 Trace amine-associated receptor-7 (agonist) EVOLVESGGGLVOAGGSLRLSCAASGFTFDEYAIGWFROAPGKEREGWS Trace amine-associated receptor-8 (agonist) CISRSDGSTYYAPSVKGRFTISSDNAKNTVYLOMNSLKPEDTAVYYCAVD Trace amine-associated receptor-9 (agonist) LEYGSNCYEYDSWGOGTOVTVSS (agonist), Cannabinoid receptor (agonist), 58-1OH SEQ ID NO 524 2O Cannabinoid CB1 receptor (agonist), EVOLVESGGGLVOPGGSMRLSCAASGSSLDAYGLGWFROAPGKEREGW Cannabinoid CB2 receptor (agonist), SCISGSDNSTYYADSVKGRFSISRDIAKNTVYLOMNGLKPEDTAVYYCAA SEE, E} (agonist) KFLGFRORSPTWFSRSWSEGSDYOYWGOGTOVTVSS Lysophosphatidate-1 receptor (agonist) Sphingosine-1-phosphate receptor-3 (agonist), 60-1OE SEQ ID NO 525 25 Lysophosphatidate-2 receptor (agonist) EVOLVESGGGLVOPGGSLRLSCAASGFTLDDYAIGWFROAPGKEREGWS Sphingosine-1-phosphate receptor-2 (agonist) CITSSDGSTYYADSVKGRFTISRDNAKNTVYLOINSLKPEDTAVYYCAAD Sphingosine-1-phosphate receptor-4 (agonist), LGVGTOCDEYDYWGOGTOVTVSS Lysophosphatidate-3 receptor (agonist) Sphingosine-1-phosphate receptor-5 (agonist) Class A hormone protein GPCR (agonist), 30 FSH (agonist), TABLE C Luteinizing hormone receptor (agonist), TSH (agonist), non-limiting list of Some therapeutically relevant Leukotriene (agonist), GPCRs (and desired action of an amino acid sequence, Leukotriene BLT receptor (agonist), a Nanobody or a polypeptide of the invention). Cysteinyl (agonist), 35 (agonist), Class A GPCRs Melatonin MT1 (agonist), Melatonin MT2 (agonist), (agonist), Melatonin MT3 (agonist) Muscarinic receptor (agonist), Class A nucleotide like GPCR (agonist), Muscarinic M1 receptor (agonist), (agonist), Muscarinic M2 receptor (agonist), 40 (agonist), Muscarinic M3 receptor (agonist), Class A orphan GPCR (agonist), Muscarinic M4 receptor (agonist), Ghrelin (agonist), Muscarinic M5 receptor (agonist) Class A peptide GPCR (agonist), Muscarinic receptor (partial agonist) Angiotensin receptor (agonist), Adrenoceptor (agonist), Angiotensin I receptor (agonist), Alpha adrenoceptor (agonist), Angiotensin II receptor (agonist), Alpha 1 adrenoceptor (agonist), 45 (agonist), Alpha 1A adrenoceptor (agonist), Bombesin BB1 receptor (agonist) Alpha 1B adrenoceptor (agonist) Bombesin BB2 receptor (agonist) Alpha 1D adrenoceptor (agonist) Bombesin bb3 receptor (agonist), Alpha 2 adrenoceptor (agonist), Gastrin releasing peptide ligand, Alpha 2A adrenoceptor (agonist), Neuromedin B ligand Alpha 2B adrenoceptor (agonist), 50 Neuromedin C ligand Alpha 2C adrenoceptor (agonist), (agonist), Alpha 2 adrenoceptor (partial agonist) Bradykinin B1 receptor (agonist), Alpha 3 adrenoceptor (agonist), Bradykinin B2 receptor (agonist), Beta adrenoceptor (agonist), receptor (agonist), Beta 1 adrenoceptor (agonist), C5a (agonist), Beta 2 adrenoceptor (agonist), 55 CCK receptor (agonist), Beta 3 adrenoceptor (agonist), CCK 1 receptor (agonist), (agonist), CCK 2 receptor (agonist), Dopamine D5 receptor (agonist) Gastrin (agonist), Dopamine D1 receptor (agonist), Chemokine (agonist), Dopamine D2 receptor (agonist), CC chemokine receptor (agonist), Dopamine D3 receptor (agonist), CCR1 chemokine (agonist), Dopamine D4 receptor (agonist), 60 CCR2 chemokine (agonist), (agonist), CCR3 chemokine (agonist), (agonist), CCR4 chemokine (agonist), (agonist), CCR5 chemokine (agonist), (agonist), CCR6 chemokine (agonist), (agonist), 65 CCRT chemokine (agonist) 5-HT GPCR (agonist), CCR8 chemokine (agonist), 5-HT 1 (agonist), CCR9 chemokine (agonist) US 9,512,236 B2 147 148 TABLE C-continued TABLE C-continued non-limiting list of Some therapeutically relevant non-limiting list of Some therapeutically relevant GPCRs (and desired action of an amino acid sequence, GPCRs (and desired action of an amino acid sequence, a Nanobody or a polypeptide of the invention). a Nanobody or a polypeptide of the invention). CCR10 chemokine (agonist), Vasopressin V1 (agonist), CCR11 chemokine (agonist) Vasopressin V2 (agonist), CX3C chemokine receptor (agonist), Prostanoid receptor (agonist), CX3CR1 chemokine (agonist), DP prostanoid (agonist), XCR1 chemokine (agonist) PGD2 (agonist), CXC chemokine receptor (agonist), 10 EP1 prostanoid (agonist), CXCR1 chemokine (agonist) PGE2 (agonist), CXCR3 chemokine (agonist), EP2 prostanoid (agonist), CXCR4 chemokine (agonist), PGE2 (agonist), CXCR5 chemokine (agonist) EP3 prostanoid (agonist), Adrenomedulin receptor (agonist), PGE2 (agonist), Endothelin (agonist), 15 EP4 prostanoid (agonist), Endothelin ET-A (agonist), PGE2 (agonist), Endothelin ET-B (agonist), FP prostanoid (agonist), Galanin (agonist), PGF2 alpha (agonist), Galanin GAL1 (agonist), P prostanoid (agonist), Galanin GAL2 (agonist), Prostacyclin (agonist), Galanin GAL3 (agonist) Prostanoid receptor (partial agonist) IL-9 (agonist), TP prostanoid (agonist), KiSS-1 receptor (agonist), Thromboxane A2 (agonist) Melanin concentrating hormone (agonist), Succinate receptor 1 (agonist) MCH receptor-1 (agonist) TRH (agonist), MCH receptor-2 (agonist) TRH1 (agonist) Melanocortin (agonist), TRH2 (agonist) Melanocortin MC1 (agonist), 25 Vomeronasal type-1 receptor (agonist) ACTH receptor (agonist), Vomeronasal type-1 receptor-1 (agonist) Melanocortin MC3 (agonist), Vomeronasal type-1 receptor-2 (agonist) Melanocortin MC4 (agonist), Vomeronasal type-1 receptor-3 (agonist) Melanocortin MC5 (agonist), Vomeronasal type-1 receptor-4 (agonist) NK (agonist), Vomeronasal type-1 receptor-5 (agonist) NK1 (agonist), 30 Apelin receptor (modulator), NK2 (agonist) Cannabinoid receptor (modulator), NK3 (agonist), Drugs: 1 Chemokine receptor-like 1 (modulator), Neuropeptide Y receptor (agonist), Lysosphingolipid receptor (modulator), Neuropeptide Y1 receptor (agonist) Class A hormone protein GPCR (modulator), Neuropeptide Y2 receptor (agonist), Leukotriene receptor (modulator), Neuropeptide Y4 receptor (agonist), 35 (modulator), Neuropeptide Y5 receptor (agonist), Class A nucleotide like GPCR (modulator), Neuropeptide Y6 receptor (agonist) Class A orphan GPCR (modulator), Neurotensin receptor (agonist), PAF receptor (modulator), Neurotensin NTS1 (agonist), Class A peptide GPCR (modulator), Neurotensin NTS2 (agonist) Prostanoid receptor (modulator), Orexin & neuropeptide FF receptor (agonist), Succinate receptor 1 (modulator) Orexin (agonist), 40 TRH receptor (modulator), Opioid (agonist), Vomeronasal type-1 receptor (modulator), Delta Opioid (agonist), Class B GPCRS Kappa Opioid (agonist), Mu. Opioid (agonist), -protein coupled rece ptor-3 (modulator), ORL1 receptor (agonist), -protein coupled rece or-3 (agonist) Opioid (partial agonist) 45 -protein coupled receptor-3 (antagonist), Sigma Opioid (agonist) -protein coupled receptor-6 (modulator), Orexin & neuropeptide FF receptor (agonist), -protein coupled receptor-6 (agonist) Neuropeptide FF receptor (agonist), -protein coupled receptor-6 (antagonist), Neuropeptide FF1 receptor (agonist) -protein coupled receptor-12 (modulator), Neuropeptide FF2 receptor (agonist), -protein coupled receptor-12 (agonist) Orexin (agonist), 50 -protein coupled receptor-12 (antagonist), Orexin-1 (agonist) -protein coupled receptor-14 (modulator) Orexin-2 (agonist) -protein coupled receptor-14 (agonist) Protease-activated receptor (agonist), -protein coupled receptor-14 (antagonist) Protease-activated receptor-1 (agonist), Class B GPCR (agonist), Protease-activated receptor-2 (agonist), CRF-1 receptor (agonist) Protease-activated receptor-3 (agonist) 55 CRF-2 receptor (agonist), Protease-activated receptor-4 (agonist) (modulator), receptor (agonist), Calcitonin (agonist), -1 (agonist), Calcitonin (antagonist), Prokineticin receptor-2 (agonist), ACTH releasing factor receptor (modulator), Somatostatin (agonist), CRF-1 receptor (modulator), Somatostatin 1 (agonist), CRF-1 receptor (agonist) Somatostatin 2 (agonist), 60 CRF-1 receptor (antagonist), Somatostatin 3 (agonist), CRF-2 receptor (modulator), Somatostatin 4 (agonist), CRF-2 receptor (agonist), Somatostatin 5 (agonist), CRF-2 receptor (antagonist), Urotensin II (agonist), ACTH releasing factor (agonist), Vasopressin like receptor (agonist), CRF-1 receptor (agonist) Oxytocin (agonist), 65 CRF-2 receptor (agonist), Vasopressin (agonist), ACTH releasing factor (antagonist), US 9,512,236 B2 149 150 TABLE C-continued TABLE C-continued non-limiting list of Some therapeutically relevant non-limiting list of Some therapeutically relevant GPCRs (and desired action of an amino acid sequence, GPCRs (and desired action of an amino acid sequence, a Nanobody or a polypeptide of the invention). 5 a Nanobody or a polypeptide of the invention). CRF-1 receptor (antagonist), Secretin (agonist), CRF-2 receptor (antagonist), Secretin (antagonist) Glucagon-like peptide receptor (modulator), VIP receptor (modulator), Glucagon-like peptide 1 receptor (modulator), VIP-1 receptor (modulator), Glucagon-like peptide 2 receptor (modulator), VIP-2 receptor (modulator), Glucagon-like peptide (agonist), 10 VIP (agonist), Glucagon-like peptide (antagonist), VIP (antagonist), (modulator), Class C GPCRs Glucagon (agonist), Glucagon (antagonist), Class C GPCR (modulator), GHRH receptor (modulator), Class C GPCR (agonist), GHRH (agonist), 15 GABAB receptor (agonist), Growth hormone releasing factor (antagonist), Metabotropic glutamate receptor (agonist), PACAP type I receptor (modulator), Metabotropic glutamate receptor 1 (agonist), PACAP type I receptor (agonist), Metabotropic glutamate receptor 2 (agonist), PACAP type I receptor (antagonist) Metabotropic glutamate receptor 3 (agonist), PTH receptor (modulator), Metabotropic glutamate receptor 4 (agonist), PTH-1 receptor (modulator) Metabotropic glutamate receptor 5 (agonist), PTH-2 receptor (modulator) 2O Metabotropic glutamate receptor 6 (agonist) PTH (agonist), Metabotropic glutamate receptor 7 (agonist) PTH (antagonist), Metabotropic glutamate receptor 8 (agonist) (modulator),

TABLED non-limiting list human GPCRs SHT1A HUMAN (P08908) HTR1A Serotonin type 1 Homo sapiens (Human) SHT1B HUMAN (P28222) HTR1B Serotonin type 1 Homo sapiens (Human) SHT1D HUMAN (P28221) HTR1D Serotonin type 1 Homo sapiens (Human) SHT1E HUMAN (P28566) HTR1E Serotonin type 1 Homo sapiens (Human) SHT1F HUMAN (P30939) HTR1F Serotonin type 1 Homo sapiens (Human) SHT2A HUMAN (P28223) HTR2A Serotonin type 2 Homo sapiens (Human) SHT2B HUMAN (P41595) HTR2B Serotonin type 2 Homo sapiens (Human) SHT2C HUMAN (P28335) HTR2C Serotonin type 2 Homo sapiens (Human) SHT4R HUMAN (Q13639) HTR4 Serotonin type 4 Homo sapiens (Human) SHTSA HUMAN (P47898) HTRSA Serotonin type 5 Homo sapiens (Human) SHT6R HUMAN (P50406) HTR6 Serotonin type 6 Homo sapiens (Human) 5HT7R HUMAN (P34969) HTR7 Serotonin type 7 Homo sapiens (Human) AA1R HUMAN (P30542) ADORA1 Adenosine type 1 Homo sapiens (Human) AA2AR HUMAN (P29274) ADORA2A Adenosine type 2 Homo sapiens (Human) AA2BR HUMAN (P29275) ADORA2B Adenosine type 2 Homo sapiens (Human) AA3R HUMAN (P33765) ADORA3 Adenosine type 3 Homo sapiens (Human) ACM1 HUMAN (P11229) CHRM1 Musc. acetylcholine Vertebrate type 1 Homo sapiens (Human) ACM2 HUMAN (P08172) CHRM2 Musc. acetylcholine Vertebrate type 2 Homo sapiens (Human) ACM3 HUMAN (P20309) CHRM3 Musc. acetylcholine Vertebrate type 3 Homo sapiens (Human) ACM4 HUMAN (P08173) CHRM4 Musc. acetylcholine Vertebrate type 4 Homo sapiens (Human) ACMS HUMAN (P08912) CHRMS Musc. acetylcholine Vertebrate type 5 Homo sapiens (Human) ACTHRHUMAN (Q01718) MC2R Adrenocorticotropic hormone Homo sapiens (Human) ADA1A HUMAN (P35348) ADRA1A Alpha Adrenoceptors type 1 Homo sapiens (Human) ADA1B. HUMAN (P35368) ADRA1B Alpha Adrenoceptors type 1 Homo sapiens (Human) ADA1D HUMAN (P25100) ADRA1D Alpha Adrenoceptors type 1 Homo sapiens (Human) ADA2A HUMAN (PO8913) ADRA2A Alpha Adrenoceptors type 2 Homo sapiens (Human) ADA2B HUMAN (P18089) ADRA2B Alpha Adrenoceptors type 2 Homo sapiens (Human) ADA2C HUMAN (P18825) ADRA2C Alpha Adrenoceptors type 2 Homo sapiens (Human) ADMIR HUMAN (O15218) ADMIR Adrenomedullin (G10D) Homo sapiens (Human) ADRB1 HUMAN (PO8588) ADRB Beta Adrenoceptors type 1 Homo sapiens (Human) ADRB2 HUMAN (P07550) ADRB2 Beta Adrenoceptors type 2 Homo sapiens (Human) ADRB3 HUMAN (P13945) ADRB3 Beta Adrenoceptors type 3 Homo sapiens (Human) AGTR1 HUMAN (P30556) AGTR Angiotensin type 1 Homo sapiens (Human) AGTR2 HUMAN (P50052) AGTR2 Angiotensin type 2 Homo sapiens (Human) APJ HUMAN (P35414) AGTRL1 APJ like Homo sapiens (Human) BAI1. HUMAN (O14514) BAI1 Brain-specific angiogenesis inhibitor (BAI) Homo sapiens (Human) BAI2 HUMAN (O60241) BAI2 Brain-specific angiogenesis inhibitor (BAI) Homo sapiens (Human) BAI3 HUMAN (O60242) BAI3 Brain-specific angiogenesis inhibitor (BAI) Homo sapiens (Human) BKRB1 HUMAN (P46663) BDKRB1 Bradykinin Homo sapiens (Human) BKRB2 HUMAN (P30411) BDKRB2 Bradykinin Homo sapiens (Human) BRS3 HUMAN (P32247) BRS3 Bombesin Homo sapiens (Human) C3AR HUMAN (Q16581) C3AR1 C5a anaphylatoxin Homo sapiens (Human) CSARL HUMAN (Q9P296) GPR77 C5a anaphylatoxin Homo sapiens (Human) CSAR HUMAN (P21730) CSAR1 C5a anaphylatoxin Homo sapiens (Human) CALCR HUMAN (P30988) CALCR Calcitonin Homo sapiens (Human) CALRL HUMAN (Q16602) CALCRL Calcitonin Homo sapiens (Human) CASR HUMAN (P41180) CASR Extracellular calcium-sensing Homo sapiens (Human) US 9,512,236 B2 151 152 TABLE D-continued non-limiting list human GPCRs CCBP2 HUMAN (O00590) CCBP2 C-C Chemokine type X Homo sapiens ( uman) CCKAR HUMAN (P32238) CCKAR CCK type A Homo sapiens ( uman) CCR1O HUMAN (P46092) CCR1O C-C Chemokine type 10 Homo sapiens ( uman) CCR1. HUMAN (P32246) CCR1 C-C Chemokine type 1 Homo sapiens ( uman) CC UMAN (P41597) CCR2 C-C Chemokine type 2 Homo sapiens ( uman) CC UMAN (P51677) CCR3 C-C Chemokine type 3 Homo sapiens ( uman) CC UMAN (P51679) CCR4 C-C Chemokine type 4 Homo sapiens ( uman) CC UMAN (P51681) CCR5 C-C Chemokine type 5 Homo sapiens ( uman) CC 6 UMAN (P51684) CCR6 C-C Chemokine type 6 Homo sapiens ( uman) CC UMAN (P32248) CCRT C-C Chemokine type 7 Homo sapiens ( uman) CC UMAN (P51685) CCR8 C-C Chemokine type 8 Homo sapiens ( uman) CCR9 HUMAN (P51686) CCR9 C-C Chemokine type 9 Homo sapiens ( uman) CCRL1 HUMAN (Q9NPB9) CCRL1 C-C Chemokine type 11 Homo sapiens ( uman) CD97 HUMAN (P48960) CD97 EMR1 Homo sapiens ( uman) CELR1 HUMAN (Q9NYQ6) CELSR1 Cadherin EGF LAG (CELSR) Homo sapiens ( uman) CELR2 HUMAN (Q9HCU4) CELSR2 Cadherin EGF LAG (CELSR) Homo sapiens ( uman) CELR3 HUMAN (Q9NYQ7) CELSR3 Cadherin EGF LAG (CELSR) Homo sapiens ( uman) CLTR1. HUMAN (Q9Y271) CYSLTR1 Cysteinyl leukotriene Homo sapiens ( uman) CLTR2 HUMAN (Q9NS75) CYSLTR2 Cysteinyl leukotriene Homo sapiens ( uman) CML1 HUMAN (Q99788) CMKLR1 Chemokine receptor-like 1 Homo sapiens ( uman) CML2 HUMAN (Q99527) GPR30 Chemokine receptor-like 2 Homo sapiens ( uman) CNR1 HUMAN (P21554) CNR1 Cannabinoid Homo sapiens ( uman) CNR2 HUMAN (P34972) CNR2 Cannabinoid Homo sapiens ( uman) CRFR1 HUMAN (P34998) CRHR1 Corticotropin releasing factor Homo sapiens ( uman) CRFR2 HUMAN (Q13324) CRHR2 Corticotropin releasing factor Homo sapiens ( uman) CX3C1 HUMAN (P49238) CX3CR1 C—X3–C Chemokine Homo sapiens ( uman) CXCR1 HUMAN (P25024) L8RA Interleukin-8 type A Homo sapiens ( uman) CXCR3 HUMAN (P49682) CXCR3 C—X-C Chemokine type 3 Homo sapiens ( uman) CXCR4 HUMAN (P61073) CXCR4 C—X-C Chemokine type 4 Homo sapiens ( uman) CXCR5 HUMAN (P32302) BLR1 C—X-C Chemokine type 5 Homo sapiens ( uman) CXCR6 HUMAN (O00574) CXCR6 C—X-C Chemokine type 6 (Bonzo) Homo sapiens ( uman) DRD1 HUMAN (P21728) DRD1 Dopamine Vertebrate type 1 Homo sapiens ( uman) DRD2 HUMAN (P14416) DRD2 Dopamine Vertebrate type 2 Homo sapiens ( uman) DRD3 HUMAN (P35462) DRD3 Dopamine Vertebrate type 3 Homo sapiens ( uman) DRD4 HUMAN (P21917) DRD4 Dopamine Vertebrate type 4 Homo sapiens ( uman) DRDS HUMAN (P21918) DRDS Dopamine Vertebrate type 1 Homo sapiens ( uman) DUFFY HUMAN (Q16570) DARC Duffy antigen Homo sapiens ( uman) EBI2 HUMAN (P32249) EBI2 EBV-induced Homo sapiens ( uman) EDG1 HUMAN (P21453) EDG1 Sphingosine 1-phosphate Edg-1 Homo sapiens ( uman) EDG2 HUMAN (Q92633) EDG2 Edg-2 Homo sapiens ( uman) EDG3 HUMAN (Q995.00) EDG3 Sphingosine 1-phosphate Edg-3 Homo sapiens ( uman) EDG4 HUMAN (Q9HBWO) EDG4 Lysophosphatidic acid Edg-4 Homo sapiens ( uman) EDGS HUMAN (O95136) EDG5 Sphingosine 1-phosphate Edg-5 Homo sapiens ( uman) EDG6 HUMAN (O95.977) EDG6 Sphingosine 1-phosphate Edg-6 Homo sapiens ( uman) EDG7 HUMAN (Q9UBYS) EDG7 Lysophosphatidic acid Edg-7 Homo sapiens ( uman) EDG8 HUMAN (Q9H228) EDG8 Sphingosine 1-phosphate Edg-8 Homo sapiens ( uman) EDNRA HUMAN (P25101) EDNRA Endothelin Homo sapiens ( uman) EDNRB. HUMAN (P24530) EDNRB Endothelin Homo sapiens ( uman) ELTD1 HUMAN (Q9HBW9) ELTD1 ETL receptors Homo sapiens ( uman) EMR1 HUMAN (Q14246) EMR1 EMR1 Homo sapiens ( uman) EMR2 HUMAN (Q9UHX3) EMR2 EMR1 Homo sapiens ( uman) EMR3 HUMAN (Q9BY15) EMR3 EMR1 Homo sapiens ( uman) EMR4 HUMAN (Q86SQ3) EMR4 ragments Homo sapiens ( uman) ETBR2 HUMAN (O60883) GPR37L1 GPR37 endothelin B-like Homo sapiens ( uman) FFAR1 HUMAN (O14842) FFAR1 (GP40, GP41, GP43) Homo sapiens ( uman) FFAR2 HUMAN (O15552) FFAR2 Free fatty acid receptor (GP40, GP41, GP43) Homo sapiens ( uman) FFAR3 HUMAN (O14843) FFAR3 Free fatty acid receptor (GP40, GP41, GP43) Homo sapiens ( uman) FPR1 HUMAN (P21462) FPR1 Fmet-leu-phe Homo sapiens ( uman) FPRL1 HUMAN (P25090) FPRL1 Fmet-leu-phe Homo sapiens ( uman) FPRL2 HUMAN (P25089) FPRL2 Fmet-leu-phe Homo sapiens ( uman) FSHR HUMAN (P23945) FSHR Follicle stimulating hormone Homo sapiens ( uman) FZD10 HUMAN (Q9ULW2) FZD10 rizzled Group A (FZ 1&2&4&5&7-9) Homo sapiens ( uman) FZD1 HUMAN (Q9UP38) FZD1 rizzled Group A (FZ 1&2&4&5&7-9) Homo sapiens ( uman) FZD2 HUMAN (Q14332) FZD2 rizzled Group A (FZ 1&2&4&5&7-9) Homo sapiens ( uman) FZD3 HUMAN (Q9NPG1) FZD3 rizzled Group B (FZ 3 & 6) Homo sapiens ( uman) FZD4 HUMAN (Q9ULV1) FZD4 rizzled Group A (FZ 1&2&4&5&7-9) Homo sapiens ( uman) FZD5 HUMAN (Q13467) FZD5 rizzled Group A (FZ 1&2&4&5&7-9) Homo sapiens ( uman) FZD6 HUMAN (O60353) FZD6 rizzled Group B (FZ 3 & 6) Homo sapiens ( uman) FZD7 HUMAN (O75084) FZD7 rizzled Group A (FZ 1&2&4&5&7-9) Homo sapiens ( uman) FZD8 HUMAN (Q9H461) FZD8 rizzled Group A (FZ 1&2&4&5&7-9) Homo sapiens ( uman) FZD9 HUMAN (OOO144) FZD9 rizzled Group A (FZ 1&2&4&5&7-9) Homo sapiens ( uman) G109A HUMAN (Q8TDS4) GPR109A Putativefunclassified Class A GPCRs Homo sapiens ( uman) G109B. HUMAN (P49019) GPR109B Putativefunclassified Class A GPCRs Homo sapiens ( uman) GABR1 HUMAN (Q9UBS5) GABBR1 GABA-B subtype 1 Homo sapiens ( uman) GABR2 HUMAN (O75899) GABBR2 GABA-B subtype 2 Homo sapiens ( uman) GALR1 HUMAN (P47211) GALR1 Galanin Homo sapiens ( uman) GALR2 HUMAN (O43603) GALR2 Galanin Homo sapiens ( uman) GALR3 HUMAN (O60755) GALR3 Galanin Homo sapiens ( uman) US 9,512,236 B2 153 154 TABLE D-continued non-limiting list human GPCRs GASR HUMAN CC R CCK type B Homo sapiens ( uman) GHRHR HUMAN R Growth hormone-releasing hormone Homo sapiens ( uman) GHSR HUMAN t RB Growth hormone secretagogue Homo sapiens ( uman) GIPR HUMAN Gastric inhibitory peptide Homo sapiens ( uman) GLP1R HUMAN Glucagon Homo sapiens ( uman) GLP2R HUMAN Glucagon Homo sapiens ( uman) GLR HUMAN Glucagon Homo sapiens ( uman) GNRHR HUMAN s Gonadotropin-releasing hormone type I Homo sapiens ( uman) GNRR2 HUMAN N HR 2 Gonadotropin-releasing hormone type II Homo sapiens ( uman) GP101 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GP107 HUMAN Putativefunclassified other Homo sapiens ( uman) GP110 HUMAN Putativefunclassified Class B GPCRs Homo sapiens ( uman) GP111 HUMAN Putativefunclassified Class B GPCRs Homo sapiens ( uman) GP112 HUMAN Putativefunclassified Class B GPCRs Homo sapiens ( uman) GP113 HUMAN Putativefunclassified Class B GPCRs Homo sapiens ( uman) GP114 HUMAN Putativefunclassified Class B GPCRs Homo sapiens ( uman) GP115 HUMAN Putativefunclassified Class B GPCRs Homo sapiens ( uman) GP116 HUMAN Putativefunclassified Class B GPCRs Homo sapiens ( uman) GP119 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GP120 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GP123 HUMAN Putativefunclassified Class B GPCRs Homo sapiens ( uman) GP124 HUMAN Putativefunclassified Class B GPCRs Homo sapiens ( uman) GP125 HUMAN Putativefunclassified Class B GPCRs Homo sapiens ( uman) GP126 HUMAN Putativefunclassified Class B GPCRs Homo sapiens ( uman) GP128 HUMAN Putativefunclassified Class B GPCRs Homo sapiens ( uman) GP132 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GP133 HUMAN Putativefunclassified Class B GPCRs Homo sapiens ( uman) GP135 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GP139 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GP141 HUMAN 4 1 Putativefunclassified Class A GPCRs Homo sapiens ( uman) GP142 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GP143 HUMAN Ocular albinism proteins Homo sapiens ( uman) GP144 HUMAN Putativefunclassified Class B GPCRs Homo sapiens ( uman) GP146 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GP148 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GP149 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GP150 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GP151 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GP152 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GP153 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GP154 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GP155 HUMAN Putativefunclassified other Homo sapiens ( uman) GP156 HUMAN GABA-B like Homo sapiens ( uman) GP157 HUMAN Putativefunclassified Class B GPCRs Homo sapiens ( uman) GP158 HUMAN Putativefunclassified Class C GPCRs Homo sapiens ( uman) GP160 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GP161 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GP162 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GP171 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GP173 HUMAN SREB Homo sapiens ( uman) GP174 HUMAN Purinoceptor P2RY5,8,9,10 GPR35,92,174 Homo sapiens ( uman) GP175 HUMAN Putativefunclassified other Homo sapiens ( uman) GP176 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GP179 HUMAN Putativefunclassified Class C GPCRs Homo sapiens ( uman) GPBAR HUMAN G-protein coupled bile acid receptor Homo sapiens ( uman) GPC5B. HUMAN Orphan GPRCS Homo sapiens ( uman) GPC5C HUMAN Orphan GPRCS Homo sapiens ( uman) GPCSD HUMAN Orphan GPRCS Homo sapiens ( uman) GPC6A HUMAN Orphan GPCR6 Homo sapiens ( uman) GPR12 HUMAN GPR Homo sapiens ( uman) GPR15 HUMAN GPR Homo sapiens ( uman) GPR17 HUMAN GPR Homo sapiens ( uman) GPR18 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GPR19 HUMAN GPR Homo sapiens ( uman) GPR1 HUMAN GPR Homo sapiens ( uman) GPR2O HUMAN GPR Homo sapiens ( uman) GPR21 HUMAN GPR Homo sapiens ( uman) GPR22 HUMAN GPR Homo sapiens ( uman) GPR25 HUMAN GPR Homo sapiens ( uman) GPR26 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GPR27 HUMAN SREB Homo sapiens ( uman) GPR31. HUMAN GPR Homo sapiens ( uman) GPR32 HUMAN Chemokine receptor-like 1 Homo sapiens ( uman) GPR33 HUMAN Chemokine receptor-like 1 Homo sapiens ( uman) GPR34 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GPR35 HUMAN Purinoceptor P2RY5,8,9,10 GPR35,92,174 Homo sapiens ( uman) GPR37 HUMAN GPR37 endothelin B-like Homo sapiens ( uman) GPR39 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) US 9,512,236 B2 155 156 TABLE D-continued non-limiting list human GPCRs GPR3 HUMAN GPR Homo sapiens ( uman) GPR42 HUMAN R42 Free fatty acid receptor (GP40, GP41, GP43) Homo sapiens ( uman) GPR44 HUMAN R44 Chemokine receptor-like 1 Homo sapiens ( uman) GPR45. HUMAN R45 GPR45 like Homo sapiens ( uman) GPR4 HUMAN GPR Homo sapiens ( uman) GPR52 HUMAN GPR Homo sapiens ( uman) GPR55 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GPR56 HUMAN Putativefunclassified Class B GPCRs Homo sapiens ( uman) GPR61 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GPR62 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) GPR63 HUMAN GPR45 like Homo sapiens ( uman) GPR64 HUMAN Putativefunclassified Class B GPCRs Homo sapiens ( uman) GPR6 HUMAN GPR Homo sapiens ( uman) GPR75 HUMAN R75 Putativefunclassified Class A GPCRs Homo sapiens ( uman) GPR78 HUMAN R78 Putativefunclassified Class A GPCRs Homo sapiens ( uman) GPR81 HUMAN R81 Putativefunclassified Class A GPCRs Homo sapiens ( uman) GPR82 HUMAN R82 Putativefunclassified Class A GPCRs Homo sapiens ( uman) GPR83 HUMAN R83 Neuropeptide Y other Homo sapiens ( uman) GPR84 HUMAN R84 Putativefunclassified Class A GPCRs Homo sapiens ( uman) GPR85 HUMAN R8S SREB Homo sapiens ( uman) GPR87 HUMAN R87 Purinoceptor P2RY12-14 GPR87 (UDP-Glucose) Homo sapiens ( uman) GPR88 HUMAN R88 Putativefunclassified Class A GPCRs Homo sapiens ( uman) GPR92 HUMAN R92 Purinoceptor P2RY5,8,9,10 GPR35,92,174 Homo sapiens ( uman) GPR97 HUMAN PR97 Putativefunclassified Class B GPCRs Homo sapiens ( uman) GRPR HUMAN RPR Bombesin Homo sapiens ( uman) HRH1 HUMAN RH1 Histamine type 1 Homo sapiens ( uman) HRH2 HUMAN RH2 Histamine type 2 Homo sapiens ( uman) HRH3 HUMAN RH3 Histamine type 3 Homo sapiens ( uman) HRH4 HUMAN RH4 Histamine type 4 Homo sapiens ( uman) KISSR HUMAN SS1R Kiss receptor (GPR54) Homo sapiens ( uman) LGR4 HUMAN LGR4 LGR like (hormone receptors) Homo sapiens ( uman) LGR5 HUMAN LGRS LGR like (hormone receptors) Homo sapiens ( uman) LGR6 HUMAN LGR6 LGR like (hormone receptors) Homo sapiens ( uman) LGR8 HUMAN LGR8 LGR like (hormone receptors) Homo sapiens ( uman) LPHN1 HUMAN LPHN1 type 1 Homo sapiens ( uman) LPHN2 HUMAN LPHN2 Latrophilin type 2 Homo sapiens ( uman) LPHN3 HUMAN LPHN3 Latrophilin type 3 Homo sapiens ( uman) LSHRHUMAN LHCGR. Lutropin-choriogonadotropic hormone Homo sapiens ( uman) LT4R1 HUMAN LTB4R receptor BLT1 Homo sapiens ( uman) LT4R2 HUMAN LTB4R2 BLT2 Homo sapiens ( uman) MAS1L HUMAN MAS1L. Mas proto-oncogene & Mas-related (MRGs) Homo sapiens ( uman) MASS1 HUMAN MASS1 Very large G-protein coupled receptor Homo sapiens ( uman) MAS HUMAN MAS1 Mas proto-oncogene & Mas-related (MRGs) Homo sapiens ( uman) MC3R HUMAN MC3R Melanocortin hormone Homo sapiens ( uman) MC4R HUMAN MC4R, Melanocortin hormone Homo sapiens ( uman) MC5R HUMAN MCSR Melanocortin hormone Homo sapiens ( uman) MCHR1 HUMAN MCHR1 Melanin-concentrating hormone receptors Homo sapiens ( uman) MCHR2 HUMAN MCHR2 Melanin-concentrating hormone receptors Homo sapiens ( uman) MGR1 HUMAN GRM1 Metabotropic glutamate group Homo sapiens ( uman) MGR2 HUMAN GRM2 Metabotropic glutamate group II Homo sapiens ( uman) MGR3 HUMAN GRM3 Metabotropic glutamate group II Homo sapiens ( uman) MGR4 HUMAN GRM4 Metabotropic glutamate group III Homo sapiens ( uman) MGR5 HUMAN GRMS Metabotropic glutamate group Homo sapiens ( uman) MGR6 HUMAN GRM6 Metabotropic glutamate group III Homo sapiens ( uman) MGR7 HUMAN GRM7 Metabotropic glutamate group III Homo sapiens ( uman) MGR8 HUMAN GRM8 Metabotropic glutamate group III Homo sapiens ( uman) MRGRD HUMAN MRGPRD Mas proto-oncogene & Mas-related (MRGs) Homo sapiens ( uman) MRGRE HUMAN MRGPRE fragments Homo sapiens ( uman) MRGRF HUMAN MRGPRF Putativefunclassified Class A GPCRs Homo sapiens ( uman) MRGRG HUMAN MRGPRG fragments Homo sapiens ( uman) MRGX1 HUMAN MRGPRX1 Mas proto-oncogene & Mas-related (MRGs) Homo sapiens ( uman) MRGX2 HUMAN MRGPRX2 Mas proto-oncogene & Mas-related (MRGs) Homo sapiens ( uman) MRGX3 HUMAN MRGPRX3 Mas proto-oncogene & Mas-related (MRGs) Homo sapiens ( uman) MRGX4 HUMAN MRGPRX4 Mas proto-oncogene & Mas-related (MRGs) Homo sapiens ( uman) MSHR HUMAN MC1R Melanocyte stimulating hormone Homo sapiens ( uman) MTLR HUMAN MLNR Growth hormone secretagogue like Homo sapiens ( uman) MTR1A HUMAN MTNR1A Melatonin Homo sapiens ( uman) MTR1B. HUMAN MTNR1B Melatonin Homo sapiens ( uman) MTR1L HUMAN GPR50 Melatonin Homo sapiens ( uman) NK1R HUMAN TACR1 Substance P (NK1) Homo sapiens ( uman) NK2R HUMAN TACR2 Substance K (NK2) Homo sapiens ( uman) NK3R HUMAN TACR3 Neuromedin K (NK3) Homo sapiens ( uman) NMBR HUMAN NMBR Bombesin Homo sapiens ( uman) NMUR1 HUMAN NMUR1 Neuromedin U Homo sapiens ( uman) NMUR2 HUMAN NMUR2 Neuromedin U Homo sapiens ( uman) NPBW1. HUMAN NPBWR1 GPR Homo sapiens ( uman) NPBW2 HUMAN NPBWR2 GPR Homo sapiens ( uman) NPFF1 HUMAN NPFFR1 Neuropeptide FF Homo sapiens ( uman) US 9,512,236 B2 157 158 TABLE D-continued non-limiting list human GPCRs NPFF2 HUMAN Neuropeptide FF Homo sapiens ( uman) NPY1R HUMAN Neuropeptide Y type 1 Homo sapiens ( uman) NPY2R HUMAN Neuropeptide Y type 2 Homo sapiens ( uman) NPY4R HUMAN Neuropeptide Y type 4 Homo sapiens ( uman) NPYSR HUMAN PYSR Neuropeptide Y type 5 Homo sapiens ( uman) NTR1. HUMAN TSR1 Neurotensin Homo sapiens ( uman) NTR2 HUMAN TSR2 Neurotensin Homo sapiens ( uman) OOO32S HUMAN Prostaglandin E2 subtype EP3 Homo sapiens ( uman) OOO421 HUMAN C C C-C Chemokine other Homo sapiens ( uman) O10A1 HUMAN fragments Homo sapiens ( uman) O1 OA3 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O10A4 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O10A5 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1OA6. HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1 OA7 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1 OAD HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1 OAG HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O10C1 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1 OD4 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1 OG2 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1 OG3 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1 OG4 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1 OG6 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1 OG7 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1 OG8 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1 OG9 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1 OH1 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1 OH2 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1 OH3 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1 OH4 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1 OHS HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1OJ1 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1OJ3 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1OJS HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1OJ6 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1OK1 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1OK2 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1OP1 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O10O1 HUMAN O Q Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1 OR2 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1OS1 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1OT2 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1OV1 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1OW1. HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1OX1 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O1OZ1 HUMAN Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) O11A1 HUMAN Olfactory II fam 11/MOR106,121-122 Homo sapiens ( uman) O11G2 HUMAN Olfactory II fam 11/MOR106,121-122 Homo sapiens ( uman) O11H1 HUMAN Olfactory II fam 11/MOR106,121-122 Homo sapiens ( uman) O11H4 HUMAN Olfactory II fam 11/MOR106,121-122 Homo sapiens ( uman) O11H6 HUMAN Olfactory II fam 11/MOR106,121-122 Homo sapiens ( uman) O11L1 HUMAN Olfactory II fam 11/MOR106,121-122 Homo sapiens ( uman) O12D2 HUMAN Olfactory II fam 12/MOR250 Homo sapiens ( uman) O12D3 HUMAN Olfactory II fam 12/MOR250 Homo sapiens ( uman) O13A1 HUMAN Olfactory II fam 13/MOR253 Homo sapiens ( uman) O13C2 HUMAN Olfactory II fam 13/MOR253 Homo sapiens ( uman) O13C3 HUMAN Olfactory II fam 13/MOR253 Homo sapiens ( uman) O13C4 HUMAN Olfactory II fam 13/MOR253 Homo sapiens ( uman) O13CS HUMAN Olfactory II fam 13/MOR253 Homo sapiens ( uman) O13C8 HUMAN Olfactory II fam 13/MOR253 Homo sapiens ( uman) O13C9 HUMAN Olfactory II fam 13/MOR253 Homo sapiens ( uman) O13D1 HUMAN Olfactory II fam 13/MOR253 Homo sapiens ( uman) O13F1 HUMAN Olfactory II fam 13/MOR253 Homo sapiens ( uman) O13G1 HUMAN Olfactory II fam 13/MOR253 Homo sapiens ( uman) O13H1 HUMAN Olfactory II fam 13/MOR253 Homo sapiens ( uman) O13J1 HUMAN Olfactory II fam 13/MOR253 Homo sapiens ( uman) O14694 HUMAN fragments Homo sapiens ( uman) O2A12 HUMAN Olfactory II fam 2/MOR256-262,270-285 Homo sapiens ( uman) O2A14 HUMAN Olfactory II fam 2/MOR256-262,270-285 Homo sapiens ( uman) O2A42 HUMAN Olfactory II fam 2/MOR256-262,270-285 Homo sapiens ( uman) O2AE1 HUMAN Olfactory II fam 2/MOR256-262,270-285 Homo sapiens ( uman) O2AG1 HUMAN Olfactory II fam 2/MOR256-262,270-285 Homo sapiens ( uman) O2AJ1 HUMAN Olfactory II fam 2/MOR256-262,270-285 Homo sapiens ( uman) O2AK2 HUMAN Olfactory II fam 2/MOR256-262,270-285 Homo sapiens ( uman) O2AP1 HUMAN Olfactory II fam 6/MOR103-105,107-119 Homo sapiens ( uman) O2T10 HUMAN Olfactory II fam 2/MOR256-262,270-285 Homo sapiens ( uman) O2T11 HUMAN Olfactory II fam 2/MOR256-262,270-285 Homo sapiens ( uman) O2T12 HUMAN Olfactory II fam 2/MOR256-262,270-285 Homo sapiens ( uman) US 9,512,236 B2 159 160 TABLE D-continued non-limiting list human GPCRs O2T27 HUMAN Olfactory II fam 2/MOR256-262,270-285 Homo sapiens ( uman) O2T29 HUMAN Olfactory II fam 2/MOR256-262,270-285 Homo sapiens ( uman) O2T33 HUMAN Olfactory II fam 2/MOR256-262,270-285 Homo sapiens ( uman) O2T34 HUMAN Olfactory II fam 2/MOR256-262,270-285 Homo sapiens ( uman) O2T35 HUMAN Olfactory II fam 2/MOR256-262,270-285 Homo sapiens ( uman) O43192 HUMAN Vasopressin type 2 Homo sapiens ( uman) O43200 HUMAN SHR ragments Homo sapiens ( uman) O43624 HUMAN ragments Homo sapiens ( uman) O43625 HUMAN ragments Homo sapiens ( uman) O43626 HUMAN ragments Homo sapiens ( uman) O43627 HUMAN ragments Homo sapiens ( uman) O43789 HUMAN ragments Homo sapiens ( uman) O43871 HUMAN R16-36 ragments Homo sapiens ( uman) O43872 HUMAN R16-37 ragments Homo sapiens ( uman) O43873 HUMAN R16-88 ragments Homo sapiens ( uman) O43874 HUMAN R16-89 ragments Homo sapiens ( uman) O43875 HUMAN R16-90 ragments Homo sapiens ( uman) O43876 HUMAN R17-130 ragments Homo sapiens ( uman) O43878 HUMAN R17-137 ragments Homo sapiens ( uman) O43879 HUMAN R17-15 ragments Homo sapiens ( uman) O43880 HUMAN R17-16 ragments Homo sapiens ( uman) O43886 HUMAN R7-139 ragments Homo sapiens ( uman) O43887 HUMAN R7-140 ragments Homo sapiens ( uman) O43898 HUMAN GPR45 like Homo sapiens ( uman) O4A15. HUMAN Olfactory II fam 4/MOR225-248 Homo sapiens ( uman) O4A16 HUMAN Olfactory II fam 4/MOR225-248 Homo sapiens ( uman) O4A47 HUMAN Olfactory II fam 4/MOR225-248 Homo sapiens ( uman) UMAN Olfactory II fam 4/MOR225-248 Homo sapiens ( uman) UMAN Olfactory II fam 4/MOR225-248 Homo sapiens ( uman) UMAN Olfactory II fam 4/MOR225-248 Homo sapiens ( uman) O51A2 HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) O51A4 HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) O51A7 HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) O51B2 HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) O51B4 HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) O51B5 HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) O51B6 HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) OS1D1 HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) OS1E1 HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) OS1E2 HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) UMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) OS1 G1 HUMAN K1) Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) OS1G-2 HUMAN KO) Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) UMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) UMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) HUMAN 5) Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) UMAN 8) Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) HUMAN 9) Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) O52A1 HUMAN L2) Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) O52A5 HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) O52B2 HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) O52B4 HUMAN K2) Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) O52B6 HUMAN FO) Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) O52D1 HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) O52E1 HUMAN 3) Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) O52E2 HUMAN 4) Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) O52E4 HUMAN H9) Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) O52ES HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) OS2E6 HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) O52E8 HUMAN 1) Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) OS2H1 HUMAN 2) Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) UMAN K6) Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) UMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) 3 UMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) OS2K1 HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) O52K2 HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) O52L1 HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) OS2L2 HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) O52N1 HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) O52N2 HUMAN O) Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) O52N4 HUMAN 2) Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) O52NS HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) O52P1 HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) US 9,512,236 B2 161 162 TABLE D-continued non-limiting list human GPCRs

O52R1. HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) O52W1. HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) O56A1 HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) O56A3 HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) O56A4 HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) O56B1 HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) O56B2 HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) O56B4 HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) OSAC2 HUMAN aC ory I fam 5/MOR172-224,249,254 Homo sapiens ( uman) OSAK2 HUMAN aC ory I fam 5/MOR172-224,249,254 Homo sapiens ( uman) OSAK3 HUMAN aC ory I fam 5/MOR172-224,249,254 Homo sapiens ( uman) OSAN1 HUMAN aC ory I fam 5/MOR172-224,249,254 Homo sapiens ( uman) OSAP2 HUMAN aC ory I fam 5/MOR172-224,249,254 Homo sapiens ( uman) OSAR1 HUMAN aC ory I fam 5/MOR172-224,249,254 Homo sapiens ( uman) aC ory I fam 5/MOR172-224,249,254 Homo sapiens ( uman) aC ory I fam 5/MOR172-224,249,254 Homo sapiens ( uman) aC ory I fam 5/MOR172-224,249,254 Homo sapiens ( uman) aC ory I fam 5/MOR172-224,249,254 Homo sapiens ( uman) aC ory I fam 5/MOR172-224,249,254 Homo sapiens ( uman) aC ory I fam 5/MOR172-224,249,254 Homo sapiens ( uman) O60411 HUMAN agments Homo sapiens ( uman) O75228 HUMAN BXA2R hromboxane Homo sapiens ( uman) O75307 HUMAN CRL2 C-C Chemokine other Homo sapiens ( uman) O75824 HUMAN CKBR fragments Homo sapiens ( uman) O95220 HUMAN RSD3 fragments Homo sapiens ( uman) O95499 HUMAN r89 Olfactory II fam 2/MOR256-262,270-285 Homo sapiens ( uman) O95950 HUMAN fragments Homo sapiens ( uman) PN3 HUMAN PN3 hodopsin Other Homo sapiens ( uman) PN4 HUMAN PN4 hodopsin Other Homo sapiens ( uman) PNS HUMAN PN5 hodopsin Other Homo sapiens ( uman) PRD HUMAN PRD1 pioid type D Homo sapiens ( uman) PRK HUMAN PRK1 pioid type K Homo sapiens ( uman) PRM HUMAN PRM1 pioid type M Homo sapiens ( uman) PRX HUMAN PRL1 pioid type X Homo sapiens ( uman) PSB. HUMAN PN1SW hodopsin Vertebrate type 3 Homo sapiens ( uman) PSD HUMAN hodopsin Vertebrate type 1 Homo sapiens ( uman) PSG HUMAN hodopsin Vertebrate type 2 Homo sapiens ( uman) PSR HUMAN hodopsin Vertebrate type 2 Homo sapiens ( uman) PSX HUMAN hodopsin Other Homo sapiens ( uman) A1 HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) A2 HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) B1. HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) C1 HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) D2 HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) D4 HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) DS HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) E1 HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) UMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) UMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) UMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) FC HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) G1 HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) 1 HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) 1 HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) 2 HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) 4 HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) K1 HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) L1 HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) L3 HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) L4 HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) L6 HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) L8 HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) M1 HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) N1 HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) N2 HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) Q1 HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) S1 HUMAN aC ory am 1/MOR125-138,156 Homo sapiens ( uman) S2 HUMAN aC ory am 1/MOR125-138,156 Homo sapiens ( uman) R2A2 HUMAN aC ory am 2/MOR256-262,270-285 Homo sapiens ( uman) R2A4 HUMAN aC ory am 2/MOR256-262,270-285 Homo sapiens ( uman) R2A5 HUMAN aC ory am 2/MOR256-262,270-285 Homo sapiens ( uman) R2A7 HUMAN aC ory am 2/MOR256-262,270-285 Homo sapiens ( uman) R2B2 HUMAN aC ory am 2/MOR256-262,270-285 Homo sapiens ( uman) R2B3 HUMAN aC ory am 2/MOR256-262,270-285 Homo sapiens ( uman) R2B6 HUMAN aC ory am 2/MOR256-262,270-285 Homo sapiens ( uman) R2B8 HUMAN aC ory am 2/MOR256-262,270-285 Homo sapiens ( uman) R2BB HUMAN aC ory am 2/MOR256-262,270-285 Homo sapiens ( uman) R2C1 HUMAN aC ory am 2/MOR256-262,270-285 Homo sapiens ( uman) US 9,512,236 B2 163 164 TABLE D-continued non-limiting list human GPCRs

R2C3 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2D2 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2D3 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2F1 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2F2 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2G2 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2G3 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2G6 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2H1 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R22 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R21 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R21 HUMAN 1 aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R22 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2J3 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2K1 HUMAN aC ory am 13 MOR253 Homo sapiens ( uman) R2L2 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2L3 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2L5 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2L8 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2LD HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2M2 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2M3 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2M4 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2M7 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2S1 HUMAN aC ory am 13 MOR253 Homo sapiens ( uman) R2T1 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2T2 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2T3 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2T4 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2TS HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2T6 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2V2 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2W1. HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2W3 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2Y1. HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R2Z1 HUMAN aC ory am 2 MO R256-262,270-285 Homo sapiens ( uman) R3A1 HUMAN aC ory am 3,MO R255 Homo sapiens ( uman) R3A2 HUMAN aC ory am 3,MO R255 Homo sapiens ( uman) R3A3 HUMAN aC ory am 3,MO R255 Homo sapiens ( uman) R3A4 HUMAN aC ory am 3,MO R255 Homo sapiens ( uman) R4A4 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4A5 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4B1 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4C3 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4C5 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) UMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) 4 2 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4DS HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4D6 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4D9 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4DA HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4DB. HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4E2 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4F3 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4F4 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4F5 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4F6 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4K1 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4K2 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4K3 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4K5 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4KD HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4KE HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4KF HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4KH HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4L1 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4M1 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4M2 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4N2 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4N4 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4NS HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4P4 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) US 9,512,236 B2 165 166 TABLE D-continued non-limiting list human GPCRs

R4Q3 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4S1 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4S2 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4X1 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R4X2 HUMAN aC ory am 4 MO R225-248 Homo sapiens ( uman) R5A1 HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) R5A2 HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) RSB2 HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) R5B3 HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) RSBC HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) R5BH HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) R5C1 HUMAN C 1 aC ory am SMO 72-224,249,254 Homo sapiens ( uman) RSDD HUMAN D13 aC ory am SMO 72-224,249,254 Homo sapiens ( uman) RSDE HUMAN D14 aC ory am SMO 72-224,249,254 Homo sapiens ( uman) R5DG HUMAN D16 aC ory am SMO 72-224,249,254 Homo sapiens ( uman) R5DI HUMAN D18 aC ory am SMO 72-224,249,254 Homo sapiens ( uman) RSF1 HUMAN F1 aC ory am SMO 72-224,249,254 Homo sapiens ( uman) R5H2 HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) RSH6 HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) RSI1 HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) R5J2 HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) R5K1 HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) RSK2 HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) RSL1, HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) RSL2 HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) RSM1 HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) RSM3 HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) RSM8 HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) RSM9 HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) RSMA HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) RSMB. HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) RSP2 HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) RSP3 HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) R5R1 HUMAN aC ory am 8 MO 61-171 Homo sapiens ( uman) R5T1 HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) R5T2 HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) R5T3 HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) RSU1 HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) RSV1 HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) R5W2 HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) R6A2 HUMAN aC ory am 6,MO 05, 9 Homo sapiens ( uman) R6B1 HUMAN aC ory am 6,MO 05, Homo sapiens ( uman) R6B2 HUMAN aC ory am 6,MO 05, Homo sapiens ( uman) R6B3 HUMAN aC ory am 6,MO 05, Homo sapiens ( uman) R6C1 HUMAN aC ory am 6,MO 05, Homo sapiens ( uman) R6C2 HUMAN aC ory am 6,MO 05, Homo sapiens ( uman) R6C3 HUMAN aC ory am 6,MO 05, Homo sapiens ( uman) R6C4 HUMAN aC ory am 6,MO 05, Homo sapiens ( uman) R6F1 HUMAN aC ory am 6,MO 05, Homo sapiens ( uman) R6T1 HUMAN aC ory am 6,MO 05, Homo sapiens ( uman) R6K2 HUMAN aC ory am 6,MO 05, Homo sapiens ( uman) R6K3 HUMAN aC ory am 6,MO 05, Homo sapiens ( uman) R6K6 HUMAN aC ory am 6,MO 05, Homo sapiens ( uman) R6M1 HUMAN aC ory am 6,MO 05, Homo sapiens ( uman) R6N1 HUMAN aC ory am 6,MO 05, Homo sapiens ( uman) R6N2 HUMAN aC ory am 6,MO 05, Homo sapiens ( uman) R6P1 HUMAN aC ory am 6,MO 05, Homo sapiens ( uman) R6Q1 HUMAN aC ory am 6,MO 05, Homo sapiens ( uman) R6S1 HUMAN aC ory am 6,MO 05, Homo sapiens ( uman) R6T1 HUMAN aC ory am 6,MO 05, Homo sapiens ( uman) R6V1 HUMAN aC ory am 6,MO 05, Homo sapiens ( uman) R6X1 HUMAN aC ory am 6,MO 05, Homo sapiens ( uman) R6Y1 HUMAN aC ory am 6,MO 05, Homo sapiens ( uman) R7A2 HUMAN aC ory am 7 MO 55 Homo sapiens ( uman) R7A5 HUMAN aC ory am 7 MO 55 Homo sapiens ( uman) R7AA HUMAN aC ory am 7 MO 55 Homo sapiens ( uman) R7AH HUMAN aC ory am 7 MO 55 Homo sapiens ( uman) R7C1 HUMAN aC ory am 7 MO 55 Homo sapiens ( uman) R7C2 HUMAN aC ory am 7 MO 55 Homo sapiens ( uman) R7D2 HUMAN aC ory am 7 MO 55 Homo sapiens ( uman) R7D4 HUMAN aC ory am 7 MO 55 Homo sapiens ( uman) R7G1 HUMAN aC ory am 7 MO 55 Homo sapiens ( uman) R7G2 HUMAN aC ory am 7 MO 55 Homo sapiens ( uman) R7G3 HUMAN aC ory am 7 MO 55 Homo sapiens ( uman) R8A1 HUMAN aC ory am 8 MO 71 Homo sapiens ( uman) R8B2 EHUMAN aC ory am 8 MO 71 Homo sapiens ( uman) R8B3 HUMAN aC ory am 8 MO 71 Homo sapiens ( uman) R8B4 HUMAN aC ory am 8 MO 71 Homo sapiens ( uman) US 9,512,236 B2 167 168 TABLE D-continued non-limiting list human GPCRs R8B8 HUMAN (Q15620) Olfactory II fam 8/MOR161-17 Homo sapiens ( uman) R8BC HUMAN (Q8NGG6) Olfactory II fam 8/MOR161-17 Homo sapiens ( uman) R8D1 HUMAN (Q8WZ84) Olfactory II fam 8/MOR161-17 Homo sapiens ( uman) R8D2 HUMAN (Q9GZM6) Olfactory II fam 8/MOR161-17 Homo sapiens ( uman) R8D4 HUMAN (Q8NGM9) Olfactory II fam 8/MOR161-17 Homo sapiens ( uman) R8G1 HUMAN (Q15617) Olfactory II fam 8/MOR161-17 Homo sapiens ( uman) R8G2 HUMAN (Q15614) Olfactory II fam 8/MOR161-17 Homo sapiens ( uman) R8GS HUMAN (Q8NG78) Olfactory II fam 8/MOR161-17 Homo sapiens ( uman) R8H1 HUMAN (Q8NGG4) Olfactory II fam 8/MOR161-17 Homo sapiens ( uman) R8H2 HUMAN (Q8N162) Olfactory II fam 8/MOR161-17 Homo sapiens ( uman) R8H3 HUMAN (Q8N146) Olfactory II fam 8/MOR161-17 Homo sapiens ( uman) 2 HUMAN (Q8NOY5) Olfactory II fam 8/MOR161-17 Homo sapiens ( uman) 1 HUMAN (Q8NGP2) Olfactory II fam 8/MOR161-17 Homo sapiens ( uman) R8J3 HUMAN (Q8NGGO) Olfactory II fam 8/MOR161-17 Homo sapiens ( uman) K1 HUMAN (Q8NGG5) Olfactory II fam 5/MOR172-224.249,254 Homo sapiens ( uman) R8K3 HUMAN (Q8NH51) Olfactory II fam 8/MOR161-17 Homo sapiens ( uman) R8K5 HUMAN (Q8NH5O) Olfactory II fam 8/MOR161-17 Homo sapiens ( uman) R8S1 HUMAN (Q8NH09) Olfactory unclassified class II Homo sapiens ( uman) R8U1 HUMAN (Q8NH10) Olfactory II fam 8/MOR161-17 Homo sapiens ( uman) R9A2 HUMAN (Q8NGT5) Olfactory II fam 9/MOR120 Homo sapiens ( uman) R9A4 HUMAN (Q8NGU2) Olfactory II fam 9/MOR120 Homo sapiens ( uman) R9G1 HUMAN (Q8NH87) Olfactory II fam 9/MOR120 Homo sapiens ( uman) R9G4 HUMAN (Q8NGQ1) Olfactory II fam 9/MOR120 Homo sapiens ( uman) R9GS HUMAN (Q8NGQ0) Olfactory II fam 9/MOR120 Homo sapiens ( uman) R9I1 HUMAN (Q8NGQ6) Olfactory II fam 9/MOR120 Homo sapiens ( uman) R9K2 HUMAN (Q8NGE7) Olfactory II fam 9/MOR120 Homo sapiens ( uman) R9Q1 HUMAN (Q8NGQ5) Olfactory II fam 9/MOR120 Homo sapiens ( uman) R9Q2 HUMAN (Q8NGE9) Olfactory II fam 9/MOR120 Homo sapiens ( uman) OX1R HUMAN (O43613) Orexin Homo sapiens ( uman) OX2R HUMAN (O43614) Orexin Homo sapiens ( uman) OXER1. HUMAN (Q8TDS5) Putativefunclassified Class A GPCRs Homo sapiens ( uman) OXGR1 HUMAN (Q96P68) Putativefunclassified Class A GPCRs Homo sapiens ( uman) OXYR HUMAN (P30559) Oxytocin mesotocin Homo sapiens ( uman) P2RY1. HUMAN (P47900) Purinoceptor P2RY1-4,6,11 GPR91 Homo sapiens ( uman) P2RY2 HUMAN (P41231) Purinoceptor P2RY1-4.6, 11 GPR91 Homo sapiens ( uman) P2RY4 HUMAN (P51582) Purinoceptor P2RY1-4,6,11 GPR91 Homo sapiens ( uman) P2RYS HUMAN (P43657) Purinoceptor P2RY5,8,9,10 GPR35,92,174 Homo sapiens ( uman) P2RY6 HUMAN (Q15077) Purinoceptor P2RY1-4,6,11 GPR91 Homo sapiens ( uman) P2RY8 HUMAN (Q86VZ1) Purinoceptor P2RY5,8,9,10 GPR35,92,174 Homo sapiens ( uman) P2RY9 HUMAN (Q99677) Purinoceptor P2RY5,8,9,10 GPR35,92,174 Homo sapiens ( uman) P2Y1O HUMAN (OOO398) Purinoceptor P2RY5,8,9,10 GPR35,92,174 Homo sapiens ( uman) P2Y11 HUMAN (Q96G.91) Purinoceptor P2RY1-4,6,11 GPR91 Homo sapiens ( uman) HUMAN (Q9H244) Purinoceptor P2RY12-14 GPR87 (UDP-Glucose) Homo sapiens ( uman) P2Y13 HUMAN (Q9BPV8) Purinoceptor P2RY12-14 GPR87 (UDP-Glucose) Homo sapiens ( uman) P2Y14 HUMAN (Q15391) Purinoceptor P2RY12-14 GPR87 (UDP-Glucose) Homo sapiens ( uman) P7847O HUMAN (P78470) ragments Homo sapiens ( uman) P78471 HUMAN (P78471) ragments Homo sapiens ( uman) PACR HUMAN (P41586) ADCYAP1R1 PACAP Homo sapiens ( uman) PAR1 HUMAN (P25116) F2R Homo sapiens ( uman) PAR2 HUMAN (P55085) F2RL1 Proteinase-activated Homo sapiens ( uman) PAR3 HUMAN (OOO254) F2RL2 Proteinase-activated Homo sapiens ( uman) PAR4 HUMAN (Q96RIO) F2RL3 Proteinase-activated Homo sapiens ( uman) PD2R HUMAN (Q13258) PTGDR Prostaglandin E2/D2 subtype EP2 Homo sapiens ( uman) PE2R1. HUMAN (P34995) PTGER1 Prostaglandin E2 subtype EP1 Homo sapiens ( uman) PE2R2 HUMAN (P43116) PTGER2 Prostaglandin E2/D2 subtype EP2 Homo sapiens ( uman) PE2R3 HUMAN (P43115) PTGER3 Prostaglandin E2 subtype EP3 Homo sapiens ( uman) PE2R4 HUMAN (P35408) PTGER4 Prostaglandin E2 subtype EP4 Homo sapiens ( uman) PF2R HUMAN (P43088) PTGFR Prostaglandin F2-alpha Homo sapiens ( uman) PI2R HUMAN (P43119) PTGIR Prostacyclin Homo sapiens ( uman) PKR1 HUMAN (Q8TCW9) PROKR1 Prokineticin receptors Homo sapiens ( uman) PKR2 HUMAN (Q8NFJ6) PROKR2 Prokineticin receptors Homo sapiens ( uman) PRLHR HUMAN (P49683) PRLHR Prolactin-releasing peptide (GPR10) Homo sapiens ( uman) PSYR HUMAN (Q8IYL9) GPR65 Putativefunclassified Class A GPCRs Homo sapiens ( uman) PTAFR HUMAN (P25105) PTAFR Platelet activating factor Homo sapiens ( uman) PTHR1 HUMAN (Q03431) PTHR1 Parathyroid hormone Homo sapiens ( uman) PTHR2 HUMAN (P49190) PTHR2 Parathyroid hormone Homo sapiens ( uman) Q13027 HUMAN (Q13027) ragments Homo sapiens ( uman) Q13167 HUMAN (Q13167) DRD3 Dopamine Vertebrate type 3 Homo sapiens ( uman) Q14968 HUMAN (Q14968) Putativefunclassified Class A GPCRs Homo sapiens ( uman) Q15613 HUMAN (Q15613) cr110 ragments Homo sapiens ( uman) Q15616 HUMAN (Q15616) ORSE1P ragments Homo sapiens ( uman) Q15618 HUMAN (Q15618) OR7E18P ragments Homo sapiens ( uman) Q16144 HUMAN (Q16144) CCK type B Homo sapiens ( uman) Q16292 HUMAN (Q16292) hrombin receptor fragments Homo sapiens ( uman) Q16303 HUMAN (Q16303) dopamine D4 receptor fragments Homo sapiens ( uman) Q16503 HUMAN (Q16503) ragments Homo sapiens ( uman) Q2F3K1 HUMAN (Q2F3K1) CASR ragments Homo sapiens ( uman) Q2HIZ3 HUMAN (Q2HIZ3) OR1OH3 Olfactory II fam 10/MOR263-269 Homo sapiens ( uman)

US 9,512,236 B2 171 172 TABLE D-continued non-limiting list human GPCRs Q4KKW2 HUMAN Neuropeptide Y type 4 Homo sapiens ( uman) Q4KNO4 HUMAN Taste receptors T2R Homo sapiens ( uman) Q4KN27 HUMAN Melanocortin hormone Homo sapiens ( uman) Q4KN29 HUMAN Taste receptors T2R Homo sapiens ( uman) Q4QRI5 HUMAN Substance K (NK2) Homo sapiens ( uman) Q4QRI9 HUMAN fragments Homo sapiens ( uman) Q4QRJO HUMAN Dopamine Vertebrate type 1 Homo sapiens ( uman) Q4QRJ1 HUMAN Corticotropin releasing factor Homo sapiens ( uman) Q4QRJ3 HUMAN Follicle stimulating hormone Homo sapiens ( uman) Q4QRJ4 HUMAN Corticotropin releasing factor Homo sapiens ( uman) Q4V749 HUMAN C-C Chemokine type 10 Homo sapiens ( uman) Q4V9L2 HUMAN Mas proto-oncogene & Mas-related (MRGs) Homo sapiens ( uman) Q4VAMO HUMAN LGR like (hormone receptors) Homo sapiens ( uman) Q4VAM2 HUMAN LGR like (hormone receptors) Homo sapiens ( uman) Q4VAT1 HUMAN fragments Homo sapiens ( uman) Q4VAT2 HUMAN fragments Homo sapiens ( uman) Q4VAT3 HUMAN Glucagon Homo sapiens ( uman) Q4VAT4 HUMAN fragments Homo sapiens ( uman) Q4VAV7 HUMAN Neuropeptide Y type 4 Homo sapiens ( uman) Q4VAY7 HUMAN Serotonin type 1 Homo sapiens ( uman) Q4VB06 HUMAN B Olfactory II fam 3/MOR255 Homo sapiens ( uman) Q4VBBO HUMAN B C-C Chemokine other Homo sapiens ( uman) Q4VBB4 HUMAN B GPR Homo sapiens ( uman) Q4VBK6 HUMAN B Musc. acetylcholine Vertebrate type 2 Homo sapiens ( uman) Q4VBK7 HUMAN B ragments Homo sapiens ( uman) Q4VBK8 HUMAN B ragments Homo sapiens ( uman) Q4VBLO HUMAN B Bombesin Homo sapiens ( uman) Q4VBL2 HUMAN B CMr. C-C Chemokine type 2 Homo sapiens ( uman) Q4VBL3 HUMAN B ragments Homo sapiens ( uman) Q4VBL6 HUMAN B GPR Homo sapiens ( uman) Q4VBL7 HUMAN B A. R1 Galanin Homo sapiens ( uman) Q4VBL8 HUMAN B ragments Homo sapiens ( uman) Q4VBL9 HUMAN ragments Homo sapiens ( uman) Q4VBM3 HUMAN ragments Homo sapiens ( uman) Q4VBM7 HUMAN Alpha Adrenoceptors type 1 Homo sapiens ( uman) Q4VBNO HUMAN ragments Homo sapiens ( uman) Q4VBN1 HUMAN ragments Homo sapiens ( uman) Q4VBN3 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) Q4VBN4 HUMAN ragments Homo sapiens ( uman) Q4VBNS HUMAN ragments Homo sapiens ( uman) Q4VBN6 HUMAN ragments Homo sapiens ( uman) Q4VBN7 HUMAN Purinoceptor P2RY5,8,9,10 GPR35,92,174 Homo sapiens ( uman) Q4VBPO HUMAN Somatostatin type 2 Homo sapiens ( uman) Q4VBP1 HUMAN ragments Homo sapiens ( uman) Q4VWM1 HUMAN Opioid type M Homo sapiens ( uman) Q4VWM2 HUMAN Opioid type M Homo sapiens ( uman) Q4VWM3 HUMAN Opioid type M Homo sapiens ( uman) Q4VWM4 HUMAN Opioid type M Homo sapiens ( uman) Q4VWM6 HUMAN Opioid type M Homo sapiens ( uman) Q4VWX6 HUMAN Opioid type M Homo sapiens ( uman) Q4W594 HUMAN Alpha Adrenoceptors type 2 Homo sapiens ( uman) Q4W5G7 HUMAN Neuropeptide Y type 2 Homo sapiens ( uman) Q4ZF V2 HUMAN Purinoceptor P2RY5,8,9,10 GPR35,92,174 Homo sapiens ( uman) Q4ZILO HUMAN fragments Homo sapiens ( uman) Q502U7 HUMAN PR32 Chemokine receptor-like 1 Homo sapiens ( uman) Q502U9 HUMAN PR23 Purinoceptor P2RY5,8,9,10 GPR35,92,174 Homo sapiens ( uman) Q502VO HUMAN R1 XC Chemokine Homo sapiens ( uman) Q502V1 HUMAN MCSR Melanocortin hormone Homo sapiens ( uman) Q502V2 HUMAN RXFP3 Somatostatin- and angiogenin-like peptide Homo sapiens ( uman) Q502V7 HUMAN TAS2R9 Taste receptors T2R Homo sapiens ( uman) Q502V9 HUMAN MAS1L. Mas proto-oncogene & Mas-related (MRGs) Homo sapiens ( uman) Q504X6 HUMAN MC2R Adrenocorticotropic hormone Homo sapiens ( uman) Q506J9 HUMAN Cannabinoid Homo sapiens ( uman) Q5OKD4 HUMAN Hosa (Biaka)-T2R55 fragments Homo sapiens ( uman) Q5OKD6 HUMAN Hosa (Adygei)-T2R55 fragmen Homo sapiens ( uman) Q5OKTO HUMAN Hosa (Biaka)-T2R9 fragmen Homo sapiens ( uman) Q5OKT1 HUMAN Hosa (Japanese)-T2R9 fragmen Homo sapiens ( uman) Q5OKU1 HUMAN Hosa (Biaka)-T2R8 fragmen Homo sapiens ( uman) Q5OKU2 HUMAN Hosa (Japanese)-T2R8 fragmen Homo sapiens ( uman) Q5OKV5 HUMAN Hosa (Biaka)-T2R7 fragmen Homo sapiens ( uman) Q5OKV7 HUMAN Hosa (Adygei)-T2R7 fragments Homo sapiens ( uman) Q52LG8 HUMAN PTGER2 Prostaglandin E2/D2 subtype EP2 Homo sapiens ( uman) Q52MO4 HUMAN GIPR Gastric inhibitory peptide Homo sapiens ( uman) Q52M68 HUMAN Proteinase-activated Homo sapiens ( uman) Q52R92 HUMAN fragments Homo sapiens ( uman) Q52R93 HUMAN fragments Homo sapiens ( uman) Q52R94 HUMAN fragments Homo sapiens ( uman) Q53EMO HUMAN fragments Homo sapiens ( uman) US 9,512,236 B2 173 174 TABLE D-continued non-limiting list human GPCRs Q53EZ5 HUMAN ragmen Homo sapiens ( uman) Q53F99 HUMAN ragmen Homo sapiens ( uman) Q53FAO HUMAN ragmen Homo sapiens ( uman) Q53FA1 HUMAN ragmen Homo sapiens ( uman) Q53GA6 HUMAN ragmen Homo sapiens ( uman) Q53GM2 HUMAN ragmen Homo sapiens ( uman) Q53GPO HUMAN ragments Homo sapiens ( uman) Q53PC4 HUMAN interleukin-8 type B Homo sapiens ( uman) Q53QQ5 HUMAN ragments Homo sapiens ( uman) Q53QT9 HUMAN Prokineticin receptors Homo sapiens ( uman) Q53R18 HUMAN interleukin-8 type A Homo sapiens ( uman) Q53R22 HUMAN rizzled Group A (FZ 1&2&4&5&7-9) Homo sapiens ( uman) Q53RU7 HUMAN ragments Homo sapiens ( uman) Q53RV4 HUMAN RDC1 Homo sapiens ( uman) Q53S49 HUMAN ragments Homo sapiens ( uman) Q53S59 HUMAN rizzled Group A (FZ 1&2&4&5&7-9) Homo sapiens ( uman) Q53S69 HUMAN C—X-C Chemokine type 4 Homo sapiens ( uman) Q53SF6 HUMAN ragments Homo sapiens ( uman) Q53TOO HUMAN Secretin Homo sapiens ( uman) Q53T35 HUMAN ragments Homo sapiens ( uman) Q53TAS HUMAN Putativefunclassified Class B GPCRs Homo sapiens ( uman) Q53TI1 HUMAN Serotonin type 2 Homo sapiens ( uman) Q53TQ2 HUMAN ragments Homo sapiens ( uman) Q53TR1. HUMAN ragments Homo sapiens ( uman) Q53TS5 HUMAN ragments Homo sapiens ( uman) Q53XJ8 HUMAN Proteinase-activated Homo sapiens ( uman) Q53XVO HUMAN Thrombin Homo sapiens ( uman) Q53XV5 HUMAN Leukotriene B4 receptor BLT1 Homo sapiens ( uman) Q53XZ3 HUMAN Musc. acetylcholine Vertebrate type 1 Homo sapiens ( uman) Q53Y09 HUMAN Vasoactive intestinal polypeptide Homo sapiens ( uman) Q53YA1 HUMAN CCBP2 C-C Chemokine type X Homo sapiens ( uman) Q53YJ4 HUMAN GALR3 Galanin Homo sapiens ( uman) Q53YYO HUMAN AGTR1 Angiotensin type 1 Homo sapiens ( uman) Q53ZR7 HUMAN SSTR3 Somatostatin type 3 Homo sapiens ( uman) Q541EO HUMAN Somatostatin type 5 Homo sapiens ( uman) Q546Q1 HUMAN HTR4 Serotonin type 4 Homo sapiens ( uman) Q548M6 HUMAN HRH3 Histamine type 3 Homo sapiens ( uman) Q548YO HUMAN HCRTR2 Orexin Homo sapiens ( uman) Q549EO HUMAN CCR9 C-C Chemokine type 9 Homo sapiens ( uman) Q57Z87 HUMAN NTSR2 ragmen Homo sapiens ( uman) Q59EH9 HUMAN ragmen Homo sapiens ( uman) Q59ER8 HUMAN ragmen Homo sapiens ( uman) Q59ES7 HUMAN ragmen Homo sapiens ( uman) Q59FCO HUMAN ragmen Homo sapiens ( uman) Q59FW2 HUMAN ragmen Homo sapiens ( uman) Q59G39 HUMAN ragmen Homo sapiens ( uman) Q59G72 HUMAN ragmen Homo sapiens ( uman) Q59G95 HUMAN ragmen Homo sapiens ( uman) Q59GA2 HUMAN ragmen Homo sapiens ( uman) Q59GB1 HUMAN ragmen Homo sapiens ( uman) Q59GE5 HUMAN glutamate receptor homolog ragmen Homo sapiens ( uman) Q59GIO HUMAN ragmen Homo sapiens ( uman) Q59GL3 HUMAN ragmen Homo sapiens ( uman) Q59GP3 HUMAN ragmen Homo sapiens ( uman) Q59H16 HUMAN ragmen Homo sapiens ( uman) Q59HC2 HUMAN ragmen Homo sapiens ( uman) Q59HG8 HUMAN ragments Homo sapiens ( uman) Q5CZ57 HUMAN EP3-I Prostaglandin E2 subtype EP3 Homo sapiens ( uman) Q5CZ59 HUMAN EP3e Prostaglandin E2 subtype EP3 Homo sapiens ( uman) Q5CZ60 HUMAN EP3f Prostaglandin E2 subtype EP3 Homo sapiens ( uman) Q5CZ61 UMAN EP3-VI Prostaglandin E2 subtype EP3 Homo sapiens ( uman) Q5CZ62 HUMAN EP3-V Prostaglandin E2 subtype EP3 Homo sapiens ( uman) Q5CZ63 HUMAN EP3-IV Prostaglandin E2 subtype EP3 Homo sapiens ( uman) Q5CZ64 HUMAN EP3-III Prostaglandin E2 subtype EP3 Homo sapiens ( uman) Q5EGP2 HUMAN GPR112 Putativefunclassified Class B GPCRs Homo sapiens ( uman) Q5EKM8 HUMAN CCR5 C-C Chemokine type 5 Homo sapiens ( uman) Q5EKM9 HUMAN CCR5 C-C Chemokine type 5 Homo sapiens ( uman) Q5EKNO HUMAN CCR5 C-C Chemokine type 5 Homo sapiens ( uman) QSHYM4 HUMAN DKFZp686H1993 ragments Homo sapiens ( uman) QSHYQ4 HU MAN GPR173 SREB Homo sapiens ( uman) QSIFH6 HUMAN GPR24 Melanin-concentrating hormone receptors Homo sapiens ( uman) QSIFI4 HUMAN GPR24 Melanin-concentrating hormone receptors Homo sapiens ( uman) QSISU3 HUMAN PPYR1 Neuropeptide Y type 4 Homo sapiens ( uman) Q5JNZ1 HUMAN DAQB-117O11.7-001 fragments Homo sapiens ( uman) Q5.JPQ2 HUMAN GPR64 Putativefunclassified Class B GPCRs Homo sapiens ( uman) Q5.JPQ3 HUMAN GPR64 Putativefunclassified Class B GPCRs Homo sapiens ( uman) Q5.JPQ4 HUMAN GPR64 Putativefunclassified Class B GPCRs Homo sapiens ( uman) Q5.JPQ5 HUMAN GPR64 Putativefunclassified Class B GPCRs Homo sapiens ( uman) US 9,512,236 B2 175 176 TABLE D-continued non-limiting list human GPCRs GPR64 Putativefunclassified Class B GPCRs Homo sapiens ( uman) RP11-97815.6 Olfactory II fam 5/MOR172-224.249,254 Homo sapiens ( uman) CNR2 Cannabinoid Homo sapiens ( uman) GPR101 Putativefunclassified Class A GPCRs Homo sapiens ( uman) GPR112 ragments Homo sapiens ( uman) EBI2 EBV-induced Homo sapiens ( uman) GPR18 ragments Homo sapiens ( uman) TGFR Prostaglandin F2-alpha Homo sapiens ( uman) GPR112 ragments Homo sapiens ( uman) CNR1 Cannabinoid Homo sapiens ( uman) CCR5 ragments Homo sapiens ( uman) K PG 013 Putativefunc assified Class A GPCRs Homo sapiens ( uman) K PG 011 Cysteinyl leukotriene Homo sapiens ( uman) K PG 010 Purinoceptor P2RY5,8,9,10 GPR35,92,174 Homo sapiens ( uman) K PG 009 Putativefunc assified Class B GPCRs Homo sapiens ( uman) K PG 008 Putativefunc assified Class B GPCRs Homo sapiens ( uman) K PG 007 Purinoceptor P2RY5,8,9,10 GPR35,92,174 Homo sapiens ( uman) K PG OO6 Putativefunc assified Class B GPCRs Homo sapiens ( uman) K PG 005 Putativefunc assified Class A GPCRs Homo sapiens ( uman) K PG 004 Leukotriene B4 receptor BLT2 Homo sapiens ( uman) HUMA N K PG 003 ETL receptors Homo sapiens ( uman) KPG 002 Purinoceptor P2RY12-14 GPR87 (UDP-Glucose) Homo sapiens ( uman) CC C-C Chemokine type 5 Homo sapiens ( uman) CC C-C Chemokine type 5 Homo sapiens ( uman) CC s C-C Chemokine type 5 Homo sapiens ( uman) DAQB-36F16.7-002 ragments Homo sapiens ( uman) POGR Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) EDG3 Sphingosine 1-phosphate Edg-3 Homo sapiens ( uman) XXbac-BCX923.1-OO1 Olfactory II fam 5/MOR172-224.249,254 Homo sapiens ( uman) DAQB-304F3.2-001 Olfactory II fam 11/MOR106,121-122 Homo sapiens ( uman) XXbac-BCX147D4.2-001 Olfactory II fam 5/MOR172-224.249,254 Homo sapiens ( uman) OR22 Olfactory II fam 2/MOR256-262,270-285 Homo sapiens ( uman) BBR1 fragments Homo sapiens ( uman) Q5STL7 HUMAN BBR1 fragments Homo sapiens ( uman) Q5SUJ6 HUMAN XXbac-BPG171B11.5-001 Olfactory II fam 2/MOR256-262,270-285 Homo sapiens ( uman) Q5SUJ7 HUMAN XXbac-BPG171B11.3-001 Olfactory II fam 2/MOR256-262,270-285 Homo sapiens ( uman) Q5SUJ8 HUMAN GABBR1 GABA-B subtype 1 Homo sapiens ( uman) Q5SUJ9 HUMAN GABBR1 GABA-B subtype 1 Homo sapiens ( uman) Q5SUK1 HUMAN Olfactory II fam 2/MOR256-262,270-285 Homo sapiens ( uman) Q5SUL3 HUMAN GABBR1 GABA-B subtype 1 Homo sapiens ( uman) Q5SUN5 HUMAN MAS1L. Mas proto-oncogene & Mas-related (MRGs) Homo sapiens ( uman) Q5SUN6 HUMAN DAQB-12N14.4-001 Olfactory II fam 2/MOR256-262,270-285 Homo sapiens ( uman) Q5SUN7 HUMAN XXbac-BPG13B81-001 Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) Q5SUN9 HUMAN XXbac-BPG13B86-OO1 Olfactory II fam 12/MOR250 Homo sapiens ( uman) Q5SWW2 HUMAN ELTD1 ragments Homo sapiens ( uman) Q5SWW3 HUMAN ELTD1 ETL receptors Homo sapiens ( uman) Q5SXP7 HUMAN RP11-294K24.1-001 GPR37 endothelin B-like Homo sapiens ( uman) Q5SY22 HUMAN TAS1R1 ragments Homo sapiens ( uman) Q5SY23 HUMAN TAS1R1 ragments Homo sapiens ( uman) Q5SY24 HUMAN TAS1R1 ragments Homo sapiens ( uman) Q5T234 HUMAN PR123 Putativefunclassified Class B GPCRs Homo sapiens ( uman) Q5T261 HUMAN DG2 ragments Homo sapiens ( uman) Q5T2X9 HUMAN PYR1 ragments Homo sapiens ( uman) Q5T2Y7 HUMAN ELSR2 Cadherin EGF LAG (CELSR) Homo sapiens ( uman) Q5T5Y4 HUMAN DRB1 Beta Adrenoceptors type 1 Homo sapiens ( uman) Q5T6D8 HUMAN PR147 ragments Homo sapiens ( uman) Q5T6KO HUMAN BAI2 Brain-specific angiogenesis inhibitor (BAI) Homo sapiens ( uman) Q5T7Z3 HUMAN RP11-64P144-OO1 Olfactory II fam 1/MOR125-138,156 Homo sapiens ( uman) Q5T8CO HUMAN HTR2A Serotonin ty be 2 Homo sapiens ( uman) Q5T8P3 HUMAN GPR12 GPR Homo sapiens ( uman) Q5T9D2 HUMAN PHN2 ragments Homo sapiens ( uman) Q5TBXO HUMAN PRM1 Opioid type M Homo sapiens ( uman) Q5TF06 HUMAN P3-365O12.1-001 Putativefunc assified Class B GPCRs Homo sapiens ( uman) Q5TGK2 HUMAN PR161 Putativefunc assified Class A GPCRs Homo sapiens ( uman) Q5TGN7 HUMAN PR126 Putativefunc assified Class B GPCRs Homo sapiens ( uman) Q5TGZ1 HUMAN :TR6 Serotonin ty be 6 Homo sapiens ( uman) Q5TH86 HUMAN TGER3 Prostaglandin E2 subtype EP3 Homo sapiens ( uman) Q5TH88 HUMAN PTGER3 Prostaglandin E2 subtype EP3 Homo sapiens ( uman) Q5U003 HUMAN C-C Chemokine type 1 Homo sapiens ( uman) QSUOHO HUMAN Chemokine receptor-like 1 Homo sapiens ( uman) QSUSU4 HUMAN PTGER1 Prostaglandin E2 subtype EP1 Homo sapiens ( uman) Q5VSV1 HUMAN R P11-18OD21.2-001 Olfactory II fam 10 MOR263-269 Homo sapiens ( uman) QSVT13 HUMAN G PR82 Putativefunc assified Class A GPCRs Homo sapiens ( uman) QSVT14 HUMAN G PR34 Putativefunc assified Class A GPCRs Homo sapiens ( uman) QSVT23 HUMAN RP11-34P13.6-001 Olfactory II fam 4 MOR225-248 Homo sapiens ( uman) Q5VTMO HUMAN SLC31A2 Orexin Homo sapiens ( uman) Q5VTV7 HUMAN GPR145 Melanin-con centrating hormone receptors Homo sapiens ( uman) QSVUF8 HUMAN HTR2C Serotonin ty be 2 Homo sapiens ( uman) US 9,512,236 B2 177 178 TABLE D-continued non-limiting list human GPCRs QSVUF9 HUMAN HTR2C fragments Homo sapiens ( uman) QSVUK8 HUMAN NMBR Bombesin Homo sapiens ( uman) Q5VXO1 HUMAN HTR7 Serotonin type 7 Homo sapiens ( uman) Q5VXO2 HUMAN HTR7 Serotonin type 7 Homo sapiens ( uman) Q5VXO3 HUMAN HTR7 Serotonin type 7 Homo sapiens ( uman) Q5VXO4 HUMAN HTR7 Serotonin type 7 Homo sapiens ( uman) Q5VX75 HUMAN LPHN2 Latrophilin type 2 Homo sapiens ( uman) Q5VX77 HUMAN LPHN2 Latrophilin type 2 Homo sapiens ( uman) Q5VX78 HUMAN LPHN2 Latrophilin type 2 Homo sapiens ( uman) Q5VX79 HUMAN LPHN2 Latrophilin type 2 Homo sapiens ( uman) Q5VX80 HUMAN LPHN2 Latrophilin type 2 Homo sapiens ( uman) Q5VX81 HUMAN LPHN2 Latrophilin type 2 Homo sapiens ( uman) Q5VX82 HUMAN LPHN2 Latrophilin type 2 Homo sapiens ( uman) Q5VX83 HUMAN PHN2 Latrophilin type 2 Homo sapiens ( uman) Q5VXR2 HUMAN DRA1D Alpha Adrenoceptors type 1 Homo sapiens ( uman) Q5VXY3 HUMAN HRM3 ragments Homo sapiens ( uman) QSVY37 HUMAN S. 3 Brain-specific angiogenesis inhibitor (BAI) Homo sapiens ( uman) Q5VZXO HUMAN DG2 Lysophosphatidic acid Edg-2 Homo sapiens ( uman) Q5WOG9 HUMAN DNRB Endothelin Homo sapiens ( uman) Q5WON7 HUMAN P11-432E15.1 LGR like (hormone receptors) Homo sapiens ( uman) Q5Y190 HUMAN ESDA1 Cadherin EGF LAG (CELSR) Homo sapiens ( uman) Q5ZGL8 HUMAN HCTR-6 ragments Homo sapiens ( uman) Q5ZGX3 HUMAN SHT1A Serotonin type 1 Homo sapiens ( uman) Q63ZY2 HUMAN GPR30 Chemokine receptor-like 2 Homo sapiens ( uman) Q645Y1 HUMAN TAS2R7 Taste receptors T2R Homo sapiens ( uman) Q659U6 HUMAN HCTR-5 ragments Homo sapiens ( uman) Q66K38 HUMAN MC1R Melanocyte stimulating hormone Homo sapiens ( uman) Q66X57 HUMAN Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) Q684MO HUMAN Serotonin type 4 Homo sapiens ( uman) Q68CR4 HUMAN Rhodopsin Vertebrate type 2 Homo sapiens ( uman) Q68DM8 HUMAN Bradykinin Homo sapiens ( uman) Q6BOG7 HUMAN : N Cannabinoid Homo sapiens ( uman) Q6DHZ4 HUMAN PR126 Putativefunclassified Class B GPCRs Homo sapiens ( uman) Q6DJW7 HUMAN ragments Homo sapiens ( uman) Q6DKN4 HUMAN 3 ragments Homo sapiens ( uman) EG Putativefunclassified Class B GPCRs Homo sapiens ( uman) PR126 Putativefunclassified Class B GPCRs Homo sapiens ( uman) REG Putativefunclassified Class B GPCRs Homo sapiens ( uman) REG Putativefunclassified Class B GPCRs Homo sapiens ( uman) Q6FGM5 HUMAN PRL1 Opioid type X Homo sapiens ( uman) Q6FH06 HUMAN PYR1 ragments Homo sapiens ( uman) Q6FH34 HUMAN ragments Homo sapiens ( uman) Q6FHI8 HUMAN PR35 Purinoceptor P2RY5,8,9,10 GPR35,92,174 Homo sapiens ( uman) Q6FHK3 HUMAN DORA1 ragments Homo sapiens ( uman) Q6FHL1 HUMAN PR30 Chemokine receptor-like 2 Homo sapiens ( uman) Q6FHU6 HUMAN PR30 ragments Homo sapiens ( uman) Q6GMT1 HUMAN ragments Homo sapiens ( uman) Q6GMT4 HUMAN DRB2 Beta Adrenoceptors type 2 Homo sapiens ( uman) Q6GPG7 HUMAN Lysophosphatidic acid Edg-2 Homo sapiens ( uman) Q6GTR7 HUMAN PYSR Neuropeptide Y type 5 Homo sapiens ( uman) Q61939 HUMAN R17-219 ragments Homo sapiens ( uman) Q6I940 HUMAN R17-2O7 ragments Homo sapiens ( uman) Q6I941 HUMAN R17-82 ragments Homo sapiens ( uman) Q6IBH2 HUMAN PR19 PR Homo sapiens ( uman) Q6IET8 HUMAN P1-15413.4-001 actory am 13 MOR253 Homo sapiens ( uman) Q6IET9 HUMAN R12D2 actory II fam 12/MOR250 Homo sapiens ( uman) Q6IEUO HUMAN actory II fam 2/MOR256-262,270-285 Homo sapiens ( uman) Q6 UMAN actory II fam 5/MOR172-224,249,254 Homo sapiens ( uman) Q6 UMAN actory II fam 9/MOR120 Homo sapiens ( uman) Q6 UMAN actory II fam 9/MOR120 Homo sapiens ( uman) Q6 UMAN actory am 10 MOR263-269 Homo sapiens ( uman) Q6IEW6 HUMAN actory am 8 MOR161-171 Homo sapiens ( uman) Q6IEW7 HUMAN actory am 8 MOR161-171 Homo sapiens ( uman) Q6 UMAN actory am 11/MOR106,121-122 Homo sapiens ( uman) Q6 UMAN actory am 4 MOR225-248 Homo sapiens ( uman) Q6IE UMAN actory am 4 MOR225-248 Homo sapiens ( uman) Q6IE UMAN actory am 4 MOR225-248 Homo sapiens ( uman) Q6IE UMAN actory am 2/MOR256-262,270-285 Homo sapiens ( uman) Q6IEZ2 HUMAN actory am 5/MOR172-224,249,254 Homo sapiens ( uman) Q6IEZ4 HUMAN actory am 4 MOR225-248 Homo sapiens ( uman) Q6IEZ6 HUMAN ORSBF1 actory am 5/MOR172-224,249,254 Homo sapiens ( uman) Q6IFO1 HUMAN actory am 6/MOR103-105,107-119 Homo sapiens ( uman) Q6IF09 HUMAN actory am 11/MOR106,121-122 Homo sapiens ( uman) Q6IF12 HUMAN actory am 4 MOR225-248 Homo sapiens ( uman) Q6IF17 HUMAN actory am 1/MOR125-138,156 Homo sapiens ( uman) Q6IF20 HUMAN RP11-1123.12-001 actory am 13 MOR253 Homo sapiens ( uman) Q6IF23 HUMAN OR12D3 actory am 12MOR250 Homo sapiens ( uman) Q6IF24 HUMAN OR22 actory am 2/MOR256-262,270-285 Homo sapiens ( uman) US 9,512,236 B2 179 180 TABLE D-continued non-limiting list human GPCRs

UMAN F25) DAQB-117O11.4-001 aC ory am 2/MOR256-262,270-285 Homo sapiens ( uman) UMAN F31) aC ory fam 51-52MOR1-42 Homo sapiens ( uman) UMAN F34) aC ory am 2/MOR256-262,270-285 Homo sapiens ( uman) UMAN F35) aC ory am 6/MOR103-105,107-119 Homo sapiens ( uman) UMAN F36) aC ory am 8 MOR161-171 Homo sapiens ( uman) UMAN F40) aC ory am 2/MOR256-262,270-285 Homo sapiens ( uman) UMAN F41) aC ory am 2/MOR256-262,270-285 Homo sapiens ( uman) UMAN F43) aC ory am 2/MOR256-262,270-285 Homo sapiens ( uman) UMAN F44) aC ory am 2/MOR256-262,270-285 Homo sapiens ( uman) UMAN F46) aC ory am 5/MOR172-224,249,254 Homo sapiens ( uman) UMAN F50) RP11-317C20.1-001 aC ory am 13 MOR253 Homo sapiens ( uman) UMAN F51) RP11-317C2O.4-001 aC ory am 13 MOR253 Homo sapiens ( uman) UMAN F52) aC ory am 13 MOR253 Homo sapiens ( uman) UMAN F53) RP11-317C20.6-001 aC ory am 13 MOR253 Homo sapiens ( uman) UMAN F54) RP11-413C10.2-001 aC ory am 13 MOR253 Homo sapiens ( uman) UMAN F55) aC ory am 2/MOR256-262,270-285 Homo sapiens ( uman) UMAN F56) aC ory am 4 MOR225-248 Homo sapiens ( uman) UMAN F57) aC ory am 4 MOR225-248 Homo sapiens ( uman) UMAN F58) aC ory am 10 MOR263-269 Homo sapiens ( uman) UMAN F59) aC ory am 10 MOR263-269 Homo sapiens ( uman) UMAN F60) aC ory am 5/MOR172-224,249,254 Homo sapiens ( uman) UMAN F61) aC ory am 5/MOR172-224,249,254 Homo sapiens ( uman) UMAN F62) aC ory am 9 MOR120 Homo sapiens ( uman) UMAN F65) aC ory am 5/MOR172-224,249,254 Homo sapiens ( uman) UMAN F66) aC ory am 5/MOR172-224,249,254 Homo sapiens ( uman) UMAN F67) aC ory am 5/MOR172-224,249,254 Homo sapiens ( uman) UMAN F68) aC ory am 5/MOR172-224,249,254 Homo sapiens ( uman) UMAN F69) aC ory am 5/MOR172-224,249,254 Homo sapiens ( uman) UMAN F70) aC ory am 6/MOR103-105,107-119 Homo sapiens ( uman) UMAN F71) aC ory am 9 MOR120 Homo sapiens ( uman) UMAN F72) aC ory am 4 MOR225-248 Homo sapiens ( uman) UMAN F73) aC ory am 4 MOR225-248 Homo sapiens ( uman) UMAN F74) aC ory am 4 MOR225-248 Homo sapiens ( uman) UMAN F77) aC ory fam 51-52MOR1-42 Homo sapiens ( uman) UMAN F78) aC ory fam 51-52MOR1-42 Homo sapiens ( uman) UMAN F79) aC ory fam 51-52MOR1-42 Homo sapiens ( uman) UMAN F85) aC ory am 5/MOR172-224,249,254 Homo sapiens ( uman) UMAN F86) aC ory am 13 MOR253 Homo sapiens ( uman) UMAN F87) aC ory am 5/MOR172-224,249,254 Homo sapiens ( uman) UMAN F88) aC ory am 5/MOR172-224,249,254 Homo sapiens ( uman) UMAN F89) aC ory am 2/MOR256-262,270-285 Homo sapiens ( uman) UMAN F91) aC ory fam 51-52MOR1-42 Homo sapiens ( uman) UMAN F93) aC ory fam 51-52MOR1-42 Homo sapiens ( uman) UMAN F94) aC ory fam 51-52MOR1-42 Homo sapiens ( uman) UMAN F95) aC ory am 4 MOR225-248 Homo sapiens ( uman) UMAN F96) aC ory am 5/MOR172-224,249,254 Homo sapiens ( uman) UMAN FA1) aC ory am 2/MOR256-262,270-285 Homo sapiens ( uman) UMAN FA2) aC ory am 4 MOR225-248 Homo sapiens ( uman) UMAN FA3) aC ory am 4 MOR225-248 Homo sapiens ( uman) UMAN FA4) aC ory am 4 MOR225-248 Homo sapiens ( uman) UMAN FA5) aC ory am 4 MOR225-248 Homo sapiens ( uman) UMAN FA7) aC ory am 4 MOR225-248 Homo sapiens ( uman) UMAN FA8) aC ory am 10 MOR263-269 Homo sapiens ( uman) UMAN FA9) aC ory am 1/MOR125-138,156 Homo sapiens ( uman) UMAN FBO) aC ory am 1/MOR125-138,156 Homo sapiens ( uman) UMAN FB4) aC ory am 4 MOR225-248 Homo sapiens ( uman) UMAN FB5) aC ory am 8 MOR161-171 Homo sapiens ( uman) UMAN FB6) aC ory am 8 MOR161-171 Homo sapiens ( uman) UMAN FB7) aC ory am 8 MOR161-171 Homo sapiens ( uman) UMAN FB8) aC ory am 5/MOR172-224,249,254 Homo sapiens ( uman) UMAN FB9) aC ory am 5/MOR172-224,249,254 Homo sapiens ( uman) UMAN FCO) aC ory am 8 MOR161-171 Homo sapiens ( uman) UMAN FC1) aC ory am 8 MOR161-171 Homo sapiens ( uman) UMAN FC2) aC ory am 8 MOR161-171 Homo sapiens ( uman) UMAN FC3) aC ory am 8 MOR161-171 Homo sapiens ( uman) UMAN FC4) aC ory am 8 MOR161-171 Homo sapiens ( uman) UMAN FC5) aC ory am 8 MOR161-171 Homo sapiens ( uman) UMAN FC7) aC ory am 5/MOR172-224,249,254 Homo sapiens ( uman) UMAN FC8) aC ory am 5/MOR172-224,249,254 Homo sapiens ( uman) UMAN FC9) aC ory am 5/MOR172-224,249,254 Homo sapiens ( uman) UMAN FDO) aC ory am 5/MOR172-224,249,254 Homo sapiens ( uman) UMAN FD1) 8. gments Homo sapiens ( uman) UMAN FD3) aC ory am 5/MOR172-224,249,254 Homo sapiens ( uman) UMAN FD4) aC ory am 5/MOR172-224,249,254 Homo sapiens ( uman) UMAN FD5) aC ory am 10 MOR263-269 Homo sapiens ( uman) UMAN FD6) aC ory am 9 MOR120 Homo sapiens ( uman) UMAN FD7) aC ory fam 51-52MOR1-42 Homo sapiens ( uman) UMAN aC ory am 10 MOR263-269 Homo sapiens ( uman) US 9,512,236 B2 181 182 TABLE D-continued non-limiting list human GPCRs

FEO HUMAN aC ory I fam 5/MOR172-224,249,254 Homo sapiens ( uman) FE1 HUMAN aC ory I fam 5/MOR172-224,249,254 Homo sapiens ( uman) FE4 HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) FES HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) FE7 HUMAN aC ory I fam 6/MOR103-105,107-119 Homo sapiens ( uman) FE8 HUMAN aC ory I fam 10 MOR263-269 Homo sapiens ( uman) FE9 HUMAN aC ory I fam 8 MOR161-171 Homo sapiens ( uman) FF2 HUMAN aC ory I fam 5/MOR172-224,249,254 Homo sapiens ( uman) FF4 HUMAN aC ory I fam 5/MOR172-224,249,254 Homo sapiens ( uman) FF6 HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) FFT HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) FF8 HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) FF9 HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) FGO HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) FG2 HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) FG3 HUMAN aC ory I fam 1/MOR125-138,156 Homo sapiens ( uman) FG4 HUMAN aC ory I fam 10 MOR263-269 Homo sapiens ( uman) FGS HUMAN aC ory I fam 1/MOR125-138,156 Homo sapiens ( uman) FG6 HUMAN aC ory I fam 10 MOR263-269 Homo sapiens ( uman) FG7 HUMAN aC ory I fam 2/MOR256-262,270-285 Homo sapiens ( uman) FG8 HUMAN aC ory I fam 2/MOR256-262,270-285 Homo sapiens ( uman) FG9 HUMAN aC ory I fam 10 MOR263-269 Homo sapiens ( uman) HO HUMAN aC ory I fam 9 MOR120 Homo sapiens ( uman) H1. HUMAN aC ory I fam 6/MOR103-105,107-119 Homo sapiens ( uman) H2 HUMAN aC ory I fam 10 MOR263-269 Homo sapiens ( uman) H6 HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) H7 HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) H8 HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) H9 HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) O HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) 1 UMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) 2 UMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) 3 UMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) UMAN aC ory I fam 9 MOR120 Homo sapiens ( uman) : HUMAN aC ory I fan 13 MOR2S3 Homo sapiens ( uman) 7 UMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) 8 UMAN aC ory I fam 2/MOR256-262,270-285 Homo sapiens ( uman) 9 UMAN aC ory I fam 1/MOR125-138,156 Homo sapiens ( uman) O UMAN aC ory I fam 10 MOR263-269 Homo sapiens ( uman) 2 UMAN aC ory I fam 10 MOR263-269 Homo sapiens ( uman) 3 UMAN aC ory I fam 1/MOR125-138,156 Homo sapiens ( uman) HUMAN aC ory I fan 7 MOR139-15S Homo sapiens ( uman) HUMAN aC ory I fan 7 MOR139-15S Homo sapiens ( uman) UMAN aC ory I fan 7 MOR139-15S Homo sapiens ( uman) HUMAN aC ory I fan 7 MOR139-15S Homo sapiens ( uman) HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) HUMAN aC ory I fam 6/MOR103-105,107-119 Homo sapiens ( uman) 1. UMAN aC ory I fam 6/MOR103-105,107-119 Homo sapiens ( uman) UMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) UMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) 7 HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) K8 HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) K9 HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) LO HUMAN aC ory fam 51-52MOR1-42 Homo sapiens ( uman) L1 HUMAN RP11-413C10.1-001 aC ory am 13 MOR253 Homo sapiens ( uman) L2 HUMAN RP11-413C10.9-001 aC ory am 13 MOR253 Homo sapiens ( uman) L7 HUMAN aC ory am 1/MOR125-138,156 Homo sapiens ( uman) L8 HUMAN aC ory am 1/MOR125-138,156 Homo sapiens ( uman) L9 HUMAN aC ory am 1/MOR125-138,156 Homo sapiens ( uman) FM2 HUMAN aC ory am 1/MOR125-138,156 Homo sapiens ( uman) FM3 HUMAN aC ory an 3 MOR2SS Homo sapiens ( uman) FM4 HUMAN aC ory an 3 MOR2SS Homo sapiens ( uman) FMS HUMAN aC ory am 1/MOR125-138,156 Homo sapiens ( uman) FM6 HUMAN aC ory an 3 MOR2SS Homo sapiens ( uman) FM7 HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) FM8 HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) FM9 HUMAN aC ory am SMO 72-224,249,254 Homo sapiens ( uman) FNO HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) FN2 HUMAN aC ory am 1 MO 25-138,156 Homo sapiens ( uman) FNS HUMAN aC ory am 7 MO 39-155 Homo sapiens ( uman) FN7 HUMAN aC ory am 2/MOR256-262,270-285 Homo sapiens ( uman) FP1 HUMAN OR7AS aC ory an 7 MOR139-15S Homo sapiens ( uman) FP2 HUMAN aC ory an 7 MOR139-15S Homo sapiens ( uman) FP3 HUMAN aC ory am 4 MOR225-248 Homo sapiens ( uman) FP4 HUMAN aC ory am 2/MOR256-262,270-285 Homo sapiens ( uman) FP6 HUMAN aC ory am 6/MOR103-105,107-119 Homo sapiens ( uman) FP7 HUMAN aC ory am 2/MOR256-262,270-285 Homo sapiens ( uman) FP9 HUMAN aC ory an 7 MOR139-15S Homo sapiens ( uman) US 9,512,236 B2 183 184 TABLE D-continued non-limiting list human GPCRs Q6IFQ0 HUMAN FQ0) Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) Q6IFQ1 HUMAN FQ1) OR1OH2 Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) Q6IFQ2 HUMAN FQ2) Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) Q6IFQ5 HUMAN FQ5) DAQB-304F3.1-001 Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) Q6IFQ6 HUMAN FQ6) OR7A17 Olfactory II fam 7/MOR139-155 Homo sapiens ( uman) Q6IFQ7 HUMAN FQ7) Olfactory II fam 8/MOR161-171 Homo sapiens ( uman) Q6IFQ8 HUMAN FQ8) Olfactory II fam 8/MOR161-171 Homo sapiens ( uman) Q6IFRO HUMAN FRO) Olfactory II fam 8/MOR161-171 Homo sapiens ( uman) Q6IFR1 HUMAN FR1) Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) Q6IFR2 HUMAN FR2) OR6N2 Olfactory II fam 6/MOR103-105,107-119 Homo sapiens ( uman) Q6IFR3 HUMAN FR3) Olfactory II fam 6/MOR103-105,107-119 Homo sapiens ( uman) Q6IFR4 HUMAN FR4) Olfactory II fam 6/MOR103-105,107-119 Homo sapiens ( uman) Q6IFR5 HUMAN FR5) Olfactory II fam 6/MOR103-105,107-119 Homo sapiens ( uman) Q6IFR6 HUMAN FR6) OR6K2 Olfactory II fam 6/MOR103-105,107-119 Homo sapiens ( uman) Q6IFR7 HUMAN FR7) Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) Q6IFS1 HUMAN FS1) Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) Q6IFS2 HUMAN FS2) OR1OK1 Olfactory II fam 10/MOR263-269 Homo sapiens ( uman) Q6IN95 HUMAN N95) IL8RA interleukin-8 type A Homo sapiens ( uman) Q6IPXO HUMAN PXO) CCRL2 C-C Chemokine other Homo sapiens ( uman) Q6ISR6 HUMAN SR6) OR6W1P ragments Homo sapiens ( uman) Q6ISR8 HUMAN SR8) GHSR Growth hormone secretagogue Homo sapiens ( uman) Q6J164 HUMAN 164) GRMS Metabotropic glutamate group I Homo sapiens ( uman) Q6KHO9 HUMAN KHO9) ORSD4 ragments Homo sapiens ( uman) Q6L5J4 HUMAN L5J4) Fmet-leu-phe Homo sapiens ( uman) Q6LAJ3 HUMAN LAJ3) gamrh Adrenomedulin (G10D) Homo sapiens ( uman) Q6LD06 HUMAN LD06) ADRA1C ragments Homo sapiens ( uman) Q6LDH7 HUMAN LDH7) DRD2 ragments Homo sapiens ( uman) Q6LEE7 HUMAN LEE7) CMKLR1 ragments Homo sapiens ( uman) Q6NO55 HUMAN DKFZp686O11112 ragments Homo sapiens ( uman) Q6NOA5 HUMAN DKFZp686I13174 rizzled Group B (FZ 3 & 6) Homo sapiens ( uman) Q6NSL8 HUMAN FZD10 rizzled Group A (FZ 1&2&4&5&7-9) Homo sapiens ( uman) Q6NSP5 HUMAN GPR23 Purinoceptor P2RY5,8,9,10 GPR35,92,174 Homo sapiens ( uman) Q6NSYO HUMAN CNR2 Cannabinoid Homo sapiens ( uman) Q6NTA9 HUMAN OR1A1 Olfactory II fam 1/MOR125-138,156 Homo sapiens ( uman) Q6NTB3 HUMAN OR2C1 Olfactory II fam 2/MOR256-262,270-285 Homo sapiens ( uman) Q6NTB5 HUMAN ORSV1 ragments Homo sapiens ( uman) Q6NTC7 HUMAN NPBWR1 GPR Homo sapiens ( uman) Q6NTD7 HUMAN ORS1B4 Olfactory I fam 51-52/MOR1-42 Homo sapiens ( uman) Q6NTI7 HUMAN GPR143 Ocular albinism proteins Homo sapiens ( uman) Q6NUM3 HUMAN CHRMS Muse. acetylcholine Vertebrate type 5 Homo sapiens ( uman) Q6NUP5 HUMAN AGTR1 Angiotensin type 1 Homo sapiens ( uman) Q6NWM4 HUMAN GPR4 GPR Homo sapiens ( uman) Q6NWM5 HUMAN GPR21 GPR Homo sapiens ( uman) Q6NWQ5 HUMAN NPBWR2 GPR Homo sapiens ( uman) Q6NWQ6 HUMAN NPBWR2 GPR Homo sapiens ( uman) Q6NWQ8 HUMAN GPR77 C5a anaphylatoxin Homo sapiens ( uman) Q6NWQ9 HUMAN GPR77 C5a anaphylatoxin Homo sapiens ( uman) Q6NWRO HUMAN GPR77 C5a anaphylatoxin Homo sapiens ( uman) Q6NWR3 HUMAN GPR83 Neuropeptide Y other Homo sapiens ( uman) Q6NWR4 HUMAN GPR83 Neuropeptide Y other Homo sapiens ( uman) Q6NWR5 HUMAN GPR68 ragments Homo sapiens ( uman) Q6NWR6 HUMAN GPR68 ragments Homo sapiens ( uman) Q6NWR7 HUMAN GPR63 GPR45 like Homo sapiens ( uman) Q6NWR8 HUMAN GPR63 GPR45 like Homo sapiens ( uman) Q6NWR9 HUMAN GPR63 GPR45 like Homo sapiens ( uman) Q6NWS6 HUMAN GPR12 ragments Homo sapiens ( uman) Q6NWS7 HUMAN GPR12 ragments Homo sapiens ( uman) Q6NWS8 HUMAN GPR12 ragments Homo sapiens ( uman) Q6NXU6 HUMAN GPR45 GPR45 like Homo sapiens ( uman) Q6P2M6 HUMAN VIPR1 Vasoactive intestinal polypeptide Homo sapiens ( uman) Q6P4D8 HUMAN ragments Homo sapiens ( uman) Q6P523 HUMAN HTR2B Serotonin type 2 Homo sapiens ( uman) Q6P5R4 HUMAN MGCf2O8O ragments Homo sapiens ( uman) Q6PSW7 HUMAN OPN3 ragments Homo sapiens ( uman) Q6P7PO HUMAN C10orf7 ragments Homo sapiens ( uman) Q6P9E5 HUMAN HRH1 Histamine type 1 Homo sapiens ( uman) Q6PK25 HUMAN LOC441453 ragments Homo sapiens ( uman) Q6RKA2 HUMAN ADCYAP1R1 ragments Homo sapiens ( uman) Q6RKA3 HUMAN ADCYAP1R1 ragments Homo sapiens ( uman) Q6RYQ6 HUMAN PTGFR Prostaglandin F2-alpha Homo sapiens ( uman) Q6S991 HUMAN ADCYAP1R1 ragments Homo sapiens ( uman) UMAN ADCYAP1R1 ragments Homo sapiens ( uman) UMAN CHRM2 ragments Homo sapiens ( uman) Q6TTN3 HUMAN PTGER3 Prostaglandin E2 subtype EP3 Homo sapiens ( uman) Q6UPP1 HUMAN OPRM1 Opioid type M Homo sapiens ( uman) Q6UQ80 HUMAN OPRM1 Opioid type M Homo sapiens ( uman) Q6UR92 HUMAN MC1R Melanocyte stimulating hormone Homo sapiens ( uman) Q6UR93 HUMAN MC1R Melanocyte stimulating hormone Homo sapiens ( uman) US 9,512,236 B2 185 186 TABLE D-continued non-limiting list human GPCRs Q6UR94 HUMAN Melanocyte stimula ing hormone Homo sapiens ( uman) Q6UR95 HUMAN Melanocyte stimula ing hormone Homo sapiens ( uman) Q6UR96 HUMAN Melanocyte stimula ing hormone Homo sapiens ( uman) Q6UR97 HUMAN Melanocyte stimula ing hormone Homo sapiens ( uman) Q6UR98 HUMAN Melanocyte stimula ing hormone Homo sapiens ( uman) Q6UR99 HUMAN Melanocyte stimula ing hormone Homo sapiens ( uman) Q6URAO HUMAN Melanocyte stimula ing hormone Homo sapiens ( uman) Q6UVH2 HUMAN ragments Homo sapiens ( uman) Q6UXT6 HUMAN Olfactory II fam 5/MOR172-224.249,254 Homo sapiens ( uman) Q6XGY1 HUMAN ragments Homo sapiens ( uman) Q6ZMHO HUMAN Putativefunclassifie Class B GPCRs Homo sapiens ( uman) Q6ZMH4 HUMAN Putativefunclassifie Class B GPCRs Homo sapiens ( uman) Q6ZMI9 HUMAN ETL receptors Homo sapiens ( uman) Q6ZMNO HUMAN EMR1 Homo sapiens ( uman) Q6ZMN6 HUMA N Olfactory II fam 13/MOR253 Homo sapiens ( uman) Q6ZMP1 HUMAN Putativefunclassifie Class B GPCRs Homo sapiens ( uman) Q6ZMP9 HUMAN Purinoceptor P2RY5,8,9,10 GPR35,92,174 Homo sapiens ( uman) Q6ZMQ2 HUMAN Metabotropic glutamate group III Homo sapiens ( uman) Q6ZN22 HUMAN Vasoactive intestina polypeptide Homo sapiens ( uman) Q6ZPBO HUMAN ragments Homo sapiens ( uman) Q6ZS44 HUMAN ragments Homo sapiens ( uman) Q6ZTE9 HUMAN ragments Homo sapiens ( uman) Q6ZW62 HUMAN ragments Homo sapiens ( uman) Q711G2 HUMAN Putativefunclassified Class A GPCRs Homo sapiens ( uman) Q712M9 HUMAN Serotonin type 4 Homo sapiens ( uman) Q71U75 HUMAN Taste receptors T2R Homo sapiens ( uman) Q71V90 HUMAN ragments Homo sapiens ( uman) Q75LHO HUMAN ragments Homo sapiens ( uman) Q76E76 HUMAN ragments Homo sapiens ( uman) Q76L88 HUMAN PCR Putativefunclassified Class A GPCRs Homo sapiens ( uman) Q7KYP5 HUMAN ragments Homo sapiens ( uman) Q7KYZ9 HUMAN alpha 1c-adrenoceptor Subtype ragments Homo sapiens ( uman) Q7 KZS6 Melanocyte stimulating hormone Homo sapiens ( uman) Q7L853 HUMAN E DG1 ragments Homo sapiens ( uman) Q7M4L8 HUMAN Melanocyte stimulating hormone Homo sapiens ( uman) Q7Z3W3 HUMAN DKFZp686N1782 Proteinase-activated Homo sapiens ( uman) Q7Z580 HUMAN HTR2B ragments Homo sapiens ( uman) Q7Z581 HUMAN GPR50 ragments Homo sapiens ( uman) Q7Z582 HUMAN GPR50 Homo sapiens ( uman) Q7Z5R9 HUMAN Homo sapiens ( uman) Q7Z7I1 HUMAN C-C Chemokine typ eX Homo sapiens ( uman) Q7Z7Q5 HUMAN ragmen Homo sapiens ( uman) Q86SE3 HUMAN ragmen Homo sapiens ( uman) Q86SF1 HUMAN ragmen Homo sapiens ( uman) Q86SF3 HUMAN ragmen Homo sapiens ( uman) Q86SF4 HUMAN ragmen Homo sapiens ( uman) Q86SGO HUMAN ragmen Homo sapiens ( uman) Q86SG8 HUMAN ragmen Homo sapiens ( uman) Q86SG9 HUMAN ragmen Homo sapiens ( uman) Q86SH1 HUMAN ragmen Homo sapiens ( uman) Q86SH3 HUMAN ragmen Homo sapiens ( uman) Q86SI3 HUMAN ragmen Homo sapiens ( uman) Q86SIS HUMAN ragmen Homo sapiens ( uman) Q86SI8 HUMAN ragmen Homo sapiens ( uman) Q86SJ4 HUMAN ragmen Homo sapiens ( uman) Q86SM2 HUMAN ragmen Homo sapiens ( uman) Q86SP4 HUMAN ragmen Homo sapiens ( uman) Q86UG6 HUMAN ragmen Homo sapiens ( uman) Q86UG7 HUMAN ragmen Homo sapiens ( uman) Q86UG8 HUMAN ragmen Homo sapiens ( uman) Q86UG9 HUMAN ragmen Homo sapiens ( uman) Q86UHO HUMAN ragments Homo sapiens ( uman) Q86UK4 HUMAN PTC Taste receptors T2R Homo sapiens ( uman) Q86UN1 HUMAN HTRSA ragments Homo sapiens ( uman) Q86UN7 HUMAN CASR ragments Homo sapiens ( uman) Q86UZ8 HUMAN FZD2 ragments Homo sapiens ( uman) Q86V80 HUMAN Opioid type M Homo sapiens ( uman) Q86XI5 HUMAN ragments Homo sapiens ( uman) Q86YF2 HUMAN ragments Homo sapiens ( uman) Q86YG3 HUMAN ragments Homo sapiens ( uman) Q86YG9 HUMAN ragments Homo sapiens ( uman) Q86YW1. HUMAN MC1R Melanocyte stimulating hormone Homo sapiens ( uman) Q8IU63. HUMAN 6M1-16 ragments Homo sapiens ( uman) Q8IV06 HUMAN GPR171 Putativefunclassified Class A GPCRs Homo sapiens ( uman) Q8IV17. HUMAN SCTR Secretin Homo sapiens ( uman) Q8IV19 HUMAN CYSLTR1 Cysteinyl leukotriene Homo sapiens ( uman) Q8IV68 HUMAN LOC442421 ragments Homo sapiens ( uman) Q8IVWO HUMAN CHRMS Musc. acetylcholine Vertebrate type 5 Homo sapiens ( uman)