USOO9695227B2
(12) United States Patent (10) Patent No.: US 9,695.227 B2 Steyaert et al. (45) Date of Patent: Jul. 4, 2017
(54) BINDING DOMAINS DIRECTED AGAINST (58) Field of Classification Search GPCRG PROTEIN COMPLEXES AND USES None DERVED THEREOF See application file for complete search history. (56) References Cited (75) Inventors: Jan Steyaert, Beersel (BE); Els Pardon, Lubbeek (BE); Toon U.S. PATENT DOCUMENTS Laeremans, Dworp (BE); Brian 5,721,121 A 2/1998 Etcheverry et al. Kobilka, Palo Alto, CA (US): Soren 2007/OO77597 A1 4/2007 Gilchrist et al. Rasmussen, Frederiksberg (DK); 2007/0231830 A1 10/2007 Gilchrist et al. Sebastian Granier, Menlo Park, CA (US); Roger K. Sunahara, Ann Arbor, FOREIGN PATENT DOCUMENTS MI (US) WO 94.04678 A1 3, 1994 WO 993.7681 A2 7, 1999 (73) Assignees: Vrije Universiteit Brussel, Brussels WO OO43507 A2 T 2000 (BE); VIB VZW, Ghent (BE); The WO O190190 A2 11/2001 Board of Trustees of the Leland WO O2O85945 A2 10, 2002 Stanford Junior Universitv, Palo Alto, WO O3O25O2O A1 3, 2003 CA (US); The Regents of the WO O3035694 A2 5, 2003 WO 2004035614 A1 4/2004 University of Michigan, Ann Arbor, WO 2004.049.794 A2 6, 2004 MI (US) WO 2006086883 A1 8, 2006 WO 2009051633 4/2009 (*) Notice: Subject to any disclaimer, the term of this WO 2009,147.196 A1 12/2009 patent is extended or adjusted under 35 WO 2010043650 A2 4/2010 WO 201OO6674.0 A1 6, 2010 U.S.C. 154(b) by 594 days. WO 2012007593 A1 1, 2012 (21) Appl. No.: 14/129,100 WO 2012175643 A2 12/2012 (22) PCT Filed: Jun. 21, 2012 OTHER PUBLICATIONS Brown et al., Tolerance of single, but not multiple, amino acid (86). PCT No.: PCT/EP2012/062O36 replacements in antibody VHCDR2: a means of minimizing B cell S 371 (c)(1), wastage from somatic hypermutation? J. Immunology 156: 3285 (2), (4) Date: Jun. 5, 2014 3291, 1996.* Vajdos et al., Comprehensive functional maps of the antigen binding site of an anti-ErbB2 antibody obtained with shotgun (87) PCT Pub. No.: WO2012/175643 scanning mutagenesis. J. Mol. Biol. 320:415-428, 2002.* PCT Pub. Date: Dec. 27, 2012 Okuma Y et al., “Immunoprecipitation of Alpha-2-Adrenergic Receptor-GTP-Binding Protein Complexes. Using GTP-Binging Protein Selective Antisera Changes in Receptor-GTP-Binding Pro (65) Prior Publication Data tein Interaction Following Agonist Binding.” Journal of Biological US 2014/0275487 A1 Sep. 18, 2014 Chemistry, vol. 267, No. 21, 1992, pp. 14826-14831. Rasmussen, Sören G. F., et al., “Structure of a nanobody-stabilized active state of the beta.2 adrenoceptor,” Nature, Nature Publishing Group, United Kingdom, vol. 469, No. 7329, Jan. 13, 2011, pp. Related U.S. Application Data 175-18O. (60) Provisional application No. 61/571,159, filed on Jun. (Continued) 21, 2011. Primary Examiner — Ruixiang Li (30) Foreign Application Priority Data (74) Attorney, Agent, or Firm — TraskBritt P.C. (57) ABSTRACT Sep. 15, 2011 (EP) ...... 11181357 The present invention relates to the field of G protein coupled receptor (GPCR) structural biology and signaling. (51) Int. Cl. In particular, the present invention relates to binding domains directed against and/or specifically binding to C07K 6/28 (2006.01) GPCR:G protein complexes. Also provided are nucleic acid C07K I4/705 (2006.01) sequences encoding Such binding domains and cells express C07K I4/47 (2006.01) ing or capable of expressing Such binding domains. The C07K I4/435 (2006.01) binding domains of the present invention can be used as GOIN 33/74. (2006.01) universal tools for the structural and functional character (52) U.S. Cl. ization of G-protein coupled receptors in complex with CPC ...... C07K 14/705 (2013.01); C07K 14/435 downstream heterotrimeric G proteins and bound to various (2013.01); C07K 14/4722 (2013.01); C07K natural or synthetic ligands, for investigating the dynamic 14/7.0571 (2013.01); C07K 16/28 (2013.01); features of G protein activation, as well as for Screening and G0IN 33/74 (2013.01); C07K 231 7/22 drug discovery efforts that make use of GPCR:G protein (2013.01); C07K 231 7/569 (2013.01); C12N complexes. 2799/026 (2013.01); G0IN 2333/726 19 Claims, 23 Drawing Sheets (2013.01); G0IN 2500/02 (2013.01) (23 of 23 Drawing Sheet(s) Filed in Color) US 9,695.227 B2 Page 2
OTHER PUBLICATIONS International Search Report for PCT/EP2012/062036, mailed Apr. 19, 2013, 7 pages. European Communication in related, copending EP application No. 12730 478.0 dated Feb. 22, 2016. Translation of Text of the First Office Action in related, copending CN application No. 2012800405859, dated Sep. 11, 2015. Japanese Notice of Rejection dated May 17, 2016, Japanes Patent Application No. 2014-516358. Staus et al., Regulation of beta2-Adrenergic Receptor Function by Conformationally Selective Single-Domain Intrabodies, Molecular Pharmacology, Mar. 2014, pp. 472-481, vol. 85, No. 3. Steyaert et al., Nanobody stabilization of G protein-coupled recep tor conformational states, Current Opinion in Structural Biology, 2011, pp. 567-572, vol. 21. * cited by examiner U.S. Patent Jul. 4, 2017 Sheet 1 of 23 US 9,695.227 B2
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''. * x . . . irr, woitire it US 9,695,227 B2 1. 2 BINDING DOMAINS DIRECTED AGAINST unlocking the secrets of functional selectivity, the ability of GPCRG PROTEIN COMPLEXES AND USES differentagonists to coerce distinct downstream effects from DERVED THEREOF a single kind of receptor. Recent structural data Support the idea that GPCRs, despite their small size, are sophisticated CROSS-REFERENCE TO RELATED 5 allosteric machines with multiple signaling outputs. APPLICATIONS GPCRs, once activated, convey their signals in a GTP dependent manner via a complex of three proteins known as This application is a national phase entry under 35 U.S.C. heterotrimeric G proteins, or Golfy. Binding of extracellular S371 of International Patent Application PCT/EP2012/ ligands to GPCRs modulates their capacity to catalyze 062036, filed Jun. 21, 2012, designating the United States of 10 GDP-GTP exchange in Golfy, thereby regulating the intra America and published in English as International Patent cellular level of secondary messengers. The inactive Golfy Publication WO 2012/175643 A2 on Dec. 27, 2012, which heterotrimer is composed of two principal elements, claims the benefit under Article 8 of the Patent Cooperation Go-GDP and the G|By heterodimer. G|By sequesters the Treaty and under 35 U.S.C. S 119(e) to U.S. Provisional switch II element on Go. Such that it is unable to interact with Patent Application Ser. No. 61/571,159, filed Jun. 21, 2011, 15 second messenger systems, such as those involving cAMP and priority under Article 8 of the PCT to European Patent diacylglycerol and calcium. Activated GPCRs catalyze the Application Serial No. 11181357.2, filed Sep. 15, 2011. release of GDP from GC, allowing GTP to bind and liberate the activated GO-GTP Subunit. In this state, Switch II forms TECHNICAL FIELD a helix stabilized by the Y-phosphate of GTP allowing it to 2O interact with effectors such as adenylyl cyclase. Although The present disclosure relates to the field of G protein much progress has been made in understanding how GC, coupled receptor (GPCR) structural biology and signaling. subunits interact with and regulate the activity of their In particular, the present disclosure relates to binding downstream targets, it is not clear how activated GPCRs domains directed against and/or specifically binding to initiate this process by catalyzing nucleotide exchange on GPCR:G protein complexes. Also provided are nucleic acid 25 GO?3y. sequences encoding Such binding domains and cells express Drug discovery efforts generally focus on Small molecule ing or capable of expressing Such binding domains. The ligands that competitively bind to a particular catalytic or binding domains of the present disclosure can be used as active site, using static models of the target as a starting universal tools for the structural and functional character point. This method has identified and validated a multitude ization of G-protein coupled receptors in complex with 30 of viable active-site therapeutics in use today. However, as downstream G proteins and bound to various natural or reflected by the high failure rate of new drug compounds synthetic ligands, for investigating the dynamic features of (only an estimated 8% of phase I clinical therapeutics G protein activation, as well as for screening and drug eventually gain Food and Drug Administration approval, at discovery efforts that make use of GPCR:G protein com a conservative cost of S800 million per drug), many efforts plexes. 35 are unsuccessful and often targets are abandoned once they are deemed undruggable (Lee & Craik, 2009). A consider STATEMENT ACCORDING TO 37 C.F.R. able part of these failures are due to the fact that the most S1.821(c) OR (e) SEQUENCE LISTING prominent conformation of the target in question does not SUBMITTED AS PDF FILE WITH A REQUEST correspond to the druggable conformation to which a drug TO TRANSFER CRF FROMPARENT 40 must bind to be effective for the therapeutic indication. For APPLICATION example, efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent Pursuant to 37 C.F.R.S 1.821(c) or (e), a file containing a instability of this state in the absence of a G protein. PDF version of the Sequence Listing has been submitted Recently it became possible to obtain structures of an active concomitant with this application, the contents of which are 45 state of a GPCR, making use of conformationally selective hereby incorporated by reference. The transmittal docu stabilizing nanobodies or XAPERONESR that mimic G ments of this application include a Request to Transfer CRF proteins and increase the affinity for agonists at the from the parent application. orthosteric site (Rasmussen et al., 2011). This demonstrates the power of XAPERONES(R) to lock the structure of the BACKGROUND 50 most challenging drug targets in a therapeutically relevant conformation (Steyaert & Kobilka, 2011) and their useful Seven-transmembrane receptors (7TMRs), also called G ness for directed drug discovery allowing to specifically protein-coupled receptors (GPCRs), are the largest class of screen for potential drugs with higher sensitivity and selec receptors in the human genome and are the most commonly tivity towards a particular target (WO2012007593). One targeted protein class for medicinal therapeutics. Substantial 55 limitation of this technological approach is that for each progress has been made over the past three decades in GPCR target a specific stabilizing nanobody needs to be understanding diverse GPCRs, from pharmacology to func identified, which is not only time-consuming and costly, but tional characterization in vivo. Recent high-resolution struc also implies the availability of different tools, like biological tural studies have provided insights into the molecular material for immunization and selection purposes, amongst mechanisms of GPCR activation and constitutive activity 60 others. (e.g., Rasmussen et al., 2011). However, the molecular details of how GPCRs interact with and regulate the activity DISCLOSURE of their downstream targets are still lacking. The structures of GPCRs in complex with their downstream proteins are of Unraveling the structural and functional features of great interest not only because these interactions are phar 65 GPCRs in complex with their downstream heterotrimeric G macologically relevant but also because the atomic under proteins and bound to various natural and synthetic ligands standing of the intermolecular interactions are key to is valuable both for understanding the mechanisms of GPCR US 9,695,227 B2 3 4 signal transduction as well as for drug discovery efforts. For Agonist binding to a GPCR promotes interactions with the example, obtaining the structure of the active state ternary GDP-bound GCfBy heterotrimer leading to exchange of GDP complex composed of the agonist, the GPCR and the G for GTP on Go, and the functional dissociation of the G protein was so far unsuccessful but highly desired in the protein into GC-GTP and G|By subunits, which is needed for structural biology of signal transduction because of its further intracellular signaling. In a specific embodiment, the poorly understood biology. Crystallogenesis of this complex binding domain as described herein specifically bind to and turned out to be extremely difficult because one partner (the stabilize a GPCR:G protein complex in the absence of receptor) needs detergents to be solubilized while the G nucleotides, more specifically the binding domains bind to protein is unstable in detergents. Also, nucleotides that are and stabilize a GPCR: G protein complex wherein the G required for the formation of the complex also dissociate the 10 complex in a transient process. protein is in a nucleotide free from. In a particular embodi Therefore, and very unexpectedly, the inventors identified ment, the binding domain as described herein will specifi tools that stabilize complexes of GPCRs and G proteins, cally bind to a conformational epitope at the interface which allowed them to capture and purify such complexes, between the alpha and beta-gamma Subunit of the G protein, and finally to crystallize such complexes. This will facilitate 15 and as such blocks the GDP/GTP binding site and interferes the identification of ligands or drug compounds by, for with GDP/GTP binding. As such, and surprisingly, the example, structure based virtual screening or design, high binding domain as described herein prevents or inhibits the throughput Screening or fragment-based drug discovery dissociation of the GPCR: G protein complex in the presence (see, e.g., Example 10). More specifically, the inventors of nucleotides, in particular guanine nucleotides or analogs have identified binding domains, in particular immuno thereof, such as GTPYS. Also, the binding domain as globulin single variable domains, Suitable for the structural described herein prevents or inhibits binding of nucleotides and functional analysis of an active state complex composed to the G protein. of an agonist, GPCR and G protein (see, e.g., Example 4-7). Preferably, the binding domain as described herein is Interestingly, it was demonstrated that some of these directed against and/or specifically binds to a complex GPCR:G protein complex-selective binding domains are 25 specifically directed against the G protein, and not against comprising a GPCR, a G protein, and one or more receptor the GPCR. For example, binding domains were identified ligands. Typically, the receptor ligand will be an agonist or that bind Gs at the interface of Gos and Gfy, without a positive allosteric modulator, or a combination thereof. making contact with the beta-adrenergic receptor (see, e.g., According to another preferred embodiment, the binding Example 3), and will thus be useful to capture and stabilize 30 domain as described herein is directed against and/or spe other Gs coupled receptors, as was demonstrated for the cifically binds to a complex comprising a Gs protein coupled arginine vasopressin receptor 2 (V2R) (see, e.g., Example receptor and a Gs protein; or to a complex comprising a Gi 9). Thus, it is a particular advantage that binding domains protein coupled receptor and a Gi protein; or to complex are directed against the G protein of a GPCR:G protein comprising a Git protein coupled receptor and a Git protein; complex, since such a binding domain can be used as 35 or to a complex comprising a Ggust protein coupled receptor generic tool to stabilize and capture active state complexes and a Ggust protein; or to a complex comprising a GZ of the range of GPCRs that interact with that particular G protein coupled receptor and a GZ protein; or to a complex protein. comprising a Golf protein coupled receptor and a Golf Thus, according to a first aspect, the disclosure relates to protein; or to a complex comprising a Gq protein coupled a binding domain that is directed against and/or specifically 40 binds to a complex comprising a GPCR and a G protein. receptor and a Gq protein; or to a complex comprising a G12 More specifically, the binding domain as described herein coupled receptor and a G12 protein; or to a complex binds with higher affinity to the GPCR:G protein complex as comprising a G13 coupled receptor and a G13 protein. The compared with binding to the G protein alone and/or to the GPCR and/or the G protein as comprised in the complex GPCR alone, respectively. Also, the binding domain as 45 may be from the same or different species, in particular from described herein enhances the affinity of a G protein for a a mammalian species. Preferably the GPCR is a human GPCR. Thus, the present disclosure provides for binding protein. domains directed against and/or specifically binding to a In general, a binding domain of the disclosure can be any conformational epitope of a complex comprising a GPCR non-naturally occurring molecule, or part thereof, that is and a G protein, and stabilizes or locks the complex in a 50 able to specifically bind to a GPCR:G protein complex. particular conformational State, more specifically an active conformational state. Particularly, the binding domain as described herein is an In general, the binding domain as described herein may immunoglobulin single variable domain comprising an bind to any conformational epitope that is made available or amino acid sequence that comprises four framework regions accessible by a complex of a GPCR and a G protein. These 55 (FR1 to FR4) and three complementarity-determining conformational epitopes may be represented by the indi regions (CDR1 to CDR3), according to the following for vidual proteins comprised in the complex, and/or may only mula (1): be represented upon formation of the complex. Further, these conformational epitopes may or may not be repre FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1). sented by the individual proteins alone. According to one 60 particular embodiment, the binding domain as described Preferably, the binding domain as described herein is an herein specifically binds to the G protein comprised in the immunoglobulin single variable domain derived from a complex, and not to the GPCR. Camelidae species, and particularly is a nanobody or V.H. Typically, in nature, G proteins are in a nucleotide-bound According to specific embodiments, the binding domain form. More specifically, G proteins (or at least the C. subunit) 65 as described herein is an immunoglobulin single variable are bound to either GTP or GDP depending on the activation domain comprising an amino acid sequence that comprises status of a particular GPCR, as described further herein. four framework regions (FR1 to FR4) and three comple US 9,695,227 B2 5 6 mentarity-determining regions (CDR1 to CDR3), according a receptor ligand, in a functional conformational state, more to the following formula (1): specifically in an active conformational state. In one specific embodiment, the binding domains as described herein can be used to prevent dissociation of the complex in the and wherein CDR1 is chosen from the group consisting of presence of nucleotides, in particular guanine nucleotides or a) SEQ ID NOs: 13-18, analogs thereof. Such as GTPyS. Binding domains as tools to b) Polypeptides that have at least 80% amino acid identity stabilize GPCR:G protein complexes and block the GPCR in with SEQ ID NOs: 13-18, a functional conformational state, preferably an active con c) Polypeptides that have 3, 2 or 1 amino acid difference formational state, are thus very useful for a range of appli with SEQ ID NOs: 13-18, 10 cations, as outlined hereafter. and wherein CDR2 is chosen from the group consisting Disclosed is the use of a binding domain as described of: herein to crystallize and/or to solve the structure of a a) SEQ ID NOs: 25-30, complex comprising a GPCR and a G protein, and optionally b) Polypeptides that have at least 80% amino acid identity a receptor ligand. with SEQ ID NOs: 25-30, 15 Also envisaged within the scope of the present disclosure c) Polypeptides that have 3, 2 or 1 amino acid difference is to use a binding domain as described herein or a cell or a with SEQ ID NOs: 25-30, membrane preparation derived thereof, as described herein, and wherein CDR3 is chosen from the group consisting to screen for compounds that modulate the signaling activity of: of the GPCR. a) SEQ ID NOs: 37-42, Further, the binding domains as described herein may be b) Polypeptides that have at least 80% amino acid identity used to capture one or more interacting proteins, in particu with SEQ ID NOs: 37-42, lar proteins that interact with the G protein and/or with the c) Polypeptides that have 3, 2 or 1 amino acid difference GPCR. with SEQ ID NOs: 37-42. According to specific embodiments, the present disclo In a particularly preferred embodiment, the present dis 25 Sure provides for a method of capturing and/or purifying a closure provides for an immunoglobulin single variable complex comprising a GPCR and a G protein, the method domain comprising an amino acid sequence that comprises comprising the steps of: four framework regions (FR1 to FR4) and three comple a) Providing a binding domain as described herein, and mentarity-determining regions (CDR1 to CDR3), according b) Allowing the binding domain to bind to a complex to the following formula (1): 30 comprising a GPCR and a G protein and optionally a receptor ligand, and (1): c) Optionally, isolating the complex formed in step b). wherein CDR1 is SEQ ID NO: 13; In another specific embodiment, the present disclosure wherein CDR2 is SEQ ID NO: 25; and relates to a method of determining the crystal structure of a wherein CDR3 is SEQ ID NO:37. 35 complex comprising a GPCR and a G protein, the method According to a very specific embodiment, the binding comprising the steps of: domain, in particular the immunoglobulin single variable a) Providing a binding domain as described herein, and domain, has an amino acid sequence selected from the group b) Allowing the binding domain to bind to a complex consisting of consisting of SEQ ID NOs: 1 to 6. comprising a GPCR and a G protein and optionally a Further the binding domains as described herein may also 40 receptor ligand, and be comprised in a polypeptide. Also, the binding domains c) Crystallizing the complex formed in step b). may be immobilized on a solid Support. Some of the binding domains as described herein may In one particular aspect, the present disclosure relates to have therapeutic utility. Thus, it is also an object of the a binding domain that is directed against and/or specifically disclosure to use the binding domains as described herein to binds to a G protein. 45 modulate GPCR receptor signaling, in particular G protein Another aspect of the disclosure envisages a complex mediated GPCR receptor signaling. comprising a binding domain as described herein. In par The present disclosure further encompasses a method of ticular, the complex comprises a GPCR, a G protein, and producing a binding domain directed against and/or specifi optionally a receptor ligand. In certain applications, the cally binding to a complex comprising a GPCR and a G complex as described herein is crystalline. 50 protein, the method comprising the steps of Further, the disclosure also provides for nucleic acid a) Expressing in a Suitable cellular expression system a sequences, in particular a nucleic acid sequence encoding nucleic acid as described herein, and optionally, any amino acid sequence of any of the binding domains as b) Isolating and/or purifying the binding domain. described herein, as well as recombinant vectors comprising Another aspect of the present disclosure relates to a any of the nucleic acid sequences as described herein. 55 method of Screening for binding domains directed against Particularly preferred aspects of the disclosure are cells and/or specifically binding to a complex comprising a GPCR comprising any of the vectors or nucleic acids as described and a G protein, the method comprising the steps of: herein, and as such, that can express or are capable of a) Providing a plurality of binding domains, and expressing a GPCR and/or a G protein. Cell cultures of cells b) Screening the plurality of binding domains for a according to the disclosure as well as membrane prepara 60 binding domain that binds to a complex comprising a tions derived thereof are also within the scope of the present GPCR and a G protein, and invention. c) Isolating the binding domain that binds to the complex. The herein described binding domains, complexes and cells may be useful in a variety of contexts and applications. BRIEF DESCRIPTION OF THE DRAWINGS Thus, accordingly, one aspect of the disclosure relates to the 65 use of a binding domain as described herein to stabilize a The patent or application file contains at least one drawing complex comprising a GPCR and a G protein, and optionally executed in color. Copies of this patent or patent application US 9,695,227 B2 7 8 publication with color drawing(s) will be provided by the critical micelle concentration (CMC) of the detergent as Office upon request and payment of the necessary fee. assayed by 3H-dihydro alprenolol (3H-DHA) saturation FIG. 1: G protein cycle for the BAR:Gs complex. Panel binding. Diluting B2AR maintained in DDM 1000-fold a. Extracellular agonist binding to the BAR leads to con below the CMC cause loss in 3H-DHA binding (black data formational rearrangements of the cytoplasmic ends of 5 points) after 20 sec. In contrast 32AR in MNG-3 diluted transmembrane segments that enable the Gs heterotrimer (C. 1000-fold below the CMC maintained full ability to bind the B, and Y) to bind the receptor (R,R). GDP is released from radioligand after 24 hrs. the C. subunit upon formation of R:G complex. The GTP FIG. 5: Purity and homogeneity of the R:G complex. binds to the nucleotide-free C. Subunit resulting in dissocia Panel a) Analytical SDS-PAGE/Coomassie blue stain of tion of the C. and By subunits from the receptor. The subunits 10 samples obtained at various stages of T4L-BAR:Gs purifi regulate their respective effector proteins adenylyl cyclase cation. BI167107 agonist bound, dephosphorylated, and (AC) and Ca" channels. The Gs heterotrimer reassembles deglycosylated receptor in excess amount of Gis heterotrimer from C. and By subunits following hydrolysis of GTP to GDP is used for optimal coupling efficiency with the functional in the C. subunit. Panel b. The purified nucleotide-free fraction of the G protein. Functional purification of Gs is BAR:Gs protein complex maintained in detergent micelles. 15 archived through its interaction with the immobilized recep The GSC. subunit consists of two domains, the Ras domain tor on the M1 resin while non-functional/non-binding Gs (C.Ras) and the C-helical domain (CAH). Both are involved washes out. Panel b) A representative elution profile of one in nucleotide binding. In the nucleotide-free state, the CAH of four consecutive preparative gel filtrations with fraction domain has a variable position relative the C.Ras domain. ation indicated in red. Fractions containing the R:G complex FIG. 2: Formation of a stable AR:Gs complex. A stable were pooled within the indicated dashed lines, spin concen f3AR:Gs complex was achieved by the combined effects of: trated and analyzed for purity and homogeneity by SDS 1) binding a high affinity agonist to the receptor with an PAGE/Coomassie blue (a, second last lane to the right), gel extremely slow dissociation rate (as described in Rasmussen filtration in Panel c), and by anion exchange chromatogra et al., 2011); 2) formation of a nucleotide free complex in the phy in Panel d). Upper panel shows elution profile from presence of apyrase that hydrolyses released GDP prevent 25 analytical IEC of B2AR-365:Gs complex that was treated ing it from rebinding and causing a less stable R:G interac with PPase prior to complex formation in comparison with tion; and 3) detergent exchange of DDM for MNG-3 that complex which was not dephosphorylated resulting in a stabilizes the complex. heterogeneous preparation (lower panel). The less homoge FIGS. 3A-3G: Effect of nucleotide analogs, pH, and neous material in the fractions outside the indicated dashed nanobodies on the stability of the BAR:Gs complex. FIG. 30 lines in Panel b) were used for analytical gel filtration 3A, Analytical gel filtration showing that nucleotides GDP experiments of the R: G complex in presence of various and GTPYS (0.1 mM) causes dissociation of the BAR-365: chemicals (examples in FIG. 1). Gs complex. FIG. 3B. The phosphates pyrophosphate and FIG. 6: Flow-chart of the purification procedures for forcarnet (used at 5 mM) resembling the nucleotide phos preparing R:G complex with Nb35. phate groups did not cause disruption of the complex. They 35 FIG. 7: Purification of Nb35 and determination of R:G:Nb served as additives as they improved crystal growth of both mixing ratio. Panel a) Preparative ion exchange chromatog the T4L-f2AR:Gs complex (without nanobodies), T4L raphy following nickel affinity chromatography purification B2AR:Gs:Nb37, and T4L-B2AR:Gs:Nb35. FIG.3C. The pH of Nanobody 35 (Nb35). The nanobody eluted in two limit was determined to guide the preparation of crystalli populations (shown in red) as a minor peak and a major Zation screens. For the same purpose the effect of ionic 40 homogeneous peak which was collected, spin concentrated, strength (data not shown) was determined using NaCl at and used for crystallography following determination of various concentrations. The complex is stable in 20, 100, proper mixing ratio with the R:G complex as shown in Panel and 500 mM but dissociates at 2.5 M NaCl. FIG. 3D, b). Panel b) The agonist bound T4L-B2AR:Gs complex was Nanobody 35 (Nb35, red broken line) binds to the T4L mixed with slight excess of Nb35 (1 to 1.2 molar ratio of |B2AR:Gs: BI167107 ternary complex (blue solid line) to 45 R:G complex to Nb35) on the basis of their protein concen form the R:G:Nb35 complex (red solid line) which is trations and verified by analytical gel filtration. insensitive to GTPYS treatment (green solid line) in contrast FIG. 8: Crystals of the T4L-B2AR:Gs:Nb35 complex in to the treated R: G complex (green broken line). Nb35 and sponge-like mesophase. Nb37 bind separate epitopes on the Gs heterotrimer to form FIG.9: Overall structure of the BAR:Gs complex. Panel a R:G:Nb35:Nb37 complex (purple solid line). FIG. 3E, 50 a, Lattice packing of the complex shows alternating layers of Nanobody 36 (Nb36, red broken line) binds to the to the R:G receptor and G protein within the crystal. Abundant contacts complex (black solid line) to form the R:G:Nb36 complex are formed among proteins within the aqueous layers. Panel (red solid line) which is less sensitive to GTPYS treatment b. The overall structure of the asymmetric unit contents (green solid line). Nb36 and Nb37 bind separate epitopes on shows the faR (green) bound to an agonist (yellow the Gs heterotrimer to form a R:G:Nb36:Nb37 complex 55 spheres) and engaged in extensive interactions with GSC. (purple solid line). FIG.3F, Nanobody 37 (Nb37, green line) (orange). GCS together with GB (cyan) and GY (purple) binds to the to the R:G complex (black solid line) to form the constitute the heterotrimeric G protein Gs. A Gs binding R:G:Nb37 complex (red solid line). FIG. 3G, The R:G:Nb37 nanobody (Nb35, red) binds the G protein between the C. and complex is insensitive to GTPYS treatment (blue solid line) B subunits. The nanobody (Nb35) facilitates crystallization, in contrast to the treated R:G complex (blue broken line). 60 as does T4 lysozyme (magenta) fused to the amino terminus FIG. 4: Stabilizing effect of MNG-3 on the R:G complex. of the BAR. Panel c, The biological complex omitting Panel a) Analytical gel filtration of BAR-365:Gs complexes crystallization aids, showing its location and orientation purified in DDM (in black), MNG-3 (in blue), or two within a cell membrane. MNG-3 analogs (in red and green) following incubation for FIGS. 10A-10C: Interactions of Nb35 with GS within the 48 hrs at 4°C. In contrast to DDM the R:G complexes are 65 BI-167107 bound T4L-B2AR:Gs:Nb35 complex. FIG. 10A, stable in the MNG detergents. Panel b) Effect of diluting Two representative views on the interactions of CDR1 unliganded purified B2AR in DDM or MNG-3 below the (space filling representation) of Nb35 (red) with GP (space US 9,695,227 B2 9 10 filling, cyan). FIG. 10B. Two representative views on the structure. Panel d, The B1-C1 and B6-C.5 loops are rear interactions of CDR3 (space filling representation) of Nb35 ranged in the nucleotide-free faR:Gs structure. (red) with GCS (space filling, orange) and GB (space filling, FIG. 16: Nb37 inhibits GTPYS binding to Gos. Bodipy cyan). By interacting with GCS and GP. Nb35 may reduce GTPYS (100 nM) was incubated with 1 uM purified Gos and the conformational flexibility of the complex. FIG. 10C. the fluorescence increase measured in real time in the Two representative views on the interactions of the frame presence of increasing concentrations of Nb37. work regions of Nb35 (space filling representation, red) with FIG. 17: Nb35 does not affect GTPYS binding to Gos. GCS (orange). Bodipy-GTPYS (100 nM) was incubated with 1 uM purified FIGS. 11A and 11B: Crystal contacts between Nb35 and GCS and the fluorescence increase measured in real time in GCS Subunits of adjacent complexes. Crystal contacts 10 the presence of increasing concentrations of Nb35. involving Nb35 (red, space-filling representation) and GoS FIG. 18: Nb35 inhibits GTPYS binding to the Gsofy (orange) of the -X.y-1/2.-Z+1 symmetry related complex heterotrimer. Bodipy-GTPYS (100 nM) was incubated with 1 uM purified GSOfy heterotrimer and the fluorescence (FIG. 11A) and the x,y-1.Z symmetry related complex (FIG. increase measured in real time in the presence of increasing 11B). 15 concentrations of Nb35. FIG. 12: Comparison of active and inactive f32AR struc FIG. 19: Purification of a stable AVP:NT4LV2R:Gs com tures. Panel a, Side and cytoplasmic views of the B2AR:Gs plex. Panel A, schematic representation of the purification of structure (green) compared to the inactive carazolol-bound the AVP:NT4LV2R:Gs complex using Ni-NTA followed by B2AR structure (blue; Rosenbaum et al., 2007). Significant FLAG-tag affinity purification. Panel b, SEC chromatogram structural changes are seen for the intracellular domain of of the affinity purified AVP:NT4LV2R:Gs. Panel c, SDS TM5 and TM6. TM5 is extended by two helical turns while page to monitor the purification scheme. lane 1: flow trough TM6 is moved outward by 14 A as measured at the C-car of the FLAG-tag affinity column; lane 2: mix of AVP bons of Glu268 (yellow arrow) in the two structures. Panel NT4LV2R and Gs, prior to purification; lane 3: molecular b, B2AR:Gs compared with the nanobody-stabilized active marker; lane 4: AVP:NT4LV2R:Gs complex eluted from state B2AR:Nb80 structure (orange, Rasmussen et al., 2011). 25 SEC; lane 5: AVP:NT4LV2R:Gs complex after Ni-NTA Panel c, The positions of residues in the E/DRY and NPXXY followed by FLAG-tag affinity purification. motifs and other key residues of the B2AR:Gs and B2AR: FIG. 20: Stability of the AVP:NT4LV2R:Gs complex Nb80 structures as seen from the cytoplasmic side. All monitored by SEC. Dashed line: SEC chromatogram of the residues occupy very similar positions except Arg131 which AVP:NT4LV2R:Gs complex after 24 hours incubation on in the B2AR:Nb80 structure interacts with the nanobody. 30 ice. Blue line: SEC chromatogram of the AVP:NT4LV2R:Gs FIG. 13: Views of electron density for residues in the R:G complex after 48 hours incubation on ice. Red line, SEC interface. Panel a) The D/ERY motif at the cytoplasmic end chromatogram of AVP:NT4LV2R:Gs after incubation of the of TM3. Panel b) Packing interaction between Arg131 of the complex with 10 uM of the antagonist SR 121463. E/DRY motif and Tyr391 of C-terminal Go.S. Panel c) The NPXXY in the cytoplasmic end of TM7. Paneld) Interactions 35 DETAILED DESCRIPTION of Thr68 and Tyr141 with Asp130 of the E/DRY motif. Phel39 of IL2 is buried in a hydrophobic pocket in GC.S. Definitions Panel e) The B1-C1 loop (P-loop) of GCS involved in The present disclosure will be described with respect to nucleotide binding. Electron density maps are 2Fo-Fc maps particular embodiments and with reference to certain draw contoured at 1 sigma. 40 ings but the disclosure is not limited thereto but only by the FIG. 14: Receptor:G protein interactions. Panels a and b: claims. Any reference signs in the claims shall not be The C.5-helix of GCS docks into a cavity formed on the construed as limiting the scope. The drawings described are intracellular side of the receptor by the opening of trans only schematic and are non-limiting. In the drawings, the membrane helices 5 and 6. Panel a: Within the transmem size of Some of the elements may be exaggerated and not brane core, the interactions are primarily non-polar. An 45 drawn on scale for illustrative purposes. Where the term exception involves packing of Tyr391 of the C.5-helix “comprising is used in the present description and claims, against Arg131 of the conserved DRY sequence in TM3 (see it does not exclude other elements or steps. Where an also FIG. 13). Arg131 also packs against Tyr of the con indefinite or definite article is used when referring to a served NPXXY sequence in TM7. Panel b: As C.5-helix exits singular noun, e.g., “a” “an,” or “the, this includes a plural the receptor it forms a network of polar interactions with 50 of that noun unless something else is specifically stated. TM5 and TM3. Panel c: Receptor residues Thr68 and Furthermore, the terms first, second, third and the like in the Asp130 interact with the IL2 helix of the BAR via Tyr141, description and in the claims, are used for distinguishing positioning the helix so that Phe 139 of the receptor docks between similar elements and not necessarily for describing into a hydrophobic pocket on the G protein surface, thereby a sequential or chronological order. It is to be understood structurally linking receptor-G protein interactions with the 55 that the terms so used are interchangeable under appropriate highly conserved DRY motif of the BAR. circumstances and that the embodiments of the disclosure FIG. 15: Conformational changes in GC.S. Panel a, A described herein are capable of operation in other sequences comparison of GCS in the BAR:Gs complex (orange) with than described or illustrated herein. the GTPYS-bound Gos (grey) (PDB ID: 1 AZT: Sunahara et Unless otherwise defined herein, scientific and technical al., 1997). GTPYS is shown as spheres. The helical domain 60 terms and phrases used in connection with the present of GCS (GCSAH) exhibits a dramatic displacement relative disclosure shall have the meanings that are commonly to its position in the GTPYS-bound state. Panel b, The understood by those of ordinary skill in the art. Generally, C.5-helix of GCS is rotated and displaced toward the BAR, nomenclatures used in connection with, and techniques of perturbing the B6-O.5 loop which otherwise forms part of the molecular and cellular biology, genetics and protein and GTPYS binding pocket. Panel c, The B1-C1 loop (P-loop) 65 nucleic acid chemistry and hybridization described herein and 36-O.5 loop of GOs interact with the phosphates and are those well known and commonly used in the art. The purine ring, respectively, of GTPYS in the GTPyS-GOs methods and techniques of the present disclosure are gen US 9,695,227 B2 11 12 erally performed according to conventional methods well The term “conformation' or “conformational state' of a known in the art and as described in various general and protein refers generally to the range of structures that a more specific references that are cited and discussed protein may adopt at any instant in time. One of skill in the throughout the present specification unless otherwise indi art will recognize that determinants of conformation or cated. See, for example, Sambrook et al., Molecular Clon conformational State include a protein's primary structure as ing: A Laboratory Manual. 2d ed., Cold Spring Harbor reflected in a protein’s amino acid sequence (including Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel modified amino acids) and the environment Surrounding the et al., Current Protocols in Molecular Biology, Greene protein. The conformation or conformational state of a Publishing Associates (1992, and Supplements to 2002). protein also relates to structural features such as protein 10 secondary structures (e.g., C.-helix, B-sheet, among others), The term “binding domain or “protein binding domain tertiary structure (e.g., the three dimensional folding of a refers generally to any non-naturally occurring molecule, or polypeptide chain), and quaternary structure (e.g., interac part thereof, that is able to bind a protein or peptide using tions of a polypeptide chain with other protein subunits). specific intermolecular interactions. A variety of molecules Post-translational and other modifications to a polypeptide can function as protein binding domains, including, but not 15 chain Such as ligand binding, phosphorylation, Sulfation, limited to, proteinaceous molecules (protein, peptide, pro glycosylation, or attachments of hydrophobic groups, tein-like or protein containing), nucleic acid molecules among others, can influence the conformation of a protein. (nucleic acid, nucleic acid-like, nucleic acid containing), and Furthermore, environmental factors, such as pH, salt con carbohydrate molecules (carbohydrate, carbohydrate-like, centration, ionic strength, and osmolality of the Surrounding carbohydrate containing). A more detailed description can Solution, and interaction with other proteins and co-factors, be found further in the specification. among others, can affect protein conformation. The confor The terms “polypeptide.” “protein, or “peptide' are used mational state of a protein may be determined by either interchangeably herein, and refer to a polymeric form of functional assay for activity or binding to another molecule amino acids of any length, which can include coded and or by means of physical methods such as X-ray crystallog non-coded amino acids, chemically or biochemically modi 25 raphy, NMR, or spin labeling, among other methods. For a fied or derivatized amino acids, and polypeptides having general discussion of protein conformation and conforma modified peptide backbones. tional states, one is referred to Cantor and Schimmel, As used herein, the term “protein complex” or simply Biophysical Chemistry, Part I: The Conformation of Bio “complex,” refers to a group of two or more associated logical Macromolecules, W.H. Freeman and Company, polypeptide chains. Proteins in a protein complex are linked 30 1980, and Creighton, Proteins: Structures and Molecular by non-covalent protein-protein interactions. The "quater Properties, W.H. Freeman and Company, 1993. A “specific nary structure' is the structural arrangement of the associ conformation” or “specific conformational state' is any ated folded proteins in the protein complex. It will be Subset of the range of conformations or conformational understood that the complex may be a multimeric complex states that a protein may adopt. comprising two, three, four, five, six or more polypeptides. 35 As used herein, a “functional conformation' or a “func Also, the complex may additionally comprise a non-pro tional conformational state.” refers to the fact that proteins teinaceous molecule. possess different conformational states having a dynamic As used herein, the terms “nucleic acid molecule,” “poly range of activity, in particular ranging from no activity to nucleotide.” “polynucleic acid,” or “nucleic acid are used maximal activity. Examples of functional conformational interchangeably and refer to a polymeric form of nucleotides 40 states include active conformations and inactive conforma of any length, either deoxyribonucleotides or ribonucle tions. It should be clear that “a functional conformational otides, or analogs thereof. Polynucleotides may have any state' is meant to cover any conformational state of a GPCR, three-dimensional structure, and may perform any function, having any activity, including no activity; and is not meant known or unknown. Non-limiting examples of polynucle to cover the denatured States of proteins. A particular class otides include a gene, a gene fragment, exons, introns, 45 of functional conformations that is envisaged here is a messenger RNA (mRNA), transfer RNA, ribosomal RNA, 'druggable conformation' and generally refers to a unique ribozymes, cDNA, recombinant polynucleotides, branched therapeutically relevant conformational state of a target polynucleotides, plasmids, vectors, isolated DNA of any protein. As an illustration, the active conformation of the B2 sequence, control regions, isolated RNA of any sequence, adrenergic receptor corresponds to the druggable conforma nucleic acid probes, and primers. The nucleic acid molecule 50 tion of this receptor for the treatment of asthma. It will thus may be linear or circular. be understood that drugability is confined to particular As used herein, the term “ligand’ or “receptor ligand conformations depending on the therapeutic indication. means a molecule that specifically binds to a GPCR, either The wording “locking' or “trapping or “fixing or intracellularly or extracellularly. A ligand may be, without “freezing with respect to a functional conformational state the purpose of being limitative, a protein, a (poly)peptide, a 55 of a GPCR (as defined herein), as used herein, refers to the lipid, a small molecule, a protein scaffold, an antibody, an retaining or holding of a GPCR in a subset of the possible antibody fragment, a nucleic acid, a carbohydrate. A ligand conformations that it could otherwise assume, due to the may be synthetic or naturally occurring. The term “ligand effects of the interaction of the GPCR:G protein complex includes a “native ligand’ which is a ligand that is an with the binding domain according to the invention. Accord endogenous, natural ligand for a native GPCR. In most 60 ingly, a protein that is "conformationally trapped' or “con cases, a ligand is a “modulator that increases or decreases formationally fixed' or “conformationally locked' or “con an intracellular response when it is in contact with, for formationally frozen,” as used herein, is one that is held in example binds to, a GPCR that is expressed in a cell. a subset of the possible conformations that it could other Examples of ligands that are modulators include agonists, wise assume, due to the effects of the interaction of the partial agonists, inverse agonists, and antagonists, of which 65 GPCR:G protein complex with the binding domain accord a more detailed description can be found further in the ing to the invention. Within this context, a binding domain specification. that specifically or selectively binds to a specific conforma US 9,695,227 B2 13 14 tion or conformational state of a protein refers to a binding different antigen, and does not necessarily imply high affin domain that binds with a higher affinity to a protein in a ity (as defined further herein). A binding domain, in par Subset of conformations or conformational states than to ticular an immunoglobulin, such as an antibody, or an other conformations or conformational states that the protein immunoglobulin fragment, Such as a nanobody, that can may assume. One of skill in the art will recognize that specifically bind to and/or that has affinity for a specific binding domains that specifically or selectively bind to a antigen or antigenic determinant (e.g., epitope) is said to be specific conformation or conformational state of a protein "against” or “directed against the antigen or antigenic will stabilize this specific conformation or conformational determinant. A binding domain according to the disclosure State. is said to be “cross-reactive' for two different antigens or As used herein, the terms "complementarity-determining 10 region' or “CDR' within the context of antibodies refer to antigenic determinants if it is specific for both these different variable regions of either H (heavy) or L (light) chains (also antigens or antigenic determinants. abbreviated as V and V, respectively) and contains the The term “affinity,” as used herein, refers to the degree to amino acid sequences capable of specifically binding to which a binding domain, in particular an immunoglobulin, antigenic targets. These CDR regions account for the basic 15 Such as an antibody, or an immunoglobulin fragment, Such specificity of the antibody for a particular antigenic deter as a nanobody, binds to an antigen so as to shift the minant structure. Such regions are also referred to as “hyper equilibrium of antigen and binding domain toward the variable regions.” The CDRs represent non-contiguous presence of a complex formed by their binding. Thus, for stretches of amino acids within the variable regions but, example, where an antigen and antibody (fragment) are regardless of species, the positional locations of these criti combined in relatively equal concentration, an antibody cal amino acid sequences within the variable heavy and light (fragment) of high affinity will bind to the available antigen chain regions have been found to have similar locations So as to shift the equilibrium toward high concentration of within the amino acid sequences of the variable chains. The the resulting complex. The dissociation constant (K) is variable heavy and light chains of all canonical antibodies commonly used to describe the affinity between the protein each have three CDR regions, each non-contiguous with the 25 binding domain and the antigenic target. Typically, the others (termed L1, L2, L3, H1, H2, H3) for the respective dissociation constant is lower than 10 M. Preferably, the light (L) and heavy (H) chains. In particular, immunoglobu dissociation constant is lower than 10 M, more preferably, lin single variable domains, such as nanobodies (as defined lower than 107 M. Most preferably, the dissociation con further herein), generally comprise a single amino acid chain stant is lower than 10M. that comprises four “framework sequences or regions' or 30 The terms “specifically bind' and “specific binding, as FRs (termed FR1, FR2, FR3, FR4) and three complemen tarity-determining regions” or CDRs (termed CDR1, CDR2, used herein, generally refers to the ability of a binding CDR3), each non-contiguous with the others. The delinea domain, in particular an immunoglobulin, Such as an anti tion of the CDR sequences (and thus also of the FR body, or an immunoglobulin fragment, such as a nanobody, sequences) is based on the IMGT unique numbering system 35 to preferentially bind to a particular antigen that is present in for V-domains and V-like domains (Lefranc et al., 2003). a homogeneous mixture of different antigens. In certain An "epitope,” as used herein, refers to an antigenic embodiments, a specific binding interaction will discrimi determinant of a polypeptide. An epitope could comprise nate between desirable and undesirable antigens in a sample, three amino acids in a spatial conformation, which is unique in some embodiments more than about 10 to 100-fold or to the epitope. Generally an epitope consists of at least 4, 5, 40 more (e.g., more than about 1000- or 10,000-fold). 6, 7 such amino acids, and more usually, consists of at least The terms “specifically bind,” “selectively bind,” “pref. 8, 9, 10 such amino acids. Methods of determining the erentially bind.” and grammatical equivalents thereof, are spatial conformation of amino acids are known in the art, used interchangeably herein. The terms “conformational and include, for example, X-ray crystallography and multi specific' or “conformational selective' are also used inter dimensional nuclear magnetic resonance. 45 changeably herein. A “conformational epitope,” as used herein, refers to an A “deletion' is defined here as a change in either amino epitope comprising amino acids in a spatial conformation acid or nucleotide sequence in which one or more amino that is unique to a folded 3-dimensional conformation of the acid or nucleotide residues, respectively, are absent as polypeptide. Generally, a conformational epitope consists of compared to an amino acid sequence or nucleotide sequence amino acids that are discontinuous in the linear sequence 50 of a parental polypeptide or nucleic acid. Within the context that come together in the folded structure of the protein. of a protein or a fragment thereof, a deletion can involve However, a conformational epitope may also consist of a deletion of about 2, about 5, about 10, up to about 20, up to linear sequence of amino acids that adopts a conformation about 30 or up to about 50 or more amino acids. A protein that is unique to a folded 3-dimensional conformation of the or a fragment thereof may contain more than one deletion. polypeptide (and not present in a denatured State). In protein 55 Within the context of a GPCR, a deletion may in particular complexes, conformational epitopes consist of amino acids be a loop deletion, or an N- and/or C-terminal deletion. that are discontinuous in the linear sequences of one or more An “insertion” or “addition' is that change in an amino polypeptides that come together upon folding of the different acid or nucleotide sequence which has resulted in the folded polypeptides and their association in a unique qua addition of one or more amino acid or nucleotide residues, ternary structure. Similarly, conformational epitopes may 60 respectively, as compared to an amino acid sequence or here also consist of a linear sequence of amino acids of one nucleotide sequence of a parental protein. “Insertion' gen or more polypeptides that come together and adopt a con erally refers to addition to one or more amino acid residues formation that is unique to the quaternary structure. within an amino acid sequence of a polypeptide, while The term “specificity,” as used herein, refers to the ability “addition' can be an insertion or refer to amino acid residues of a binding domain, in particular an immunoglobulin, Such 65 added at an N- or C-terminus, or both termini. Within the as an antibody, or an immunoglobulin fragment. Such as a context of a protein or a fragment thereof, an insertion or nanobody, to bind preferentially to one antigen, Versus a addition is usually of about 1, about 3, about 5, about 10, up US 9,695,227 B2 15 16 to about 20, up to about 30 or up to about 50 or more amino other experimental biophysical structure determination acids. A protein or fragment thereof may contain more than methods that can include electron diffraction (also known as one insertion. electron crystallography) and nuclear magnetic resonance A “substitution, as used herein, results from the replace (NMR) methods. In yet other embodiments, atomic coordi ment of one or more amino acids or nucleotides by different 5 nates can be obtained using molecular modeling tools which amino acids or nucleotides, respectively as compared to an can be based on one or more of ab initio protein folding amino acid sequence or nucleotide sequence of a parental algorithms, energy minimization, and homology-based protein or a fragment thereof. It is understood that a protein modeling. These techniques are well known to persons of or a fragment thereof may have conservative amino acid ordinary skill in the biophysical and bioinformatic arts. substitutions which have substantially no effect on the 10 “Solving the structure' as used herein refers to determin protein's activity. By conservative substitutions is intended ing the arrangement of atoms or the atomic coordinates of a combinations such as gly, ala; Val, ile, leu, met; asp, glu; asn, protein, and is often done by a biophysical method. Such as gln, ser, thr; lys, arg: cys, met; and phe, tyr, trp. X-ray crystallography. The term “sequence identity” as used herein refers to the The term “compound' or “test compound” or “candidate extent that sequences are identical on a nucleotide-by- 15 compound' or "drug candidate compound as used herein nucleotide basis or an amino acid-by-amino acid basis over describes any molecule, either naturally occurring or Syn a window of comparison. Thus, a percentage of sequence thetic that is tested in an assay, such as a screening assay or identity” is calculated by comparing two optimally aligned drug discovery assay. As such, these compounds comprise sequences over the window of comparison, determining the organic and inorganic compounds. The compounds include number of positions at which the identical nucleic acid base 20 polynucleotides, lipids or hormone analogs that are charac (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., terized by low molecular weights. Other biopolymeric Ala, Pro, Ser. Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, organic test compounds include Small peptides or peptide His, Asp, Glu, Asn., Gln, Cys and Met) occurs in both like molecules (peptidomimetics) comprising from about 2 sequences to yield the number of matched positions, divid to about 40 amino acids and larger polypeptides comprising ing the number of matched positions by the total number of 25 from about 40 to about 500 amino acids, such as antibodies, positions in the window of comparison (i.e., the window antibody fragments or antibody conjugates. Test compounds size), and multiplying the result by 100 to yield the percent can also be protein scaffolds. For high-throughput purposes, age of sequence identity. Determining the percentage of test compound libraries may be used, such as combinatorial sequence identity can be done manually, or by making use or randomized libraries that provide a sufficient range of of computer programs that are available in the art. 30 diversity. Examples include, but are not limited to, natural "Crystal' or “crystalline structure,” as used herein, refers compound libraries, allosteric compound libraries, peptide to a solid material, whose constituent atoms, molecules, or libraries, antibody fragment libraries, synthetic compound ions are arranged in an orderly repeating pattern extending libraries, fragment-based libraries, phage-display libraries, in all three spatial dimensions. The process of forming a and the like. A more detailed description can be found crystalline structure from a fluid or from materials dissolved 35 further in the specification. in the fluid is often referred to as “crystallization” or As used herein, the terms “determining,”99 “measuring.&g s "crystallogenesis.” Protein crystals are almost always grown 'assessing.” “monitoring.” and “assaying are used inter in Solution. The most common approach is to lower the changeably and include both quantitative and qualitative solubility of its component molecules gradually. Crystal determinations. growth in Solution is characterized by two steps: nucleation 40 The term “biologically active,” with respect to a GPCR, of a microscopic crystallite (possibly having only 100 mol refers to a GPCR having a biochemical function (e.g., a ecules), followed by growth of that crystallite, ideally to a binding function, a signal transduction function, or an ability diffraction-quality crystal. to change conformation as a result of ligand binding) of a X-ray crystallography,” as used herein, is a method of naturally occurring GPCR. determining the arrangement of atoms within a crystal, in 45 The terms “therapeutically effective amount,”99 “therapeu&g which a beam of X-rays strikes a crystal and diffracts into tically effective dose' and “effective amount,” as used many specific directions. From the angles and intensities of herein, mean the amount needed to achieve the desired result these diffracted beams, a crystallographer can produce a or results. three-dimensional picture of the density of electrons within The term “pharmaceutically acceptable,” as used herein, the crystal. From this electron density, the mean positions of 50 means a material that is not biologically or otherwise the atoms in the crystal can be determined, as well as their undesirable, i.e., the material may be administered to an chemical bonds, their disorder and various other informa individual along with the compound without causing any tion, as will be known by those skilled in the art. undesirable biological effects or interacting in a deleterious The term "atomic coordinates, as used herein, refers to a manner with any of the other components of the pharma set of three-dimensional co-ordinates for atoms within a 55 ceutical composition in which it is contained. molecular structure. In one embodiment, atomic-co-ordi nates are obtained using X-ray crystallography according to DETAILED DESCRIPTION methods well-known to those of ordinarily skill in the art of biophysics. Briefly described, X-ray diffraction patterns can Despite the great diversity of ligands that may activate be obtained by diffracting X-rays off a crystal. The diffrac 60 GPCRs, they interact with a relatively small number of tion data are used to calculate an electron density map of the intracellular proteins to induce profound physiological unit cell comprising the crystal; the maps are used to change. Heterotrimeric G proteins, B-arrestins and GPCR establish the positions of the atoms (i.e., the atomic co kinases are well known for their ability to specifically ordinates) within the unit cell. Those skilled in the art recognize GPCRs in their active state, though poorly under understand that a set of structure co-ordinates determined by 65 stood both from a structural as well as functional point of X-ray crystallography contains standard errors. In other view. Therefore, Surprisingly and advantageously, binding embodiments, atomic co-ordinates can be obtained using domains were identified that specifically bind to GPCR:G US 9,695,227 B2 17 18 protein complexes and are capable of stabilizing or locking gamma (Y) and their classification is largely based on the the complex in a functional conformational state, in particu identity of their distinct C. subunits, and the nature of the lar an active conformational state. Moreover, the binding subsequent transduction event. Further classification of G domains are generic tools for stabilization and capturing of proteins has come from cDNA sequence homology analysis. a GPCR of choice in its G protein-bound state, which is 5 G proteins bind either guanosine diphosphate (GDP) or generally assumed to represent an active state of a GPCR (as guanosine triphosphate (GTP), and possess highly homolo defined herein). gous guanine nucleotide binding domains and distinct Accordingly, a first aspect of the disclosure relates to a domains for interactions with receptors and effectors. Dif binding domain that is directed against and/or specifically ferent Subclasses of GC proteins, such as GCS, Goi, God and binds to a complex comprising a GPCR and a G protein. 10 GC 12, amongst others, signal through distinct pathways The binding domain of the present disclosure can be any involving second messenger molecules such as cAMP. inosi non-naturally occurring molecule or part thereof (as defined tol triphosphate (IP3), diacylglycerol, intracellular Ca" and hereinbefore) that is capable of specifically binding to a RhoA GTPases. To illustrate this further, the C. subunit complex comprising a GPCR and a G protein. According to (39-46 kDa) contains the guanine nucleotide binding site a preferred embodiment, the binding domains as described 15 and possesses GTPase activity; the B (37 kDa) and Y (8 kDa) herein are protein scaffolds. The teen “protein scaffold Subunits are tightly associated and function as a By heterodi refers generally to folding units that form structures, par mer. There are 23 types (including some splicing isoforms) ticularly protein or peptide structures, that comprise frame of C. subunits, 6 of B, and 11 of Y currently described. The works for the binding of another molecule, for instance a classes of G protein and Subunits are Subscripted: thus, for protein (See, e.g., Skerra (2000), for review). A binding example, the C. subunit of Gs protein (which activates domain can be derived from a naturally occurring molecule, adenylate cyclase) is Gisa; other G proteins include Gi. e.g., from components of the innate or adaptive immune which differs from Gis structurally (different type of C. system, or it can be entirely artificially designed. A binding subunit) and inhibits adenylate cyclase. Further examples domain can be immunoglobulin-based or it can be based on are provided in Table 1. domains present in proteins, including but limited to micro 25 Typically, in nature, G proteins are in a nucleotide-bound bial proteins, protease inhibitors, toxins, fibronectin, lipoca form. More specifically, G proteins (or at least the C. subunit) lins, single chain antiparallel coiled coil proteins or repeat are bound to either GTP or GDP depending on the activation motif proteins. Examples of binding domains which are status of a particular GPCR. Agonist binding to a GPCR known in the art include, but are not limited to: antibodies, promotes interactions with the GDP-bound Go. By het heavy chain antibodies (hcAb), single domain antibodies 30 erotrimer leading to the exchange of GDP for GTP on GO. (sdAb), minibodies, the variable domain derived from cam and the functional dissociation of the G protein into elid heavy chain antibodies (VHH or nanobodies), the Go-GTP and GBy subunits. The separate Go-GTP and GBY variable domain of the new antigen receptors derived from Subunits can modulate, either independently or in parallel, shark antibodies (VNAR), alphabodies, protein A, protein G, downstream cellular effectors (channels, kinases or other designed ankyrin-repeat domains (DARPins), fibronectin 35 enzymes, see Table 1). The intrinsic GTPase activity of Gy type III repeats, anticalins, knottins, engineered CH2 leads to hydrolysis of GTP to GDP and the re-association of domains (nanoantibodies), peptides and proteins, lipopep Go-GDP and Gfy subunits, and the termination of signaling. tides (e.g., pepducins), DNA, and RNA (see, e.g., Thus, G proteins serve as regulated molecular Switches Gebauer & Skerra, 2009; Skerra, 2000; Starovasnik et al., capable of eliciting bifurcating signals through C. and BY 1997; Binz et al., 2004, Koide et al., 1998; Dimitrov, 2009: 40 subunit effects. The switch is turned on by the receptor and Nygren et al., 2008: WO2010.066740). Frequently, when it turns itself off within a few seconds, a time sufficient for generating a particular type of binding domain using selec considerable amplification of signal transduction. tion methods, combinatorial libraries comprising a consen “G-protein coupled receptors,” or “GPCRs, as used sus or framework sequence containing randomized potential herein, are polypeptides that share a common structural interaction residues are used to screen for binding to a 45 motif, having seven regions of between 22 to 24 hydropho molecule of interest, such as a protein. bic amino acids that form seven alpha helices, each of which The binding domain of the present disclosure may be spans the membrane. Each span is identified by number, i.e., directed against and/or specifically bind any GPCR:G pro transmembrane-1 (TM1), transmembrane-2 (TM2), etc. The tein complex of choice. Preferred target complexes are transmembrane helices are joined by regions of amino acids complexes of a GPCR and a G protein that occur in nature 50 between transmembrane-2 and transmembrane-3, trans or, alternatively, for example in case of non-naturally occur membrane-4 and transmembrane-5, and transmembrane-6 ring variants (as described further herein) of GPCRs and G and transmembrane-7 on the exterior, or “extracellular side, protein, complexes wherein the GPCR and the G protein will of the cell membrane, referred to as “extracellular regions associate under the appropriate physiological conditions. It 1, 2 and 3 (EC1, EC2 and EC3), respectively. The trans will be understood by the person skilled in the art that the 55 membrane helices are also joined by regions of amino acids structural relationship between GPCR and G protein deter between transmembrane-1 and transmembrane-2, trans mines whether a particular GPCR: G protein complex can be membrane-3 and transmembrane-4, and transmembrane-5 formed, which will be detailed further below for members of and transmembrane-6 on the interior, or “intracellular side, the G protein family and members of the GPCR family. of the cell membrane, referred to as “intracellular regions With “G proteins are meant the family of guanine 60 1, 2 and 3 (IC1, IC2 and IC3), respectively. The “carboxy” nucleotide-binding proteins involved in transmitting chemi (“C”) terminus of the receptor lies in the intracellular space cal signals outside the cell, and causing changes inside the within the cell, and the “amino” (“N”) terminus of the cell. G proteins are key molecular components in the intra receptor lies in the extracellular space outside of the cell. cellular signal transduction following ligand binding to the Any of these regions are readily identifiable by analysis of extracellular domain of a GPCR. They are also referred to as 65 the primary amino acid sequence of a GPCR. "heterotrimeric G proteins,” or “large G proteins.” G pro GPCR structure and classification is generally well known teins consist of three subunits: alpha (C.), beta (B), and in the art and further discussion of GPCRs may be found in US 9,695,227 B2 19 20 Probst et al., 1992; Marchese et al., 1994: Lagerstrom & that of rhodopsin. Ligands for these GPCRs are hormones, Schioth, 2008; Rosenbaum et al., 2009; and the following Such as glucagon, secretin, gonadotropin-releasing hormone books: Jurgen Wess (Ed) Structure-Function Analysis of G and parathyroid hormone. The Glutamate family receptors Protein-Coupled Receptors published by Wiley-Liss (1 st (Class C or Class 3 receptors) also have a large extracellular edition; Oct. 15, 1999); Kevin R. Lynch (Ed) Identification domain, which functions like a “Venus fly trap' since it can and Expression of G Protein-Coupled Receptors published open and close with the agonist bound inside. Family by John Wiley & Sons (March 1998) and Tatsuya Haga (Ed), members are the metabotropic glutamate, the Ca"-sensing G Protein-Coupled Receptors, published by CRC Press and the y-aminobutyric acid (GABA)-B receptors. (Sep. 24, 1999); and Steve Watson (Ed) G-Protein Linked GPCRs include, without limitation, serotonin olfactory Receptor Factsbook, published by Academic Press (1st 10 receptors, glycoprotein hormone receptors, chemokine edition: 1994). GPCRs can be grouped on the basis of receptors, adenosine receptors, biogenic amine receptors, sequence homology into several distinct families. Although melanocortin receptors, neuropeptide receptors, chemotactic all GPCRs have a similar architecture of seven membrane receptors, somatostatin receptors, opioid receptors, mela spanning C-helices, the different families within this recep tonin receptors, calcitonin receptors, PTH/PTHrP receptors, tor class show no sequence homology to one another, thus 15 glucagon receptors, secretin receptors, latrotoxin receptors, Suggesting that the similarity of their transmembrane metabotropic glutamate receptors, calcium receptors, domain structure might define common functional require GABA-B receptors, pheromone receptors, the protease ments. A comprehensive view of the GPCR repertoire was activated receptors, the rhodopsins and other G-protein possible when the first draft of the human genome became coupled seven transmembrane segment receptors. GPCRs available. Fredriksson and colleagues divided 802 human also include these GPCR receptors associated with each GPCRs into families on the basis of phylogenetic criteria. other as homomeric or heteromeric dimers or as higher This showed that most of the human GPCRs can be found order oligomers. The amino acid sequences (and the nucleo in five main families, termed Rhodopsin, Adhesion, Secre tide sequences of the cDNAs which encode them) of GPCRs tin, Glutamate, Frizzled/Taste2 (Fredriksson et al., 2003). are readily available, for example by reference to GenBank Members of the Rhodopsin family (corresponding to class 25 (on the World Wide Web at ncbi.nlm.nih.gov/entrez). A (Kolakowski, 1994) or Class 1 (Foord et al. (2005) in Thus, according to specific embodiments, the present older classification systems) only have Small extracellular disclosure provides for binding domains that are directed loops and the interaction of the ligands occurs with residues against and/or specifically bind GPCR: G protein complexes within the transmembrane cleft. This is by far the largest wherein the G protein is selected from the group consisting group (>90% of the GPCRs) and contains receptors for 30 of Gs, Gi. Go, Gt, Ggust, GZ, Golf, Gq, G12 and G|B. In one odorants, Small molecules such as catecholamines and preferred embodiment, the G protein is Gs. In another amines, (neuro)peptides and glycoprotein hormones. Rho preferred embodiment, the G protein is Gi. In still another dopsin, a representative of this family, is the first GPCR for preferred embodiment, the G protein is Gt, more specifically which the structure has been solved (Palczewski et al., transducin. In correspondence thereto, the GPCR comprised 2000). B2-AR, the first receptor interacting with a diffusible 35 within the complex is selected from the group consisting of ligand for which the structure has been solved (Rosenbaum a GS coupled receptor, a Gi coupled receptor, a Go coupled et al., 2007) also belongs to this family. Based on phyloge receptor, a Git coupled receptor, a Ggust coupled receptor, a netic analysis, class B GPCRs or Class 2 (Foord et al., 2005) Golf coupled receptor, a Gq coupled receptor, a G12 coupled receptors have recently been subdivided into two families: receptor and a G13 coupled receptor. In one preferred adhesion and secretin (Fredriksson et al., 2003). Adhesion 40 embodiment, the GPCR is a Gs coupled receptor. In another and secretin receptors are characterized by a relatively long preferred embodiment, the GPCR is a Gi coupled receptor. amino terminal extracellular domain involved in ligand In still another preferred embodiment, the GPCR is a Git binding. Little is known about the orientation of the trans coupled receptor, more specifically rhodopsin. Particular membrane domains, but it is probably quite different from non-limiting examples are provided in Table 1. TABLE 1. Non-limiting examples of the relationship of G protein-coupled receptors and signaling pathways. G protein Effectors. Signaling family C. Subunit pathways Use/Receptors Gi family Gi Ci Inhibition of Acetylcholine M2 & M4 receptors Go CO adenylate cyclase Adenosine A1 & A3 receptors (cAMP) Adrenergic C2A, C.2B, & C2C receptors Closing Ca" Apelin receptors channels (Ca") Calcium-sensing receptor Chemokine CXCR4 receptor Dopamine D2, D3, D4 GABAB receptor Glutamate molluR2, mGluR3, mGluR4, mGluR6, mGluR7, & mCluR8 receptors Histamine H2 & H3 & H4 receptors Melatonin MT1, MT2, & MT3 receptors Muscarinic M2 & M4 receptors Opioid 6, K, L, & nociceptin receptors Prostaglandin EP1, EP3, FP, & TP receptors Serotonin 5-HT1 & 5-HT5 receptors US 9,695,227 B2 21 22 TABLE 1-continued Non-limiting examples of the relationship of G protein-coupled receptors and signaling pathways. G protein Effectors. Signaling family C. Subunit pathways Use/Receptors Gt Ot Activation Rhodopsin (transducin) phosphodiesterase 6 (vision) Ggust Clgust Activation Taste receptors (gustoducin) phosphodiesterase 6 (vision) GZ CZ Inhibition adenylate unknown cyclase (cAMP) Gs family Gs CS Activation adenylate 5-HT receptors types 5-HT4 and 5-HT7 cyclase (cAMP ) ACTH receptor Adenosine receptor types A2a and A2b Arginine vasopressin receptor 2 B-adrenergic receptors types 1, 32 and 33 Calcitonin receptor Calcitonin gene-related peptide receptor Corticotropin-releasing hormone receptor Dopamine receptors D1-like family (D1 and D5) FSH-receptor Gastric inhibitory polypeptide receptor Glucagon receptor Histamine H2 receptor Luteinizing hormone? choriogonadotropin receptor Melanocortin receptor Parathyroid hormone receptor 1 Prostaglandin receptor types D2 and I2 Secretin receptor Thyrotropin receptor Golf Colf Activation adenylate Olfactory receptors cyclase (cAMP ) Gq family Gq Cq. Activation of 5-HT2 serotonergic receptors phospholipase C Alpha-1 adrenergic receptor (IP. f.) Vasopressin type 1 receptor Angiotensin II receptor type 1 Calcitonin receptor Histamine H1 receptor Metabotropic glutamate receptor, Group I M1, M3, and M5 muscarinic receptors G12.13 family
G12 C12 G13 C13 Na/H* exchange By subunit By Opening K' channels (K' ) By Adenylate cyclase (cAMP) or , By Phospholipase C (IP)
Generally, binding domains of the disclosure will at least GPCRs or G proteins are found in nature. The term “non bind to those forms of GPCR:G protein complexes that are naturally occurring,” as used herein, means a GPCR or a G most relevant from a biological and/or therapeutic point of 55 protein that is not naturally occurring. In certain circum view, as will be clear to the skilled person. It will thus be stances, it may be advantageous that the GPCR and/or G understood that, depending on the purpose and application, protein comprised in the complex are non-naturally occur the GPCR and G protein comprised in the target complex ring proteins. For example, and for illustration purposes may be naturally occurring or non-naturally occurring (i.e., only, to increase the probability of obtaining crystals of a altered by man). The term “naturally occurring,” as used GPCR:G protein complex stabilized by the binding domains herein, means a GPCR or a G protein that are naturally of the present invention, it might be desired to perform some produced. In particular, wild type polymorphic variants and protein engineering without or only minimally affecting isoforms of GPCRs and G proteins, as well as orthologs ligand binding affinity. Or, alternatively or additionally, to across different species are examples of naturally occurring increase cellular expression levels of a GPCR and/or a G proteins, and are found for example, and without limitation, protein, or to increase the stability, one might also consider in a mammal, more specifically in a human, or in a virus, or to introduce certain mutations in the GPCR and/or the G in a plant, or in an insect, amongst others). Thus, Such protein of interest. Non-limiting examples of non-naturally US 9,695,227 B2 23 24 occurring GPCRs include, without limitation, GPCRs that Some particular examples of isoforms of G protein sub have been made constitutively active through mutation, units are provided in Table 5. The skilled person will GPCRs with a loop deletion, GPCRs with an N- and/or appreciate that the amino acid sequences of the different G C-terminal deletion, GPCRs with a substitution, an insertion protein subunits are almost 100%, if not 100%, conserved or addition, or any combination thereof, in relation to their 5 across species and organisms. Notably, sequence alignment amino acid or nucleotide sequence, or other variants of of the amino acid sequence of the human, bovine, rat and naturally occurring GPCRs. Similarly, non-limiting mouse B subunit of the G protein reveals that the amino acid examples of non-naturally occurring G proteins include, sequences between these organisms are 100% conserved. without limitation, G proteins with an N- and/or C-terminal Analogously, the amino acid sequences of the human, 10 mouse, and bovine Y subunit of the G protein are 100% deletion, G proteins with a substitution, an insertion or identical. The rat and mouse GCS amino acid sequences are addition, or any combination thereof, in relation to their also 100% identical, whereas human and bovine Gos only amino acid or nucleotide sequence, or other variants of differ in 1 or 2 amino acids, respectively. Thus, the binding naturally occurring G proteins. Also comprised within the domains directed against and/or specifically binding to a scope of the present disclosure are target GPCR:G protein 15 GPCR:G protein complex, and particularly binding to the G complexes comprising a chimeric or hybrid GPCR, for protein comprised in the complex, are expected to be cross example a chimeric GPCR with an N- and/or C-terminus reactive. It will also be clear that the GPCR and G protein from one GPCR and loops of a second GPCR, or comprising comprised in the target complex may be from the same or a GPCR fused to a moiety, such as T4 lysozyme (see also different species. Preferably, the GPCR and/or the G protein Example section). is a mammalian protein, or a plant protein, or a microbial According to specific embodiments, a non-naturally protein, or a viral protein, or an insect protein. More occurring GPCR or G protein, as comprised in the GPCR:G preferably, the GPCR is a human protein. protein complex, may have an amino acid sequence that is It is also expected that binding domains of the disclosure at least 80% identical to, at least 90% identical to, at least will generally be capable of binding to GPCR:G protein 95% identical to, at least 97% identical to, or at least 99% 25 complexes comprising all naturally occurring or synthetic identical to, a naturally occurring GPCR or G protein. To analogs, variants, mutants, alleles, parts, fragments, and illustrate this further, and taking the B2-adrenergic receptor isoforms of a particular GPCR and/or G protein comprised as a particular non-limiting example of a GPCR within the in the complex; or at least to those analogs, variants, scope hereof, it should be clear from the above that in mutants, alleles, parts, fragments, and isoforms of a particu addition to the human fB adrenergic receptor (e.g., the 30 lar GPCR and/or G protein comprised in the complex that sequence described by GenBank accession number contain one or more antigenic determinants or epitopes that NP 000015), the mouse B adrenergic receptor (e.g., as are essentially the same as the antigenic determinant(s) or described by GenBank accession no. NM007420) or other epitope(s) to which the binding domains of the disclosure mammalian B adrenergic receptor are encompassed. Also bind to a particular GPCR and/or G protein comprised in the envisaged are wild-type polymorphic variants and certain 35 complex. other active variants of the B adrenergic receptor from a Various methods may be used to determine specific bind particular species. For example, "human B adrenergic ing (as defined hereinbefore) between the binding domain receptor has an amino acid sequence that is at least 80% and a target GPCR: G protein complex, including for identical to, at least 90% identical to, at least 95% identical example, enzyme linked immunosorbent assays (ELISA), to, at least 97% identical to, or at least 99% identical to the 40 Surface Plasmon resonance assays, phage display, and the naturally occurring "human B adrenoreceptor of GenBank like, which are common practice in the art, for example, in accession number NP 000015. discussed in Sambrook et al. (2001), Molecular Cloning. A Analogously, and taking GCS, GOi and Got as particular Laboratory Manual. Third Edition. Cold Spring Harbor non-limiting examples of Subunits of G proteins within the Laboratory Press, Cold Spring Harbor, N.Y., and are further scope of the present invention, it should be clear from the 45 illustrated in the Example section. It will be appreciated that above that in addition to the human Gos or Goli or Gott, the for this purpose often a unique label or tag will be used. Such mouse GCS or Goli or Got proteins or other mammalian Gos as a peptide label, a nucleic acid label, a chemical label, a or GCi or Got proteins are encompassed. Also envisaged are fluorescent label, or a radio frequency tag, as described wild-type polymorphic variants and certain other active further herein. variants of the Gos or Goi or Got from a particular species. 50 According to a specific embodiment, the binding domain For example, a “human Gos' or a “human Goi' or a “human against a GPCR: G protein complex binds with higher affin Got has an amino acid sequence that is at least 80% ity to the complex compared with binding to the G protein identical to, at least 90% identical to, at least 95% identical alone and/or to the GPCR alone, respectively. In one to, at least 97% identical to, or at least 99% identical to the embodiment, the binding domain against a GPCR:G protein naturally occurring “human Gos' or “human Goi' or 55 complex specifically binds to the GPCR comprised in the “human Got of Genbank accession number P63092, complex, and not to the G protein. Preferably, in another P63096 and P1 1488, respectively. Further, many isoforms of embodiment, the binding domain against a GPCR:G protein G protein Subunits exist, including for example isoforms of complex specifically binds to the G protein comprised in the Gs and Gi proteins (Gos: GNAS; Go,0: GNAO1: Goi: complex, and not to the GPCR. More specifically, the GNAI1 or GNAI2 or GNAI3; G|B: GNB1 or GNB2 or GNB3 60 binding domain against a GPCR: G protein complex specifi or GNB4 or GNB5 or GNB1L or GNB2L: GY: GNGT1 or cally binds to the G protein which is a Gs protein comprised GNGT2 or GNG2 or GNG3 or GNG4 or GNG5 or GNG-7 in a complex of a GS coupled receptor and a Gs protein. or GNG8 or GNG10 or GNG11 or GNG12 or GNG 13: It is well-known that GPCRs are conformationally com according to HGNC standardized nomenclature to human plex membrane proteins that exhibit a spectrum of func genes; accession numbers of different isoforms from differ 65 tional behavior in response to natural and synthetic ligands. ent organisms are available from the World Wide Web at In nature, a ligand-bound GPCR may associate with a G uniprot.org). protein into a complex which will represent a particular US 9,695,227 B2 25 26 functional conformational state, more specifically an active sity of activation or deactivation, while allowing the body to conformational state, resulting in a particular biological retain its natural control over initiating receptor activation, activity. The present disclosure offers the particular advan by altering the affinity of the receptor for its (endogenous) tage that the binding domains as described herein can ligand. Allosteric regulators that enhance the protein’s activ stabilize various of these active conformations of GPCRs in ity are referred to herein as “allosteric activators' or “posi complex with a G protein and bound to various natural or tive allosteric modulators,' whereas those that decrease the synthetic ligands. One of skill in the art will recognize that protein's activity are referred to herein as “allosteric inhibi binding domains that specifically bind to a ligand:GPCR:G tors' or otherwise “negative allosteric modulators.” Thus, in protein complex, will stabilize the specific conformation of one particular embodiment, the binding domain of the the GPCR comprised in the complex. In a preferred embodi 10 disclosure is directed against and/or specifically binds to a ment, the binding domain is capable of Stabilizing, or complex comprising a GPCR, a G protein and a receptor otherwise, increasing the stability of a particular functional ligand, wherein the receptor ligand is an allosteric modula conformational state of a GPCR:G protein complex, pref tor, preferably a positive allosteric modulator. More specifi erably wherein the GPCR is in an active conformational cally, the positive allosteric modulator binds the receptor at state. Generally, a functional conformation state of the 15 an allosteric site. GPCR can be a basal conformational state, or an active As explained, the canonical view of how GPCRs regulate conformational state or an inactive conformational state. cellular physiology is that the binding of ligands (such as Preferably, the binding domain of the disclosure is capable hormones, neurotransmitters or sensory stimuli) stabilizes of stabilizing a GPCR in its active conformational state an active conformational state of the receptor, thereby allow and/or is capable of locking the GPCR in an active confor ing interactions with heterotrimeric G proteins. In addition mational state upon binding the GPCR:G protein complex, to interacting with G proteins, agonist-bound GPCRs asso whether or not in the presence of a receptor ligand. ciate with GPCR kinases (GRKs), leading to receptor phos In a particularly preferred embodiment, it is envisaged phorylation. A common outcome of GPCR phosphorylation that the binding domain is directed against and/or specifi by GRKs is a decrease in GPCR interactions with G proteins cally binds to a complex comprising a GPCR, a G protein 25 and an increase in GPCR interactions with arrestins, which and a receptor ligand (as defined herein). More preferably, sterically interdict further G protein signaling, resulting in the binding domain is directed against and/or specifically receptor desensitization. As B-arrestins turn off G protein binds to a complex consisting of a GPCR, a G protein and signals, they can simultaneously initiate a second, parallel a receptor ligand. A receptor ligand may be a small com set of signal cascades, such as the MAPK pathway. GPCRs pound, a peptide, an antibody, or an antibody fragment, and 30 also associate with various proteins outside the families of the like, which triggers a response upon binding. Receptor general GPCR-interacting proteins (G proteins, GRKs, ligands, or simply ligands, as used herein may be orthosteric arrestins and other receptors). These GPCR-selective part ligands (both natural and synthetic) that bind to the recep ners can mediate GPCR signaling, organize GPCR signaling tor's active site and are classified according to their efficacy through G proteins, direct GPCR trafficking, anchor GPCRs or in other words to the effect they have on receptor 35 in particular subcellular areas and/or influence GPCR phar signaling through a specific pathway. As used herein, an macology (Ritter and Hall 2009). In this regard, ligands as “agonist” refers to a ligand that, by binding a receptor, used herein may also be “biased ligands' with the ability to increases the receptor's signaling activity. Full agonists are selectively stimulate a Subset of a receptor's signaling capable of maximal receptor stimulation; partial agonists are activities, for example the selective activation of G protein unable to elicit full activity even at Saturating concentra 40 or B-arrestin function. Such ligands are known as "biased tions. Partial agonists can also function as “blockers” by ligands.” “biased agonists,” or “functionally selective ago preventing the binding of more robust agonists. An “antago nists.” More particularly, ligand bias can be an imperfect nist” refers to a ligand that binds a receptor without stimu bias characterized by a ligand stimulation of multiple recep lating any activity. An 'antagonist’ is also known as a tor activities with different relative efficacies for different “blocker because of its ability to prevent binding of other 45 signals (non-absolute selectivity) or can be a perfect bias ligands and, therefore, block agonist-induced activity. Fur characterized by a ligand stimulation of one receptor activity ther, an “inverse agonist” refers to an antagonist that, in without any stimulation of another known receptor activity. addition to blocking agonist effects, reduces receptors basal Thus, in one particular embodiment, the binding domain of or constitutive activity below that of the unliganded recep the disclosure is directed against and/or specifically binds to tor. Preferably, the binding domain of the disclosure is 50 a complex comprising a GPCR, a G protein and a receptor directed against and/or specifically binds to a complex ligand, wherein the receptor ligand is a biased ligand. comprising a GPCR, a G protein and a receptor ligand, Further, according to a preferred embodiment, it is par wherein the receptor ligand is an agonist. More specifically, ticularly envisaged that the binding domain of the disclosure the agonist binds the receptor at the orthosteric site. directed against and/or specifically binding to a GPCR:G The signaling activities of GPCRs (and thus their confor 55 protein complex, as described hereinbefore, is derived from mational behavior) may also be affected by the binding of an innate or adaptive immune system. Preferably, the bind another kind of ligands known as allosteric regulators. ing domain is derived from an immunoglobulin. Preferably, “Allosteric regulators' or otherwise “allosteric modulators' the binding domain according to the disclosure is an anti or “effector molecules' bind at an allosteric site of a GPCR body or an antibody fragment. The term “antibody’ (Ab) (that is, a regulatory site physically distinct from the pro 60 refers generally to a polypeptide encoded by an immuno tein's active site). In contrast to orthosteric ligands, allos globulin gene, or a functional fragment thereof, that spe teric modulators are non-competitive because they bind cifically binds and recognizes an antigen, and is known to receptors at a different site and modify receptor function the person skilled in the art. A conventional immunoglobulin even if the endogenous ligand also is binding. Because of (antibody) structural unit comprises a tetramer. Each this, allosteric modulators are not limited to simply turning 65 tetramer is composed of two identical pairs of polypeptide a receptor on or off, the way most drugs are. Instead, they act chains, each pair having one “light” (about 25 kDa) and one more like a dimmer switch, offering control over the inten “heavy chain (about 50-70 kDa). The N-terminus of each US 9,695,227 B2 27 28 chain defines a variable region of about 100 to 110 or more Therefore, Nbs can be used in many applications including amino acids primarily responsible for antigen recognition. drug discovery and therapy (Saerens et al., 2008) but also as The terms variable light chain (VL) and variable heavy a versatile and valuable tool for purification, functional chain (VH) refer to these light and heavy chains respec study and crystallization of proteins (Conrath et al., 2009). tively. The term “antibody' is meant to include whole A particular class of nanobodies that act as crystallization antibodies, including single-chain whole antibodies, and chaperones binding conformational epitopes of native tar antigen-binding fragments. In some embodiments, antigen gets are called XAPERONESTM and are particularly envis binding fragments may be antigen-binding antibody frag aged here. XAPERONESTM are unique tools in structural ments that include, but are not limited to, Fab, Fab' and biology. XAPERONETM is a trademark of VIB and VUB F(ab')2, Fd, single-chain Fvs (sclv), single-chain antibodies, 10 (Belgium). Major advantages for the use of camelid anti disulfide-linked FVS (dsEv) and fragments comprising or body fragments as crystallization aid are that XAPER consisting of either a VL or VH domain, and any combina ONESTM (1) bind cryptic epitopes and lock proteins in tion of those or any other functional portion of an immu unique native conformations, (2) increase the stability of noglobulin peptide capable of binding to the target antigen. soluble proteins and solubilized membrane proteins, (3) The term “antibodies' is also meant to include heavy chain 15 reduce the confoimational complexity of soluble proteins antibodies, or functional fragments thereof. Such as single and solubilized membrane proteins, (4) increase the polar domain antibodies, more specifically, immunoglobulin Surface enabling the growth of diffracting crystals, (5) single variable domains such as VHHS or nanobodies, as sequester aggregative or polymerizing Surfaces, (6) allow to defined further herein. affinity-trap active protein. Preferably, the binding domain of the disclosure is an Thus, the immunoglobulin single variable domains immunoglobulin single variable domain. More preferably, hereof, in particular the nanobodies hereof, generally com the binding domain is an immunoglobulin single variable prise a single amino acid chain that typically comprises 4 domain that comprises an amino acid sequence comprising “framework sequences” or FRs and three “complementarity four framework regions (FR1 to FR4) and three comple deter mining regions' or CDRS according to formula (1): mentarity-determining regions (CDR1 to CDR3), preferably 25 according to the following formula (1): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1). The term "complementarity-determining region' or FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1), “CDR' refers to variable regions in immunoglobulin single or any suitable fragment thereof (which will then usually variable domains and contains the amino acid sequences contain at least Some of the amino acid residues that form at 30 capable of specifically binding to antigenic targets. These least one of the complementarity-determining regions). CDR regions account for the basic specificity of the nano Binding domains comprising 4 FRs and 3 CDRs are body for a particular antigenic determinant structure. Such known to the person skilled in the art and have been regions are also referred to as “hypervariable regions.” The described, as a non-limiting example, in Wesolowski et al. immunoglobulin single variable domains have 3 CDR (2009, Med. Microbiol. Immunol. 198:157). Typical, but 35 regions, each non-contiguous with the others (termed CDR1. non-limiting, examples of immunoglobulin single variable CDR2, CDR3). It should be clear that framework regions of domains include light chain variable domain sequences immunoglobulin single variable domains may also contrib (e.g., a V, domain sequence), or heavy chain variable ute to the binding of their antigens (Desmyter et al., 2002: domain sequences (e.g., a V. domain sequence), which are Korotkov et al., 2009). Non-limiting examples of such usually derived from conventional four-chain antibodies. 40 immunoglobulin single variable domains according to the Preferably, the immunoglobulin single variable domains are present disclosure as well as particular combinations of FRS derived from camelid antibodies, preferably from heavy and CDRs are as described herein (see Tables 2 and 3). The chain camelid antibodies, devoid of light chains, and are delineation of the CDR sequences (and thus also the FR known as V.H domain sequences or nanobodies (as sequences) is based on the IMGT unique numbering system described further herein). 45 for V-domains and V-like domains (Lefranc et al., 2003). The term “nanobody’ (Nb), as used herein, refers to the Alternatively, the delineation of the FR and CDR sequences Smallest antigen binding fragment or single variable domain can be done by using the Kabat numbering system as applied (VH) derived from naturally occurring heavy chain anti to V.H domains from Camelids in the article of Riechmann body and is known to the person skilled in the art. They are and Muyldermans (2000). As will be known by the person derived from heavy chain only antibodies, seen in camelids 50 skilled in the art, the immunoglobulin single variable (Hamers-Casterman et al., 1993: Desmyter et al., 1996). In domains, in particular the nanobodies, can in particular be the family of “camelids' immunoglobulins devoid of light characterized by the presence of one or more Camelidae polypeptide chains are found. “Camelids' comprise old hallmark residues in one or more of the framework world camelids (Camelus bactrianus and Camelus drom sequences (according to Kabat numbering), as described for edarius) and new world camelids (for example, Lama pac 55 example in WO 08/020079, on page 75, Table A-3, incor COS, Lama glama, Lama guanicoe and Lama vicugna). The porated herein by reference). single variable domain heavy chain antibody is herein In a preferred embodiment, the disclosure provides immu designated as a Nanobody or a VH antibody. NanobodyTM noglobulin single variable domains with an amino acid and NanobodiesTM are trademarks of Ablynx NV (Belgium). sequence selected from the group consisting of amino acid The small size and unique biophysical properties of Nbs 60 sequences that essentially consist of four framework regions excel conventional antibody fragments for the recognition of (FR1 to FR4, respectively) and three complementarity uncommon or hidden epitopes and for binding into cavities determining regions (CDR1 to CDR3, respectively), in or active sites of protein targets. Further, Nbs can be which the CDR sequences of the amino acid sequences have designed as multispecific and/or multivalent antibodies or at least 70% amino acid identity, preferably at least 80% attached to reporter molecules (Conrath et al., 2001). Nbs 65 amino acid identity, more preferably at least 90% amino acid are stable and rigid single domain proteins that can easily be identity, such as at least 95%, at least 96%, at least 97%, at manufactured and Survive the gastro-intestinal system. least 98%, at least 99%, or even 100% amino acid identity US 9,695,227 B2 29 30 with the CDR sequences (see Table 3) of at least one of the blood sample, or any sample of B-cells), and by generating immunoglobulin single variable domains of SEQ ID NO:s VH sequences directed against a target complex, starting 1-6, preferably SEQID NO: 1 and/or 4. It will be understood from the sample, using any Suitable technique known perse. that for determining the degree of amino acid identity of the Such techniques will be clear to the skilled person. Alter amino acid sequences of the CDRS of one or more sequences natively, such naturally occurring VH domains can be of the immunoglobulin single variable domains, the amino obtained from naive libraries of Camelid VH sequences, acid residues that from the framework regions are disre for example by Screening such a library using a target garded. Some preferred, but non-limiting, examples of complex or at least one part, fragment, antigenic determinant immunoglobulin single variable domains of the disclosure or epitope thereof using one or more screening techniques are given in SEQ ID NO:s 1 to 6, preferably SEQ ID NO: 10 known perse. Such libraries and techniques are for example 1 and/or SEQ ID NO: 4 (see Table 2). It should be noted that the immunoglobulin single vari described in WO993.7681, WOO190190, WO03025020 and able domains, in particular the nanobodies, of the disclosure WO03035694. Alternatively, improved synthetic or semi in their broadest sense are not limited to a specific biological synthetic libraries derived from naive VH libraries may be Source or to a specific method of preparation. For example, 15 used, such as VH libraries obtained from naive VH the immunoglobulin single variable domains hereof, in libraries by techniques such as random mutagenesis and/or particular the nanobodies, can generally be obtained: (1) by CDR shuffling, as for example described in WO0043507. isolating the VH domain of a naturally occurring heavy Yet another technique for obtaining VH sequences directed chain antibody; (2) by expression of a nucleotide sequence against a target involves Suitably immunizing a transgenic encoding a naturally occurring VH domain; (3) by mammal that is capable of expressing heavy chain antibod “humanization' of a naturally occurring VH domain or by ies (i.e., so as to raise an immune response and/or heavy expression of a nucleic acid encoding a Such humanized chain antibodies directed against a target), obtaining a VH domain; (4) by “camelization of a naturally occurring Suitable biological sample from the transgenic mammal VH domain from any animal species, and in particular from (such as a blood sample, or any sample of B-cells), and then a mammalian species, such as from a human being, or by 25 generating VHSequences directed against a target starting expression of a nucleic acid encoding such a camelized VH from the sample, using any Suitable technique known perse. domain; (5) by "camelization of a “domain antibody” or For example, for this purpose, the heavy chain antibody “Dab' as described in the art, or by expression of a nucleic expressing mice and the further methods and techniques acid encoding Such a camelized VII domain; (6) by using described in WOO2O85945 and in WOO4049794 can be synthetic or semi-synthetic techniques for preparing pro 30 teins, polypeptides or other amino acid sequences known per used. se; (7) by preparing a nucleic acid encoding a nanobody A particularly preferred class of immunoglobulin single using techniques for nucleic acid synthesis known per se, variable domains hereof, in particular nanobodies hereof, followed by expression of the nucleic acid thus obtained: comprises immunoglobulin single variable domains with an and/or (8) by any combination of one or more of the 35 amino acid sequence that corresponds to the amino acid foregoing. sequence of a naturally occurring VH domain, but that has One preferred class of immunoglobulin single variable been "humanized, i.e., by replacing one or more amino acid domains corresponds to the VH domains of naturally residues in the amino acid sequence of the naturally occur occurring heavy chain antibodies directed against a target ring VH sequence (and in particular in the framework complex of a GPCR and a G protein. Although naive or 40 sequences) by one or more of the amino acid residues that synthetic libraries of immunoglobulin single variable occur at the corresponding position(s) in a VH domain from domains may contain conformational binders against the a conventional 4-chain antibody from a human being. This target complex, a preferred embodiment of this disclosure can be performed in a manner known per se, which will be includes the immunization of a Camelidae with a target clear to the skilled person, for example on the basis of the complex to expose the immune system of the animal with 45 further description herein and the prior art on humanization the conformational epitopes that are unique to the complex. referred to herein. Again, it should be noted that such Animals can be immunized with mixtures of the interacting humanized immunoglobulin single variable domains of the monomers. Optionally, the complex can be stabilized by disclosure can be obtained in any Suitable manner known per chemical cross-linking or by adding cooperative/allosteric se (i.e., as indicated under points (1)–(8) above) and thus are ligands/metabolites that stabilize the complex (orthosteric 50 not strictly limited to polypeptides that have been obtained agonists, allosteric activators, Ca", ATP, etc.). The complex using a polypeptide that comprises a naturally occurring can also be stabilized by covalent modification (phospho VH domain as a starting material. Humanized immuno rylation, etc.) of (one of) the members of the complex. In an globulin single variable domains, in particular nanobodies, alternatively embodiment, one might also immunize Cam may have several advantages, such as a reduced immuno elidae with the GPCR and/or the G protein alone, thus not 55 genicity, compared to the corresponding naturally occurring in complex with each other. Optionally, the GPCR and/or the VH domains. Such humanization generally involves G protein may also be stabilized, for example, by adding replacing one or more amino acid residues in the sequence cooperative/allosteric ligands/metabolites that stabilize the of a naturally occurring VH with the amino acid residues GPCR and/or G protein (orthosteric agonists, allosteric that occur at the same position in a human VH domain, Such activators, Ca", ATP, amongst others). 60 as a human VH3 domain. The humanizing Substitutions Thus, such VH sequences can generally be generated or should be chosen Such that the resulting humanized immu obtained by Suitably immunizing a species of Camelid with noglobulin single variable domains still retain the favorable a target complex comprising a GPCR and a G protein, or properties of immunoglobulin single variable domains as with either one or both of its constituting member proteins defined herein. The skilled person will be able to select (i.e., so as to raise an immune response and/or heavy chain 65 humanizing Substitutions or Suitable combinations of antibodies directed against a target complex), by obtaining a humanizing Substitutions which optimize or achieve a Suitable biological sample from the Camelid (such as a desired or suitable balance between the favorable properties US 9,695,227 B2 31 32 provided by the humanizing Substitutions on the one hand Also within the scope of the disclosure are natural or and the favorable properties of naturally occurring VH synthetic analogs, mutants, variants, alleles, parts or frag domains on the other hand. ments (herein collectively referred to as “variants') of the Another particularly preferred class of immunoglobulin immunoglobulin single variable domains, in particular the single variable domains hereof, in particular nanobodies nanobodies, of the disclosure as defined herein, and in hereof, comprises immunoglobulin single variable domains particular variants of the immunoglobulin single variable with an amino acid sequence that corresponds to the amino domains of SEQ ID NOs: 1-6 (see Tables 2-3). Thus, acid sequence of a naturally occurring VH domain, but that according to one embodiment hereof, the term “immuno has been "camelized, i.e., by replacing one or more amino globulin single variable domain hereof or “nanobody acid residues in the amino acid sequence of a naturally 10 hereof in its broadest sense also covers such variants. occurring VH domain from a conventional 4-chain antibody Generally, in Such variants, one or more amino acid residues by one or more of the amino acid residues that occur at the may have been replaced, deleted and/or added, compared to corresponding position(s) in a VH domain of a heavy chain the immunoglobulin single variable domains of the disclo antibody. Such "camelizing substitutions are preferably 15 Sure as defined herein. Such substitutions, insertions or inserted at amino acid positions that form and/or are present deletions may be made in one or more of the FRs and/or in at the VH-VL interface, and/or at the so-called Camelidae one or more of the CDRs and, in particular, variants of the hallmark residues, as defined herein (see for example FRs and CDRs of the immunoglobulin single variable WO9404678). Preferably, the VH sequence that is used as a domains of SEQID NOS:1-6 (see Tables 2 and 3). Variants, starting material or starting point for generating or designing as used herein, are sequences wherein each or any frame the camelized nanobody is preferably a VH sequence from work region and each or any complementarity-determining a mammal, more preferably the VH sequence of a human region shows at least 80% identity, preferably at least 85% being, such as a VH3 sequence. However, it should be noted identity, more preferably 90% identity, even more preferably that Such camelized immunoglobulin single variable 95% identity or, still even more preferably 99% identity with domains of the disclosure can be obtained in any suitable 25 the corresponding region in the reference sequence (i.e., manner known perse (i.e., as indicated under points (1)–(8) FR 1 variant versus FR1 reference, CDR1 variant versus above) and thus are not strictly limited to polypeptides that CDR1 reference, FR2 variant versus FR2 reference, have been obtained using a polypeptide that comprises a CDR2 variant versus CDR2 reference, FR3 variant versus naturally occurring VH domain as a starting material. FR3 reference, CDR3 variant versus CDR3 reference, For example both “humanization' and “camelization' can 30 FR4 variant versus FR4 reference), as can be measured be performed by providing a nucleotide sequence that electronically by making use of algorithms such as PILEUP encodes a naturally occurring VH domain or VH domain, and BLAST (50, 51). Software for performing BLAST respectively, and then changing, in a manner known per se, analyses is publicly available through the National Center one or more codons in the nucleotide sequence in Such away for Biotechnology Information (on the World Wide Web at that the new nucleotide sequence encodes a “humanized' or 35 incbi.nlm.nih.gov). It will be understood that for determining "camelized immunoglobulin single variable domains the degree of amino acid identity of the amino acid hereof, respectively. This nucleic acid can then be expressed sequences of the CDRS of one or more sequences of the in a manner known per se, so as to provide the desired immunoglobulin single variable domains, the amino acid immunoglobulin single variable domains hereof. Alterna residues that form the framework regions are disregarded. tively, based on the amino acid sequence of a naturally 40 Similarly, for determining the degree of amino acid identity occurring VH domain or VH domain, respectively, the of the amino acid sequences of the FRs of one or more amino acid sequence of the desired humanized or camelized sequences of the immunoglobulin single variable domains immunoglobulin single variable domains hereof, respec hereof, the amino acid residues that form the complemen tively, can be designed and then synthesized de novo using tarity regions are disregarded. Such variants of immuno techniques for peptide synthesis known per se. Also, based 45 globulin single variable domains may be of particular advan on the amino acid sequence or nucleotide sequence of a tage since they may have improved potency/affinity. naturally occurring VH domain or VH domain, respec By means of non-limiting examples, a Substitution may tively, a nucleotide sequence encoding the desired human for example be a conservative substitution (as described ized or camelized immunoglobulin single variable domains herein) and/or an amino acid residue may be replaced by hereof, respectively, can be designed and then synthesized 50 another amino acid residue that naturally occurs at the same de novo using techniques for nucleic acid synthesis known position in another VH domain. Thus, any one or more per se, after which the nucleic acid thus obtained can be Substitutions, deletions or insertions, or any combination expressed in a manner known per se, so as to provide the thereof, that either improve the properties of the immuno desired immunoglobulin single variable domains hereof. globulin single variable domains of the disclosure or that at Other suitable methods and techniques for obtaining the 55 least do not detract too much from the desired properties or immunoglobulin single variable domains of the disclosure from the balance or combination of desired properties of the and/or nucleic acids encoding the same, starting from natu nanobody of the disclosure (i.e., to the extent that the rally occurring VHSequences or preferably VH sequences, immunoglobulin single variable domains is no longer Suited will be clear from the skilled person, and may for example for its intended use) are included within the scope hereof. A comprise combining one or more parts of one or more 60 skilled person will generally be able to determine and select naturally occurring VH sequences (such as one or more FR suitable substitutions, deletions or insertions, or suitable sequences and/or CDR sequences), one or more parts of one combinations of thereof, based on the disclosure herein and or more naturally occurring VH sequences (such as one or optionally after a limited degree of routine experimentation, more FR sequences or CDR sequences), and/or one or more which may for example involve introducing a limited num synthetic or semi-synthetic sequences, in a Suitable manner, 65 ber of possible substitutions and determining their influence So as to provide a nanobody of the disclosure or a nucleotide on the properties of the immunoglobulin single variable sequence or nucleic acid encoding the same. domains thus obtained. US 9,695,227 B2 33 34 According to particularly preferred embodiments, vari Examples of modifications, as well as examples of amino ants of the immunoglobulin single variable domains, in acid residues within the binding domain, in particular the particular the nanobodies, of the present disclosure may immunoglobulin single variable domain of the disclosure have a substitution, deletion or insertion, of 1, 2 or 3 amino that can be modified (i.e., either on the protein backbone but acids in either one, two or three of the CDRs, more specifi preferably on a side chain), methods and techniques that can cally a substitution, deletion or insertion of 1, 2 or 3 amino be used to introduce Such modifications and the potential acids (i) in CDR1 or CDR2 or CDR3; (ii) in CDR1 and uses and advantages of Such modifications will be clear to CDR2, or, in CDR1 and CDR3, or, in CDR2 and CDR3; (iii) the skilled person. For example, Such a modification may in CDR1 and CDR2 and CDR3, of which the amino acid involve the introduction (e.g., by covalent linking or in sequences of CDRs are as listed in Table 3. More preferably, 10 another Suitable manner) of one or more functional groups, variants of the immunoglobulin single variable domains, in residues or moieties into or onto the binding domain, in particular the nanobodies, of the present disclosure may particular the immunoglobulin single variable domain have a conservative substitution (as defined herein) of 1, 2 hereof, and in particular of one or more functional groups, or 3 amino acids in one, two or three of the CDRs, more residues or moieties that confer one or more desired prop specifically (i) in CDR1 or CDR2 or CDR3; (ii) in CDR1 15 erties or functionalities to the binding domain hereof. and CDR2, or, in CDR1 and CDR3, or, in CDR2 and CDR3; Examples of Such functional groups and of techniques for (iii) in CDR1 and CDR2 and CDR3, as listed in Table 3. introducing them will be clear to the skilled person, and can According to a specific embodiment, the present disclo generally comprise all functional groups and techniques Sure provides for an immunoglobulin single variable domain mentioned in the art as well as the functional groups and comprising an amino acid sequence that comprises four techniques known per se for the modification of pharma framework regions (FR1 to FR4) and three complementar ceutical proteins, and in particular for the modification of ity-determining regions (CDR1 to CDR3), according to the antibodies or antibody fragments (including SchvS and following formula (1): single domain antibodies), for which reference is for example made to Remington's Pharmaceutical Sciences, 25 16th ed., Mack Publishing Co., Easton, Pa. (1980). Such functional groups may for example be linked directly (for and wherein CDR1 is chosen from the group consisting example covalently) to the binding domain, in particular the of: immunoglobulin single variable domain hereof, or option a) SEQ ID NOs: 13-18, ally via a Suitable linker or spacer, as will again be clear to b) Polypeptides that have at least 80% amino acid identity 30 the skilled person. with SEQ ID NOs: 13-18, One of the most widely used techniques for increasing the c) Polypeptides that have 3, 2 or 1 amino acid difference half-life and/or reducing immunogenicity of pharmaceutical with SEQ ID NOs: 13-18, proteins comprises attachment of a suitable pharmacologi and wherein CDR2 is chosen from the group consisting cally acceptable polymer, Such as poly(ethylene glycol) of: 35 (PEG) or derivatives thereof (such as methoxypoly(ethylene a) SEQ ID NOs: 25-30, glycol) or mPEG). Generally, any suitable form of pegy b) Polypeptides that have at least 80% amino acid identity lation can be used. Such as the pegylation used in the art for with SEQ ID NOs: 25-30, antibodies and antibody fragments (including but not limited c) Polypeptides that have 3, 2 or 1 amino acid difference to (single) domain antibodies and ScHVS); reference is made with SEQ ID NOs: 25-30, 40 to for example Chapman, Nat. Biotechnol. 54, 531-545 and wherein CDR3 is chosen from the group consisting (2002); by Veronese and Harris, Adv. Drug Deliv. Rev. 54, of: 453-456 (2003), by Harris and Chess, Nat. Rev. Drug. a) SEQ ID NOs: 37-42, Discov. 2, (2003) and in WO04060965. Various reagents for b) Polypeptides that have at least 80% amino acid identity pegylation of proteins are also commercially available, for with SEQ ID NOs: 37-42, 45 example from Nektar Therapeutics, USA. Preferably, site c) Polypeptides that have 3, 2 or 1 amino acid difference directed pegylation is used, in particular via a cysteine with SEQ ID NOs: 37-42. residue (see for example Yang et al., Protein Engineering, In a particularly preferred embodiment, the present dis 16, 10, 761-770 (2003). For example, for this purpose, PEG closure provides for an immunoglobulin single variable may be attached to a cysteine residue that naturally occurs domain comprising an amino acid sequence that comprises 50 in a immunoglobulin single variable domain hereof, a four framework regions (FR1 to FR4) and three comple immunoglobulin single variable domain of the disclosure mentarity-determining regions (CDR1 to CDR3), according may be modified so as to suitably introduce one or more to the following formula (1): cysteine residues for attachment of PEG, or an amino acid sequence comprising one or more cysteine residues for 55 attachment of PEG may be fused to the N- and/or C-termi wherein CDR1 is SEQ ID NO: 13; wherein CDR2 is SEQ nus of a nanobody hereof, all using techniques of protein ID NO: 25; and wherein CDR3 is SEQ ID NO:37. engineering known per se to the skilled person. Preferably, Further, and depending on the host organism used to for the immunoglobulin single variable domain hereof, a express the binding domain, in particular the immunoglobu PEG is used with a molecular weight of more than 5000, lin single variable domain hereof, deletions and/or substi 60 such as more than 10,000 and less than 200,000, such as less tutions may be designed in Such a way that one or more sites than 100,000; for example in the range of 20,000-80,000. for post-translational modification (such as one or more Another, usually less preferred modification comprises glycosylation sites) are removed, as will be within the ability N-linked or O-linked glycosylation, usually as part of co of the person skilled in the art. Alternatively, substitutions or translational and/or post-translational modification, depend insertions may be designed so as to introduce one or more 65 ing on the host cell used for expressing the binding domain, sites for attachment of functional groups (as described in particular the immunoglobulin single variable domain herein), for example to allow site-specific pegylation. hereof. Another technique for increasing the half-life of a US 9,695,227 B2 35 36 binding domain may comprise the engineering into bifunc Targetting, 8, 4, 257 (2000). Such binding pairs may also be tional binding domains (for example, one immunoglobulin used to link a therapeutically active agent to the binding single variable domain against the target GPCR:G protein domain hereof. complex and one against a serum protein Such as albumin) Also encompassed within the scope of the present disclo or into fusions of binding domains, in particular immuno Sure are the binding domains, in particular the immuno globulin single variable domains, with peptides (for globulin single variable domains of the disclosure that are in example, a peptide against a serum protein such as albumin). a “multivalent” form and are formed by bonding, chemically Yet another modification may comprise the introduction or by recombinant DNA techniques, together two or more of one or more detectable labels or other signal-generating monovalent immunoglobulin single variable domains. Non 10 limiting examples of multivalent constructs include “biva groups or moieties, depending on the intended use of the lent” constructs, “trivalent” constructs, “tetravalent” con labeled binding domain. Suitable labels and techniques for structs, and so on. The immunoglobulin single variable attaching, using and detecting them will be clear to the domains comprised within a multivalent construct may be skilled person, and for example include, but are not limited identical or different. In another particular embodiment, the to, fluorescent labels (such as fluorescein, isothiocyanate, 15 immunoglobulin single variable domains of the disclosure rhodamine, phycoerythrin, phycocyanin, allophycocyanin, are in a “multi-specific' form and are formed by bonding o-phthaldehyde, and fluorescamine and fluorescent metals together two or more immunoglobulin single variable Such as Eu or others metals from the lanthanide series), domains, of which at least one with a different specificity. phosphorescent labels, chemiluminescent labels or biolumi Non-limiting examples of multi-specific constructs include nescent labels (such as luminal, isoluminol, theromatic “bi-specific' constructs, "tri-specific' constructs, “tetra-spe acridinium ester, imidazole, acridinium salts, oxalate ester, cific' constructs, and so on. To illustrate this further, any dioxetane or GFP and its analogs), radio-isotopes, metals, multivalent or multispecific (as defined herein) immuno metals chelates or metallic cations or other metals or metal globulin single variable domain of the disclosure may be lic cations that are particularly Suited for use in in Vivo, in Suitably directed against two or more different epitopes on vitro or in situ diagnosis and imaging, as well as chro 25 the same antigen, for example against two or more different mophores and enzymes (such as malate dehydrogenase, parts of the G protein as comprised in the GPCR:G protein staphylococcal nuclease, delta-V-steroid isomerase, yeast complex; or may be directed against two or more different alcohol dehydrogenase, alpha-glycerophosphate dehydroge antigens, for example against an epitope of the GPCR and an nase, triose phosphate isomerase, biotinavidin peroxidase, epitope of the G protein. In particular, a monovalent immu horseradish peroxidase, alkaline phosphatase, asparaginase, 30 noglobulin single variable domain of the disclosure is such that it will bind to the target GPCR: G protein complex (as glucose oxidase, beta-galactosidase, ribonuclease, urease, described herein) with an affinity less than 500 nM, prefer catalase, glucose-VI-phosphate dehydrogenase, glucoamy ably less than 200 nM, more preferably less than 10 nM, lase and acetylcholine esterase). such as less than 500 pM. Multivalent or multispecific Other suitable labels will be clear to the skilled person, 35 immunoglobulin single variable domains of the disclosure and for example include moieties that can be detected using may also have (or be engineered and/or selected for) NMR or ESR spectroscopy. Such labeled binding domains increased avidity and/or improved selectivity for the desired of the disclosure may for example be used for in vitro, in GPCR:G protein complex, and/or for any other desired Vivo or in situ assays (including immunoassays known per property or combination of desired properties that may be se such as ELISA, RIA, EIA and other “sandwich assays.” 40 obtained by the use of such multivalent or multispecific etc.) as well as in vivo diagnostic and imaging purposes, immunoglobulin single variable domains. depending on the choice of the specific label. As will be clear Further, the binding domain hereof, in particular the to the skilled person, another modification may involve the immunoglobulin single variable domain hereof, may gener introduction of a chelating group, for example to chelate one ally be directed against or specifically binding to any con of the metals or metallic cations referred to above. Suitable 45 formational epitope that is represented by or accessible on or chelating groups for example include, without limitation, part of a complex comprising a GPCR and a G protein. A diethyl-enetriaminepentaacetic acid (DTPA) or ethylenedi binding domain that specifically binds to a “three dimen aminetetraacetic acid (EDTA). sional epitope or “conformational epitope is a binding Yet another modification may comprise the introduction domain that specifically binds to a tertiary or quaternary of a functional group that is one part of a specific binding 50 structure of a folded protein or protein complex. Such a pair, such as the biotin-(strept)avidin binding pair. Such a binding domain binds at much reduced (i.e., by a factor of functional group may be used to link the binding domain of at least 2, 5, 10, 50 or 100) affinity to the linear (i.e., the disclosure to another protein, polypeptide or chemical unfolded, denatured) polypeptide chain. The structure to compound that is bound to the other half of the binding pair, which Such a binding domain binds usually contains amino i.e., through formation of the binding pair. For example, a 55 acids that are discontinuous in the linear sequence of the immunoglobulin single variable domain of the disclosure protein (complex). In other words, binding of Such a binding may be conjugated to biotin, and linked to another protein, domain to a polypeptide is dependent upon the polypeptide polypeptide, compound or carrier conjugated to avidin or being folded into a particular three dimensional conforma streptavidin. For example, such a conjugated immunoglobu tion. It should be clear that the conformational epitopes lin single variable domain may be used as a reporter, for 60 selectively recognized by the binding domains of the dis example in a diagnostic system where a detectable signal closure can be GPCR specific epitopes, or G protein specific producing agent is conjugated to avidin or streptavidin. Such epitopes, or otherwise GPCR:G protein complex-specific binding pairs may for example also be used to bind the epitopes, which are only formed upon association of the immunoglobulin single variable domain of the disclosure to constituting proteins, and thus by combining amino acid a carrier, including carriers suitable for pharmaceutical 65 residues of the GPCR and the G protein. In one embodiment, purposes. One non-limiting example are the liposomal for the binding domain hereof, in particular the immunoglobulin mulations described by Cao and Suresh, Journal of Drug single variable domain, specifically binds to any conforma US 9,695,227 B2 37 38 tional epitope of any desired G protein or parts thereof. In stabilizing an agonist-bound GPCR:G protein complex and/ another embodiment, the conformational epitope can be part or enhances the affinity of an agonist for a GPCR: G protein of an intracellular or extracellular region, or an intramem complex. Preferably, the binding domain is capable of braneous region, or a domain or loop structure of any desired increasing the affinity of the G protein for the GPCR and/or GPCR. It is clear that some of these conformational epitopes 5 the affinity of the agonist for the GPCR:G protein complex will be accessible in the GPCR and/or the G protein in the at least twofold, at least fivefold, and more preferably at least non-associated form, while others will only be accessible tenfold as measured by a decrease in K. Alternatively, the once the complex is formed. According to one specific binding domain is capable of inducing a shift in ECso or ICso embodiment, the binding domain hereof, in particular the by at least twofold, at least fivefold, and more preferably at immunoglobulin single variable domain, specifically binds a 10 least tenfold in any assay set-up comparing the interaction conformational epitope at the interface between the C. and B strength of the receptor and the G protein in presence of the Subunit of a G protein, as is specified as a non-limiting complex stabilizing binding domain versus a condition example further herein (see Example section). where this binding domain is absent or any other measure of According to other specific embodiments, the binding affinity or potency known to one of skill in the art. domain binds to a GPCR:G protein complex wherein the G 15 The term “a functional conformational state,” as used protein is in its nucleotide free from. According to further herein, refers to the fact that proteins, in particular mem specific embodiments, the binding domain hereof, in par brane proteins such as GPCRs, possess many different ticular the immunoglobulin single variable domain, inhibits conformational states having a dynamic range of activity, in or prevents the dissociation of the GPCR:G protein in the particular ranging from no activity to maximal activity presence of nucleotides, in particular guanine nucleotides, (reviewed in Kobilka and Deupi, 2007). It should be clear such as GDP or GTP, or analogs thereof, such as nonhydro that “a functional conformational state' is not meant to lyzable GTP analogs, such as GTPYS, or GDP in combina cover the denatured States of proteins. For example, a basal tion with aluminum or beryllium fluoride species, or mini conformational state can be defined as a low energy state of mal nucleotide analogs such as pyrophosphate or forcarnet. the receptor in the absence of a ligand. The probability that In the absence of the binding domains hereof, dissociation of 25 a protein will undergo a transition to another conformational the GPCR:G protein complex normally occurs in the pres state is a function of the energy difference between the two ence of these nucleotides. states and the height of the energy barrier between the two According to one particular aspect, the disclosure also states. In the case of a receptor protein, such as a GPCR, the provides for a binding domain that is directed against or energy of ligand binding can be used either to alter the specifically binds to a G protein (i.e., a G protein alone, thus 30 energy barrier between the two states, or to change the not in complex with a GPCR). In specific embodiments, the relative energy levels between the two states, or both. binding domain as described herein is directed against Changing of the energy barrier would have an effect on the and/or specifically binds to a Gs protein. According to rate of transition between the two states, whereas changing specific embodiments of this aspect, the binding domain of the energy levels would have an effect on the equilibrium the disclosure prevents or inhibits the binding of nucleo 35 distribution of receptors in two states. Binding of an agonist tides, in particular guanine nucleotides or analogs (as or partial agonist would lower the energy barrier and/or described hereinbefore), to the G protein. Or also, the reduce the energy of the more active conformational State binding domain of the disclosure is capable of displacing a relative to the inactive conformational state. An inverse guanine nucleotide or analog from the G protein. Non agonist would increase the energy barrier and/or reduce the limiting examples of assays to determine the degree of 40 energy of the inactive state conformation relative to the inhibition of binding or displacement of guanine nucleotides active conformation. Coupling of the receptor to its G to a protein are provided in the Example section, for protein could further alter the energy landscape. Cooperative example in Example 3 and 8. Further, it will be appreciated interactions of B2AR and Gs observed in ligand binding that all particular embodiments related to the binding assays formed the foundation of the ternary complex model domains hereof, as described hereinbefore, also apply for 45 of GPCR activation (Delean et al., 1980). In the ternary this particular aspect hereof. complex consisting of agonist, receptor, and G protein, the The functional versatility of GPCRs is inherently coupled affinity of the receptor for agonist is enhanced and the to the flexibility of these proteins resulting in a spectrum of specificity of the G protein for guanine nucleotides changes conformations. The confoimational energy landscape is in favor of GTP over GDP. intrinsically coupled to such factors as the presence of bound 50 It should be noted that the activities of integral membrane ligands (effector molecules, agonists, antagonists, inverse proteins, including GPCRs are also affected by the structures agonists, etc.), the lipid environment or the binding of of the lipid molecules that surround them in the membrane. interacting proteins. Thus, in one embodiment, the binding Membrane proteins are not rigid entities, and deform to domains hereof, in particular the immunoglobulin single ensure good hydrophobic matching to the Surrounding lipid variable domains increase the stability of the complex 55 bilayer. One important parameter is the hydrophobic thick comprising a GPCR and a G protein upon binding of the ness of the lipid bilayer, defined by the lengths of the lipid binding domain. histill a further embodiment, binding of the fatty acyl chains. Also, the structure of the lipid headgroup binding domain of the disclosure to any conformational region is likely to be important in defining the structures of epitope that is represented by or accessible on or part of a those parts of a membrane protein that are located in the complex comprising a GPCR:G protein complex, induces 60 lipid headgroup region. Among other lipids, palmitoylation the formation of a functional conformational state of a and binding of cholesterol to GPCRs may also play a GPCR, in particular an active confoimational state of the structural role inside monomeric receptors and contribute to GPCR. More specifically, the binding domain of the disclo the formation/stabilization of receptor oligomers (Lee 2004: sure is capable of stabilizing the active state of the GPCR Chini and Parenti 2009). comprised in the GPCR:G protein complex, by increasing 65 A further aspect of the present disclosure relates to a the affinity of the G protein for the receptor. Likewise, the complex comprising a binding domain hereof. More spe binding domain of the present disclosure is capable of cifically, a complex is provided comprising a binding US 9,695,227 B2 39 40 domain hereof, a GPCR, a G protein, and optionally a 93061607 (CAMR, Salisbury, Wiltshire, UK), RR-CHOK1 receptor ligand. As a non-limiting example, a stable complex designated ECACC 92052129 (CAMR, Salisbury, Wilt may be purified by gel filtration, as was done for example for shire, UK), dihydrofolate reductase negative CHO cells the quaternary complex containing a nanobody, a GPCR, a (CHO/-DHFR, Urlaub and Chasin, 1980, Proc. Natl. Acad. G protein, and a receptor ligand (see Example section). In a Sci. USA, 77:4216), and dp12.CHO cells (U.S. Pat. No. particular embodiment, the complex can be crystalline. 5,721,121); monkey kidney CV1 cells transformed by SV40 Accordingly, a crystal of the complex is also provided, as (COS cells, COS-7, ATCC CRL-1651); human embryonic well as methods of making the crystal, which are described kidney cells (e.g., 293 cells, or 293T cells, or 293 cells in more detail further herein. Subcloned for growth in Suspension culture, Graham et al., In another aspect, a nucleic acid sequence encoding an 10 1977, J. Gen. Virol. 36:59, or GnTI KO HEK293S cells, amino acid sequence of any of the binding domains hereof, Reeves et al., 2002, PNAS,99: 13419); baby hamster kidney in particular immunoglobulin single variable domains, is cells (BHK, ATCC CCL-10); monkey kidney cells (CV1, also part of the present disclosure and non-limiting examples ATCC CCL-70); African green monkey kidney cells are provided in Table 4. According to preferred embodi (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); ments, the disclosure relates to nucleic acid sequences of 15 mouse sertoli cells (TM4, Mather, 1980, Biol. Reprod. binding domains hereof, in particular immunoglobulin 23:243-251); human cervical carcinoma cells (HELA, single variable domains, in which the sequences have more ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL than 80%, preferably more than 90%, more preferably more 34); human lung cells (W138, ATCC CCL-75); human than 95%, such as 99% or more sequence identity (as hepatoma cells (HEP-G2, HB 8065); mouse mammary defined herein) with the sequences of at least one of the tumor cells (MMT 060562, ATCCCCL-51); buffalo rat liver nucleic acid sequences of the binding domains of SEQ ID cells (BRL3A, ATCC CRL-1442); TRI cells (Mather, 1982, NO:s 49-54 (see Table 4). For the calculation of the per Annals N.Y. Acad. Sci., 383:44-68); MCR5 cells: FS4 cells. centage sequence identity, the nucleic acid sequences of tags According to a particular embodiment, the cells are mam (e.g., His tag or EPEA tag) should be disregarded. Also, the malian cells selected from Hek293 cells or COS cells. nucleic acid sequences as described herein may be com 25 Exemplary non-mammalian cell lines include, but are not prised in a nucleic acid sequence. limited to, Sf9 cells, baculovirus-insect cell systems (e.g., Further, the present disclosure also envisages expression review Jarvis, Virology Volume 310, Issue 1, 25 May 2003, vectors comprising nucleic acid sequences encoding any of Pages 1-7), plant cells Such as tobacco cells, tomato cells, binding domains hereof, in particular immunoglobulin maize cells, algae cells, or yeasts such as Saccharomyces single variable domains, as well as host cells expressing 30 species, Schizosaccharomyces species, Hansenula species, Such expression vectors. Suitable expression systems Yarrowia species or Pichia species. According to particular include constitutive and inducible expression systems in embodiments, the eukaryotic cells are yeast cells from a bacteria or yeasts, virus expression systems, such as bacu Saccharomyces species (e.g., Saccharomyces cerevisiae), lovirus, Semliki forest virus and lentiviruses, or transient Schizosaccharomyces sp. (for example Schizosaccharomy transfection in insect or mammalian cells. The cloning, 35 ces pombe), a Hansenula species (e.g., Hansenula polymor expression and/or purification of the binding domains pha), a Yarrowia species (e.g., Yarrowia lipolytica), a hereof, in particular the immunoglobulin single variable Kluyveromyces species (e.g., Kluyveromyces lactis), a domains, can be done according to techniques known by the Pichia species (e.g., Pichia pastoris), or a Komagataella skilled person in the art. species (e.g., Komagataella pastoris). According to a spe Thus, the present disclosure encompasses a cell or a 40 cific embodiment, the eukaryotic cells are Pichia cells, and culture of cells expressing a binding domain hereof, in in a most particular embodiment Pichia pastoris cells. particular an immunoglobulin single variable domain that is Transfection of target cells (e.g., mammalian cells) can be directed against and/or capable of specifically binds to a carried out following principles outlined by Sambrook and complex comprising a GPCR and a G protein. The cells Russel (Molecular Cloning, A Laboratory Manual, 3" Edi according to the present disclosure can be of any prokaryotic 45 tion, Volume 3, Chapter 16, Section 16.1-16.54). In addition, or eukaryotic organism. Preferably, cells are eukaryotic viral transduction can also be performed using reagents such cells, for example yeast cells, or insect cells, or cultured cell as adenoviral vectors. Selection of the appropriate viral lines, for example mammalian cell lines, preferably human vector system, regulatory regions and host cell is common cell lines, that endogenously or recombinantly express a knowledge within the level of ordinary skill in the art. The GPCR and/or G protein of interest. The nature of the cells 50 resulting transfected cells are maintained in culture or frozen used will typically depend on the ease and cost of producing for later use according to standard practices. the native protein(s), the desired glycosylation properties, Accordingly, another aspect of the disclosure relates to a the origin of the target protein, the intended application, or method for producing a binding domain according to the any combination thereof. Eukaryotic cell or cell lines for invention, the method comprising at least the steps of: protein production are well known in the art, including cell 55 a) Expressing in a suitable cellular expression system (as lines with modified glycosylation pathways, and non-limit defined hereinabove) a nucleic acid according to the ing examples will be provided hereafter. invention, and optionally Animal or mammalian host cells Suitable for harboring, b) Isolating and/or purifying the binding domain. expressing, and producing proteins for Subsequent isolation The herein described binding domains, complexes, cells and/or purification include Chinese hamster ovary cells 60 or cell lines can be can be used in a variety of contexts and (CHO), such as CHO-K1 (ATCCCCL-61), DG44 (Chasin et applications, for example, and without limitation, for cap al., 1986, Som. Cell Molec. Genet., 12:555-556; and turing and/or purification of GPCR: G protein complexes, Kolkekar et al., 1997, Biochemistry, 36:10901-10909), and in crystallization studies and high-resolution structural CHO-K1 Tet-On cell line (Clontech), CHO designated analysis of GPCR: G protein complexes. It is thus one of the ECACC 85.050302 (CAMR, Salisbury, Wiltshire, UK), 65 aims of the disclosure to use the binding domain according CHO clone 13 (GEIMG, Genova, IT), CHO clone B to the invention, in particular immunoglobulin single vari (GEIMG, Genova, IT), CHO-K1/SF designated ECACC able domains, such as nanobodies, as tools to stabilize US 9,695,227 B2 41 42 GPCR:G protein complexes and further to use these binding preferred embodiment, the binding domain is capable of domains as co-crystallization aids for GPCRs in complex increasing the thermal stability of a GPCR:G protein com with a G protein, or in other words to facilitate crystallo plex with a receptor ligand, more specifically an agonist or genesis of GPCR:G protein complexes. Additionally, and/or positive allosteric modulator of the GPCR dependent sig alternatively, the binding domains and preferably cellular naling pathway. According to another preferred embodi systems expressing the binding domains, as described ment, the binding domain according to the disclosure is herein, can be useful for other applications such as ligand capable of increasing the stability of a GPCR:G protein screening, drug discovery, immunization, all of which will complex in the presence of a detergent or a chaotrope. be described into further detail below. Preferably, the binding domain is capable of increasing the Stabilization of a GPCR:G Protein Complex and Locking 10 the GPCR in the G Protein-Bound State stability of a GPCR:G protein complex to denaturation Thus, according to one aspect, the disclosure relates to the induced by thermal or chemical approaches. In relation to an use of a binding domain as described hereinbefore to stabi increased Stability to heat, a detergent or to a chaotrope, lize a complex comprising a GPCR and a G protein. Accord typically the GPCR:G protein is incubated for a defined time ing to a preferred embodiment, the complex that is stabilized 15 in the presence of a test detergent or a test chaotropic agent further comprises a receptor ligand, more specifically an and the stability is detennined using, for example, ligand agonist. The term “stabilize.” “stabilizing,” or “increasing binding or a spectroscoptic method, optionally at increasing the stability,” as used herein, refers to increasing the stability temperatures as discussed above. According to still another of a GPCR: G protein complex with respect to the structure preferred embodiment, the binding domain according to the (conformational state) and/or particular biological activity disclosure is capable of increasing the Stability to extreme (intracellular signaling activity) of one or both of the con pH of a functional conformational state of a GPCR. Pref stituting proteins of the complex, in particular the GPCR erably, the binding domain is capable of increasing the and/or the G protein. In one particularly preferred embodi stability of a GPCR: G protein complex to extreme pH. In ment, the binding domain of the disclosure can be used to relation to an extreme of pH, a typical test pH would be stabilize the GPCR:G protein complex so that the GPCR is 25 chosen for example in the range 6 to 8, the range 5.5 to 8.5, locked or fixed in an active or G protein-bound state. A the range 5 to 9, the range 4.5 to 9.5, more specifically in the GPCR that adopts such an active or G protein-bound state range 4.5 to 5.5 (low pH) or in the range 8.5 to 9.5 (high pH). will exert its biological activity in nature. Ways to determine In a particularly preferred embodiment, the binding the (increased) stability of a GPCR: G protein complex have domain according to the disclosure can be used to prevent been described hereinbefore and are further illustrated in the 30 the dissociation of the complex in the presence of nucleo Examples section. tides, in particular guanine nucleotides or analogs thereof. It will be appreciated that having increased stability with More specifically, guanine nucleotides include GDP and respect to structure and/or a particular biological activity of GTP, and analogs of guanine nucleotides include, without a GPCR includes the stability to other denaturants or dena being limitative, GTPYS or GDP in combination with alu turing conditions including heat, a detergent, a chaotropic 35 minum or beryllium fluoride species or nucleotide fragments agent and an extreme pH. Accordingly, in a further embodi Such as pyrophosphate or foscarnet. ment, the binding domain according to the disclosure is Capturing and/or Purifying a GPCR:G Protein Complex capable of increasing the stability of a GPCR:G protein It will thus be understood that the ability of forming a complex under non-physiological conditions induced by stable GPCR: G protein complex is particularly useful for dilution, concentration, buffer composition, heating, cool 40 capturing and/or purifying a GPCR: G protein complex, ing, freezing, detergent, chaotropic agent, pH, amongst which will allow Subsequent crystallization, ligand charac others. Accordingly, the term “thermostabilize.” “thermo terization and compound screening, immunizations, stabilizing,” “increasing the thermostability of refers to the amongst others. Moreover, it is of particular advantage that functional rather than to the thermodynamic properties of a the binding domains of the disclosure can be useful generic GPCR:G protein complex and to the constituting protein's 45 tools that may be applicable for a range of GPCR:G protein resistance to irreversible denaturation induced by thermal complexes. and/or chemical approaches including but not limited to Accordingly, the present disclosure also envisages a heating, cooling, freezing, chemical denaturants, pH, deter method of capturing and/or purifying a complex comprising gents, salts, additives, proteases or temperature. Irreversible a GPCR and a G protein, the method comprising the steps denaturation leads to the irreversible unfolding of the func 50 of: tional conformations of the protein, loss of biological activ a) Providing a binding domain according to the invention, ity and aggregation of the denaturated protein. The term and “(thermo)stabilize.” “(thermo)stabilizing,” “increasing the b) Allowing the binding domain to bind to a complex (thermo)stability of” as used herein, applies to GPCR:G comprising a GPCR and a G protein, and protein complexes embedded in lipid particles or lipid layers 55 c) Optionally, isolating the complex foimed in step b). (for example, lipid monolayers, lipid bilayers, and the like) In a specific embodiment, the disclosure provides for a and to GPCR:G protein complexes that have been solubi method of capturing a complex comprising a GPCR and a G lized in detergent. protein comprising the steps of: In relation to an increased stability to heat, this can be a) applying a solution containing a plurality of GPCRs readily determined by measuring ligand binding or by using 60 and G proteins to a solid Support possessing an immo spectroscopic methods such as fluorescence, CD or light bilized binding domain according to the invention, and scattering that are sensitive to unfolding at increasing tem b) Forming a complex of the binding domain, the GPCR peratures. It is preferred that the binding domain is capable and the G protein, and of increasing the stability as measured by an increase in the c) Removing weakly bound or unbound molecules, thermal stability of a GPCR:G protein complex with at least 65 The present disclosure also envisages a method of puri 2 C., at least 5°C., at least 8° C., and more preferably at fying a complex comprising a GPCR and a G protein, the least 10° C. or 15° C. or 20° C. According to another method comprising the steps of US 9,695,227 B2 43 44 a) Contacting a solution containing a GPCR and a G affinity, solubility, and chemical stability may not be com protein with a binding domain according to the inven patible with crystallization trials. Therefore, it was surpris tion, and ingly found that the binding domains of the present disclo b) Forming a complex comprising the binding domain, sure can be used as tools to increase the probability of the GPCR and the G protein, and obtaining well-ordered crystals by minimizing the confor c) Isolating the complex of step b) mational heterogeneity in the target GPCR:G protein com wherein a complex of a GPCR and a G protein is plex by binding to a specific conformation of G protein. essentially purified. Crystallization of GPCRs for high-resolution structural According to a particular embodiment, the binding studies is particularly difficult because of the amphipathic domain as described herein can also be used to capture a 10 surface of these membrane proteins. Embedded in the mem target GPCR: G protein complex further comprising a recep brane bilayer, the contact sites of the protein with the acyl tor ligand and/or one or more other interacting proteins. chains of the phospholipids are hydrophobic, whereas the The above methods for capturing/purifying target polar Surfaces are exposed to the polar head groups of the GPCR:G protein complexes include, without limitation, lipids and to the aqueous phases. To obtain well-ordered affinity-based methods such as affinity chromatography, 15 three-dimensional crystals—a prerequisite to X-ray struc affinity purification, immunoprecipitation, protein detection, tural analysis at high resolution—GPCRs are solubilized immunochemistry, Surface-display, amongst others, and are with the help of detergents and purified as protein—deter all well-known in the art. gent complexes. The detergent micelle covers the hydro Crystallization and Resolving the Structure of a GPCR:G phobic surface of the membrane protein in a belt-like Protein Complex manner (Hunte and Michel 2002; Ostermeier et al., 1995). Crystallization of membrane proteins including GPCRs GPCR detergent complexes form three-dimensional crys remains a formidable challenge. Although expression and tals in which contacts between adjacent protein molecules purification methods are appearing that allow for the gen are made by the polar surfaces of the protein protruding from eration of milligram quantities, achieving stability with the detergent micelle (Day et al., 2007). Obviously, the these molecules is perhaps the most difficult hurdle to 25 detergent micelle requires space in the crystal lattice. overcome. First of all, binding domains according to the Although attractive interactions between the micelles might disclosure may increase the stability of detergent solubilized stabilize the crystal packing (Rasmussen et al., 2007; Dunn GPCR:G protein complexes, protecting them from pro et al., 1997), these interactions do not lead to rigid crystal teolytic degradation and/or aggregation and facilitating the COntactS. purification and concentration of homogenous samples of 30 Because many membrane proteins, including GPCRs correctly folded proteins. Persons of ordinary skill in the art contain relatively small or highly flexible hydrophilic will recognize that such samples are the preferred starting domains, a strategy to increase the probability of getting point for the generation of diffracting crystals. well-ordered crystals is to enlarge the polar surface of the Crystallization is another major bottleneck in the process protein and/or to reduce their flexibility. The most physi of macromolecular structure determination by X-ray crys 35 ologic approach is to use a native signaling partner Such as tallography. Successful crystallization requires the forma a G protein or arrestin. Unfortunately, interactions of GPCRs tion of nuclei and their Subsequent growth to crystals of with G proteins or arrestins are highly lipid dependent, and Suitable size. Crystal growth generally occurs spontaneously it has been difficult to form complexes of sufficient stability in a Supersaturated Solution as a result of homogenous for crystallography. So, the binding domains of the present nucleation. Proteins may be crystallized in a typical sparse 40 disclosure can be used to enlarge the polar Surfaces of the matrix screening experiment, in which precipitants, addi GPCRs through binding of a G protein, supplementing the tives and protein concentration are sampled extensively, and amount of protein Surface that can facilitate primary contacts Supersaturation conditions Suitable for nucleation and crys between molecules in the crystal lattice with the polar tal growth can be identified for a particular protein. Related surfaces of the G protein and the nanobody. Binding to the sparse matrix screening approach is to generate 45 domains of the present disclosure can also reduce the structural variation in the protein itself, for example by flexibility of its extracellular regions to grow well-ordered adding ligands that bind the protein, or by making different crystals. Immunoglobulin single variable domains, includ mutations, preferentially in Surface residues of the target ing nanobodies, are especially Suited for this purpose protein or by trying to crystallize different species ortho because they bind conformational epitopes and are com logues of the target protein (Chang 1998). One unexpected 50 posed of one single rigid globular domain, devoid of flexible finding of the present disclosure is the usefulness of binding linker regions unlike conventional antibodies or fragments domains that specifically bind to a GPCR: G protein complex derived such as Fabs. to introduce a degree of structural variation upon binding Thus, according to a preferred embodiment, the present while preserving the overall fold of the complex. disclosure provides for binding domains useful as tools to Because crystallization involves an unfavorable loss of 55 crystallize a complex comprising a GPCR and a G protein, conformational entropy in the molecule to be assembled in and eventually to solve the structure. More preferably, the the crystal lattice, methods that reduce the conformational complex which is crystallized by making using of a binding entropy of the target while still in solution should enhance domain of the present disclosure further comprises a recep the likelihood of crystallization by lowering the net entropic tor ligand, more specifically an agonist. In a particularly penalty of lattice formation. The “surface entropy reduction 60 preferred embodiment, the GPCR comprised in the complex approach has proved to be highly effective (Derewenda is in an active state or conformation. 2004). Likewise, binding partners such as ions, Small mol Thus, the binding domain in complex with the GPCR:G ecule ligands, and peptides can reduce the confoimational protein complex and optionally receptor ligand may be heterogeneity by binding to and stabilizing a Subset of crystallized using any of a variety of specialized crystalli conformational states of a protein. Although Such binding 65 Zation methods for membrane proteins, many of which are partners are effective, not all proteins have a known binding reviewed in Caffrey (2003 & 2009). In general terms, the partner, and even when a binding partner is known, its methods are lipid-based methods that include adding lipid to US 9,695,227 B2 45 46 the complex prior to crystallization. Such methods have allosteric regulators and/or G proteins are needed. The previously been used to crystallize other membrane proteins. present disclosure particularly provides general tools to Many of these methods, including the lipidic cubic phase obtain crystals of GPCR:G protein complexes. In particular, crystallization method and the bicelle crystallization agonist-bound GPCR:G protein complex crystals may pro method, exploit the spontaneous self-assembling properties vide three-dimensional representations of the active states of of lipids and detergent as vesicles (vesicle-fusion method), GPCRs. These structures will help clarify the conforma discoidal micelles (bicelle method), and liquid crystals or tional changes connecting the ligand-binding and G protein mesophases (in meso or cubic-phase method). Lipidic cubic interaction sites, and lead to more precise mechanistic phases crystallization methods are described in, for hypotheses and eventually new therapeutics. Given the example: Landau et al., 1996; Gouaux 1998: Rummel et al., 10 conformational flexibility inherent to ligand-activated 1998; Nollert et al., 2004, Rasmussen et al., 2011, which GPCRs and the greater heterogeneity exhibited by agonist publications are incorporated by reference for disclosure of bound receptors, stabilizing Such a state is not easy. Thus, those methods. Bicelle crystallization methods are described such efforts can benefit from the stabilization of a complex in, for example: Faham et al., 2005; Faham et al., 2002, of an agonist-bound receptor conformation bound to its which publications are incorporated by reference for disclo 15 heterotrimeric G protein by the addition of binding domains sure of those methods. that are specific for Such a complex. Especially Suited are According to another embodiment, the disclosure relates binding domains that bind to the G protein that forms part of to the use of a binding domain as described herein to solve Such a complex, since these binding domains can be used as a structure of a target complex comprising a GPCR and a G general tools to stabilize all GPCRs that signal through the protein, and optionally further comprising a receptor ligand. same G protein (e.g., Gs coupled receptors, Gi coupled “Solving the structure' as used herein refers to determining receptors, etc.). the arrangement of atoms or the atomic coordinates of a According to an alternative embodiment, the present protein, and is often done by a biophysical method. Such as disclosure encompasses a method of determining the crystal X-ray crystallography. structure of a complex comprising a GPCR and a G protein, In X-ray crystallography, the diffraction data when prop 25 the method comprising the steps of: erly assembled gives the amplitude of the 3D Fourier a) Providing a binding domain according to the invention, transform of the molecule's electron density in the unit cell. and If the phases are known, the electron density can be simply b) Allowing the binding domain to bind to a complex obtained by Fourier synthesis. For a protein complex, the comprising a GPCR and a G protein, and Success to derive phase information from molecular replace 30 c) Crystallizing the complex formed in step b). ment (MR) alone is questionable when the fraction of In particular embodiments of the above method of deter proteins with a known structure (the search models) is low mining the crystal structure, the target complex comprising (less than 50% of the amino acid content) and/or when the a GPCR and a G protein further comprises a receptor ligand, crystals exhibit limited diffraction quality. While the com more specifically an agonist, bound to the GPCR. bination of multiple isomorphous replacement (MIR) and 35 The determining of the crystal structure may be done by MR phasing has proven Successful for protein complexes a biophysical method such as X-ray crystallography. The (e.g., Ostermeier et al., 1995; Li et al., 1997; Hunte et al., method may further comprise a step for obtaining the atomic 2000), the requirement of producing a good heavy atom coordinates of the crystal (as defined hereinbefore). derivative is almost always problematic. Over the past Identification of Compounds Targeting a GPCR: G Protein decade, classical MIR approaches have generally been 40 Complex Superseded by the use of anomalous dispersion data princi In the process of compound screening, drug discovery and pally using selenomethionine (SeMet) incorporation (MAD lead optimization, there is a requirement for faster, more or SAD) (Hendrickson 1991). In fact, the anomalous experi effective, less expensive and especially information-rich mental data using Se-edge energies generally provide Supe screening assays that provide simultaneous information on rior and less biased phase information compared with either 45 various compound characteristics and their affects on vari MIR or model-based MR phasing data. Accordingly, one ous cellular pathways (i.e., efficacy, specificity, toxicity and specific embodiment relates to the use of a binding domain drug metabolism). Thus, there is a need to quickly and according to the disclosure for the phasing of GPCR:G inexpensively screen large numbers of compounds in order complexes by MR or MAD. In particular, immunoglobulin to identify new specific ligands of a GPCR of interest, single variable domains, including nanobodies, generally 50 preferably conformation specific ligands, which may be express robustly and are suitable for SeMet incorporation. potential new drug candidates. The present disclosure solves To illustrate this further, and without being limitative, phas this problem by providing binding domains that stabilize a ing a complex comprising a GPCR, G protein, and a GPCR:G protein complex in a functional conformational nanobody by introducing all the SeMet sites in the nanobody state, that can then be used as immunogen or selection alone circumvents the need to incorporate SeMet sites in the 55 reagent for Screening in a variety of contexts. GPCR or the G protein. A major advantage of the binding domains according to In many cases, obtaining a diffraction-quality crystal is the disclosure is that the GPCR as comprised in the GPCR:G the chief barrier to solving its atomic-resolution structure. protein complex can be kept in a stabilized functional Thus, according to specific embodiments, the herein conformation, particularly in an active state conformation. described binding domains can be used to improve the 60 For example, library compounds that selectively bind this diffraction quality of the crystals so that the crystal structure active conformation of the receptor have a higher propensity of the target complex can be solved. to behave as agonists because orthosteric or allosteric sta Further, obtaining structural information of GPCR targets, bilization of the active conformation of the GPCR elicits for example to help guide GPCR drug discovery, is highly biological responses. desired. Beyond the crystallization of more GPCRs, espe 65 Another advantage is that the binding domain increases cially methods for acquiring structures of receptors bound to the thermostability of the active conformation of the GPCR different classes of ligands including agonists, antagonists, comprised in the complex, thus protecting the GPCR against US 9,695,227 B2 47 48 irreversible or thermal denaturation induced by the non (iii) Providing a test compound, and native conditions used in compound screening and drug (iv) Evaluating whether the test compound binds to the discovery, without the need to rely on mutant GPCRs with complex, and increased stability. (v) Selecting a compound that selectively binds to the Another major advantage of the conformation-selective complex. binding domains according to the disclosure is that they It will be clear that the binding domain as used in any of allow to quickly and reliably screen for and differentiate the above methods is capable of stabilizing the functional between receptor agonists, inverse agonists, antagonists conformational state of the GPCR:G protein complex and and/or modulators as well as inhibitors of GPCRs and prevents the dissociation of the complex. Preferably, the 10 GPCR:G protein complex is in an active conformational GPCR-dependent pathways, so increasing the likelihood of state (as defined hereinbefore). According to particularly identifying a ligand with the desired pharmacological prop preferred embodiments of the above screening methods, the erties. GPCR:G protein complex further comprises a receptor To illustrate this further, it is a well-established concept ligand. that most GPCRs exhibit higher agonist binding affinity 15 It should be noted that particularly preferred embodiments when complexed with G protein. This is attributed to the of the binding domains are as described hereinbefore with cooperative interaction between agonist occupied receptor respect to the earlier aspects of the disclosure. and G protein. Binding domains of the present disclosure Thus, the binding domains of the present disclosure can that inhibit the dissociation of a complex of a GPCR and a be useful in screening assays. Screening assays for drug G protein thus will stabilize an active conformational state discovery can be solid phase or Solution phase assays, e.g., of the R:G protein complex, thus increasing the affinity of a binding assay, such as radioligand binding assays. It will the GPCR for agonists and decreasing the affinity for inverse be appreciated that in some instances high throughput agonists. It follows that binding domains that recognize the screening of test compounds is preferred and that the meth active functional conformation of the R:G complex can for ods as described above may be used as a “library screening example be used in high-throughput Screening assays to 25 method, a term well known to those skilled in the art. Thus, screen for agonists because they increase the affinity of the the test compound may be a library of test compounds. In receptor for agonists, relative to inverse agonists. Binding particular, high-throughput screening assays for therapeutic domains that recognize the active functional conformation compounds Such as agonists, antagonists or inverse agonists of the G.R complex can also be used in high-throughput and/or modulators form part hereof. For high-throughput screening assays to screen for biased agonists with the 30 purposes, compound libraries may be used such as allosteric ability to selectively stimulate a subset of a receptors compound libraries, peptide libraries, antibody libraries, signaling activities, for example the selective activation of G fragment-based libraries, synthetic compound libraries, protein, relative to 3-arrestin function. natural compound libraries, phage-display libraries and the According to a specific embodiment, a binding domain like. which specifically binds to the G protein in complex with a 35 Methodologies for preparing and screening Such libraries GPCR (e.g., Tables 2 and 3) can be used as a universal tool are known in the art. In one preferred embodiment, high for Screening programs targeting a plurality of GPCRs, since throughput screening methods involve providing a combi a particular G protein (e.g., Gs) will form a complex with a natorial chemical or peptide library containing a large num plurality of GPCRs (e.g., Gs coupled receptors, including ber of potential therapeutic ligands. Such “combinatorial 5-HT receptors types 5-HT, and 5-HT, ACTH receptor, 40 libraries' or “compound libraries' are then screened in one Adenosine receptor types A and A. Arginine vasopressin or more assays, as described herein, to identify those library receptor 2. B-adrenergic receptors types f, B and B, members (particular chemical species or subclasses) that Calcitonin receptor, Calcitonin gene-related peptide recep display a desired characteristic activity. A "compound tor, Corticotropin-releasing hormone receptor, Dopamine library' is a collection of stored chemicals usually used receptors D-like family (D, and Ds), FSH-receptor, Gastric 45 ultimately in high-throughput screening A "combinatorial inhibitory polypeptide receptor, Glucagon receptor, Hista library' is a collection of diverse chemical compounds mine H receptor, Luteinizing hormone/choriogonadotropin generated by either chemical synthesis or biological synthe receptor, Melanocortin receptor, Parathyroid hormone sis, by combining a number of chemical “building blocks' receptor 1, Prostaglandin receptor types D and I, Secretin Such as reagents. Preparation and screening of combinatorial receptor, Thyrotropin receptor, etc.; see also Table 1). 50 libraries are well known to those of skill in the art. The Thus, another aspect according to the present disclosure compounds thus identified can serve as conventional "lead encompasses the use of a binding domain, or the use of a compounds' or can themselves be used as potential or actual complex, a cell, a membrane preparation comprising a therapeutics. Thus, in one further embodiment, the screening binding domain, all as described hereinbefore, in screening methods as described herein above further comprises modi and/or identification programs for conformation-specific 55 fying a test compound which has been shown to bind to a binding partners of a GPCR: G protein complex, which conformationally active GPCR:G protein complex, and ultimately might lead to potential new drug candidates. determining whether the modified test compound binds to According to one embodiment, the disclosure envisages a the GPCR when residing in the particular conformation. method of identifying compounds capable of selectively In cases where high-throughput Screening of target binding to a GPCR:G protein complex, the method com 60 GPCR:G protein complexes for conformation-specific bind prising the steps of ing partners will be preferred, this will be facilitated by (i) Providing a complex comprising a GPCR and a G immobilization of the binding domain according to the protein invention, or the GPCR:G protein complex stabilized by the (ii) Contacting the complex with a binding domain that is binding domain, onto a Suitable solid Surface or Support that directed against and/or specifically binds to the com 65 can be arrayed or otherwise multiplexed. Non-limiting plex and allowing the binding domain to bind to the examples of Suitable solid Supports include beads, columns, complex, and slides, chips or plates. More specifically, the Solid Supports US 9,695,227 B2 49 50 may be particulate (e.g. beads or granules, generally used in example ELISA, or immunoaffinity purification processes extraction columns) or in sheet form (e. g. membranes or by contacting the immobilized proteins according to the filters, glass or plastic slides, microtiter assay plates, dip disclosure with a test sample (i.e., comprising the test stick, capillary fill devices or such like) which can be flat, compound, amongst other) according to standard methods pleated, or hollow fibers or tubes. The following matrices are conventional in the art. Alternatively, and particularly for given as examples and are not exhaustive. Such examples high-throughput purposes, the immobilized proteins can be could include silica (porous amorphous silica), i. e. the arrayed or otherwise multiplexed. Preferably, the immobi FLASH series of cartridges containing 60 A irregular silica lized proteins according to the disclosure are used for the (32-63 um or 35-70 um) supplied by Biotage (a division of screening and selection of compounds that specifically bind Dyax Corp.), agarose or polyacrylamide Supports, for 10 to a conformationally stabilized GPCR:G protein complex, example the Sepharose range of products Supplied by Amer wherein in particular the GPCR is in an active conforma sham Pharmacia Biotech, or the Affi-Gel supports supplied tional state. by Bio-Rad. In addition there are macroporous polymers, It will be appreciated that either the binding domain, or such as the pressure-stable Afli-Prep supports as supplied by the (conformationally stabilized) GPCR:G protein complex, Bio-Rad. Other supports that could be utilized include: 15 or its constituting proteins may be immobilized, depending dextran, collagen, polystyrene, methacrylate, calcium alg on the type of application or the type of screening that needs inate, controlled pore glass, aluminium, titanium and porous to be done. Also, the choice of the GPCR:G protein stabi ceramics. Alternatively, the solid Surface may comprise part lizing binding domain (targeting a particular conformational of a mass dependent sensor, for example, a Surface plasmon epitope of the GPCR: G protein complex), will determine the resonance detector. Further examples of commercially avail orientation of the proteins and accordingly, the desired able Supports are discussed in, for example, Protein Immo outcome of the compound identification, e.g., compounds bilization, R. F. Taylor ed., Marcel Dekker, Inc., New York, specifically binding to extracellular parts, intramembranal (1991). parts or intracellular parts of the conformationally stabilized Immobilization may be either non-covalent or covalent. GPCR or compounds specifically binding to the conforma In particular, non-covalent immobilization or adsorption on 25 tionally stabilized G protein. a solid surface of the binding domain, or the GPCR:G Alternatively, the test compound (or a library of test protein complex stabilized by the binding domain, according compounds) may be immobilized on a solid Surface. Such as to the disclosure may occur via a Surface coating with any a chip surface, whereas the binding domain and the GPCR:G of an antibody, or streptavidin or avidin, or a metal ion, protein complex are provided, for example, in a detergent recognizing a molecular tag attached to the binding domain 30 Solution or in a membrane-like preparation (see below). or the GPCR, according to standard techniques known by Most preferably, neither the binding domain, nor the the skilled person (e.g., biotin tag. Histidine tag, etc.). GPCR:G protein complex or its constituting proteins, nor Alternatively, the binding domain, or the GPCR:G protein the test compound are immobilized, as is the case for complex stabilized by the binding domain, according to the example in phage-display selection protocols in Solution, or invention, may be attached to a solid Surface by covalent 35 radioligand binding assays. In a preferred embodiment, the cross-linking using conventional coupling chemistries. A binding domain, the GPCR:G protein complex (or sepa Solid Surface may naturally comprise cross-linkable residues rately, the constituting proteins) as used in any of the above suitable for covalent attachment or it may be coated or screening methods, are provided as whole cells, or cell derivatized to introduce Suitable cross-linkable groups (organelle) extracts such as membrane extracts or fractions according to methods well known in the art. 40 thereof, or may be incorporated in lipid layers or vesicles In one particular embodiment, sufficient functionality of (comprising natural and/or synthetic lipids), high-density the immobilized protein is retained following direct covalent lipoparticles, or any nanoparticle, such as nanodisks, or are coupling to the desired matrix via a reactive moiety that does provided as VLPs, so that sufficient functionality of the not contain a chemical spacer aim. Further examples and respective proteins is retained. Preparations of GPCRs more detailed information on immobilization methods of 45 formed from membrane fragments or membrane-detergent antibody (fragments) on Solid Supports are discussed in Jung extracts are reviewed in detail in Cooper (2004), incorpo et al. (2008); similarly, membrane receptor immobilization rated herein by reference. Alternatively, binding domains, methods are reviewed in Cooper (2004); both herein incor GPCR:G protein complexes, or the constituting proteins porated by reference. Noteably, the mutation of a particular may also be solubilized in detergents. Non-limiting amino acid (in a protein with known or inferred structure) to 50 examples of solubilized receptor preparations are further alysine or cysteine (or other desired amino acid) can provide provided in the Example section. a specific site for covalent coupling, for example. It is also Various methods may be used to determine binding possible to reengineer a specific protein to alter the distri between the stabilized GPCR:G protein complex and a test bution of surface available amino acids involved in the compound, including for example, enzyme linked immu chemical coupling (Kalliwass et al., 1993), in effect control 55 nosorbent assays (ELISA), Surface Plasmon resonance ling the orientation of the coupled protein. A similar assays, chip-based assays, immunocytofluorescence, yeast approach can be applied to the binding domains according to two-hybrid technology and phage display which are com the invention, as well as to the conformationally stabilized mon practice in the art, for example, in Sambrook et al. GPCR:G protein complexes, so providing a means of ori (2001), Molecular Cloning, A Laboratory Manual. Third ented immobilization without the addition of other peptide 60 Edition. Cold Spring Harbor Laboratory Press, Cold Spring tails or domains containing either natural or unnatural amino Harbor, N.Y. Other methods of detecting binding between a acids. In case of an antibody or an antibody fragment, Such test compound and a GPCR include ultrafiltration with ion as a nanobody, introduction of mutations in the framework spray mass spectroscopy/HPLC methods or other (bio) region is preferred, minimizing disruption to the antigen physical and analytical methods. Fluorescence Energy Reso binding activity of the antibody (fragment). 65 nance Transfer (FRET) methods, for example, well known Conveniently, the immobilized proteins may be used in to those skilled in the art, may also be used. It will be immunoadsorption processes Such as immunoassays, for appreciated that a bound test compound can be detected US 9,695,227 B2 51 52 using a unique label or tag associated with the compound, bind to the target complex in such a way that they inhibit or Such as a peptide label, a nucleic acid label, a chemical label, enhance the assembly of GPCR functional homomers or a fluorescent label, or a radio frequency tag, as described heteromers. further herein. Also, cell-based assays are critical for assessing the The efficacy of the compounds and/or compositions com mechanism of action of new biological targets and biologi prising the same, can be tested using any Suitable in vitro cal activity of chemical compounds. Current cell-based assay, cell-based assay, in vivo assay and/or animal model assays for GPCRs include measures of pathway activation known perse, or any combination thereof, depending on the (Ca" release, cAMP generation or transcriptional activity); specific disease or disorder involved. measurements of protein trafficking by tagging GPCRs and 10 downstream elements with GFP, and direct measures of In one particular embodiment, it is determined whether interactions between proteins using Fórster resonance the compound alters the binding of the GPCR to a receptor energy transfer (FRET), bioluminescence resonance energy ligand (as defined herein). Binding of a GPCR to its ligand transfer (BRET) or yeast two-hybrid approaches. Introduc can be assayed using standard ligand binding methods ing the binding domains of the present invention, inside the known in the art as described herein. For example, a ligand 15 cell to the relevant compartment of the cell (intra- or may be radiolabeled or fluorescently labeled. The assay may extracellularly) by any means well known and commonly be carried out on whole cells or on membranes obtained used in the art, may lead to new or better cell-based assays. from the cells or aqueous solubilized receptor with a deter In particular, there is a need to “de-orphanize” those gent. The compound will be characterized by its ability to GPCRs for which a natural activating ligand has not been alter the binding of the labeled ligand (see also Example identified. The stabilization of GPCRs in a functional con section). The compound may decrease the binding between formational State using the binding domains according to the the GPCR and its ligand, or may increase the binding disclosure enables screening approaches that may be used to between the GPCR and its ligand, for example by a factor of identify ligands of “orphan' GPCRs where the natural at least 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 30 fold, ligand is unknown. For example, various approaches to 50 fold, 100 fold. Thus, according to more specific embodi 25 "de-orphanization' have been adopted including array ments, the complex as used in any of the above Screening screening against families of known ligands. Ligands of methods further comprises a receptor ligand. Preferably, the orphan GPCRs may be identified from biological samples. receptor ligand is chosen from the group comprising a small Thus, in a particular embodiment, the test compound is molecule, a polypeptide, an antibody or any fragment provided as a biological sample. In particular, the sample 30 can be any suitable sample taken from an individual. For derived thereof, a natural product, and the like. More pref example, the sample may be a body fluid sample Such as erably, the receptor ligand is a full agonist, or a partial blood, serum, plasma, spinal fluid. Alternatively, the sample agonist, or an inverse agonist, or an antagonist, as described is tissue or cell extract. hereinbefore. The test compound as used in any of the above Screening In addition to establishing binding to a target GPCR:G 35 methods may be selected from the group comprising a protein complex in a functional conformational State, it will polypeptide, a peptide, a Small molecule, a natural product, also be desirable to determine the functional effect of a a peptidomimetic, a nucleic acid, a lipid, lipopeptide, a compound on the GPCR: G protein complex, in particular on carbohydrate, an antibody or any fragment derived thereof, the biological activity of the GPCR and downstream inter such as Fab, Fab' and F(ab')2, Fd, single-chain Fvs (sclv), acting partners. In particular, the binding domains according 40 single-chain antibodies, disulfide-linked FVs (dsEv) and to the disclosure can be used to Screen for compounds that fragments comprising either a VL or VH domain, a heavy modulate (increase or decrease) the biological activity of the chain antibody (hcAb), a single domain antibody (sdAb), a GPCR:G protein complex, or its constituents, being the minibody, the variable domain derived from camelid heavy GPCR or the G protein. The desired modulation in biologi chain antibodies (VHH or nanobody), the variable domain cal activity will depend on the GPCR of choice. The 45 of the new antigen receptors derived from shark antibodies compounds may bind to the target GPCR:G protein com (VNAR), a protein scaffold including an alphabody, protein plex, in particular to one or both of its constituents, resulting A, protein G, designed ankyrin-repeat domains (DARPins), in the modulation (activation or inhibition) of downstream fibronectin type III repeats, anticalins, knottins, engineered receptor signaling. This modulation of GPCR signaling can CH2 domains (nanoantibodies), as defined hereinbefore. occur ortho- or allosterically. The compounds may bind to 50 The test compound may optionally be covalently or the target complex comprising a GPCR bound to a G protein non-covalently linked to a detectable label. Suitable detect or its constituents so as to activate or increase receptor able labels and techniques for attaching, using and detecting signaling; or alternatively so as to decrease or inhibit recep them will be clear to the skilled person, and include, but are tor signaling. The compounds may also bind to the target not limited to, any composition detectable by spectroscopic, complex in such a way that they block off the constitutive 55 photochemical, biochemical, immunochemical, electrical, activity of the GPCR. The compounds may also bind to the optical or chemical means. Useful labels include magnetic target complex in Such a way that they mediate allosteric beads (e.g., dynabeads), fluorescent dyes (e.g., all Alexa modulation (e.g., bind to the GPCR or G protein at an Fluor dyes, fluorescein isothiocyanate, Texas red, rhod allosteric site). In this way, the compounds may modulate amine, green fluorescent protein and the like), radiolabels the receptor function by binding to different regions in the 60 (e.g., H, °I, S, ''C or P), enzymes (e.g., horse radish GPCR;G protein complex (e.g., at allosteric sites). Refer peroxidase, alkaline phosphatase), and colorimetric labels ence is for example made to George et al. (2002), Kenakin Such as colloidal gold or colored glass or plastic (e.g., (2002) and Rios et al. (2001). The compounds of the polystyrene, polypropylene, latex, etc.) beads. Means of disclosure may also bind to the target complex in Such a way detecting such labels are well known to those of skill in the that they prolong the duration of the GPCR-mediated sig 65 art. Thus, for example, radiolabels may be detected using naling or that they enhance receptor signaling by increasing photographic film or Scintillation counters, fluorescent receptor-ligand affinity. Further, the compounds may also markers may be detected using a photodetector to detect US 9,695,227 B2 53 54 emitted illumination. Enzymatic labels are typically detected Therapeutic and Diagnostic Applications by providing the enzyme with a substrate and detecting the Certain of the above-described binding domains may have reaction product produced by the action of the enzyme on therapeutic utility and may be administered to a subject the substrate, and colorimetric labels are detected by simply having a condition in order to treat the subject for the visualizing the colored label. Other suitable detectable labels condition. The therapeutic utility for a binding domain is were described earlier within the context of the first aspect determined by the target GPCR: G protein complex to which of the disclosure relating to a binding domain. the binding domain binds in that signaling via that GPCR is According to a specifically preferred embodiment, the test linked to the condition. hi certain cases, the GPCR may be compound is an antibody or any fragment derived thereof, as activated in the condition by binding to a ligand. In other described above, including a nanobody. For example, and 10 embodiments, the GPCR may be mutated to make it con without the purpose of being limitative, test compounds may stitutively active, for example. A Subject binding domain be antibodies (as defined herein in its broadest sense) that may be employed for the treatment of a GPCR-mediated have been raised against a GPCR:G protein complex stabi condition Such as Schizophrenia, migraine headache, reflux, lized by a binding domain according to the invention. asthma, bronchospasm, prostatic hypertrophy, ulcers, epi Methods for raising antibodies in vivo are known in the art. 15 lepsy, angina, allergy, rhinitis, cancer, e.g., prostate cancer, Preferably, immunization of an animal will be done in a glaucoma and stroke. Further exemplary GPCR-related con similar way as described herein before. The disclosure also ditions at the On-line Mendelian Inheritance in Man data relates to methods for selecting antibodies specifically bind base found at the world wide website of the NCBI. So, a ing to a conformationally stabilized GPCR:G protein com particular embodiment of the present disclosure also envis plex, involving the screening of expression libraries encod ages the binding domain hereof, or a pharmaceutical com ing immunoglobulin genes, or portions thereof, expressed in position comprising the binding domain, for use in the bacteria, yeast, filamentous phages, ribosomes or ribosomal treatment of a GPCR-related disease or disorder. It will be subunits or other display systems on the GPCR:G protein appreciated that the therapeutic utility will also depend on complex. the particular conformational epitope of the GPCR: G protein A particular aspect of the present disclosure relates to a 25 complex against which the binding domain is directed to. Solid Support to which is immobilized a binding domain A Subject binding domain may be mixed with another according to the invention. Such a solid Support (as drug Substance in a fixed pharmaceutical composition or it described hereinbefore) may thus be used in any of the may be administered separately, before, simultaneously with above Screening methods. or after the other drug Substance. In general terms, these Modulating GPCR Receptor Signaling 30 protocols involve administering to an individual Suffering The binding domains of the present disclosure can also be from a GPCR-related disease or disorder an effective used to modulate GPCR signaling, in particular G protein amount of a binding domain that modulates the signaling mediated GPCR signaling, including abolishing G protein activity of a GPCR in the host and treat the individual for the mediated GPCR signaling. The terms “modulating,” “modu disorder. lation,” and “modulated” means an increase or decrease in 35 In some embodiments, where a reduction in activity of a activity of a protein or a protein complex, in particular a certain GPCR is desired, one or more compounds that GPCR:G protein complex. In particular, the binding decrease the activity of the GPCR may be administered, domains of the present disclosure can be allosteric modu whereas when an increase in activity of a certain GPCR is lators or allosteric inhibitors. The terms “allosteric modula desired, one or more compounds that increase the activity of tor” or “allosteric inhibitor” in the context of the present 40 the GPCR activity may be administered. disclosure refer to noncompetitive modulators or inhibitors, A variety of individuals are treatable according to the which exert their effect by binding to a site other than the Subject methods. Generally Such individuals are mammals active site of the receptor, and modulate the activity of the or mammalian, where these terms are used broadly to receptor or render the receptor ineffective in terms of signal describe organisms which are within the class mammalia, transduction. A "positive allosteric modulator (PAM) 45 including the orders carnivore (e.g., dogs and cats), rodentia increases signal transduction, whereas a “negative allosteric (e.g., mice, guinea pigs, and rats), and primates (e.g., modulator (NAM) reduces signal transduction. In particu humans, chimpanzees, and monkeys). In many embodi lar, an allosteric inhibitor may also abolish signal transduc ments, the individuals will be humans. Subject treatment tion. Assays to evaluate the modulation in GPCR signaling methods are typically performed on individuals with such by the binding domains of the disclosure are as described 50 disorders or on individuals with a desire to avoid such hereinbefore. disorders. In that regard, according to a specific embodiment, the In another aspect, the disclosure also relates to a phar binding domains of the present invention, in particular maceutical composition comprising a therapeutically effec immunoglobulin single variable domains, can also be useful tive amount of the binding domains of the disclosure and at for lead identification and the design of peptidomimetics. 55 least one phaii iaceutically acceptable carrier, adjuvant or Using a biologically relevant peptide or protein structure as diluent. a starting point for lead identification represents one of the A “carrier' or “adjuvant,” in particular, a “pharmaceuti most powerful approaches in modern drug discovery. Pep cally acceptable carrier' or “phaiinaceutically acceptable tidomimetics are compounds whose essential elements adjuvant.” is any suitable excipient, diluent, carrier and/or (pharmacophore) mimic a natural peptide or protein in 3D 60 adjuvant which, by themselves, do not induce the production space and which retain the ability to interact with the of antibodies haiinful to the individual receiving the com biological target and produce the same biological effect. position nor do they elicit protection. So, pharmaceutically Peptidomimetics are designed to circumvent some of the acceptable carriers are inherently non-toxic and nonthera problems associated with a natural peptide: for example peutic, and they are known to the person skilled in the art. stability against proteolysis (duration of activity) and poor 65 Suitable carriers or adjuvantia typically comprise one or bioavailability. Certain other properties, such as receptor more of the compounds included in the following non selectivity or potency, often can be substantially improved. exhaustive list: large slowly metabolized macromolecules US 9,695,227 B2 55 56 Such as proteins, polysaccharides, polylactic acids, polygly fractionated coconut oil, oily esters such as esters of glyc colic acids, polymeric amino acids, amino acid copolymers erine, propylene glycol, or ethyl alcohol; preservatives, for and inactive virus particles. Carriers or adjuvants may be, as example methyl or propyl p-hydroxybenzoate or Sorbic acid, a non-limiting example, Ringer's Solution, dextrose solution and if desired conventional flavoring or coloring agents. or Hank's Solution. Non aqueous solutions such as fixed oils 5 Oral formulations also include conventional Sustained and ethyl oleate may also be used. A preferred excipient is release formulations. Such as tablets or granules having an 5% dextrose in saline. The excipient may contain minor enteric coating. amounts of additives Such as Substances that enhance iso Preferably, compositions for inhalation are presented for tonicity and chemical stability, including buffers and pre administration to the respiratory tract as a Snuff oran aerosol servatives. 10 or solution for a nebulizer, or as a microfine powder for The administration of a binding domain as described insufflation, alone or in combination with an inert carrier herein or a pharmaceutically acceptable salt thereof may be Such as lactose. In Such a case the particles of active by way of oral, inhaled or parenteral administration. In compound suitably have diameters of less than 50 microns, particular embodiments the nanobody is delivered through preferably less than 10 microns, for example between 1 and intrathecal or intracerebroventricular administration. The 15 5 microns, such as between 2 and 5 microns. A favored active compound may be administered alone or preferably inhaled dose will be in the range of 0.05 to 2 mg, for formulated as a pharmaceutical composition. An amount example 0.05 to 0.5 mg, 0.1 to 1 mg or 0.5 to 2 mg. effective to treat a certain disease or disorder that express the For parenteral administration, fluid unit dose forms are antigen recognized by the binding domain depends on the prepared containing a compound of the present disclosure usual factors such as the nature and severity of the disorder and a sterile vehicle. The active compound, depending on being treated and the weight of the mammal. However, a unit the vehicle and the concentration, can be either Suspended or dose will normally be in the range of 0.01 to 50 mg, for dissolved. Parenteral solutions are normally prepared by example 0.01 to 10 mg, or 0.05 to 2 mg of binding domain dissolving the compound in a vehicle and filter sterilizing or a pharmaceutically acceptable salt thereof. Unit doses will before filling into a suitable vial or ampoule and sealing. normally be administered once or more than once a day, for 25 Advantageously, adjuvants such as a local anesthetic, pre example 2, 3, or 4 times a day, more usually 1 to 3 times a servatives and buffering agents are also dissolved in the day, Such that the total daily dose is normally in the range of vehicle. To enhance the stability, the composition can be 0.0001 to 1 mg/kg; thus a suitable total daily dose for a 70 frozen after filling into the vial and the water removed under kg adult is 0.01 to 50 mg, for example 0.01 to 10 mg or more vacuum. Parenteral Suspensions are prepared in Substantially usually 0.05 to 10 mg. It is greatly preferred that the 30 the same manner except that the compound is suspended in compound or a pharmaceutically acceptable salt thereof is the vehicle instead of being dissolved and sterilized by administered in the form of a unit-dose composition, such as exposure to ethylene oxide before suspending in the sterile a unit dose oral, parenteral, or inhaled composition. Such vehicle. Advantageously, a Surfactant or wetting agent is compositions are prepared by admixture and are Suitably included in the composition to facilitate uniform distribution adapted for oral, inhaled or parenteral administration, and as 35 of the active compound. Where appropriate, Small amounts Such may be in the form of tablets, capsules, oral liquid of bronchodilators for example sympathomimetic amines preparations, powders, granules, lozenges, reconstitutable Such as isoprenaline, isoetharine, Salbutamol, phenylephrine powders, injectable and infusable solutions or Suspensions and ephedrine; Xanthine derivatives such as theophylline and or Suppositories or aerosols. aminophylline and corticosteroids such as prednisolone and Tablets and capsules for oral administration are usually 40 adrenal stimulants such as ACTH may be included. As is presented in a unit dose, and contain conventional excipients common practice, the compositions will usually be accom Such as binding agents, fillers, diluents, tabletting agents, panied by written or printed directions for use in the medical lubricants, disintegrants, colorants, flavorings, and wetting treatment concerned. agents. The tablets may be coated according to well-known Delivery of binding domains, in particular immunoglobu methods in the art. Suitable fillers for use include cellulose, 45 lin single variable domains, into cells may be performed as mannitol, lactose and other similar agents. Suitable disinte described for peptides, polypeptides and proteins. If the grants include starch, polyvinylpyrrolidone and starch antigen is extracellular or an extracellular domain, the derivatives such as sodium starch glycollate. Suitable lubri binding domain may exert its function by binding to this cants include, for example, magnesium Stearate. Suitable domain, without need for intracellular delivery. The binding pharmaceutically acceptable wetting agents include sodium 50 domains of the present disclosure as described herein may lauryl Sulphate. These Solid oral compositions may be pre target intracellular conformational epitopes of GPCR:G pro pared by conventional methods of blending, filling, tablet teins of interest. To use these binding domains as effective ting or the like. Repeated blending operations may be used and safe therapeutics inside a cell, intracellular delivery may to distribute the active agent throughout those compositions be enhanced by protein transduction or delivery systems employing large quantities of fillers. Such operations are, of 55 know in the art. Protein transduction domains (PTDs) have course, conventional in the art. attracted considerable interest in the drug delivery field for Oral liquid preparations may be in the form of, for their ability to translocate across biological membranes. The example, aqueous or oily Suspensions, solutions, emulsions, PTDs are relatively short (11-35 amino acid) sequences that syrups, or elixirs, or may be presented as a dry product for confer this apparent translocation activity to proteins and reconstitution with water or other suitable vehicle before 60 other macromolecular cargo to which they are conjugated, use. Such liquid preparations may contain conventional complexed or fused (Sawant and Torchilin 2010). The additives Such as Suspending agents, for example Sorbitol, HIV-derived TAT peptide (YGRKKRRQRRR), for syrup, methyl cellulose, gelatin, hydroxyethylcellulose, car example, has been used widely for intracellular delivery of boxymethyl cellulose, aluminium Stearate gel or hydroge various agents ranging from Small molecules to proteins, nated edible fats, emulsifying agents, for example lecithin, 65 peptides, range of pharmaceutical nanocarriers and imaging Sorbitan monooleate, or acacia; non-aqueous vehicles agents. Alternatively, receptor-mediated endocytic mecha (which may include edible oils), for example, almond oil, nisms can also be used for intracellular drug delivery. For US 9,695,227 B2 57 58 example, the transferrin receptor-mediated internalization Eroglu et al. (2003), among others. In certain cases, the pathway is an efficient cellular uptake pathway that has been GPCR and phospholipids may be reconstituted at high exploited for site-specific delivery of drugs and proteins density (e.g., 1 mg receptor per mg of phospholipid). In (Qian et al., 2002). This is achieved either chemically by many cases, a GPCR may be present in the phospholipid conjugation of transferrin with therapeutic drugs or proteins 5 vesicle in both orientations (in the normal orientation, and in or genetically by infusion of therapeutic peptides or proteins the “upside down orientation in which the intracellular into the structure of transferrin. Naturally existing proteins loops are on the outside of the vesicle). Other immunization (such as the iron-binding protein transferrin) are very useful methods with a GPCR include, without limitation, the use of in this area of drug targeting since these proteins are complete cells expressing a GPCR and/or a G protein or biodegradable, nontoxic, and non-immunogenic. Moreover, 10 they can achieve site-specific targeting due to the high membranes derived thereof. amounts of their receptors present on the cell surface. Still In a particular embodiment, the animal is immunized with other delivery systems include, without the purpose of being a target complex that is cross-linked with a bifunctional limitative, polymer- and liposome-based delivery systems. cross-linker (see also Example section). Chemical crosslink The efficacy of the binding domains hereof, and of 15 ing can be done using standard techniques that are well compositions comprising the same, can be tested using any known by the skilled person in the art (see, e.g., Heimanson, Suitable in vitro assay, cell-based assay, in vivo assay and/or G. T. (2008) Bioconjugate Techniques, 2nd ed., Elsevier animal model known per se, or any combination thereof, Inc., 1202 pages). depending on the specific disease or disorder involved. Any suitable animal, e.g., a warm-blooded animal, in Screening, Selection, Production of Binding Domains particular a mammal such as a rabbit, mouse, rat, camel, In still another aspect, the disclosure also encompasses a sheep, cow or pig or a bird such as a chicken or turkey, may method of Screening for binding domains directed against be immunized using any of the techniques well known in the and/or specifically binding to a complex comprising a GPCR art Suitable for generating an immune response. and a G protein, comprising the steps of The screening for binding domains specifically binding to a) Providing a plurality of binding domains, and 25 a conformational epitope of a target complex may for b) Screening the plurality of binding domains for a example be performed by Screening a set, collection or binding domain that binds to a complex comprising a library of cells that express the binding domains on their GPCR and a G protein, and Surface (e.g., B-cells obtained from a Suitably immunized c) Isolating the binding domain that binds to the complex. Camelid), by Screening of a (naive or immune) library of In a preferred embodiment of this aspect hereof, binding 30 binding domains, or by screening of a (naive or immune) domains are generated and screened for their specific bind library of nucleic acid sequences that encode amino acid ing to a complex comprising a GPCR and a G protein and sequences of the binding domains, which may all be per optionally a receptor ligand. The binding domains may also formed in a manner known per se, and which method may be generated and Screened for their specific binding to a G optionally further comprise one or more other Suitable steps, protein. As described herein, binding domains can be gen 35 Such as, for example and without limitation, a step of affinity erated in many ways. In the case of immunoglobulin single maturation, a step of expressing the desired amino acid variable domains, such as nanobodies, typically, immuniza sequence, a step of Screening for binding and/or for activity tion of an animal will be done with a target complex against the desired antigen, a step of determining the desired comprising a GPCR bound to a G protein and a receptor amino acid sequence or nucleotide sequence, a step of ligand, as described hereinbefore (e.g., for VH sequences, 40 introducing one or more humanizing Substitutions, a step of as a non-limiting example) and also exemplified further formatting in a suitable multivalent and/or multispecific herein. format, a step of screening for the desired biological and/or For the immunization of an animal with a target complex, physiological properties (i.e., using a suitable assay known the proteins of the target complex (i.e., GPCR and G protein) in the art), and/or any combination of one or more of Such may be produced and purified using conventional methods 45 steps, in any Suitable order. that may employ expressing a recombinant form of the Yet another aspect of the disclosure relates to a kit proteins in a host cell, and purifying the proteins using comprising a binding domain according to the invention. affinity chromatography and/or antibody-based methods. In The kit may further comprise a combination of reagents such particular embodiments, the bactulovirus/Sf-9 system may as buffers, molecular tags, vector constructs, reference be employed for expression, although other expression 50 sample material, as well as a suitable solid Supports, cells, systems (e.g., bacterial, yeast or mammalian cell systems) nucleic acids, and the like. Such a kit may be useful for any may also be used. Exemplary methods for expressing and of the applications of the present disclosure as described purifying GCPRs are described in, for example, Kobilka herein. For example, the kit may comprise (a library of) test (1995), Eroglu et al. (2002), Chelikani et al. (2006) and the compounds useful for compound screening applications. book “Identification and Expression of G Protein-Coupled 55 Finally, a last aspect of the disclosure is the use of any Receptors' (Kevin R. Lynch (Ed.), 1998), among many binding domain according to the disclosure to isolate amino others. A functional GPCR:G protein complex may also be acid sequences that are responsible for specific binding to a reconstituted by using purified receptor (e.g., B2-AR or conformational epitope of a GPCR: G protein complex and MOR) reconstituted into recombinant HDL particles with a to construct artificial binding domains based on the amino stoichiometric excess of the G protein (Gs or Gi) as 60 acid sequences. It will be appreciated that in the binding described in Whorton et al. (2009) for B2-AR:Gs or in domains according to the invention, the framework regions Kuszak et al. (2009) for MOR:Gi. A GPCR may also be and the complementarity-determining regions are known, reconstituted in phospholipid vesicles and loaded with a and the study of derivatives of the binding domain, binding stoichiometric excess of the G protein. Likewise, methods to the same conformational epitope of a GPCR:G protein for reconstituting an active GPCR in phospholipid vesicles 65 complex, will allow deducing the essential amino acids are known, and are described in: Luca et al. (2003), Mansoor involved in binding the conformational epitope. This knowl et al. (2006), Niu et al. (2005), Shimada et al. (2002), and edge can be used to construct a minimal binding domain and US 9,695,227 B2 59 60 to create derivatives thereof, which can routinely be done by (FIG. 5, Panel a) and gel filtration (FIG. 5, Panel c). To techniques known by the skilled in the art. confirm a pure, homogeneous, and dephosphorylated prepa The following examples are intended to promote a further ration, the R:G complex was routinely analyzed by ion understanding of the present invention. While the present exchange chromatography (FIG. 5, Panel d). disclosure is described herein with reference to illustrated embodiments, it should be understood that the disclosure is Example 2 not limited hereto. Those having ordinary skill in the art and access to the teachings herein will recognize additional Generation of Nanobodies Binding to the modifications and embodiments within the scope thereof. Agonist: B2AR:Gs Ternary Complex Therefore, the present disclosure is limited only by the 10 claims attached herein. From negative stain EM imaging (data not shown), we observed that the alpha helical domain of Gos was flexible. EXAMPLES Targeted stabilization of this domain was addressed by generating nanobodies that bind to the agonist-B2AR-GS Example 1 15 ternary complex. Nanobodies are single domain antibodies, Formation and Purification of a Stable derived from heavy chain only antibodies from llamas Agonist-B2AR-Gs Ternary Complex (Muyldermans, 2001). To identify Nanobodies that bind the (agonist loaded) receptor coupled GS-protein, we immu Formation of a stable complex (see FIG. 2) was accom nized two llamas (Llama glama) with the bis(SulfoSuccin plished by mixing Gs heterotrimer at approximately 100 uM imidyl)glutarate (BS2G, Pierce) cross-linked 2AR:Gs: concentration with BI-167107 bound T4L-BAR (or BAR BI167107 ternary complex. Both animals were immunized 365) in molar excess (approximately 130 uM) in 2 ml buffer with 4 bi-weekly shots of 50 to 100 ug. After completing the (10 mM HEPES, pH 7.5, 100 mM NaCl, 0.1% DDM, 1 mM immunization, peripheral blood lymphocytes were isolated EDTA, 3 mM MgCl, 10uM BI-167107) and incubating for 25 from the immunized animals to extract total RNA and 3 hours at room temperature. BI-167107, which was iden prepare cDNA. Total RNA was isolated from about 107 tified from screening and characterizing approximately 50 lymphocytes as described by Chomczynski and Sacchi different fAR agonists, has a dissociation half-time of (1987). First strand cDNA synthesis was prepared using a approximately 30 hours providing higher degree of stabili dN6 primer and the superscript RT according to the manu zation to the active G protein-bound receptor than other full 30 facturers (Invitrogen) instructions. Fragments encoding agonists such as isoproterenol (Rasmussen et al., 2011). To VHH genes were amplified from this cDNA by PCR using maintain the high-affinity nucleotide-free state of the com specific primers as described previously (Conrad et al., plex, apyrase (25 mU/ml, NEB) was added after 90 minto 2001). hydrolyze residual GDP released from GCs upon binding to Using nested PCR, Pstl and BstEH were engineered at the the receptor. GMP resulting from hydrolysis of GDP by 35 start and the end of the VHH open reading frame, respec apyrase has very poor affinity for the G protein in the tively. VHHs were cloned as Pstl-BstEII fragments into the complex. Rebinding of GDP can cause dissociation of the phage display vector pMESy4. For each llama, a separate R:G complex (FIG. 3A). phage display library was constructed harboring the respec The R:G complex in DDM shows significant dissociation tive Nanobody repertoire as a geneIII fusion (Domanska et after 48 hours at 4°C. (FIG. 4, Panel a). We screened and 40 al., 2011). R:G complex specific nanobodies were enriched characterized over 50 amphiphiles (data not shown) and by two rounds of biopanning on i) the B2AR:Gs: BI167107 identified MNG-3 (NG-310, Affymetrix-Anatrace; Chae et ternary complex embedded in Apoll biotinylated high-den al., 2011) and its closely related analogs as detergents that sity lipoprotein particles (rhDL, Whorton et al., 2007) or ii) substantially stabilize the complex (FIG. 4, Panels a and b). on the BS2G cross-linked B2AR:Gs:BI167107 ternary com The complex was exchanged into MNG-3 by adding the R:G 45 plex. For the first biopanning strategy, biotinylated rhDL mixture (2 ml) to 8 ml buffer (20 mM HEPES, pH 7.5, 100 particles containing the 2AR:Gs:BI167107 ternary com mM NaCl, 10 uM BI-167107) containing 1% MNG-3 for 1 plex were immobilized on a neutravidin coated Maxisorp hour at room temperature. plate (Nunc) at 1 g/well in 20 mM Hepes (pH 8.0), 100 mM At this stage the mixture contains the R:G complex, NaCl, 1 mM EDTA, 100 uMTCEP, and 100 nM BI167107. non-functional Gs, and an excess of BAR. To separate 50 For the second biopanning strategy the BS2G cross-linked functional R:G complex from non-functional Gs, and to B2AR:Gs: BI167107ternary complex was solid phase coated complete the detergent exchange, the R:G complex was on a Maxisorp plate at 1 Lug?well. For each round of immobilized on M1 Flag resin and washed in buffer (20 mM biopanning, 10' phage was added to immobilized antigens HEPES, pH 7.5, 100 mM NaCl, 10 uM BI-167107, and 3 and incubated for one to two hours. Next, non-bound phage mM CaCl) containing 0.2% MNG-3. To prevent cysteine 55 was removed from the antigen containing wells and the bridge-mediated aggregation of R:G complexes, 100 LM wells were washed 14 times with 20 mM HEPES, 100 mM TCEP was added to the eluted protein prior to concentrating NaCl, pH8 and finally incubated for 10 minutes with 200 uL it with a 50 kDa MWCO Millipore concentrator. The final of 20 mM Hepes (pH 8.0), 100 mM NaCl, 1 mM EDTA, 100 size exclusion chromatography procedure to separate excess pM TCEP and 100 nM BI167107 to remove aspecific free receptor from the R: G complex (FIG. 5, Panel b) was 60 phages. To elute complex specific phage, the wells were performed on a Superdex 200 10/300 GL column (GE treated with trypsin, phage was recovered and used to infect Healthcare) equilibrated with buffer containing 0.02% exponentially growing TG1 cells (OD600+0.5). MNG-3, 10 mM HEPES pH 7.5, 100 mM NaCl, 10 uM From each enriched library, 48 colonies were randomly BI-167107, and 100 uMTCEP. Peak fractions were pooled picked and grown in 1 ml 2XTY containing amplicillin and (FIG. 5, Panel b) and concentrated to approximately 90 mg 65 glucose. Cultures were induced with IPTG to induce the ml with a 100 kDa MWCO Viva-spin concentrator and expression of the nanobodies and periplasmatic extracts analyzed by SDS-PAGE/Coomassie brilliant blue staining containing a partially purified nanobody were prepared. US 9,695,227 B2 61 62 Nanobodies contained in these periplasmic extracts were to solubilize and purify these proteins (Example 1). It was analyzed for binding to the agonist: B2AR:GS ternary com found that a relatively stable BAR:Gs complex could be plex by ELISA. prepared by mixing purified GDP-Gs (approximately 100 Nanobodies enriched on biotinylated rHDL particles con uM final concentration) with a molar excess of purified taining the B2AR:Gs: BI167107 ternary complex were ana BAR bound to a high affinity agonist (BI167107: Rasmus lyzed by comparative ELISA on the same complex immo Sen et al., 2011) in dodecylmaltoside solution. Apyrase, a bilized on neutravidin coated Maxisorb plates versus empty non-selective purine pyrophosphatase, was added to hydro rHDL particles. Nanobodies enriched on solid phase coated lyze GDP released from Gs on forming a complex with the BS2G cross-linked B2AR:Gs:BI167107 ternary complex fAR. The complex was subsequently purified by sequential were analyzed by comparative ELISA on the same solid 10 antibody affinity chromatography and size exclusion chro phase coated complex versus non-coated wells. From colo matography. The stability of the complex was enhanced by nies that scored positive in comparative ELISA, single exchanging it into a recently developed maltose neopentyl clones were prepared, DNA was extracted and the sequences glycol detergent (NG-310, Anatrace) (Chae et al., 2010). of the encoded nanobody genes were analyzed using routine This complex could be incubated at room temperature for 24 methods (amino acid sequences shown in Tables 2 and 3). 15 hours without any noticeable degradation; however, initial For Nb35, Nb36 and Nb37, binding to the B2AR:Gs: efforts to crystallize the complex using sparse matrix screens BI167107 ternary complex was further confirmed by ana in detergent micelles, bicelles and lipidic cubic phase (LCP) lytical gel filtration (FIGS. 3D, 3E, 3F, 3G). failed. To further assess the quality of the complex, the protein Example 3 was analyzed by single particle electron microscopy (EM). The results confirmed that the complex was monodisperse Nb35, Nb36 and Nb37 Bind to Gs and Prevent (data not shown), but revealed other possible bottlenecks for Dissociation of the Complex by GTPYS obtaining diffraction of quality crystals. First, the detergent used to stabilize the complex formed a large micelle, leaving To determine if the Nanobodies raised against the B2AR: 25 little polar surface on the extracellular side of the BAR:Gs Gs:BI167107 ternary complex (Table 2) bind to the receptor complex for the formation of crystal lattice contacts. There or to Gs, we next monitored binding of these nanobodies in fore, we replaced the unstructured amino terminus of the ELISA on purified receptor alone. All nanobodies from RAR with T4 lysozyme (T4L). We previously used T4L to Table 2 scored negative in solid phase coated (Maxisorb, facilitate crystallogenesis of the inactive f3AR by inserting Nunc) agonist-bound B2AR-356 reconstituted at high den 30 T4L between the cytoplasmic ends of transmembrane seg sity into phospholipid vesicles (Rasmussen et al., 2011). ments (TMs) 5 and 6 (Rosenbaum et al., 2007). This fusion Nb80, a B2AR specific nanobody (Rasmussen et al., 2011) protein (T4L-BAR) exhibited normal ligand binding and Gs scored positive in this ELISA. None of the B2AR:Gs: coupling properties. Crystallization trials were carried out in BI167107 binders described in Table 2 bind the reconstituted LCP using a modified monolein (7.7 MAG, provided by receptor alone, indicating that they bind epitopes contained 35 Martin Caffrey) designed to accommodate the large hydro on Gs. Size exclusion chromatography shows that Nb35 and philic component of the T4L-BAR:Gs complex (Misquitta Nb37 bind separate epitopes on the Gs heterotrimer to form et al., 2004). Although we were able to obtain small crystals a R:G:Nb35:Nb37 complex (FIG. 3D). Similarly, Nb36 and that diffracted to 7 A, we were unable to improve their Nb37 bind separate epitopes on the Gs heterotrimer to form quality through the use of additives and other modifications. a R:G:Nb36:Nb37 complex (FIG. 3E). 40 Another possible problem for crystallogenesis revealed GDP, GTP and non-hydrolyzable GTP analogs disrupt the by single particle EM analysis was increased variability in B2AR:Gs complex (FIG. 3A), causing dissociation of GPCR the positioning of the C-helical component of the GCS G protein complexes in vitro and in vivo. The mutual effects subunit. Gois consists of two domains, the ras-like GTPase of Nbs and the non-hydrolyzable GTP analog GTPYS on the domain (GOsRas), which interacts with the BAR and the integrity of the agonist: B2AR:GS ternary complex was ana 45 G|B subunit, and the C-helical domain (GCSAH) (Sprang et lyzed by analytical size exclusion chromatography in the al., 1997). The interface of the two GC.s subdomains forms presence and absence of GTPyS. It was found that nano the nucleotide-binding pocket (FIG. 1), and EM 2D averages bodies 35, 36 and 37 protect the B2AR:Gs: BI167107 com and 3D reconstructions Suggest that in the absence of plex from dissociation by GTPYS (FIGS. 3D, 3E and 3G). guanine nucleotide, GCSAH has a variable position relative 50 to the complex of T4L-BAR-GCSRAS-G BY (FIG. 1, Panel Example 4 b). In an effort to further facilitate crystallogenesis of the Nanobody-Aided Crystallization of the AR-Gs complex, we tried co-crystallization of the complex with Complex nanobody 35. Nb35 protects the complex from dissociation 55 by GTPYS, suggestive of a stabilizing Gs:Nb interaction G protein-coupled receptors (GPCRs) are responsible for (FIG. 3A). BI-167107 bound T4L-B2AR:Gs complex and the majority of cellular responses to hormones and neu Nb35 were mixed in 1:1.2 molar ratio (see FIGS. 6 and 7). rotransmitters as well as the senses of sight, olfaction and The small molar excess of Nb35 was verified by analytical taste. The paradigm of GPCR signaling is the activation of gel filtration (FIG. 7, Panel b). The mixture incubated for 1 a heterotrimeric GTP binding protein (G protein) by an 60 hour at ROOM TEMPERATURE prior to mixing with 7.7 agonist-occupied receptor. In an effort to understand the MAG (provided by Martin Caffrey) containing 10% choles structural basis for GPCR signaling, we crystallized the terol (C8667, Sigma) in 1:1 protein to lipid ratio (w/w) using fAR-Gs complex to solve its structure by X-ray crystal the twin-syringe mixing method reported previously (Caf lography. frey 2009). Concentration of R:G:Nb complex in 7.7 MAG One challenge for crystallogenesis was to prepare a stable 65 was approximately 25 mg/ml. The protein:lipid mixture was BAR:Gs complex in detergent Solution. The (BAR and GS delivered through a LCP dispensing robot (Gryphon, Art couple efficiently in lipid bilayers, but not in detergents used Robbins Instruments) in 40 nil drops to either 24-well or US 9,695,227 B2 63 64 96-well glass sandwich plates and overlaid en-bloc with 0.8 the second intracellular loop (ICL2), which forms an ul precipitant solution. Multiple crystallization leads were extended loop in the inactive f3AR structure and an O.-helix initially identified using in-house screens partly based on in the BAR:Gs complex. This helix is also observed in the reagents from the StockOptions Salt kit (Hampton |BAR-Nb80 structure (FIG. 12, Panel b); however, it may Research). Crystals for data collection were grown in 18 to not be a feature that is unique to the active state, since it is 22% PEG 400, 100 mM MES pH 6.5 (FIG. 1, Panel c), 350 also observed in the inactive structure of the highly homolo to 450 mM potassium nitrate, 10 mM foscarnet (FIG. 3B), gous avian BAR (Warne et al., 2008). 1 mM TCEP and 10 uM BI167107. Crystals reached full The quality of the electron density maps for the AR is size within 3-4 days at 20°C. (FIG. 8) and were picked from highest at this faR-GCSRas interface, and much weaker a sponge-like mesophase and flash-frozen without additional 10 for the extracellular half, possibly due to the lack of crystal cryo-protectant in liquid nitrogen. lattice contacts with the extracellular surface (FIG. 9, Panel a). As a result, we cannot confidently model the high-affinity Example 5 agonist (BI-167107) in the ligand-binding pocket. However, the overall structure of the BAR in the T4L-BAR:Gs Nb35 Facilitates Crystal Formation of the R:G 15 complex is very similar to our recent active-state structure of Complex fAR stabilized by a G protein mimetic nanobody (Nb80). These structures deviate primarily at the cytoplasmic ends of The BI-167107 bound T4L-B2AR:Gs:Nb35 complex TMs 5 and 6 (FIG. 12, Panelb), possibly due to the presence crystallized in space group P2, with a single complex in of T4L that replaces ICL3 in the BAR-Nb80 structure. each asymmetric unit. FIG. 9, Panel a, shows the crystallo Nonetheless, the BAR-Nb80 complex exhibits the same graphic packing interactions. FIG. 9, Panel b, shows the high affinity for the agonist isoproterenol as does the faR: structure of the complete complex including T4L and Nb35, Gs complex (Rasmussen et al., 2011), consistent with high and FIG. 9, Panel c shows the BAR:Gs complex alone. structural homology around the ligand binding pocket. The BI-167107 bound T4L-R2AR:Gs:Nb35 complexes are electron density maps for the BAR-Nb80 crystals provide a arrayed in alternating aqueous and lipidic layers with lattice 25 more reliable view of the conformational rearrangements of contacts formed almost exclusively between soluble com amino acids around the ligand-binding pocket and between ponents of the complex, leaving receptor molecules Sus the ligand-binding pocket and the Gs-coupling interface pended between G protein layers and widely separated from (Rasmussen et al., 2011). one another in the plane of the membrane. Extensive lattice FIG. 12, Panel c, shows the position of the highly con contacts are formed among all the soluble proteins, likely 30 served sequence motifs including D/ERY and NPXXY in the accounting for the strong overall diffraction and remarkably BAR:Gs complex compared with the AR-Nb80 complex clear electron density for the G protein. (see also FIG. 13). These conserved sequences have been Nb35 and T4L facilitated crystal formation of the proposed to be important for activation or for maintaining BI-167107 bound T4L-B2AR:Gs:Nb35 complex. Nb35 the receptor in the inactive state (Hofmann et al., 2009). The binds a conformational epitope on Gs and packs at the 35 positions of these amino acids are essentially identical in interface of Gf and GC. subunits with complementarity these two structures demonstrating that Nb80 is a very good determining region (CDR, defined according to IMTG num G protein surrogate. Only Arg131 differs between these two bering; Lefranc, 2003) 1 interacting primarily with G|B (FIG. structures. In the BAR-Nb80 structure Arg131 interacts 10A) and a long CDR3 loop interacting with both G|B and with Nb80, whereas in the AR:Gs structure Arg131 packs Go. subunits (FIG. 10B). Some framework regions of Nb35 40 against Tyr391 of Gos (FIG. 13). also interact with GC. from the same complex (FIG. 10C). The active state of the BAR is stabilized by extensive Other framework regions from one complex interact with interactions with (GCSFRas) (FIG. 14). There are no direct GC. subunits from two adjacent complexes (FIGS. 11A and interactions with GB or GY subunits. The total buried surface 11B), contributing considerably to the crystal contacts of the BAR-GosRas interface is 2576 A (1300 A for within the crystal lattice. T4L forms relatively sparse inter 45 Goskas and 1276 A for the RAR). This interface is formed actions with the amino terminus of the receptor, but packs by ICL2, TM5 and TM6 of the BAR, and by C.5-helix, the against the amino terminus of the GB subunit of one com CN-B1 junction, the top of the B3-strand, and the C4-helix plex, the carboxyl terminus of the GY subunit of another of GCSRas (see Table 6 for specific interactions). The BAR complex, and the GCSubunit of yet another complex. sequences involved in this interaction have been shown to 50 play a role in G protein coupling; however, there is no clear Example 6 consensus sequence for Gs-coupling specificity when these segments are aligned with other GPCRs. Perhaps this is not Structure of the Active-State BAR Surprising considering that the BAR also couples to Gi and that many GPCRs couple to more than one G protein The BAR:Gs structure provides the first high-resolution 55 isoform. The structural basis for G protein coupling speci insight into the mechanism of signal transduction across the ficity must therefore involve more subtle features of the plasma membrane by a GPCR, and the structural basis for secondary and tertiary structure. Nevertheless, a noteworthy the functional properties of the ternary complex. FIG. 12, interaction involves Phel39, which is located at the begin Panel a, compares the structures of the agonist-bound recep ning of the ICL2 helix and sits in a hydrophobic pocket tor in the BAR:Gs complex and the inactive carazolol 60 formed by Gos His41 at the beginning of the 1-strand, bound AR. The largest difference between the inactive Val213 at the start of the B3-strand and Phe376, Arg,380 and and active structures is a 14 A outward movement of TM6 Ile383 in the C.5-helix (FIG. 14, Panel c). The BAR mutant when measured at the Co. carbon of E268. There is a smaller F139A displays severely impaired coupling to Gs (Moro et outward movement and extension of the cytoplasmic end of al., 1993). The residue corresponding to Phe 139 is a Phe or the TM5 helix by 7 residues. A stretch of 26 amino acids in 65 Leu on almost all Gs coupled receptors, but is more variable the third intracellular loop (ICL3) is disordered. Another in GPCRs known to couple to other G proteins. Of interest, notable difference between inactive and active structures is the ICL2 helix is stabilized by an interaction between US 9,695,227 B2 65 66 Asp130 of the conserved DRY sequence and Tyr141 in the be noted that Nb35 binds at the interface between GosRas middle of the ICL2 helix (FIG. 14, Panel c). Tyr141 has been and GP (FIG. 2, Panel b). Therefore, we cannot exclude the shown to be a substrate for the insulin receptor tyrosine possibility that Nb35 may influence the relative orientation kinase (Baltensperger et al., 1996); however, the functional of the GosRas-G BY interface in the crystal structure. How significance of this phosphorylation is currently unknown. ever, single particle EM studies provide evidence that Nb35 does not disrupt interactions between GCSAH and GosRas Example 7 (data not shown). Structure of Activated Gs Example 8 10 The most Surprising observation in the BAR:Gs complex Nb35 and Nb37 Bind Different Epitopes on Gs and is the large displacement of the GCSAH relative to GCsPRas Inhibit Nucleotide Binding (an approximately 180° rotation about the junction between the domains) (FIG. 15, Panel a). In the crystal structure of To investigate the effect of nanobodies (Nb35 and Nb37) GOs, the nucleotide-binding pocket is formed by the inter 15 on Gs alone, nanobodies were added together with bodipy face between GosRas and Got SAH. Guanine nucleotide GTPYS-FL and various Gs protein preparations in 20 mM binding stabilizes the interaction between these two Tris-HCl, Ph 8.0, 3 mM MgCl, 1 mM DTT in a final domains. The loss of this stabilizing effect of guanine volume of 200 uL. Samples containing a heterotrimeric Gs nucleotide binding is consistent with the high flexibility protein also included 0.1% DDM. Bodipy-GTPYS-FL is a observed for GCSAH in single particle EM analysis of the stable fluorescent GTP analog (ws470 nm, was515 nm). detergent solubilized complex (data not shown). It is also in Its fluorescence intensity increases upon G protein binding agreement with the increase in deuterium exchange at the and Bodipy-GTPYS-FL can therefore be used for real-time interface between these two domains upon formation of the measurements of nucleotide binding to G proteins (McEwen complex (data not shown). Recently Hamm, Hubbell and et al., 2001). Fluorescence was measured in a 96-well colleagues, using double electron electron resonance 25 microtiter plate format on a M5 fluorescence plate reader (DEER) spectroscopy, documented large (up to 20 A) (Molecular Precision). changes in distance between nitroxide probes positioned on In a first experiment (FIG. 16), increasing amounts of the Ras and C.-helical domains of Gi upon formation of a Nb37 were incubated with 1 uM purified GOS and 100 nM complex with light-activated rhodopsin (Van Eps 2011). Bodipy-GTPYS-FL and the fluorescence increase was mea Therefore, it is perhaps not surprising that GCSAH is dis 30 sured on a short time scale (300 seconds) to minimize the placed relative to GCSERas; however, its location in this accumulation of the hydrolysis product bodipy-phosphate crystal structure likely reflects the influence of crystal pack (Jameson et al., 2005). The Gos subunit of the heterotrim ing interactions rather than a physiological conformation. eric Gs protein was purified as described previously (Suna The conformational links between the AR and the hara et al., 1997). From this experiment it appears that Nb37 nucleotide-binding pocket primarily involve the amino and 35 blocks GTPYS binding to GSO. alone in a dose dependent carboxyl terminal helices of Gos (FIG. 14). FIG. 15, Panel manner. These results also indicate that the binding epitope b, focuses on the region of GCSRAS that undergoes the of Nb37 is confined to the GSC. subunit of the heterotrimeric largest conformational change when comparing the structure Gs protein. In a similar experiment (FIG. 17), increasing of GCSRAS from the Gs-fAR complex with that from the amounts of Nb35 were incubated with 1 uM purified GOS Gos-GTPYS complex (Sunahara et al., 1997). The largest 40 and 100 nM Bodipy-GTPYS-FL. Consistent with the obser difference is observed for the C.5 helix, which is displaced 6 vation that Nb35 binds an epitope composed of elements of A towards the receptor and rotated as the carboxyl terminal Gosk AS and G? (see Example 7), Nb35 has no effect on end projects into transmembrane core of the BAR. Associ GTPYS binding to the GCS subunit alone. ated with this movement, the B6-C5 loop, which interacts In another experiment (FIG. 18), increasing amounts of with the guanine ring in the Gos-GTPYS structure, is dis 45 Nb35 were incubated with 1 uM of the purified Gs O?3y placed outward, away from the nucleotide-binding pocket heterotrimer and 100 nM Bodipy-GTPYS-FL. This experi (FIG. 15, Panels b-d). The movement of C5 helix is also ment indicates that Nb35 blocks GTPYS binding to the free associated with changes in interactions between this helix GSC?y heterotrimer in a dose dependent manner. and the B6 sheet, the CN-B1 loop, and the al helix. The B1 strand forms another link between the BAR and the nucleo 50 Example 9 tide-binding pocket. The C-terminal end of this strand changes conformation around Gly47, and there are further Nb35 Stabilizes Other Agonist-GPCR-Gs changes in the B1-C1 loop (P-loop) that coordinates the Complexes Y-phosphate in the GTP-bound form (FIG. 15, Panels b-d). The observations in the crystal structure are in agreement 55 The Gs alpha subunit (or Gs protein) is a heterotrimeric G with deuterium exchange experiments where there is protein subunit that activates the cAMP-dependent pathway enhanced deuterium exchange in the 31 sheet and the amino by activating adenylate cyclase. The G protein-coupled terminal end of the O5 helix upon formation of the nucleo receptors that couple to Gs include: 5-HT receptors types tide-free BAR:Gs complex (data not shown). 5-HT4 and 5-HT7, ACTH receptor, Adenosine receptor The structure of a Gs heterotrimer has not been deter 60 types A2a and A2b, Arginine vasopressin receptor 2, mined, so it is not possible to directly compare the GCS-GBY B-adrenergic receptors types f1, f2 and B3, Calcitonin interface before and after formation of the BAR:Gs com receptor, Calcitonin gene-related peptide receptor, Corti plex. Based on the structure of the GDP bound Gi het cotropin-releasing hormone receptor, Dopamine receptors erotrimer (Wall et al., 1995), we do not observe large D1-like family (D1 and D5), FSH-receptor, Gastric inhibi changes in interactions between GCSRAS and GBY upon 65 tory polypeptide receptor, Glucagon receptor, Glucagon-like formation of the complex with BAR. This is also consistent peptide 1 receptor (GLP1-R), Histamine H2 receptor, with deuterium exchange studies (data not shown). It should Luteinizing hormone/choriogonadotropin receptor, Melano US 9,695,227 B2 67 68 cortin receptor, Parathyroid hormone receptor 1, Prostaglan the interface of GCs and G|By subunits. An example of a din receptor types D2 and 12, Secretin receptor, Thyrotropin screening assay to identify ligands that selectively interact receptor, amongst others. with the low abundant active state conformer of B2AR can To determine if Nanobodies that bind to Gs in the B2AR: be a radioligand assay using Nb35 to shift f2AR population Gs:BI167107 also stabilize other GPCR:Gs:agonist com more towards its active state. Such radioligand assay can be plexes, we prepared a complex of the Arginine vasopressin executed similarly as the one described by Seifert and receptor 2 (Accession number P30518; V2R HUMAN) in co-workers (1998) with minor modifications. We describe complex with Gs, Nb35 and Arginine vasopressin (AVP: here an assay involving B2AR as the target of choice NT4LV2R:Gs) and demonstrated the stability of this com replacing B2AR by any other GPCR interacting with the plex in SEC. Arginine vasopressin (AVP), also known as 10 GCS Subunit, allow implementation of similar screening vasopressin, argipressin or antidiuretic hormone, is a natural ligand that activates Arginine vasopressin receptor 2. methods to identify agonistic ligands against that particular Formation of a stable complex (FIG. 19, Panel a) was GPCR. A small molecule (compound MW typically between accomplished by mixing His tagged Gs heterotrimer at 250 and 1000 Da) or even a fragment based (compound MW approximately 90 uM concentration with AVP bound 15 typically <250 Da) library can be screened to identify NT4LV2R (90 uM) and NB35 (100 uM) in 0.1 ml buffer (10 candidate agonists. The stabilization of the non-prominent mM HEPES, pH 7.5, 100 mM NaCl, 0.1% DDM, 1 mM conformer will increase the performance of the fragment EDTA, 3 mM MgCl, 10uMAVP) and incubated for 2 hours library Screens considerably, especially because the initial at room temperature. Next, the AVP:NT4LV2R:Gs complex hits in fragment based drug screening typically have a low was purified in two Successive affinity purification steps. The potency/affinity. Nb35 will selectively increase the affinity purification was monitored by SDS-PAGE (FIG. 19). First, of those compounds that are specific for the selective, the complex was applied on a Ni-NTA column after adding druggable conformer thus having a profound effect on the 300 all of 1% MNG in 10 mM HEPES, pH 7.5, 100 mM identification of de novo fragments. NaCl buffer onto the reaction mix. Following extensive An appropriate amount (typically 10 ug) of human B2AR washing with buffer, the complex was eluted in 0.2% MNG, 25 homogenized membrane extracts from HEK293T cells con 10 mM HEPES, pH 7.5, 100 mM. NaCl AVP 10 uM with 200 taining the membrane anchored G protein subunits are mM imidazole. Next the complex was applied on a FLAG incubated in parallel with Nb35 or a non-related Nanobody tag affinity column, washed extensively in the same buffer (which does not stabilize the active GPCR conformation) for containing 0.01% MNG and eluted with the FLAG-peptide. 1 hour at 30° C. in incubation buffer (50 mM Hepes pH 7.4, This procedure was monitored by SDS-PAGE (FIG. 19, 30 1 mM CaCl2, 5 mM MgCl2, 100 mM NaCl and 0.5% w/v. Panel b) and shows that a complex containing NT4LVTR, BSA). Nanobodies are exogenously supplied in large molar GO.S, GB, GY and Nb35 can be purified accordingly. This excess (e.g., 21 M) versus the adrenergic receptor. Subse complex was further purified by SEC on a superdex200 quently, the Nanobody-bound membranes are added to column in 10 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% 96-well plates containing library compounds and 2 nM of MNG, 1 mM EDTA, 3 mM MgCl, 1 uM AVP). This 35 procedure was monitored by SDS-PAGE (FIG. 19, Panel c) 3H-dihydroalprenolol (DHA) antagonistic radioligand. The and shows that a monodisperse complex containing total volume per well is adjusted with incubation buffer to NT4LVTR, GCS, G|B, Gy and Nb35 can be purified accord 100 ul and the reaction mixture is further incubated for ingly. another hour at 30° C. Subsequently, membrane-bound To confirm the stability of the AVP:NT4LV2R:Gs com 40 radioligand is harvested using GF/C glass fiber 96-well filter plex, we incubated the purified sample for 24 hours on ice plate (Perkin Elmer) presoaked in 0.3% polyethylenimine. and reapplied it to SEC to confirm its monodisperse char Filter plates are washed with ice-cold wash buffer (50 mM acter and its MW (FIG. 20). As expected, an excess amount Tris-HCl pH 7.4), and dried for 30 minutes at 50° C. After of the antagonist SR 121463 (10 disrupts the AVP: adding 25 ul of scintillation fluid (MICROSCINTTM-O, NT4LV2R:Gs complex. 45 Perkin Elmer), radioactivity (cpm) is measured in a Wallac MicroBeta Trillux scintillation counter. Those library com Example 10 pounds that significantly decrease the cpm in presence of Nb35 while not using the non-related Nanobody are con Improved Screening for Agonists or Positive sidered agonistic ligands. Candidate agonistic hits identified Allosteric Modulators. Using Nanobodies 50 via the primary library Screening will be rescreened in a dose Stabilizing GPCR:G Protein Complexes response manner. IC50 values of the % radioligand displace A Nanobody that selectively stabilizes a non-prominent ment curves for each candidate agonist in presence of Nb35 conformer of GPCRs will allow more efficient screening for and the non-related Nanobody will be calculated using the ligands that selectively interact with this particular low 55 Graphpad Prism software. To prove effective agonism of the abundancy conformer. Besides, conformer selective Nano identified de novo compounds, the dose dependent effect of bodies could also be used to unmask allosteric or hidden these compounds in a cellular B2AR signaling assay will be druggable sites or inversely mask undesired binding pockets evaluated. One example of Such assay relies on the detection for drug screening. In case the particular conformer is an of secondary messenger molecules such as cAMP after GCS active state, the identified ligands will have a high probabil 60 mediated signaling (e.g., HitHunter cAMP assay technology, ity to behave as agonists, supported by in silico docking DiscoverX). Instead of using membrane extracts and exog experiments described by Costanzi & Vilar (2011). Indeed, enously applied Nb35, the radioligand assay could be per their results indicate that activated structures favor identifi formed on a B2AR expressing cell line co-transfected with cation of agonists over antagonists, whereas inactive struc Nb35 as an intrabody (or derived membranes) in order to tures favor identification of receptor blockers over agonists. 65 shift the B2AR population to its active state. Alternatively, Evidence is provided that Nb35 stabilizes the complex recombinant G protein and B2AR can be used to stabilize via between the activated 2AR and the G protein by binding Nb35 the active state of the B2AR. US 9,695,227 B2 69 70 Material and Methods to the Examples mM NaCl, 1.25 mM MgCl, 6.25 mM imidazole, 0.2% Expression and Purification of B2AR, Gs Heterotrimer, and Anzergent 3-12, 1xPTT, 1XLS, 5 mM B-ME, 10 uMGDP), Nanobody-35 taking care to add the buffer slowly to avoid dropping the An N-terminally fused T4 lysozyme-f2AR construct cholate concentration below its critical micelle concentra truncated in position 365 (T4L-B2AR, described in detail tion too quickly. 3 ml of Ni-NTA resin (Qiagen) pre below) was expressed in Sf-9 insect cell cultures infected equilibrated in Ni-NTA wash buffer 1 (20 mM HEPES pH with recombinant baculovirus (BestBac, Expression Sys 8.0, 300 mM. NaCl, 2 mM MgCl, 5 mM imidazole, 0.2% tems), and solubilized in n-Dodecyl-?3-D-maltoside (DDM) Cholate, 0.15% Anzergent 3-12, 1xPTT, 1XLS, 5 mM B-ME, according to methods described previously (Kobilka et al., 10 uM GDP) per liter culture volume was added and the 1995) (see FIG. 6 for purification overview). A B2AR 10 sample was stirred on ice for 20 minutes. The resin was construct truncated after residue 365 (B2AR-365: SEQ ID collected into a gravity column and washed with 4.x column NO:55) was used for the majority of the analytical experi volumes of Ni-NTA wash buffer 1, Ni-NTA wash buffer 2 ments and for deuterium exchange experiments. M1 Flag (20 mM HEPES pH 8.0, 50 mM NaCl, 1 mM MgCl, 10 affinity chromatography (Sigma) served as the initial puri mMimidazole, 0.15% Anzergent 3-12, 0.1% DDM, 1xPTT, fication step followed by alprenolol-Sepharose chromatog 15 1xLS, 5 mM B-ME, 10 uM GDP), and Ni-NTA wash buffer raphy for selection of functional receptor. A subsequent M1 3 (20 mM HEPES pH 8.0, 50 mM. NaCl, 1 mM MgCl, 5 Flag affinity chromatography step was used to exchange mMimidazole, 0.1% DDM, 1xPTT, 1XLS, 5 mM B-ME, 10 receptor-bound alprenolol for high-affinity agonist uM GDP). The protein was eluted with Ni-NTA elution BI-167107. The agonist-bound receptor was eluted, dialyzed buffer (20 mM HEPES pH 8.0, 40 mM. NaCl, 1 mM MgCl, against buffer (20 mM HEPES pH7.5, 100 mM NaCl, 0.1% 200 mM imidazole, 0.1% DDM, 1XPTT, 1XLS, 5 mM DDM and 10 uM BI-167107), treated with lambda phos B-ME, 10 uM GDP). Protein-containing fractions were phatase (New England Biolabs), and concentrated to pooled and MnCl2 was added to a final concentration of 100 approximately 50 mg ml with a 50 kDa molecular weight uM. 50 g of purified lambda protein phosphatase (prepared cut off (MWCO) Millipore concentrator. Prior to spin con in house) was added per liter of culture volume and the elute centration the B2AR-365 construct, but not T4L-32AR, was 25 was incubated on ice with stirring for 30 minutes. The elute treated with PNGaseF (New England Biolabs) to remove was passed through a 0.22 um filter and loaded directly onto amino-terminal N-linked glycosylation. The purified recep a Mono O HR 16/10 column (GE Healthcare) equilibrated in tor was routinely analyzed by SDS-PAGE/Coomassie bril MonoO buffer A (20 mM HEPES pH 8.0, 50 mM NaCl, 100 liant blue staining (see FIG. 5, Panel a). uM MgCl, 0.1% DDM, 5 mM 13-ME, 1XPTT). The Bovine GC short, rat Gf fused to a His tag, and rat Gy 30 column was washed with 150 ml buffer A at 5 ml/min and (see Table 5) were expressed in High 5 insects cells (Invit bound proteins were eluted over 350 ml with a linear rogen) grown in Insect Xpress serum-free media (Lonza). gradient up to 28% MonoQ buffer B (same as buffer A Cultures were grown to a density of 1.5 million cells per ml except with 1 M NaCl). Fractions were collected in tubes and then infected with three separate Autographa Californica spotted with enough GDP to make a final concentration of nuclear polyhedrosis virus each containing the gene for one 35 10 uM. The Gs containing fractions were concentrated to 2 of the G protein subunits at a 1:1 multiplicity of infection ml using a stirred ultrafiltration cell with a 10 kDa NMWL (the viruses were a generous gift from Dr. Alfred Gilman). regenerated cellulose membrane (Millipore). The concen After 40-48 hours of incubation the infected cells were trated sample was run on a Superdex 200 prep grade XK harvested by centrifugation and resuspended in 75 ml lysis 16/70 column (GE Healthcare) equilibrated in S200 buffer buffer (50 mM HEPES pH 8.0, 65 mM NaCl, 1.1 mM 40 (20 mM HEPES pH 8.0, 100 mM NaCl, 1.1 mM MgCl, 1 MgCl, 1 mM EDTA, 1xPTT (35 ug/ml phenylmethanesul mM EDTA, 0.012% DDM, 100 uMTCEP. 2 uMGDP). The fonyl fluoride, 32 g/ml tosyl phenylalanyl chloromethyl fractions containing pure Gs were pooled, glycerol was ketone, 32 ug/ml tosyl lysyl chloromethyl ketone), 1xLS added to 10% final concentration, and then the protein was (3.2 g/ml leupeptin and 3.2 Lug/ml Soybean trypsin inhibi concentrated to at least 10 mg/ml using a 30 kDa MWCO tor), 5 mM 13-ME, and 10 uM GDP) per liter of culture 45 regenerated cellulose Amicon centrifugal ultrafiltration volume. The suspension was pressurized with 600 psig N. device. The concentrated sample was then aliquoted, flash for 40 minutes in a nitrogen cavitation bomb (Parr Instru frozen, and stored at -80. A typical yield of final, purified ment Company). After depressurization, the lysate was Gs heterotrimer from 8 liters of cell culture volume was 6 centrifuged to remove nuclei and unlysed cells, and then ng. ultracentrifuged at 180,000xg for 40 minutes. The pelleted 50 Nanobody-35 (Nb35) (SEQ ID NO: 1) was expressed in membranes were resuspended in 30 ml wash buffer (50 mM the periplasm of Escherichia coli strain WK6, extracted, and HEPES pH 8.0, 50 mM NaCl, 100 uM MgCl, 1XPTT, purified by nickel affinity chromatography according to 1xLS, 5 mM 13-ME, 10 uM GDP) per liter culture volume previously described methods (Rasmussen et al., 2011) using a Dounce homogenizer and centrifuged again at followed by ion-exchange chromatography (FIG. 7a) using 180,000xg for 40 minutes. The washed pellet was resus 55 a Mono S 10/100 GL column (GE Healthcare). Selected pended in a minimal volume of wash buffer and flash frozen Nb35 fractions were dialysis against buffer (10 mM HEPES with liquid nitrogen. pH 7.5, 100 mM NaCl) and concentrated to approximately The frozen membranes were thawed and diluted to a total 65 mg ml with a 10 kDa MWCO Millipore concentrator. protein concentration of 5 mg/ml with fresh wash buffer. Protein Engineering Sodium cholate detergent was added to the Suspension at a 60 To increase the probability of obtaining crystals of the final concentration of 1.0%, MgCl, was added to a final R:G complex we set out to increase the polar surface area of concentration of 5 mM, and 0.05 mg of purified protein the receptors extracellular Surface using two strategies. The phosphatase 5 (prepared in house) was added per liter of approach was to replace the flexible and presumably culture volume. The sample was stirred on ice for 40 unstructured N-terminus with the globular protein T4 minutes, and then centrifuged at 180,000xg for 40 minutes 65 lysozyme (T4L) used previously to crystallize and solve the to remove insoluble derbies. The supernatant was diluted carazolol bound receptor (Rosenbaum et al., 2007). The 5-fold with Ni-NTA load buffer (20 mM HEPES pH 8.0,363 construct used here (T4L-f32AR) contained the cleavable US 9,695,227 B2 71 72 signal sequence followed by the M1 Flag epitope refinement and manual adjustments, the structure was (DYKDDDDA: SEQ ID NO:70), the TEV protease recog refined in CNS using the DEN method. Although the reso nition sequence (ENLYFQG: SEQ ID NO:71), bacterio lution of this structure exceeds that for which DEN is phage T4 lysozyme from N2 through Y161 including C54T typically most useful, the presence of several poorly and C97A mutations, and a two residue alanine linker fused 5 resolved regions indicated that the incorporation of addi to the human BAR sequence D29 through G365 (T4L tional information to guide refinement could provide better |B2AR fusion construct defined by SEQ ID NO:69). The results. The DEN reference models used were those indi PNGaseF inaccessible glycosylation site of the BAR at cated above as molecular replacement search models, with N187 was mutated to Glu. M96 and M98 in the first the exception of NB35, which was well ordered and for extracellular loop were each replaced by Thr to increase the 10 which no higher resolution structure is available. Figures otherwise low expression level of T4L-f32AR. The threonine were prepared using PyMOL (The PyMOL Molecular mutations did not affect ligand binding affinity for H-di Graphics System, Version 1.3, Schrödinger, LLC.). Refine hydro-alprenolol, but caused a small, approximately two ment statistics are given in Table 7. fold decrease in affinity for isoproterenol (data not shown). Binding Note that the wild type reference B2AR that is used here is 15 Membranes expressing the 2AR or the BAR-Gspeptide defined by SEQ ID NO:72. fusion were prepared from baculovirus-infected Sf9 cells Microcrystallography Data Collection and Processing. and H-dihydroalprenolol (H-DHA) binding performed as Data collection was performed at the Advanced Photon previously described (Swaminath et al., 2002). For compe Source beamline 23 ID-B. Hundreds of crystals were tition binding, membranes were incubated with H-DHA screened, and a final dataset was compiled using diffraction (1.1 nM final) and increasing concentrations of (-)-isopro wedges of typically 10 degrees from 20 strongly diffracting terenol (ISO) for 1 hour before harvesting onto GF/B filters. crystals. All data reduction was performed using HKL2000 Competition data were fitted to a two-site binding model and (Otwinowski et al., 1997). Although in many cases diffrac ISO high and low Kis and fractions calculated using Graph tion to beyond 3 A was seen in initial frames, radiation Pad prism. damage and anisotropic diffraction resulted in low complete 25 Purification of NT4LV2R ness in higher resolution shells. Analysis of the final dataset The N-terminally fused T4L V2R construct (NT4L-V2R: by the UCLA diffraction anisotropy server (Strong et al., SEQ ID NO:73) was expressed in Sf9 cells using the 2006) indicated that diffraction along the a reciprocal axis baculovirus system (PfastBac). Cells were infected at a was superior to that in other directions. On the basis of an density of 4x10 cells per ml and culture flasks were shaken F/sigF cutoff of 3 along each reciprocal space axis, reflec 30 at 27°C. for 48 hours. After harvesting, cells were lysed by tions were subjected to an anisotropic truncation with reso osmotic shock in a buffer comprised of 10 mM Tris-HCl pH lution limits of 2.9, 3.2, and 3.2 Angstroms along a, b, and 7.5, 1 mM EDTA, 1 M Tolvaptain (Sigma) and 2 mg ml c prior to use in refinement. Due to the low completeness iodoacetamide to block reactive cysteines. Extraction of in high-resolution shells, we report this structure to an NT4L-V2R from Sf9 membranes was done with a Dounce overall resolution of only 3.3 A, although it should be noted 35 homogenizer in a solubilization buffer comprised of 0.5% that some diffraction data to 2.9 A was included during dodecyl maltoside (DDM), 0.3% Cholate, 0.03% cholesterol refinement and map calculation. hemisuccinate (CHS), 20 mM HEPES pH 7.5, 0.5 M NaCl, Structure Solution and Refinement 30% v/v glycerol, 2 mgml iodoacetamide and 1 uM The structure was solved by molecular replacement using Tolvaptan. After centrifugation, nickel-NTA agarose was Phaser (McCoy et al., 2007a, b). The order of the molecular 40 added to the Supernatant, stirred for 2 hours, and then replacement search was found to be critical in solving the washed in batch with 100 g spins for 5 minutes each with a structure. In the order used, search models were: the struc washing buffer of 0.1% DDM, 0.03% Cholate, 0.01% CHS, ture of beta and gamma subunits from the structure of a Gi 20 mM HEPES pH 7.5 and 0.5 M NaCl. The resin was heterotrimeric G protein (PDB ID: 1GP2), Gs alpha ras poured into a glass column and bound receptor was eluted in domain (PDB ID: 1AZT), active-state beta2 adrenergic 45 washing buffer supplemented with 300 mM imidazole. We receptor (PDB ID: 3POG), beta2 binding nanobody (PDB used anti-Flag M1 affinity resin to purify NT4L-V2R further ID: 3POG), T4 lysozyme (PDB ID: 2RH1), Gs alpha helical and to exchange the ligand with the agonist AVP. Ni-NTA domain (PDB ID: 1AZT). Following the determination of resin eluate was loaded onto anti-Flag M1 resin and washed the initial structure by molecular replacement, rigid body extensively in the presence of 10 uM AVP Receptor was refinement and simulated annealing were performed in 50 then eluted from the anti-Flag M1 affinity resin with 0.2 Phenix (Alfonine et al., 2005) and BUSTER (Blanc et al., mgml' Flag peptide and 2 mM EDTA in the presence of 1 2004), followed by restrained refinement and manual uM AVP and concentrated using a 100 kDa MWCO con rebuilding in Coot (Emsley et al., 2004). After iterative Centrator. TABLE 2
List of nanobodies Nanobody Nanobody SEQ reference short ID Sequence number notation NO: (including C-terminal Histidine tag and EPEA tag) CA4 435 No.35 1. QVOLOESGGGLVOPGGSLRLSCAASGFTFSNYKMNWVR OAPGKGLEWVSDISOSGASISYTGSVKGRFTISRDNAKNT LYLOMNSLKPEDTAVYYCARCPAPFTRDCFDVTSTTYAY RGOGTOVTVSSHHHHHHEPEA
CA4 433 No.33 2 QVOLOESGGGLVOPGGSLRLSCAASGFTFSNYWMNWVR OAPGKGLEWVSDISNGGGTTSYASSVKGRFTISRDNAKN
US 9,695,227 B2 81 82 TABLE 6 TABLE 7-continued
Potential intermolecular interactions within the R:G interface Data collection and refinement statistics Gy 1-4, 63-68 atom in 32AR atom in GSC. distance (A T4 lysozyme 161 Average B-factors (A) ARG 131 CG TYR391 CE2) 3.6 ALA 134 Ol HIS 387 ND1 3.1 32 adrenergic receptor 133.5 ALA 134 CB HIS 387 ND1 3.8 G. C., ras domain 82.8 ALA 134 CB HIS 387 CG) 3.8 G. C., helical domain 123.0 TM3 ILE 135 O) GLN 384 NE2) 2.9 10 G. B 642 ILE135 CD1 LEU 388 CD1 C.5 3.5 Gy 85.2 Nanobody 35 60.7 ILE135 CD1 LEU 393 CD1) 3.5 T4 lysozyme 113.7 THR 136 Ol ARG 380 NH2) 3.0 R.m.S. deviation from ideality PRO 138 Ol LE383 CD1 3.4 PRO 138 CG GLN 384 CG 3.3 15 Bond length (A) O.OO7 Bond angles () 0.72 PHE 139 CD2 HIS 41 NE2 - B1 3.6 Ramachandran statistics PHE 139 CB VAL 217 CG2 - C4 3.7 PHE 139 CE1 PHE 376 CZ 3.3 Favored regions (%) 95.8 | PHE 139 CZ (CYS379 C) 3.9 Allowed regions (%) 4.2 PHE 139 CZ ARG 38ON C.5 3.2 Outliers (%) O TYR 141 CD2 HIS387 NE2) 3.9 *Highest shell statistics are in parentheses. GLN 142 OE1 HIS387 NE2) 3.5 These regions were omitted from the model due to poorly resolved electron density, SER 143 OG ALA 39 CB - CN 3.6 Unmodeled purification tags are not included in these residue ranges, Residues 128 of B2AR were omitted from the construct and T4L was fused to the amino terminus of transmembrane helix 1 to facilitate crystallization. VAL 222 CG1 LEU 393 CD1) 3.7 Residue of T4L was omitted from the construct, GLU 225 OE2 (GLN 384 NE2) 3.0 25 As defined by MolProbity. ALA 226 CA LEU 388 CD2 3.6 ALA 226 CB LEU 393 O 3.8 GLN 229 NE2 ASP 381 OD1 3.6 REFERENCES GLN 229 NE2 (GLN 384 OE1 C.5 2.8 TM5 GLN 229 OE1) ARG 385 NE 3.4 30 Afonine, P. V., Grosse-Kunstleve, R. W., & Adams, P. D. GLN 229 CG LEU 388 CD2 3.9 (2005). A robust bulk-solvent correction and anisotro LEU 230 CG LEU 394 CD1 3.5 pic scaling procedure. Acta Crystallographica. Section LYS 232 NZ ASP 381 OD1 3.1 D, Biological crystallography, 61, 850-5. ILE233 CD1 TYR358 OH - B6 3.4 ILE233 CG1 ARG385 NH1) - c.5 3.5 Baltensperger, K. et al., The beta-adrenergic receptor is a 35 substrate for the insulin receptor tyrosine kinase. J Biol ARG 239 NE THR350 OG1 - C4 3.5 Chem 271, 1061-1064 (1996). ALA271 CB LEU 393 O 3.0 Binz et al., Nature Biotech., 22:575-582 (2004) THR 274 CG2 (GLU 392 O 3.4 Blanc, E., Roversi, P. Vonrhein, C., Flensburg, C., Lea, S. TM6 THR 274 OG1, LEU 393 CD2' 3.3 M., Bricogne, G. et al. (2004). Refinement of severely LEU 275 CD2 LEU 393 CD2 3.6 40 incomplete structures with maximum likelihood in BUSTER-TNT. Acta crystallographica. Section D, Biological crystallography, 60, 2210-21. Caffrey (2003). Membrane protein crystallization. J TABLE 7 Struct. Biol. 2003 142:108-32. Data collection and refinement statistics 45 Caffrey, M. & Cherezov, V. Crystallizing membrane pro teins using lipidic mesophases. Nat Protoc 4, 706-731, Data collection* (2009). Number of crystals Chae, P. S. et al., Maltose-neopentyl glycol (MNG) Space group amphiphiles for solubilization, stabilization and crys Cell dimensions 50 tallization of membrane proteins. Nat Methods 7, 1003 a, b, c (A) 119.3, 64.6, 131.2 1008 (2010). Cl, f, Y () 90.0, 91.7, 90.0 Chelikani et al., Protein Sci. 2006 15:1433-40 Resolution (A) 41-3.2 (3.26-3.20) Chini, B., & Parenti, M. (2009). G-protein-coupled recep Rmerge (%) 15.6 (55.3) tors, cholesterol and palmitoylation: facts about fats. <>, SEQUENCE LISTING <16 Os NUMBER OF SEO ID NOS: 73 SEO ID NO 1 LENGTH: 138 TYPE PRT ORGANISM: Lama glama <4 OOs SEQUENCE: 1 Glin Wall Glin Lieu. Glin Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly 1. 5 15 Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Gly Phe Thir Phe Ser Asn Tyr 2O 25 3 O Llys Met Asn Trp Val Arg Glin Ala Pro Gly Lys Gly Luell Glu Trp Val 35 4 O 45 Ser Asp Ile Ser Glin Ser Gly Ala Ser Ile Ser Tyr Thir Gly Ser Wall SO 55 60 US 9,695,227 B2 87 88 - Continued Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thir Luell Tyr 65 70 7s Lieu. Glin Met Asn. Ser Lieu Lys Pro Glu Asp Thir Ala Wall Tyr Tyr 85 90 95 Ala Arg Cys Pro Ala Pro Phe Thir Arg Asp Phe Asp Wall Thir Ser 1OO 105 11 O Thir Thr Tyr Ala Tyr Arg Gly Glin Gly Thir Glin Wall Thir Wall Ser Ser 115 12 O 125 His His His His His His Glu Pro Glu Ala 13 O 135 <210s, SEQ ID NO 2 &211s LENGTH: 138 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 2 Glin Wall Glin Lieu. Glin Glu Ser Gly Gly Gly Luell Wall Glin Pro Gly Gly 1. 5 15 Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Gly Phe Thir Phe Ser Asn 2O 25 Val Met Asn Trp Val Arg Glin Ala Pro Gly Lys Gly Lell Glu Trp Wall 35 4 O 45 Ser Asp Ile Ser Asn Gly Gly Gly Thir Thir Ser Tyr Ala Ser Ser Wall SO 55 6 O Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Thir Luell Tyr 65 70 75 Lieu. Glin Met Asin Gly Lieu Lys Pro Ala Asp Thir Ala Wall Tyr 85 90 95 Ala Arg Cys Pro Ala Pro Phe Thir Asn Asp Met Asp Ile Thir Ser 1OO 105 11 O Thir Thr Tyr Ala Tyr Arg Gly Glin Gly Thir Glin Wall Thir Wall Ser Ser 115 12 O 125 His His His His His His Glu Pro Glu Ala 13 O 135 <210s, SEQ ID NO 3 &211s LENGTH: 129 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 3 Glin Wall Glin Lieu. Glin Glu Ser Gly Gly Gly Ser Wall Glin Ala Gly Gly 1. 5 1O 15 Ser Lieu. Arg Lieu. Ser Cys Thr Wall Ser Gly Thir Ile Phe Ser Wall Thir 2O 25 3O Val Met Gly Trp Tyr Arg Glin Ala Pro Gly Glin Arg Glu Luell Wall 35 4 O 45 Ala Gly Phe Thr Asn Thr Arg Asn Thir Asn Tyr Wall Asp Ser Wall SO 55 6 O Gly Arg Phe Thr Ile Ser Lys Asp Ser Ala Lys Asn Thir Met Luell 65 70 8O Gln Met Asn Ser Lieu Lys Pro Glu Asp Thir Ala Wall Cys Asn 85 90 95 Val Arg Arg Trp Gly Gly Thr Asn Trp Asn Asp Trp Gly Glin Gly 1OO 105 11 O US 9,695,227 B2 89 - Continued Thr Glin Wall Thr Wal Ser Ser His His His His His His Glu Pro Glu. 115 12 O 125 Ala <210s, SEQ ID NO 4 &211s LENGTH: 129 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 4 Glin Val Glin Lieu. Glin Glu Ser Gly Gly Gly Phe Val Glin Ala Gly Gly 1. 5 1O 15 Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Lys Asn 2O 25 3O Thir Met Ala Trp Phe Arg Glin Ala Pro Gly Lys Glu Arg Glu Lieu Val 35 4 O 45 Ala Ala Ser Pro Thr Gly Gly Ser Thr Ala Tyr Lys Asp Ser Val Lys SO 55 6 O Gly Arg Phe Thir Ile Ser Arg Asp Ser Ala Lys Asn Thr Val Lieu. Lieu. 65 70 7s 8O Gln Met Asn Val Lieu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys His 85 90 95 Lieu. Arg Glin Asn. Asn Arg Gly Ser Trp Phe His Tyr Trp Gly Glin Gly 1OO 105 11 O Thr Glin Wall Thr Wal Ser Ser His His His His His His Glu Pro Glu. 115 12 O 125 Ala <210s, SEQ ID NO 5 &211s LENGTH: 129 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 5 Glin Val Glin Lieu. Glin Glu Ser Gly Gly Gly Lieu Val Glin Ala Gly Gly 1. 5 1O 15 Ser Lieu. Arg Lieu Ser Cys Ala Val Ser Gly Thr Ile Phe Asp Ile Thr 2O 25 3O Pro Met Gly Trp Tyr Arg Glin Thr Pro Gly Lys Glin Arg Glu Val Val 35 4 O 45 Ala Asp Lieu. Thir Ser Arg Gly. Thir Thr Asn Tyr Ala Asp Ser Val Lys SO 55 6 O Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Lys Llys Met Lieu. Tyr Lieu. 65 70 7s 8O Glin Met Asn. Ser Lieu Lys Ser Asp Asp Thr Gly Val Tyr Tyr Cys Asn 85 90 95 Val Lys Arg Trp Gly Gly Ile Gly Trp Asin Asp Tyr Trp Gly Glin Gly 1OO 105 11 O Thr Glin Wall Thr Wal Ser Ser His His His His His His Glu Pro Glu. 115 12 O 125 Ala <210s, SEQ ID NO 6 &211s LENGTH: 129 212. TYPE: PRT <213> ORGANISM: Lama glama US 9,695,227 B2 91 92 - Continued <4 OOs, SEQUENCE: 6 Glin Val Glin Lieu. Glin Glu Ser Gly Gly Gly Lieu Val Glin Ser Gly Gly 1. 5 1O 15 Ser Lieu. Arg Lieu. Ser Cys Val Ala Ser Gly Phe Arg Phe Ser Asn. Phe 2O 25 3O Pro Met Met Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Val 35 4 O 45 Ser Lieu. Ile Ser Ile Gly Gly Ser Thr Thr Asn Tyr Ala Asp Ser Val SO 55 6 O Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Lys Asn. Thir Lieu. Phe 65 70 7s 8O Lieu Gln Met Asn Ser Lieu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Lieu. Gly Arg Lieu Val Pro Pro Thir Thr Glu Gly Glin Gly 1OO 105 11 O Thr Glin Wall Thr Wal Ser Ser His His His His His His Glu Pro Glu. 115 12 O 125 Ala <210s, SEQ ID NO 7 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OO > SEQUENCE: 7 Gln Val Glin Lieu Gln Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly 1. 5 1O 15 Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser 2O 25 <210s, SEQ ID NO 8 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 8 Glin Val Glin Lieu. Glin Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly 1. 5 1O 15 Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser 2O 25 <210s, SEQ ID NO 9 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 9 Glin Val Glin Lieu. Glin Glu Ser Gly Gly Gly Ser Val Glin Ala Gly Gly 1. 5 1O 15 Ser Lieu. Arg Lieu Ser Cys Thr Val Ser 2O 25 <210s, SEQ ID NO 10 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 10 Glin Val Glin Lieu. Glin Glu Ser Gly Gly Gly Phe Val Glin Ala Gly Gly US 9,695,227 B2 93 94 - Continued 1. 5 1O 15 Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser 2O 25 <210s, SEQ ID NO 11 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 11 Glin Wall Glin Lieu. Glin Glu Ser Gly Gly Gly Lieu Val Glin Ala Gly Gly 1. 5 15 Ser Lieu. Arg Lieu. Ser Cys Ala Wall Ser 2O 25 <210s, SEQ ID NO 12 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 12 Glin Wall Glin Lieu. Glin Glu Ser Gly Gly Gly Lieu Val Glin Ser Gly Gly 1. 5 15 Ser Lieu. Arg Lieu. Ser Cys Val Ala Ser 2O 25 <210s, SEQ ID NO 13 &211s LENGTH: 8 212. TYPE PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 13 Gly Phe Thr Phe Ser Asn Tyr 1. 5 <210s, SEQ ID NO 14 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 14 Gly Phe Thr Phe Ser Asn Tyr Wall 1. 5 <210s, SEQ ID NO 15 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 15 Gly. Thir Ile Phe Ser Val Thr Wall 1. 5 <210s, SEQ ID NO 16 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 16 Gly Ser Ile Phe Ser Lys Asn Thir 1. 5 <210s, SEQ ID NO 17 US 9,695,227 B2 95 96 - Continued &211s LENGTH: 8 212. TYPE: PRT &213s ORGANISM: Lama glama <4 OOs, SEQUENCE: 17 Gly. Thir Ile Phe Asp Ile Thr Pro 1. 5 <210s, SEQ ID NO 18 &211s LENGTH: 8 212. TYPE: PRT &213s ORGANISM: Lama glama <4 OOs, SEQUENCE: 18 Gly Phe Arg Phe Ser Asn. Phe Pro 1. 5 <210s, SEQ ID NO 19 &211s LENGTH: 17 212. TYPE: PRT &213s ORGANISM: Lama glama <4 OOs, SEQUENCE: 19 Met Asn Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Val Ser 1. 5 15 Asp <210s, SEQ ID NO &211s LENGTH: 17 212. TYPE PRT &213s ORGANISM: Lama glama <4 OOs, SEQUENCE: Met Asn Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Val Ser 1. 5 15 Asp <210s, SEQ ID NO 21 &211s LENGTH: 17 212. TYPE: PRT &213s ORGANISM: Lama glama <4 OOs, SEQUENCE: 21 Met Gly Trp Tyr Arg Glin Ala Pro Gly Lys Glin Arg Glu Lieu Val Ala 1. 5 15 Gly <210s, SEQ ID NO 22 &211s LENGTH: 17 212. TYPE: PRT &213s ORGANISM: Lama glama <4 OOs, SEQUENCE: 22 Met Ala Trp Phe Arg Glin Ala Pro Gly Lys Glu Arg Glu Lieu Val Ala 1. 5 15 Ala <210s, SEQ ID NO 23 &211s LENGTH: 17 212. TYPE: PRT &213s ORGANISM: Lama glama <4 OOs, SEQUENCE: 23 US 9,695,227 B2 97 98 - Continued Met Gly Trp Tyr Arg Glin Thr Pro Gly Lys Glin Arg Glu Val Val Ala 1. 5 15 Asp <210s, SEQ ID NO 24 &211s LENGTH: 17 212. TYPE: PRT &213s ORGANISM: Lama glama <4 OOs, SEQUENCE: 24 Met Met Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Val Ser 1. 5 15 Lell <210s, SEQ ID NO 25 &211s LENGTH: 8 212. TYPE: PRT &213s ORGANISM: Lama glama <4 OOs, SEQUENCE: 25 Ile Ser Glin Ser Gly Ala Ser Ile 1. 5 <210s, SEQ ID NO 26 &211s LENGTH: 8 212. TYPE: PRT &213s ORGANISM: Lama glama < 4 OO SEQUENCE: 26 Ile Ser Asn Gly Gly Gly. Thir Thr 1. 5 <210s, SEQ ID NO 27 &211s LENGTH: 7 212. TYPE: PRT &213s ORGANISM: Lama glama <4 OOs, SEQUENCE: 27 Phe Thir Asn. Thir Arg Asn Thr 1. 5 <210s, SEQ ID NO 28 &211s LENGTH: 7 212. TYPE: PRT &213s ORGANISM: Lama glama <4 OOs, SEQUENCE: 28 Ser Pro Thr Gly Gly Ser Thr 1. 5 <210s, SEQ ID NO 29 &211s LENGTH: 7 212. TYPE: PRT &213s ORGANISM: Lama glama <4 OOs, SEQUENCE: 29 Lieu. Thir Ser Arg Gly. Thir Thr 1. 5 <210s, SEQ ID NO 3 O &211s LENGTH: 8 212. TYPE: PRT &213s ORGANISM: Lama glama US 9,695,227 B2 99 100 - Continued <4 OOs, SEQUENCE: 30 Ile Ser Ile Gly Gly Ser Thr Thr 1. 5 <210s, SEQ ID NO 31 &211s LENGTH: 38 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 31 Ser Tyr Thr Gly Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1. 5 1O 15 Ala Lys Asn. Thir Lieu. Tyr Lieu Gln Met Asn. Ser Lieu Lys Pro Glu Asp 2O 25 3O Thr Ala Val Tyr Tyr Cys 35 <210s, SEQ ID NO 32 &211s LENGTH: 38 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 32 Ser Tyr Ala Ser Ser Val Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn 1. 5 1O 15 Ala Lys Asn. Thir Lieu. Tyr Lieu Gln Met Asin Gly Lieu Lys Pro Ala Asp 2O 25 3O Thr Ala Val Tyr Tyr Cys 35 <210s, SEQ ID NO 33 &211s LENGTH: 38 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 33 Asn Tyr Val Asp Ser Val Lys Gly Arg Phe Thir Ile Ser Lys Asp Ser 1. 5 1O 15 Ala Lys Asn. Thir Met Tyr Lieu Gln Met Asn. Ser Lieu Lys Pro Glu Asp 2O 25 3O Thr Ala Val Tyr Tyr Cys 35 <210s, SEQ ID NO 34 &211s LENGTH: 38 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 34 Ala Tyr Lys Asp Ser Val Lys Gly Arg Phe Thir Ile Ser Arg Asp Ser 1. 5 1O 15 Ala Lys Asn. Thr Val Lieu. Lieu Gln Met Asin Val Lieu Lys Pro Glu Asp 2O 25 3O Thr Ala Val Tyr Tyr Cys 35 <210s, SEQ ID NO 35 &211s LENGTH: 38 212. TYPE: PRT <213> ORGANISM: Lama glama US 9,695,227 B2 101 102 - Continued <4 OOs, SEQUENCE: 35 Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn 1. 5 1O 15 Ala Lys Llys Met Lieu. Tyr Lieu Gln Met Asn. Ser Lieu Lys Ser Asp Asp 2O 25 3O Thr Gly Val Tyr Tyr Cys 35 <210s, SEQ ID NO 36 &211s LENGTH: 38 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 36 Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn 1. 5 1O 15 Ala Lys Asn. Thir Lieu. Phe Lieu Gln Met Asn. Ser Lieu Lys Pro Glu Asp 2O 25 3O Thr Ala Val Tyr Tyr Cys 35 <210s, SEQ ID NO 37 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OO > SEQUENCE: 37 Ala Arg Cys Pro Ala Pro Phe Thr Arg Asp Cys Phe Asp Val Thr Ser 1. 5 1O 15 Thir Thr Tyr Ala Tyr 2O <210s, SEQ ID NO 38 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 38 Ala Arg Cys Pro Ala Pro Phe Thr Asn Asp Cys Met Asp Ile Thr Ser 1. 5 1O 15 Thir Thr Tyr Ala Tyr 2O <210s, SEQ ID NO 39 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 39 Asn Val Arg Arg Trp Gly Gly Thr Asn Trp Asn Asp Tyr 1. 5 1O <210s, SEQ ID NO 4 O &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 4 O His Lieu. Arg Glin Asn. Asn Arg Gly Ser Trp Phe His Tyr 1. 5 1O US 9,695,227 B2 103 104 - Continued <210s, SEQ ID NO 41 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 41 Asn. Wall Lys Arg Trp Gly Gly Ile Gly Trp Asn Asp Tyr 1. 5 1O <210s, SEQ ID NO 42 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 42 Ala Lys Tyr Lieu. Gly Arg Lieu Val Pro Pro Thir Thr 1. 5 1O <210s, SEQ ID NO 43 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 43 Arg Gly Glin Gly Thr Glin Val Thr Val Ser Ser 1. 5 1O <210s, SEQ ID NO 44 &211s LENGTH: 11 212. TYPE PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 44 Arg Gly Glin Gly Thr Glin Val Thr Val Ser Ser 1. 5 1O <210s, SEQ ID NO 45 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 45 Trp Gly Glin Gly Thr Glin Val Thr Val Ser Ser 1. 5 1O <210s, SEQ ID NO 46 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 46 Trp Gly Glin Gly Thr Glin Val Thr Val Ser Ser 1. 5 1O <210s, SEQ ID NO 47 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 47 Trp Gly Glin Gly Thr Glin Val Thr Val Ser Ser 1. 5 1O <210s, SEQ ID NO 48 US 9,695,227 B2 105 106 - Continued &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 48 Glu Gly Glin Gly Thr Glin Val Thr Val Ser Ser 1. 5 1O <210s, SEQ ID NO 49 &211s LENGTH: 417 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 49 Cys Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Cys Ala Gly Gly 1. 5 1O 15 Ala Gly Thr Cys Thr Gly Gly Ala Gly Gly Ala Gly Gly Cys Thr Thr 2O 25 3O Gly Gly Thr Gly Cys Ala Gly Cys Cys Thr Gly Gly Gly Gly Gly Gly 35 4 O 45 Thr Cys Thr Cys Thr Gly Ala Gly Ala Cys Thr Cys Thr Cys Cys Thr SO 55 6 O Gly Thr Gly Cys Gly Gly Cys Cys Thr Cys Thr Gly Gly Ala Thr Thr 65 70 7s 8O Cys Ala Cys Cys Thr Thr Cys Ala Gly Cys Ala Ala Thr Thr Ala Thr 85 90 95 Ala Ala Ala Ala Thr Gly Ala Ala Cys Thr Gly Gly Gly Thr Cys Cys 1OO 105 11 O Gly Cys Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Gly Ala Ala Ala 115 12 O 125 Gly Gly Gly Gly Cys Thr Cys Gly Ala Gly Thr Gly Gly Gly Thr Cys 13 O 135 14 O Thr Cys Ala Gly Ala Thr Ala Thr Thr Thr Cys Thr Cys Ala Gly Ala 145 150 155 160 Gly Thr Gly Gly Thr Gly Cys Thr Ala Gly Cys Ala Thr Ala Ala Gly 1.65 17O 17s Thir Thr Ala Cys Ala Cys Ala Gly Gly Cys Thr Cys Cys Gly Thr Gly 18O 185 19 O Ala Ala Gly Gly Gly Cys Cys Gly Ala Thir Thr Cys Ala Cys Cys Ala 195 2OO 2O5 Thir Cys Thr Cys Cys Ala Gly Ala Gly Ala Cys Ala Ala Cys Gly Cys 21 O 215 22O Cys Ala Ala Gly Ala Ala Cys Ala Cys Gly Cys Thr Gly. Thir Ala Thr 225 23 O 235 24 O Cys Thr Ala Cys Ala Ala Ala Thr Gly Ala Ala Cys Ala Gly Cys Cys 245 250 255 Thr Gly Ala Ala Gly Cys Cys Thr Gly Ala Gly Gly Ala Cys Ala Cys 26 O 265 27 O Gly Gly Cys Cys Gly Thr Cys Thr Ala Thr Thr Ala Cys Thr Gly Thr 27s 28O 285 Gly Cys Cys Ala Gly Ala Thr Gly Thr Cys Cys Gly Gly Cys Cys Cys 29 O 295 3 OO Cys Ala Thr Thr Cys Ala Cys Gly Ala Gly Ala Gly Ala Thr Thr Gly 3. OS 310 315 32O Thir Thir Thr Thr Gly Ala Cys Gly Thr Gly Ala Cys Thr Ala Gly Thr 3.25 330 335 US 9,695,227 B2 107 108 - Continued Ala Ala Gly Thir Ala Thir Gly Cys Cys Thir Ala Cys Ala 34 O 345 35. O Gly Gly Gly Gly Ala Gly Gly Gly Gly Ala Cys Cys Cys Ala 355 360 365 Gly Gly Thir Ala Cys Cys Gly Thir Thir Cys Cys Thr Cys Ala 37 O 375 Cys Ala Ala Cys Ala Thir Ala Cys Ala Thr Cys 385 390 395 4 OO Ala Gly Ala Ala Cys Thir Gly Ala Ala Gly Cys Thr Ala 4 OS 41O 415 Gly <210s, SEQ ID NO 50 &211s LENGTH: 417 212. TYPE : PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: SO Cys Ala Gly Gly Thir Gly Cys Ala Gly Cys Thir Gly Ala Gly Gly 1. 5 1O 15 Ala Gly Thir Thir Gly Gly Ala Gly Gly Gly Gly Gly Cys Thr Thir 25 3O Gly Gly Thir Gly Ala Gly Cys Thir Gly Gly Gly Gly Gly Gly 35 4 O 45 Thir Cys Thir Thir Gly Ala Gly Ala Thir Cys Thir Thir 50 55 60 Gly Thir Gly Ala Gly Thir Thir Gly Gly Ala Thr Thir 65 70 Ala Thir Thir Thir Ala Gly Thir Ala Ala Thir Ala Thir 85 90 95 Gly Thir Ala Thir Gly Ala Ala Cys Thir Gly Gly Gly Thr Cys 105 11 O Gly Cys Ala Gly Gly Thir Ala Gly Gly Ala Ala Ala 115 12 O 125 Gly Gly Gly Gly Thir Cys Gly Ala Gly Thir Gly Gly Gly Thr 13 O 135 14 O Thir Ala Gly Ala Thir Ala Thir Thir Thir Cys Thir Ala Ala Thr Gly 145 150 155 160 Gly Gly Gly Thir Gly Gly Thir Ala Cys Ala Ala Ala Gly 1.65 17O 17s Thir Thir Ala Thir Gly Ala Ala Gly Thir Gly Thr Gly 18O 185 19 O Ala Ala Gly Gly Gly Gly Ala Thir Thir Ala Ala 195 2O5 Thir Cys Thir Ala Gly Ala Gly Ala Ala Ala 21 O 215 22O Cys Ala Ala Ala Ala Ala Gly Cys Thir Gly Thir Ala Thir 225 23 O 235 24 O Thir Gly Ala Ala Ala Thir Gly Ala Ala Gly Gly Cys 245 250 255 Thir Gly Ala Ala Gly Thir Gly Gly Gly Ala Cys Ala 26 O 265 27 O Gly Gly Cys Gly Thir Thir Ala Thir Thir Ala Cys Thr Gly Thir 27s 28O 285 US 9,695,227 B2 109 110 - Continued Gly Cys Ala Ala Gly Ala Thir Thir Cys Gly Gly Cys Cys 29 O 295 3 OO Cys Ala Thir Thir Ala Ala Ala Cys Gly Ala Thir Thir Gly 3. OS 310 315 Thir Ala Thir Gly Gly Ala Thir Ala Ala Thir Ala Gly Thir 3.25 330 335 Ala Ala Gly Thir Thir Gly Cys Thir Ala Cys Ala 34 O 345 35. O Gly Gly Gly Gly Ala Gly Gly Gly Ala Cys Cys Ala 355 365 Gly Gly Thir Ala Cys Thir Thir Cys Thir Ala 37 O 375 Cys Ala Ala Cys Thir Ala Ala Thir Cys 385 390 395 4 OO Ala Gly Ala Ala Thir Gly Ala Ala Gly Cys Thir Ala 4 OS 41O 415 Gly <210s, SEQ ID NO 51 &211s LENGTH: 390 212. TYPE : PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 51 Cys Ala Gly Gly Thir Gly Cys Ala Gly Cys Thir Gly Ala Gly Gly 1. 5 1O 15 Ala Gly Thir Cys Gly Gly Ala Gly Gly Ala Gly Gly Thir 2O 25 Gly Gly Thir Gly Ala Gly Gly Thir Gly Gly Gly Gly Gly Gly 35 4 O 45 Thir Cys Thir Thir Gly Ala Gly Ala Thir Cys Thir Thir SO 55 6 O Gly Thir Ala Ala Gly Thir Thir Thir Gly Gly Ala 65 70 Ala Thir Thir Thir Ala Gly Thir Gly Thir Cys Thir 85 90 95 Gly Thir Thir Gly Gly Cys Thir Gly Gly Thir 105 Gly Cys Gly Gly Thir Ala Gly Ala Ala 115 12 O Gly Cys Ala Gly Cys Gly Ala Gly Thir Thir Thir 13 O 135 14 O Gly Cys Ala Thir Thir Thir Thir Ala Cys Thir Thir Ala 145 150 155 160 Thir Ala Ala Ala Ala Cys Ala Ala Thir Ala 17s Thir Gly Thir Ala Thir Cys Gly Thir Ala Ala Gly 185 19 O Gly Gly Cys Thir Thir Ala Cys Thir Thir 195 2OO Cys Ala Ala Gly Ala Ala Gly Cys Gly Cys Cys Ala Ala 21 O 215 22O Gly Ala Ala Cys Gly Ala Thir Gly Thir Ala Thir Thir Ala 225 23 O 235 24 O US 9,695,227 B2 111 112 - Continued Cys Ala Ala Ala Thr Gly Ala Ala Cys Ala Gly Cys Cys Thr Gly Ala 245 250 255 Ala Ala Cys Cys Thr Gly Ala Gly Gly Ala Cys Ala Cys Ala Gly Cys 26 O 265 27 O Cys Gly Thr Cys Thr Ala Thr Thr Ala Cys Thr Gly Thr Ala Ala Thr 27s 28O 285 Gly Thr Ala Cys Gly Thr Cys Gly Gly Thr Gly Gly Gly Gly Cys Gly 29 O 295 3 OO Gly. Thir Ala Cys Gly Ala Ala Thir Thr Gly Gly Ala Ala Thr Gly Ala 3. OS 310 315 32O Cys Thr Ala Cys Thr Gly Gly Gly Gly Cys Cys Ala Gly Gly Gly Gly 3.25 330 335 Ala Cys Cys Cys Ala Gly Gly Thr Cys Ala Cys Cys Gly Thr Cys Thr 34 O 345 35. O Cys Cys Thr Cys Ala Cys Ala Cys Cys Ala Cys Cys Ala Thir Cys Ala 355 360 365 Cys Cys Ala Thir Cys Ala Cys Gly Ala Ala Cys Cys Thr Gly Ala Ala 37 O 375 38O Gly Cys Cys Thr Ala Gly 385 390 <210s, SEQ ID NO 52 &211s LENGTH: 390 212. TYPE: PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 52 Cys Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Cys Ala Gly Gly 1. 5 1O 15 Ala Gly Thr Cys Thr Gly Gly Ala Gly Gly Gly Gly Gly Cys Thr Thr 2O 25 3O Cys Gly Thr Gly Cys Ala Gly Gly Cys Thr Gly Gly Gly Gly Gly Gly 35 4 O 45 Thr Cys Thr Cys Thr Gly Ala Gly Ala Cys Thr Cys Thr Cys Cys Thr SO 55 6 O Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly Gly Ala Ala Gly 65 70 7s 8O Cys Ala Thr Cys Thr Thr Cys Ala Gly Thr Ala Ala Gly Ala Ala Thr 85 90 95 Ala Cys Cys Ala Thr Gly Gly Cys Cys Thr Gly Gly Thr Thr Cys Cys 1OO 105 11 O Gly Cys Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Gly Gly Ala Ala 115 12 O 125 Gly Gly Ala Gly Cys Gly Ala Gly Ala Gly. Thir Thr Gly Gly Thr Cys 13 O 135 14 O Gly Cys Ala Gly Cys Thr Ala Gly Thr Cys Cys Thr Ala Cys Gly Gly 145 150 155 160 Gly Thr Gly Gly Thr Ala Gly Cys Ala Cys Ala Gly Cys Gly Thr Ala 1.65 17O 17s Thir Ala Ala Ala Gly Ala Cys Thr Cys Cys Gly Thr Gly Ala Ala Gly 18O 185 19 O Gly Gly Cys Cys Gly Ala Thir Thr Cys Ala Cys Cys Ala Thr Cys Thr 195 2OO 2O5 Cys Cys Ala Gly Ala Gly Ala Cys Ala Gly Cys Gly Cys Cys Ala Ala US 9,695,227 B2 113 114 - Continued 21 O 215 22O Gly Ala Ala Ala Cys Gly Gly Thir Gly Thir Thir Gly Thir Gly 225 23 O 235 24 O Ala Ala Ala Thir Gly Ala Ala Gly Thir Cys Cys Thir Gly Ala 245 250 255 Ala Ala Cys Thir Gly Ala Gly Gly Ala Thir Ala Thir Gly 26 O 265 27 O Gly Thir Thir Ala Thir Thir Ala Thir Gly Thir Ala Thir 27s 28O 285 Thir Ala Gly Thir Cys Ala Ala Ala Ala Thir Ala Ala 29 O 295 3 OO Gly Thir Gly Thir Thir Thir Thir Gly Gly Thir Thir Ala 3. OS 310 315 Thir Ala Thir Gly Gly Gly Gly Cys Ala Gly Gly Gly Gly 3.25 330 335 Ala Cys Ala Gly Gly Thir Cys Ala Gly Thir Thir 34 O 345 35. O Thir Ala Ala Cys Ala Ala Thir Ala 355 360 365 Cys Ala Thir Ala Cys Gly Ala Ala Cys Thir Gly Ala Ala 37 O 375 38O Gly Cys Thir Ala Gly 385 390 <210 SEQ ID NO 53 &211s LENGTH: 390 212. TYPE : PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 53 Ala Gly Gly Thir Gly Ala Gly Cys Thir Gly Ala Gly Gly 5 1O 15 Gly Thir Cys Thir Gly Gly Gly Gly Gly Ala Gly Gly Cys Thir Thir 25 3O Gly Gly Thir Gly Ala Gly Gly Thir Gly Gly Gly Gly Gly Gly 35 4 O 45 Thir Cys Gly Cys Thir Gly Ala Gly Ala Thir Cys Thir Thir Thir SO 55 6 O Gly Thir Gly Cys Ala Gly Thir Thir Thir Gly Gly Thir Ala Cys 65 70 Gly Ala Thir Cys Thir Thir Gly Ala Thir Ala Thir Ala Cys Thir 85 90 95 Thir Gly Gly Cys Thir Gly Gly Thir Ala 105 11 O Gly Cys Gly Ala Thir Ala Gly Gly Gly Ala Ala 115 12 O 125 Gly Cys Ala Gly Cys Gly Ala Ala Gly Thir Gly Gly Thir 13 O 135 14 O Gly Cys Ala Ala Thir Thir Thir Ala Cys Thir Ala Gly Thir Cys 145 150 155 160 Gly Thir Ala Ala Cys Ala Ala Ala Thir Thir Ala 1.65 17O 17s Gly Gly Ala Thir Cys Gly Thir Gly Ala Ala Gly 18O 185 19 O US 9,695,227 B2 115 116 - Continued Gly Cys Gly Gly Thir Thir Ala Cys Cys Ala Thr Cys Thir 195 2OO Ala Gly Ala Gly Ala Ala Ala Cys Gly Cys Ala Ala 215 22O Gly Ala Ala Ala Thir Gly Thir Thir Gly Thir Ala Thr Thir Gly 225 23 O 235 24 O Ala Ala Thir Ala Ala Ala Gly Cys Cys Thir Ala 245 250 Ala Thir Cys Thir Ala Gly Ala Ala Cys Ala Gly 26 O 265 27 O Thir Gly Thir Thir Thir Ala Thir Gly Thr Ala 27s 28O 285 Gly Thir Gly Ala Ala Cys Gly Gly Thir Gly Gly Gly Gly Gly 29 O 295 3 OO Gly Thir Ala Thir Thir Gly Thir Gly Gly Ala Ala Ala 3. OS 315 Thir Ala Thir Gly Gly Gly Cys Ala Gly Gly Gly 3.25 330 Ala Cys Ala Gly Thir Cys Ala Thir Thir 34 O 345 35. O Thir Ala Ala Cys Ala Cys Ala Thir Ala 355 360 365 Cys Ala Thir Ala Cys Gly Ala Ala Cys Thr Gly Ala Ala 37 O 375 38O Gly Cys Thr Ala Gly 385 390 <210s, SEQ ID NO 54 &211s LENGTH: 390 212. TYPE : PRT <213> ORGANISM: Lama glama <4 OOs, SEQUENCE: 54 Ala Gly Gly Thir Gly Ala Gly Cys Thir Gly Cys Ala Gly Gly 5 1O 15 Gly Thir Cys Thir Gly Gly Ala Gly Gly Ala Gly Gly Cys Thir Thir 2O 25 3O Gly Gly Thir Gly Ala Gly Thir Thir Gly Gly Gly Gly Gly Gly 35 4 O 45 Thir Cys Thir Thir Gly Ala Gly Ala Thir Cys Thr Thir SO 55 6 O Gly Thir Gly Thir Ala Gly Thir Thir Gly Gly Ala Thir Thir 65 70 Ala Gly Ala Thir Thir Ala Gly Thir Ala Ala Cys Thir Thir Thir 85 90 95 Thir Ala Thir Gly Ala Thir Gly Thir Gly Gly Gly Thir 1OO 105 11 O Gly Cys Ala Gly Gly Cys Ala Gly Gly Ala Ala Ala 115 12 O 125 Gly Gly Gly Gly Thir Cys Gly Ala Gly Thir Gly Gly Gly Thir 13 O 135 14 O Thir Gly Thir Gly Ala Thir Thir Ala Gly Cys Ala Thir Thir Gly 145 150 155 160 Gly Thir Gly Gly Thir Ala Gly Thir Ala Cys Ala Cys Gly Ala Ala 1.65 17O 17s US 9,695,227 B2 117 118 - Continued Thir Thir Ala Thir Gly Gly Gly Ala Cys Thr Cys Cys Gly Thir Gly 18O 185 19 O Ala Ala Gly Gly Gly Gly Ala Th Thir Cys Ala Cys Cys Ala 195 Thir Cys Thir Cys Ala Gly Ala Gly Ala Cys Ala Ala Cys Gly 21 O 215 Cys Ala Ala Ala Ala Gly Cys Thir Gly Thir Thir Thir 225 23 O 235 24 O Thir Gly Ala Ala Thir Gly Ala Ala Ala Gly Cys 250 255 Thir Gly Ala Ala Thir Gly Ala Gly Gly Ala Cys Ala 26 O 265 27 O Gly Gly Cys Thir Gly Thir Ala Th Thir Ala Cys Thir Gly Thir 27s 285 Gly Cys Ala Ala Ala Thir Ala Thir Cys Thr Thir Gly Gly Thir 29 O 295 3 OO Gly Gly Thir Gly Thir Cys Ala Cys Gly Ala Cys 3. OS 310 315 Thir Ala Thir Ala Gly Gly Gly Cys Cys Ala Gly Gly Gly Gly 330 335 Ala Cys Gly Gly Thir Cys Ala Cys Gly Thir Thir 34 O 345 35. O Thir Ala Cys Ala Cys Ala Thir Ala 355 360 365 Cys Ala Thir Cys Ala Cys Gly Ala Ala Cys Cys Thir Gly Ala Ala 37 O 375 38O Gly Thir Ala Gly 385 390 SEO ID NO 55 LENGTH: 1095 TYPE : PRT ORGANISM: Artificial FEATURE: OTHER INFORMATION: Beta2AR construct SEQUENCE: 55 Gly Gly Gly Cys Ala Gly Gly Gly Ala Cys Gly 1. 1O 15 Gly Cys Ala Gly Gly Thir Thr Cys Thir Thir Thir 2O 25 Gly Gly Cys Ala Ala Thir Ala Gly Ala 35 45 Ala Thir Gly Gly Gly Gly Ala Cys Cys Gly SO 6 O Ala Gly Thir Ala Gly Ala Gly Ala Ala Gly 65 70 7s Gly Gly Ala Gly Ala Gly Gly Thir Gly Thr Gly Thir Gly 85 90 95 Gly Thir Gly Gly Gly Ala Thir Gly Gly Gly Ala Cys Gly 1OO 105 Thir Ala Thir Gly Thir Thir Thr Cys Ala Thir Cys Gly Thir 115 12 O 125 Cys Thir Gly Gly Cys Ala Thir Cys Gly Thir Gly Thir Thir Thir 13 O 135 14 O US 9,695,227 B2 119 120 - Continued Gly Gly Ala Ala Thir Gly Thir Gly Thir Gly Gly Thr Cys Ala 145 150 155 160 Thir Ala Ala Gly Ala Thir Thir Gly Cys Cys Ala Ala 1.65 17O Gly Thir Thir Cys Gly Ala Gly Gly Thir Cys Thr Gly Cys Ala Gly 185 19 O Ala Gly Gly Thir Ala Cys Ala Ala Cys Thr Ala Cys Thir 195 2O5 Thir Cys Ala Thir Ala Cys Thir Thir Ala Cys Thr Gly Gly 21 O 215 22O Cys Thir Gly Thir Gly Cys Thir Gly Ala Thir Cys Thr Gly Gly Thir Cys 225 23 O 235 24 O Ala Thir Gly Gly Gly Thir Gly Gly Ala Gly Thir Gly Gly 245 250 255 Thir Gly Cys Thir Thir Thir Gly Gly Gly Gly Cys Cys Gly 26 O 265 27 O Ala Thir Ala Thir Thir Cys Thir Thir Ala Thr Gly Ala Ala Ala 285 Ala Thir Gly Thir Gly Gly Ala Thir Thir Thir Thr Gly Gly Ala 29 O 295 3 OO Ala Thir Thir Thir Gly Gly Thir Gly Cys Gly Ala Gly Thir Thir 3. OS 310 315 Thir Thir Gly Gly Ala Thir Thir Cys Ala Th Thir Gly Ala Thir 3.25 330 335 Gly Thir Gly Cys Thir Gly Thir Gly Cys Gly Thir Cys Ala Cys Gly Gly 34 O 345 35. O Ala Gly Ala Thir Thir Gly Ala Gly Ala Cys Thir 355 360 365 Gly Thir Gly Gly Thir Gly Ala Thir Gly Cys Ala Thir Gly 37 O 375 38O Gly Ala Thir Gly Cys Thir Ala Thir Thir Thr Gly Ala 385 390 395 4 OO Thir Thir Ala Thir Thir Ala Cys Thir Th Thir Ala Ala 4 OS 415 Gly Thir Ala Cys Ala Gly Ala Gly Thr Gly Cys Thir Gly 42O 425 43 O Ala Cys Ala Ala Gly Ala Ala Thir Ala Ala Gly Gly 435 44 O 445 Gly Gly Gly Thir Gly Ala Thir Ala Thir Thir Cys Thr Gly Ala Thir 450 45.5 460 Gly Gly Thir Gly Thir Gly Gly Ala Thir Thir Gly Thr Gly Thir Ala 465 470 47s 48O Gly Gly Thir Thir Ala Thir Cys Thr Thir Cys Thir 485 490 495 Thir Gly Cys Ala Thir Thir Cys Ala Gly Ala Thr Gly Ala SOO 505 51O Thir Gly Gly Thir Ala Cys Gly Gly Gly Ala 515 52O 525 Ala Ala Gly Gly Ala Ala Gly Cys Ala Thir Ala 53 O 535 54 O Ala Thir Gly Thir Ala Thir Gly Cys Gly Ala Gly Gly Ala 5.45 550 555 560 US 9,695,227 B2 121 122 - Continued Gly Ala Thir Gly Thir Gly Thir Gly Ala Cys Thir Thr 565 st O sts Thir Thir Ala Gly Ala Ala Cys Ala Ala Gly Cys Cys Thir 58O 585 59 O Ala Thir Gly Ala Thir Thir Gly Cys Thir Cys Thir Thir 595 6OO 605 Ala Thir Thir Gly Thir Thir Thir Cys Thir Ala 610 615 Gly Thir Thir Cys Thir Gly Gly Thir Gly Ala Thir Cys Ala 625 630 635 64 O Thir Gly Gly Thir Cys Thir Thir Gly Thir Cys Thir Ala Cys Thir 645 650 655 Ala Gly Gly Thir Thir Thir Thir Cys Ala Gly Gly Ala 660 665 67 O Gly Cys Ala Ala Ala Gly Gly Ala Gly Cys Thir Cys 675 68O 685 Ala Gly Ala Ala Ala Thir Thir Gly Ala Cys Ala Ala Ala Thir 69 O. 695 7 OO Thir Gly Ala Gly Gly Gly Thir Thir Cys Ala Thir 7 Os 72O Gly Thir Gly Ala Cys Thir Thir Ala Gly Cys 73 O 73 Ala Gly Gly Thir Gly Gly Cys Ala Gly Gly Ala Thir Gly 740 74. 7 O Gly Gly Gly Gly Gly Gly Cys Ala Thr Gly Gly 760 765 Thir Gly Ala Thir Cys Thir Thir Cys Cys 770 Ala Gly Thir Thir Thir Thir Thir Gly Ala Ala Gly Gly 78s 79 O 79. Gly Ala Ala Ala Gly Cys Cys Thir Ala Ala 805 810 815 Ala Gly Thir Thir Ala Gly Cys Ala Thir Ala Thir Cys 82O 825 83 O Thir Gly Gly Gly Ala Thir Thir Thir Cys Ala Cys Cys Cys Thir 835 84 O 845 Thir Gly Thir Gly Gly Thir Gly Cys Cys Thir Thir 850 855 860 Thir Thir Ala Thir Cys Gly Thir Thir Ala Ala Ala Thir Thir Gly 865 88O Thir Ala Thir Gly Thir Gly Ala Thir Cys Ala Gly Gly Ala 885 890 895 Thir Ala Cys Thir Ala Thir Cys Thir Ala Ala Gly 9 OO 905 91 O Gly Ala Gly Thir Thir Thir Ala Ala Thir Cys Thir Cys 915 92 O 925 Thir Ala Ala Thir Thir Gly Gly Ala Thir Ala Gly Gly Cys Thir Ala 935 94 O Thir Thir Ala Ala Thir Thir Thir Gly Gly Thir Thir Thir Cys 945 950 955 96.O Ala Thir Cys Thir Thir Ala Thir Thir Ala Cys Thir 965 97. Gly Gly Gly Ala Gly Cys Ala Gly Ala Thir Thir Thir US 9,695,227 B2 123 124 - Contin lued 98O 985 99 O Ala Gly Gly Ala Thr Thr Gly Cys Cys Thr Thr Cys Cys Ala Gly 995 1OOO 1005 Gly Cys Thir Thr Cys Thir Gly Thir Gly Cys Cys Thr Gly O15 Cys Ala Gly Gly Thr Cys Thir Thir Cys Thr Gly Ala Gly Gly Cys Thr Al a Thr Gly Gly Gly Ala Ala Thr Gly Thir Ala Ala Gly Cys Ala Ala Cys Gly Ala Ala Ala Gly Gly Gly Gly Ala Gly Gly Ala Gly Thr Gly Gl y Ala Thir Ala Ala <210s, SEQ ID NO 56 &211s LENGTH: 212. TYPE : PRT &213s ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 56 Met Gly Cys Lieu. Gly Asn Ser Lys Thir Glu Asp Glin Arg Asn Glu Glu 1. 5 1O 15 Lys Ala Glin Arg Glu Ala Asn Lys Lys Ile Glu Lys Glin Lieu. Glin 25 3O Asp Glin Wall Arg Ala Thr His Arg Luell Lieu Lleu Luell Gly Ala 35 4 O 45 Gly Glu Ser Gly Ser Thir Ile Wall Glin Met Arg Ile Luell His SO 55 6 O Wall Asn Gly Phe Asn Gly Asp Ser Glu Ala Thr Lys Val Glin Asp 65 70 Ile Asn Asn Lell Glu Ala Ile Glu Thir Ile Wall Ala Ala Met 85 90 95 Ser Asn Luell Wall Pro Pro Wall Glu Luell Ala ASn Pro Glu Asn Glin Phe 105 11 O Arg Wall Asp Ile Lell Ser Wall Met Asn Wall Pro Asp Phe Asp Phe 115 12 O 125 Pro Pro Glu Phe Glu His Ala Ala Luell Trp Glu Asp Glu Gly 13 O 135 14 O Wall Arg Ala Glu Arg Ser Asn Glu Tyr Gln Lieu. Ile Asp Cys 145 150 155 160 Ala Glin Phe Lell Asp Lys Ile Asp Wall Ile Lys Glin Ala Asp 1.65 17O 17s Wall Pro Ser Asp Glin Asp Lieu. Luell Arg Arg Wall Lieu. Thir Ser Gly 18O 185 19 O Ile Phe Glu Thir Phe Glin Wall Asp Wall ASn Phe His Met Phe 195 2OO 2O5 Asp Wall Gly Gly Glin Arg Asp Glu Arg Arg Trp Ile Glin Phe 21 O 215 22O Asn Asp Wall Thir Ala Ile Ile Phe Wall Wall Ala Ser Ser Ser Asn 225 23 O 235 24 O Met Wall Ile Arg Glu Asp ASn Glin Thir Asn Arg Lieu. Glin Glu Ala Luell 245 250 255 US 9,695,227 B2 125 126 - Continued Asn Lieu. Phe Llys Ser Ile Trp Asn Asn Arg Trp Lieu. Arg Thr Ile Ser 26 O 265 27 O Wall Ile Lieu. Phe Lieu. Asn Lys Glin Asp Luell Luell Ala Glu Lys Wall Luell 27s 28O 285 Ala Gly Lys Ser Lys Ile Glu Asp Phe Pro Glu Phe Ala Arg 29 O 295 3 OO Thir Thr Pro Glu Asp Ala Thr Pro Glu Pro Gly Glu Asp Pro Arg Wall 3. OS 310 315 32O Thir Arg Ala Lys Tyr Phe Ile Arg Asp Glu Phe Lell Arg Ile Ser Thir 3.25 330 335 Ala Ser Gly Asp Gly Arg His Tyr Cys Pro His Phe Thir Ala 34 O 345 35. O Wall Asp Thr Glu Asn. Ile Arg Arg Wall Phe ASn Asp Cys Arg Asp Ile 355 360 365 Ile Glin Arg Met His Lieu. Arg Glin Tyr Glu Luell Lell 37 O 375 38O <210s, SEQ ID NO 57 &211s LENGTH: 354 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO > SEQUENCE: 57 Met Gly Cys Thir Lieu. Ser Ala Glu Asp Lys Ala Ala Wall Glu Arg Ser 1. 5 1O 15 Lys Met Ile Asp Arg Asn Lieu. Arg Glu Asp Gly Glu Ala Ala Arg 2O 25 30 Glu Val Llys Lieu Lleu Lleu Lieu. Gly Ala Gly Glu Ser Gly Ser Thir 35 4 O 45 Ile Val Lys Gln Met Lys Ile Ile His Glu Ala Gly Tyr Ser Glu Glu SO 55 6 O Glu Cys Lys Glin Tyr Lys Ala Val Wall Ser Asn Thir Ile Glin Ser 65 70 Ile Ile Ala Ile Ile Arg Ala Met Gly Arg Luell Ile Asp Phe Gly 85 90 95 Asp Ser Ala Arg Ala Asp Asp Ala Arg Glin Luell Phe Wall Luell Ala Gly 1OO 105 11 O Ala Ala Glu Glu Gly Phe Met Thr Ala Glu Luell Ala Gly Wall Ile 115 12 O 125 Arg Lieu. Trp Lys Asp Ser Gly Val Glin Ala Phe Asn Arg Ser Arg 13 O 135 14 O Glu Tyr Glin Lieu. Asn Asp Ser Ala Ala Tyr Lell Asn Asp Luell Asp 145 150 155 160 Arg Ile Ala Glin Pro Asn Tyr Ile Pro Thir Glin Glin Asp Wall Luell Arg 1.65 17O 17s Thir Arg Val Lys Thr Thr Gly Ile Wall Glu Thir His Phe Thir Phe 18O 185 19 O Asp Lieu. His Phe Lys Met Phe Asp Wall Gly Gly Glin Arg Ser Glu 195 2OO Lys Trp Ile His Cys Phe Glu Gly Wall Thir Ala Ile Ile Phe 21 O 215 22O Wall Ala Lieu. Ser Asp Tyr Asp Lieu Wall Luell Ala Glu Asp Glu Glu Met 225 23 O 235 24 O Asn Arg Met His Glu Ser Met Lys Luell Phe Asp Ser Ile Asn Asn 245 250 255 US 9,695,227 B2 127 128 - Continued Trp Phe Thir Asp Thr Ser Ile Ile Luell Phe Lieu. Asn Lys Asp 26 O 265 27 O Lell Phe Glu Glu Lys Ile Llys Llys Ser Pro Luell Thir Ile Cys Pro 28O 285 Glu Tyr Ala Gly Ser Asn Thr Tyr Glu Glu Ala Ala Ala Tyr Ile Glin 29 O 295 3 OO Cys Glin Phe Glu Asp Lieu. Asn Lys Arg Asp Thir Glu Ile Tyr 3. OS 310 315 Thir His Phe Thir Cys Ala Thr Asp Thir Lys ASn Wall Glin Phe Wall Phe 3.25 330 335 Asp Ala Wall Thir Asp Wal Ile Ile Lys Asn ASn Lell Asp Gly 34 O 345 35. O Lell Phe <210s, SEQ ID NO 58 &211s LENGTH: 350 212. TYPE : PRT &213s ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 58 Met Gly Ala Gly Ala Ser Ala Glu Glu Lys His Ser Arg Glu Luell Glu 1. 5 1O 15 Lys Lys Luell Lys Glu Asp Ala Glu Lys Asp Ala Arg Thir Wall Luell 25 3O Lell Luell Luell Gly Ala Gly Glu Ser Gly Ser Thir Ile Wall Glin 35 4 O 45 Met Lys Ile Ile His Glin Asp Gly Ser Luell Glu Glu Luell Glu SO 55 6 O Phe Ile Ala Ile Ile Tyr Gly Asn Thir Luell Glin Ser Ile Luell Ala Ile 65 70 Wall Arg Ala Met Thir Thir Lieu. Asn Ile Glin Tyr Gly Asp Ser Ala Arg 85 90 95 Glin Asp Asp Ala Arg Llys Lieu Met His Met Ala Asp Thir Ile Glu Glu 105 11 O Gly Thir Met Pro Lys Glu Met Ser Asp Ile Ile Glin Arg Luell Trp 115 12 O 125 Asp Ser Gly Ile Glin Ala Cys Phe Glu Arg Ala Ser Glu Tyr Glin Luell 13 O 135 14 O Asn Asp Ser Ala Gly Tyr Tyr Lieu. Ser Asp Luell Glu Arg Luell Wall Thir 145 150 155 160 Pro Gly Tyr Wall Pro Thr Glu Glin Asp Wall Luell Arg Ser Arg Wall 1.65 17O 17s Thir Thir Gly Ile Ile Glu. Thir Glin Phe Ser Phe Asp Luell Asn Phe 18O 185 19 O Arg Met Phe Asp Val Gly Gly Glin Arg Ser Glu Arg Lys Trp Ile 195 2OO His Cys Phe Glu Gly Val Thr Cys Ile Ile Phe Ile Ala Ala Luell Ser 21 O 215 22O Ala Asp Met Wall Lieu Val Glu Asp Asp Glu Wall Asn Arg Met His 225 23 O 235 24 O Glu Ser Luell His Leul Phe Asn. Ser Ile Cys ASn His Arg Phe Ala 245 250 255 Thir Thir Ser Ile Wall Lieu. Phe Lieu. Asn Asp Wall Phe Phe Glu 26 O 265 27 O US 9,695,227 B2 129 130 - Continued Ile Lys Ala His Lell Ser Ile Phe Pro Asp Tyr Asp Gly 27s 28O 285 Pro Asn Thir Glu Asp Ala Gly Asn Ile Lys Wall Glin Phe Luell 29 O 295 3 OO Glu Luell Asn Met Arg Arg Asp Wall Glu Ile Tyr Ser His Met Thir 3. OS 310 315 Ala Thir Asp Thir Glin Asn Wall Phe Wall Phe Asp Ala Wall Thir 3.25 330 335 Asp Ile Ile Ile Lys Glu Asn Luell Lys Asp Gly Lell Phe 34 O 345 35. O <210s, SEQ ID NO 59 &211s LENGTH: 394 212. TYPE : PRT &213s ORGANISM: Bos talurus <4 OOs, SEQUENCE: 59 Met Gly Cys Lieu. Gly Asn Ser Thir Glu Asp Glin Arg Asn Glu Glu 1. 5 1O 15 Lys Ala Glin Arg Glu Ala Asn Lys Ile Glu Glin Luell Glin 25 Asp Glin Wall Ala Thir His Arg Luell Lell Lell Luell Gly Ala 35 4 O 45 Gly Glu Ser Gly Ser Thir Ile Wall Glin Met Arg Ile Luell His SO 55 6 O Wall Asn Gly Phe Asn Gly Glu Gly Gly Glu Glu Asp Pro Gln Ala Ala 65 70 Arg Ser Asn Ser Asp Gly Glu Ala Thir Wall Glin Asp Ile 85 90 95 Asn Asn Luell Lys Glu Ala Ile Glu Thir Ile Wall Ala Ala Met Ser Asn 105 11 O Lell Wall Pro Pro Wall Glu Lell Ala Asn Pro Glu Asn Glin Phe Arg Wall 115 12 O 125 Asp Tyr Ile Luell Ser Wall Met Asn Wall Pro Asp Phe Asp Phe Pro Pro 13 O 135 14 O Glu Phe Glu His Ala Ala Luell Trp Glu Asp Glu Gly Wall Arg 145 150 155 160 Ala Glu Arg Ser Asn Glu Glin Luell Ile Asp Ala Glin 1.65 17O Phe Luell Asp Ile Asp Wall Ile Glin Asp Asp Tyr Wall Pro 18O 185 19 O Ser Asp Glin Asp Lell Lell Arg Cys Arg Wall Luell Thir Ser Gly Ile Phe 195 Glu Thir Phe Glin Wall Asp Wall Asn Phe His Met Phe Asp Wall 21 O 215 22O Gly Gly Glin Arg Asp Glu Arg Arg Trp Ile Glin Phe Asn Asp 225 23 O 235 24 O Wall Thir Ala Ile Ile Phe Wall Wall Ala Ser Ser Ser Asn Met Wall 245 250 255 Ile Arg Glu Asp Asn Glin Thir Asn Arg Luell Glin Glu Ala Luell Asn Luell 26 O 265 27 O Phe Ser Ile Trp Asn Asn Arg Trp Luell Arg Thir Ile Ser Wall Ile 27s 285 Lell Phe Luell Asn Glin Asp Luell Luell Ala Glu Wall Luell Ala Gly US 9,695,227 B2 131 132 - Continued 29 O 295 3 OO Lys Ser Ile Glu Asp Phe Pro Glu Phe Ala Arg Thir Thir 3. OS 310 315 Pro Glu Ala Thir Pro Glu Pro Gly Glu Asp Pro Arg Wall Thir Arg 3.25 330 335 Ala Phe Ile Arg Asp Glu Phe Luell Arg Ile Ser Thir Ala Ser 34 O 345 35. O Gly Asp Gly Arg His Tyr Tyr Pro His Phe Thir Cys Ala Wall Asp 355 360 365 Thir Glu Asn Ile Arg Arg Wall Phe Asn Asp Arg Asp Ile Ile Glin 37 O 375 38O Arg Met His Luell Arg Glin Tyr Glu Luell Luell 385 390 SEQ ID NO 60 LENGTH: 394 TYPE : PRT ORGANISM: Rattus norvegicus < 4 OOs SEQUENCE: 6 O Met Gly Cys Lieu. Gly Asn Ser Thir Glu Asp Glin Arg Asn Glu Glu 1. 5 1O 15 Lys Ala Glin Arg Glu Ala Asn Lys Ile Glu Glin Luell Glin 25 Asp Glin Wall Ala Thir His Arg Luell Lell Lell Luell Gly Ala 35 4 O 45 Gly Glu Ser Gly Llys Ser Thir Ile Wall Glin Met Arg Ile Luell His SO 55 6 O Wall Asn Gly Phe Asn Gly Glu Gly Gly Glu Glu Asp Pro Glin Ala Ala 65 70 Arg Ser Asn Ser Asp Gly Glu Ala Thir Wall Glin Asp Ile 85 90 95 Asn Asn Luell Lys Glu Ala Ile Glu Thir Ile Wall Ala Ala Met Ser Asn 105 11 O Lell Wall Pro Pro Wall Glu Lell Ala Asn Pro Glu Asn Glin Phe Arg Wall 115 12 O 125 Asp Tyr Ile Luell Ser Wall Met Asn Wall Pro ASn Phe Asp Phe Pro Pro 13 O 135 14 O Glu Phe Glu His Ala Ala Luell Trp Glu Asp Glu Gly Wall Arg 145 150 155 160 Ala Glu Arg Ser Asn Glu Glin Luell Ile Asp Ala Glin 1.65 17O Phe Luell Asp Lys Ile Asp Wall Ile Glin Ala Asp Tyr Wall Pro 18O 185 19 O Ser Asp Glin Asp Lieu. Luell Arg Cys Arg Wall Luell Thir Ser Gly Ile Phe 195 2O5 Glu Thir Phe Glin Wall Asp Wall Asn Phe His Met Phe Asp Wall 21 O 215 22O Gly Gly Glin Arg Asp Glu Arg Arg Trp Ile Glin Phe Asn Asp 225 23 O 235 24 O Wall Thir Ala Ile Ile Phe Wall Wall Ala Ser Ser Ser Asn Met Wall 245 250 255 Ile Arg Glu Asp ASn Glin Thir Asn Arg Luell Glin Glu Ala Luell Asn Luell 26 O 265 27 O US 9,695,227 B2 133 134 - Continued Phe Llys Ser Ile Trp Asn Asn Arg Trp Luell Arg Thir Ile Ser Wall Ile 27s 28O 285 Lell Phe Lieu. Asn Llys Glin Asp Lieu. Luell Ala Glu Lys Wall Luell Ala Gly 29 O 295 3 OO Lys Ser Lys Ile Glu Asp Tyr Phe Pro Glu Phe Ala Arg Thir Thir 3. OS 310 315 Pro Glu Asp Ala Thr Pro Glu Pro Gly Glu Asp Pro Arg Wall Thir Arg 3.25 330 335 Ala Lys Tyr Phe Ile Arg Asp Glu Phe Luell Arg Ile Ser Thir Ala Ser 34 O 345 35. O Gly Asp Gly Arg His Pro His Phe Thir Cys Ala Wall Asp 355 360 365 Thir Glu Asn. Ile Arg Arg Val Phe Asn Asp Arg Asp Ile Ile Glin 37 O 375 38O Arg Met His Lieu. Arg Gln Tyr Glu Luell Luell 385 390 <210s, SEQ ID NO 61 &211s LENGTH: 394 212. TYPE: PRT &213s ORGANISM: Mus musculus <4 OOs, SEQUENCE: 61 Met Gly Cys Lieu. Gly Asn Ser Lys Thir Glu Asp Glin Arg Asn Glu Glu 1. 5 1O 15 Lys Ala Glin Arg Glu Ala Asn Lys Lys Ile Glu Glin Luell Glin 2O 25 30 Asp Lys Glin Val Tyr Arg Ala Thr His Arg Luell Lell Lell Luell Gly Ala 35 4 O 45 Gly Glu Ser Gly Lys Ser Thir Ile Wall Glin Met Arg Ile Luell His SO 55 6 O Wall Asin Gly Phe Asn Gly Glu Gly Gly Glu Glu Asp Pro Glin Ala Ala 65 70 Arg Ser Asn. Ser Asp Gly Glu Lys Ala Thir Wall Glin Asp Ile 85 90 95 Asn Asn Lieu Lys Glu Ala Ile Glu Thir Ile Wall Ala Ala Met Ser Asn 1OO 105 11 O Lell Wall Pro Pro Wall Glu Lieu. Ala Asn Pro Glu Asn Glin Phe Arg Wall 115 12 O 125 Asp Tyr Ile Leu Ser Wal Met Asn Wall Pro ASn Phe Asp Phe Pro Pro 13 O 135 14 O Glu Phe Tyr Glu. His Ala Lys Ala Luell Trp Glu Asp Glu Gly Wall Arg 145 150 155 160 Ala Cys Tyr Glu Arg Ser Asn. Glu Tyr Glin Luell Ile Asp Ala Glin 1.65 17O Phe Lieu. Asp Llys Ile Asp Val Ile Glin Ala Asp Tyr Wall Pro 18O 185 19 O Ser Asp Glin Asp Lieu Lieu. Arg Cys Arg Wall Luell Thir Ser Gly Ile Phe 195 2OO 2O5 Glu Thr Llys Phe Glin Val Asp Llys Wall Asn Phe His Met Phe Asp Wall 21 O 215 22O Gly Gly Glin Arg Asp Glu Arg Arg Trp Ile Glin Phe Asn Asp 225 23 O 235 24 O Wall Thir Ala Ile Ile Phe Wal Wall Ala Ser Ser Ser Asn Met Wall 245 250 255 US 9,695,227 B2 135 136 - Continued Ile Arg Glu Asp Asn Glin Thir Asn Arg Luell Glin Glu Ala Luell Asn Luell 26 O 265 27 O Phe Ser Ile Trp Asn Asn Arg Trp Luell Arg Thir Ile Ser Wall Ile 27s 285 Lell Phe Luell Asn Glin Asp Luell Luell Ala Glu Lys Wall Luell Ala Gly 29 O 295 3 OO Lys Ser Ile Glu Asp Phe Pro Glu Phe Ala Arg Thir Thir 3. OS 310 315 Pro Glu Ala Thir Pro Glu Pro Gly Glu Asp Pro Arg Wall Thir Arg 3.25 330 335 Ala Phe Ile Arg Asp Glu Phe Luell Arg Ile Ser Thir Ala Ser 34 O 345 35. O Gly Asp Gly Arg His Tyr Pro His Phe Thir Cys Ala Wall Asp 355 360 365 Thir Glu Asn Ile Arg Arg Wall Phe Asn Asp Arg Asp Ile Ile Glin 37 O 375 38O Arg Met His Luell Arg Glin Tyr Glu Luell Luell 385 390 SEQ ID NO 62 LENGTH: 34 O TYPE : PRT ORGANISM: Bos talurus < 4 OOs SEQUENCE: 62 Met Ser Glu Lieu. Asp Gln Lieu Arg Gln Glu Ala Glu Gln Luell Lys Asn 1. 5 15 Glin Ile Arg Asp Ala Arg Ala Cys Ala Asp Ala Thir Luell Ser Glin 25 3O Ile Thir Asn Asn Ile Asp Pro Wall Gly Arg Ile Glin Met Arg Thir Arg 35 4 O 45 Arg Thir Luell Arg Gly His Lell Ala Ile Ala Met His Trp Gly SO 55 6 O Thir Asp Ser Arg Lell Lell Wall Ser Ala Ser Glin Asp Gly Luell Ile 65 70 Ile Trp Asp Ser Tyr Thir Thir Asn Wall His Ala Ile Pro Luell Arg 85 90 95 Ser Ser Trp Wall Met Thir Ala Tyr Ala Pro Ser Gly Asn Wall 105 11 O Ala Gly Gly Lell Asp Asn Ile Ser Ile Asn Luell Thir 115 12 O 125 Arg Glu Gly Asn Wall Arg Wall Ser Arg Glu Luell Ala Gly His Thir Gly 13 O 135 14 O Tyr Luell Ser Arg Phe Luell Asp Asp ASn Glin Ile Wall Thir Ser 145 150 155 160 Ser Gly Asp Thir Thir Ala Luell Trp Asp Ile Glu Thir Gly Glin Glin 1.65 17O 17s Thir Thir Thir Phe Thir Gly His Thir Gly Asp Wall Met Ser Luell Ser Luell 18O 185 19 O Ala Pro Asp Thir Arg Lell Phe Wall Ser Gly Ala Asp Ala Ser Ala 195 Luell Trp Asp Wall Arg Glu Gly Met Arg Glin Thir Phe Thir Gly 21 O 215 22O His Glu Ser Asp Ile Asn Ala Ile Phe Phe Pro Asn Gly Asn Ala US 9,695,227 B2 137 138 - Continued 225 23 O 235 24 O Phe Ala Thr Gly Ser Asp Asp Ala Thir Cys Arg Lell Phe Asp Luell Arg 245 250 255 Ala Asp Glin Glu Lieu Met Thr Tyr Ser His Asp Asn Ile Ile Gly 26 O 265 27 O Ile Thr Ser Val Ser Phe Ser Lys Ser Gly Arg Lell Lell Luell Ala Gly 27s 28O 285 Tyr Asp Asp Phe Asn. Cys Asn. Wall Trp Asp Ala Lell Ala Asp Arg 29 O 295 3 OO Ala Gly Val Lieu Ala Gly His Asp Asn Arg Wall Ser Luell Gly Wall 3. OS 310 315 Thir Asp Asp Gly Met Ala Val Ala Thir Gly Ser Trp Asp Ser Phe Luell 3.25 330 335 Lys Ile Trp Asn 34 O <210s, SEQ ID NO 63 &211s LENGTH: 34 O 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 63 Met Ser Glu Lieu. Asp Gln Lieu. Arg Glin Glu Ala Glu Glin Luell Lys Asn 1. 5 15 Glin Ile Arg Asp Ala Arg Lys Ala Cys Ala Asp Ala Thir Luell Ser Glin 2O 25 3O Ile Thr Asn Asn Ile Asp Pro Val Gly Arg Ile Glin Met Arg Thir Arg 35 4 O 45 Arg Thr Lieu. Arg Gly. His Lieu Ala Ile Ala Met His Trp Gly SO 55 6 O Thir Asp Ser Arg Lieu. Lieu Val Ser Ala Ser Glin Asp Gly Luell Ile 65 70 Ile Trp Asp Ser Tyr Thr Thr Asn Wall His Ala Ile Pro Luell Arg 85 90 95 Ser Ser Trp Val Met Thr Cys Ala Tyr Ala Pro Ser Gly Asn Tyr Wall 1OO 105 11 O Ala Cys Gly Gly Lieu. Asp Asn. Ile Ser Ile Asn Luell Thir 115 12 O 125 Arg Glu Gly Asn Val Arg Val Ser Arg Glu Luell Ala Gly His Thir Gly 13 O 135 14 O Tyr Lieu. Ser Cys Cys Arg Phe Lieu Asp Asp ASn Glin Ile Wall Thir Ser 145 150 155 160 Ser Gly Asp Thir Thr Cys Ala Lieu Trp Asp Ile Glu Thir Gly Glin Glin 1.65 17O 17s Thir Thr Thr Phe Thr Gly His Thr Gly Asp Wall Met Ser Luell Ser Luell 18O 185 19 O Ala Pro Asp Thr Arg Lieu. Phe Val Ser Gly Ala Asp Ala Ser Ala 195 2OO Llys Lieu. Trp Asp Val Arg Glu Gly Met Arg Glin Thir Phe Thir Gly 21 O 215 22O His Glu Ser Asp Ile Asn Ala Ile Phe Phe Pro Asn Gly Asn Ala 225 23 O 235 24 O Phe Ala Thr Gly Ser Asp Asp Ala Thir Cys Arg Lell Phe Asp Luell Arg 245 250 255 US 9,695,227 B2 139 140 - Continued Ala Asp Glin Glu Lell Met Thir Ser His Asp Asn Ile Ile Cys Gly 26 O 265 27 O Ile Thir Ser Wall Ser Phe Ser Lys Ser Gly Arg Lell Lell Luell Ala Gly 27s 28O 285 Asp Asp Phe Asn Asn Wall Trp Asp Ala Lell Ala Asp Arg 29 O 295 3 OO Ala Gly Wall Luell Ala Gly His Asp Asn Arg Wall Ser Luell Gly Wall 3. OS 310 315 Thir Asp Asp Gly Met Ala Wall Ala Thir Gly Ser Trp Asp Ser Phe Luell 3.25 330 335 Ile Trp Asn 34 O SEQ ID NO 64 LENGTH: 34 O TYPE : PRT ORGANISM: Rattus norvegicus < 4 OOs SEQUENCE: 64 Met Ser Glu Lieu. Asp Glin Lell Arg Glin Glu Ala Glu Glin Luell Lys Asn 1. 5 15 Glin Ile Arg Asp Ala Arg Ala Cys Ala Asp Ala Thir Luell Ser Glin 25 3O Ile Thir Asn Asn Ile Asp Pro Wall Gly Arg Ile Glin Met Arg Thir Arg 35 4 O 45 Arg Thir Luell Arg Gly His Lell Ala Ile Tyr Ala Met His Trp Gly 50 55 60 Thir Asp Ser Arg Lell Lell Wall Ser Ala Ser Glin Asp Gly Luell Ile 65 70 Ile Trp Asp Ser Tyr Thir Thir Asn Wall His Ala Ile Pro Luell Arg 85 90 95 Ser Ser Trp Wall Met Thir Ala Tyr Ala Pro Ser Gly Asn Wall 105 11 O Ala Gly Gly Lell Asp Asn Ile Ser Ile Asn Luell Thir 115 12 O 125 Arg Glu Gly Asn Wall Arg Wall Ser Arg Glu Luell Ala Gly His Thir Gly 13 O 135 14 O Tyr Luell Ser Arg Phe Luell Asp Asp ASn Glin Ile Wall Thir Ser 145 150 155 160 Ser Gly Asp Thir Thir Ala Luell Trp Asp Ile Glu Thir Gly Glin Glin 1.65 17O 17s Thir Thir Thir Phe Thir Gly His Thir Gly Asp Wall Met Ser Luell Ser Luell 18O 185 19 O Ala Pro Asp Thir Arg Lell Phe Wall Ser Gly Ala Asp Ala Ser Ala 195 2OO Luell Trp Asp Wall Arg Glu Gly Met Arg Glin Thir Phe Thir Gly 21 O 215 His Glu Ser Asp Ile Asn Ala Ile Phe Phe Pro Asn Gly Asn Ala 225 23 O 235 24 O Phe Ala Thir Gly Ser Asp Asp Ala Thir Cys Arg Lell Phe Asp Luell Arg 245 250 255 Ala Asp Glin Glu Lell Met Thir Ser His Asp Asn Ile Ile Gly 26 O 265 27 O Ile Thir Ser Wall Ser Phe Ser Lys Ser Gly Arg Lell Lell Luell Ala Gly 27s 28O 285 US 9,695,227 B2 141 142 - Continued Tyr Asp Asp Phe Asn Cys Asn Val Trp Asp Ala Lieu Lys Ala Asp Arg 29 O 295 3 OO Ala Gly Val Luell Ala Gly His Asp Asn Arg Wall Ser Cys Lieu. Gly Wall 3. OS 310 315 32O Thir Asp Asp Gly Met Ala Wall Ala Thir Gly Ser Trp Asp Ser Phe Luell 3.25 330 335 Ile Trp Asn 34 O <210s, SEQ ID NO 65 &211s LENGTH: 34 O 212. TYPE : PRT &213s ORGANISM: Mus musculus <4 OOs, SEQUENCE: 65 Met Ser Glu Luell Asp Glin Lell Arg Glin Glu Ala Glu Glin Luell Lys Asn 1. 5 15 Glin Ile Arg Asp Ala Arg Ala Cys Ala Asp Ala Thir Luell Ser Glin 25 3O Ile Thir Asn Asn Ile Asp Pro Wall Gly Arg Ile Glin Met Arg Thir Arg 35 4 O 45 Arg Thir Luell Arg Gly His Lell Ala Ile Ala Met His Trp Gly SO 55 6 O Thir Asp Ser Arg Lell Lell Wall Ser Ala Ser Glin Asp Gly Luell Ile 65 70 Ile Trp Asp Ser Tyr Thr Thr Asn Lys Wall His Ala Ile Pro Lieu Arg 85 90 95 Ser Ser Trp Wall Met Thir Ala Tyr Ala Pro Ser Gly Asn Wall 105 11 O Ala Gly Gly Lell Asp Asn Ile Ser Ile Asn Luell Thir 115 12 O 125 Arg Glu Gly Asn Wall Arg Wall Ser Arg Glu Luell Ala Gly His Thir Gly 13 O 135 14 O Tyr Luell Ser Arg Phe Luell Asp Asp ASn Glin Ile Wall Thir Ser 145 150 155 160 Ser Gly Asp Thir Thir Ala Luell Trp Asp Ile Glu Thir Gly Glin Glin 1.65 17O 17s Thir Thir Thir Phe Thir Gly His Thir Gly Asp Wall Met Ser Luell Ser Luell 18O 185 19 O Ala Pro Asp Thir Arg Lell Phe Wall Ser Gly Ala Asp Ala Ser Ala 195 Luell Trp Asp Wall Arg Glu Gly Met Arg Glin Thir Phe Thir Gly 21 O 215 22O His Glu Ser Asp Ile Asn Ala Ile Phe Phe Pro Asn Gly Asn Ala 225 23 O 235 24 O Phe Ala Thir Gly Ser Asp Asp Ala Thir Cys Arg Lell Phe Asp Luell Arg 245 250 255 Ala Asp Glin Glu Lell Met Thir Ser His Asp Asn Ile Ile Gly 26 O 265 27 O Ile Thir Ser Wall Ser Phe Ser Lys Ser Gly Arg Lell Lell Luell Ala Gly 27s 28O 285 Asp Asp Phe Asn Asn Wall Trp Asp Ala Lell Ala Asp Arg 29 O 295 3 OO Ala Gly Wall Luell Ala Gly His Asp Asn Arg Wall Ser Luell Gly Wall US 9,695,227 B2 143 144 - Continued 3. OS 310 315 32O Thir Asp Asp Gly Met Ala Val Ala Thr Gly Ser Trp Asp Ser Phe Lieu. 3.25 330 335 Lys Ile Trp Asn 34 O <210s, SEQ ID NO 66 &211s LENGTH: 71 212. TYPE: PRT &213s ORGANISM: Bos taurus <4 OOs, SEQUENCE: 66 Met Ala Ser Asn. Asn. Thir Ala Ser Ile Ala Glin Ala Arg Llys Lieu Val 1. 5 1O 15 Glu Gln Lieu Lys Met Glu Ala Asn. Ile Asp Arg Ile Llys Val Ser Lys 2O 25 3O Ala Ala Ala Asp Lieu Met Ala Tyr Cys Glu Ala His Ala Lys Glu Asp 35 4 O 45 Pro Leu Lieu. Thr Pro Val Pro Ala Ser Glu Asn Pro Phe Arg Glu Lys SO 55 6 O Llys Phe Phe Cys Ala Ile Lieu. 65 70 <210s, SEQ ID NO 67 &211s LENGTH: 71 212. TYPE: PRT <213s ORGANISM: Mus musculus <4 OO > SEQUENCE: 67 Met Ala Ser Asn. Asn. Thir Ala Ser Ile Ala Glin Ala Arg Llys Lieu Val 1. 5 1O 15 Glu Gln Lieu Lys Met Glu Ala Asn. Ile Asp Arg Ile Llys Val Ser Lys 2O 25 3O Ala Ala Ala Asp Lieu Met Ala Tyr Cys Glu Ala His Ala Lys Glu Asp 35 4 O 45 Pro Leu Lieu. Thr Pro Val Pro Ala Ser Glu Asn Pro Phe Arg Glu Lys SO 55 6 O Llys Phe Phe Cys Ala Ile Lieu. 65 70 <210s, SEQ ID NO 68 &211s LENGTH: 71 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 68 Met Ala Ser Asn. Asn. Thir Ala Ser Ile Ala Glin Ala Arg Llys Lieu Val 1. 5 1O 15 Glu Gln Lieu Lys Met Glu Ala Asn. Ile Asp Arg Ile Llys Val Ser Lys 2O 25 3O Ala Ala Ala Asp Lieu Met Ala Tyr Cys Glu Ala His Ala Lys Glu Asp 35 4 O 45 Pro Leu Lieu. Thr Pro Val Pro Ala Ser Glu Asn Pro Phe Arg Glu Lys SO 55 6 O Llys Phe Phe Cys Ala Ile Lieu. 65 70 <210s, SEQ ID NO 69 US 9,695,227 B2 145 146 - Continued &211s LENGTH: 514 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: T4L-Beta2AR fusion construct <4 OOs, SEQUENCE: 69 Asp Tyr Lys Asp Asp Asp Asp Ala Glu Asn Lieu. Tyr Phe Glin Gly Asn 1. 5 1O 15 Ile Phe Glu Met Lieu. Arg Ile Asp Glu Gly Lieu. Arg Lieu Lys Ile Tyr 2O 25 3O Lys Asp Thr Glu Gly Tyr Tyr Thr Ile Gly Ile Gly His Leu Lieu. Thr 35 4 O 45 Llys Ser Pro Ser Lieu. Asn Ala Ala Lys Ser Glu Lieu. Asp Lys Ala Ile SO 55 6 O Gly Arg Asn. Thir Asn Gly Val Ile Thir Lys Asp Glu Ala Glu Lys Lieu. 65 70 7s 8O Phe Asin Glin Asp Wall Asp Ala Ala Val Arg Gly Ile Lieu. Arg Asn Ala 85 90 95 Llys Lieu Lys Pro Val Tyr Asp Ser Lieu. Asp Ala Val Arg Arg Ala Ala 1OO 105 11 O Lieu. Ile Asn Met Val Phe Gln Met Gly Glu Thr Gly Val Ala Gly Phe 115 12 O 125 Thir Asn. Ser Lieu. Arg Met Lieu. Glin Glin Lys Arg Trp Asp Glu Ala Ala 13 O 135 14 O Val Asn Lieu Ala Lys Ser Arg Trp Tyr Asn Glin Thr Pro Asn Arg Ala 145 150 155 16 O Lys Arg Val Ile Thr Thr Phe Arg Thr Gly. Thir Trp Asp Ala Tyr Ala 1.65 17O 17s Ala Asp Glu Val Trp Val Val Gly Met Gly Ile Val Met Ser Lieu. Ile 18O 185 19 O Val Lieu Ala Ile Val Phe Gly Asn Val Lieu Val Ile Thr Ala Ile Ala 195 2OO 2O5 Llys Phe Glu Arg Lieu Gln Thr Val Thr Asn Tyr Phe Ile Thr Ser Lieu. 21 O 215 22O Ala Cys Ala Asp Lieu Val Met Gly Lieu Ala Val Val Pro Phe Gly Ala 225 23 O 235 24 O Ala His Ile Lieu. Thir Lys Thir Trp Thr Phe Gly Asn Phe Trp Cys Glu 245 250 255 Phe Trp Thr Ser Ile Asp Val Lieu. Cys Val Thr Ala Ser Ile Glu Thr 26 O 265 27 O Lieu. Cys Val Ile Ala Val Asp Arg Tyr Phe Ala Ile Thr Ser Pro Phe 27s 28O 285 Llys Tyr Glin Ser Lieu. Lieu. Thir Lys Asn Lys Ala Arg Val Ile Ile Lieu. 29 O 295 3 OO Met Val Trp Ile Val Ser Gly Lieu. Thir Ser Phe Leu Pro Ile Glin Met 3. OS 310 315 32O His Trp Tyr Arg Ala Thr His Glin Glu Ala Ile Asn. Cys Tyr Ala Glu 3.25 330 335 Glu Thr Cys Cys Asp Phe Phe Thr Asn Glin Ala Tyr Ala Ile Ala Ser 34 O 345 35. O Ser Ile Val Ser Phe Tyr Val Pro Leu Val Ile Met Val Phe Val Tyr 355 360 365 Ser Arg Val Phe Glin Glu Ala Lys Arg Glin Lieu. Glin Lys Ile Asp Llys 37 O 375 38O US 9,695,227 B2 147 148 - Continued Ser Glu Gly Arg Phe His Wall Glin Asn Luell Ser Glin Wall Glu Glin Asp 385 390 395 4 OO Gly Arg Thir Gly His Gly Lell Arg Arg Ser Ser Phe Luell 4 OS 415 Glu His Ala Lell Thir Luell Gly Ile Ile Met Gly Thir Phe Thir 425 43 O Lell Trp Luell Pro Phe Phe Ile Wall Asn Ile Wall His Wall Ile Glin 435 44 O 445 Asp Asn Luell Ile Arg Glu Wall Ile Luell Lell Asn Trp Ile Gly 450 45.5 460 Tyr Wall Asn Ser Gly Phe Asn Pro Luell Ile Tyr Cys Arg Ser Pro Asp 465 470 Phe Arg Ile Ala Phe Glin Glu Luell Luell Cys Luell Arg Arg Ser Ser Luell 485 490 495 Ala Gly Asn Gly Ser Ser Asn Gly Asn Thir Gly Glu Glin SOO 505 51O Ser Gly SEQ ID NO 70 LENGTH: 8 TYPE : PRT ORGANISM: Artificial FEATURE: OTHER INFORMATION: M1 Flag epitope <4 OOs, SEQUENCE: 70 Asp Tyr Lys Asp Asp Asp Asp Ala 1. 5 SEO ID NO 71 LENGTH: 7 TYPE : PRT ORGANISM: Artificial FEATURE: OTHER INFORMATION: TEV protease recognition sequence <4 OOs, SEQUENCE: 71. Glu Asn Lieu. Tyr Phe Glin Gly 1. 5 <210s, SEQ ID NO 72 &211s LENGTH: 413 212. TYPE : PRT &213s ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 72 Met Gly Glin Pro Gly Asn Gly Ser Ala Phe Luell Lell Ala Pro Asn Gly 1. 5 1O 15 Ser His Ala Pro Asp His Asp Wall Thir Glin Glu Arg Asp Glu Wall Trp 25 Wall Wall Gly Met Gly Ile Wall Met Ser Luell Ile Wall Lell Ala Ile Wall 35 4 O 45 Phe Gly Asn Wall Lell Wall Ile Thir Ala Ile Ala Lys Phe Glu Arg Luell SO 55 6 O Glin Thir Wall Thir Asn Tyr Phe Ile Thir Ser Luell Ala Ala Asp Luell 65 70 Wall Met Gly Luell Ala Wall Wall Pro Phe Gly Ala Ala His Ile Luell Met 85 90 95 US 9,695,227 B2 149 150 - Continued Met Trp Thir Phe Gly Asn Phe Trp Glu Phe Trp Thir Ser Ile 1OO 105 11 O Asp Wall Luell Wall Thir Ala Ser Ile Glu Thir Lell Cys Wall Ile Ala 115 12 O 125 Wall Asp Arg Phe Ala Ile Thir Ser Pro Phe Lys Tyr Glin Ser Luell 13 O 135 14 O Lell Thir Asn Ala Arg Wall Ile Ile Luell Met Wall Trp Ile Wall 145 150 155 160 Ser Gly Luell Thir Ser Phe Lell Pro Ile Glin Met His Trp Arg Ala 1.65 17O Thir His Glin Glu Ala Ile Asn Tyr Ala ASn Glu Thir Cys Asp 18O 185 19 O Phe Phe Thir Asn Glin Ala Ala Ile Ala Ser Ser Ile Wall Ser Phe 195 2OO Wall Pro Luell Wall Ile Met Wall Phe Wall Ser Arg Wall Phe Glin 21 O 215 22O Glu Ala Arg Glin Lell Glin Ile Asp Lys Ser Glu Gly Arg Phe 225 23 O 235 24 O His Wall Glin Asn Lell Ser Glin Wall Glu Glin Asp Gly Arg Thir Gly His 245 250 255 Gly Luell Arg Arg Ser Ser Phe Cys Luell Lys Glu His Lys Ala Luell 26 O 265 27 O Thir Luell Gly Ile Ile Met Gly Thir Phe Thir Lell Cys Trp Luell Pro 285 Phe Phe Ile Wall Asn Ile Wall His Wall Ile Gln Asp Asn Luell Ile Arg 29 O 295 3 OO Lys Glu Wall Ile Lell Lell Asn Trp Ile Gly Tyr Wall Asn Ser Gly 3. OS 310 315 Phe Asn Pro Luell Ile Arg Ser Pro Asp Phe Arg Ile Ala Phe 3.25 330 335 Glin Glu Luell Luell Lell Arg Arg Ser Ser Luell Ala Tyr Gly Asn 34 O 345 35. O Gly Tyr Ser Ser Asn Gly Asn Thir Gly Glu Glin Ser Gly His Wall 355 360 365 Glu Glin Glu Glu Asn Lys Luell Luell Glu Asp Lell Pro Gly Thir 37 O 375 38O Glu Asp Phe Wall Gly His Glin Gly Thir Wall Pro Ser Asp Asn Ile Asp 385 390 395 4 OO Ser Glin Gly Arg Asn Cys Ser Thir Asn Asp Ser Lell Lell 4 OS 41O SEO ID NO 73 LENGTH: 539 TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: N-terminal T4LW2R construct SEQUENCE: 73 Asp Tyr Lys Asp Asp Asp Asp Ala Gly Lieu Met Ala Ser Thr Thir Ser 1. 5 15 Ala Val Pro Gly His Pro Ser Leu Pro Ser Leul Pro Ser Asn Ser Ser 25 Glin Glu Arg Pro Lieu. Asp Thr Asn. Ile Phe Glu Met Lieu. Arg Ile Asp 35 4 O 45