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(12) United States Patent (10) Patent No.: US 9,695.227 B2 Steyaert Et Al

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(12) United States Patent (10) Patent No.: US 9,695.227 B2 Steyaert Et Al 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 FG, s: U.S. Patent Jul. 4, 2017 Sheet 2 of 23 US 9,695.227 B2 6 8 to 2 4 6 8 20 U.S. Patent Jul. 4, 2017 Sheet 3 of 23 US 9,695.227 B2 & 8 8 : 8 x : U.S. Patent Jul. 4, 2017 Sheet 4 of 23 US 9,695.227 B2 3 : : 3 : 3 &E. 8: &: 88: {y: 38883$38 (38A U.S. Patent Jul. 4, 2017 Sheet 7 of 23 US 9,695.227 B2 3. : . & socceedeaneroone rease econooxes beseecease sororobos sexotoacoocoooooooooooooooooooooooooooooooooooooooooooosexococoorcore 33 : 3. ;3 :8: ; : ; : U.S. Patent Jul. 4, 2017 Sheet 8 of 23 US 9,695.227 B2 FS. S. U.S. Patent Jul. 4, 2017 Sheet 9 of 23 US 9,695.227 B2 FS 8. 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A G, 8 U.S. Patent Jul. 4, 2017 Sheet 15 Of 23 US 9,695.227 B2 sigre 2 page of 2} Extracelia x88. {ytepiastic view U.S. Patent Jul. 4, 2017 Sheet 16 of 23 US 9,695.227 B2 Figure 2 page 2 of 2 F.G. 3 page of 2} U.S. Patent Jul. 4, 2017 Sheet 17 Of 23 US 9,695.227 B2 FS, 3 page 2 of 2} d U.S. Patent Jul. 4, 2017 Sheet 18 of 23 US 9,695.227 B2 F.G. & gytoplastair view tag down view U.S. Patent Jul. 4, 2017 Sheet 19 Of 23 US 9,695.227 B2 F. S U.S. Patent Jul. 4, 2017 Sheet 20 of 23 US 9,695.227 B2 S 2 S. s {f} 2. 3:3: Time (s 3. 9 S- i. S. too iss 3 80 : 3 x is: Time (sec) U.S. Patent Jul. 4, 2017 Sheet 21 of 23 US 9,695.227 B2 G, 8 (-32 - N835 or 3s eteroisires Time (s) U.S. Patent Jul. 4, 2017 Sheet 22 of 23 US 9,695.227 B2 : 83-888. 88: ::::::::::::::::::::::s U.S. Patent Jul. 4, 2017 Sheet 23 of 23 US 9,695.227 B2 28 88 *. 88: & ' ''. * 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.
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