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(12) United States Patent (10) Patent No.: US 7,947,807 B2 Kobilka Et Al

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(12) United States Patent (10) Patent No.: US 7,947,807 B2 Kobilka Et Al US007947807B2 (12) United States Patent (10) Patent No.: US 7,947,807 B2 Kobilka et al. (45) Date of Patent: May 24, 2011 (54) METHOD FOR OBTAINING G (56) References Cited PROTEIN-COUPLED RECEPTOR (GPCR) DIFFRACTION-QUALITY CRYSTALS U.S. PATENT DOCUMENTS EMPLOYING AMONOCLONAL ANTIBODY 5,026,557 A 6, 1991 Estis et al. THAT BINDS TO THE THIRD INTRACELLULAR LOOP (IL3) OTHER PUBLICATIONS Day, P.W., et al., 2007. A monoclonal antibody for Gprotein-coupled (75) Inventors: Brian Kobilka, Palo Alto, CA (US); receptor crystallography, Nat. Meth. 4(11):927-929.* Dan Rohrer, Los Gatos, CA (US); Peter b.le 2 R Ming,y proteins for structures: a trillion iny tweaks, NaI. Men. 42-434. East (SS). ASna Rosenbaum, D.M., et al., 2009. The structure and function of G-pro as00d, Saratoga, (US) tein-coupled receptors, Nature 459:356-363.* Chayen, N. E., and E. Saridakis, 2008, Protein crystallization: from (73) Assignee: The Board of Trustees of the Leland purified protein to diffraction-quality crystal, Nat. Meth. 5(2): 147 Stanford Junior University, Palo Alto, 153. CA (US) Mancia; et al., “Production and characterization of monoclonal anti bodies sensitive to conformation in the 5HT2c serotonin receptor'. c PNAS, Mar 13, 2007, 104(11):4303-43.08. (*) Notice: Subject to any disclaimer the term of this Yohannan; et al., “The evolution of transmembrane helix kinks and patent is extended or adjusted under 35 the structural diversity of G protein-coupled receptors'PNAS, Jan. U.S.C. 154(b) by 74 days. 27, 2004, 101(4):959-963. (b) by y Day; et al., “A monoclonal antibody for G protein-coupled receptor crystallography”, Nat Methods, 2007 Nov. 4 (11)927-9. (21) Appl. No.: 12/284.245 Kobilka; et al., “G protein coupled receptor structure and activation'. Biochim Biophys Acta, 2007 Apr. 1768(4) 794-807. (22) Filed: Sep.19, 2008 Whorton; etal. A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein. (65) Prior Publication Data * cited by examiner US 2009/O 14851.0 A1 Jun. 11, 2009 Primary Examiner — Jeffrey S. Parkin Related U.S. Application Data (74) Attorney, Agent, or Firm — Bozicevic, Field & Francis (60) Provisional application No. 60/980,122, filed on Oct. LLP; JaS S. KeddiCCC1 15, 2007. (57) ABSTRACT (51) Int. Cl An antibody that specifically binds a three dimensional CO 7k i4/00 (2006.01) epitope on the IC3 loop of a GPCR is provided. The antibody may be employed in a method that comprises: contacting a CI2P 2/08 (2006.01) GPCR with a monovalent version of the antibody binding- BOLD 9/00 (2006.01) conditions to form a complex; and crystallizing the complex. (52) U.S. Cl. ...................... 530/350; 530/388.22; 23/300 (58) Field of Classification Search ........................ None 8 Claims, 8 Drawing Sheets See application file for complete search history. (3 of 8 Drawing Sheet(s) Filed in Color) U.S. Patent May 24, 2011 Sheet 1 of 8 US 7,947,807 B2 | NS. g sisN sN U.S. Patent May 24, 2011 Sheet 2 of 8 US 7,947,807 B2 xxxx is (Ex: ese exie U.S. Patent May 24, 2011 Sheet 3 of 8 US 7,947,807 B2 3. 8 3. xx Y. 838888 &8388x38x888 U.S. Patent May 24, 2011 Sheet 4 of 8 US 7,947,807 B2 Nopenea U.S. Patent May 24, 2011 Sheet 5 of 8 US 7,947,807 B2 Time (min) O 40 8O 2O 60 -Yonymmemorror OO O4 O.8 12 16 Molar Ratio U.S. Patent May 24, 2011 Sheet 6 of 8 US 7,947,807 B2 U.S. Patent May 24, 2011 Sheet 7 of 8 US 7,947,807 B2 ?ensaubuxe|duosqe--avºggoºupped:: U.S. Patent May 24, 2011 Sheet 8 of 8 US 7,947,807 B2 kx & *a*s-s-s-s-s-s-xx-x-xx-xxxsssssssssssssss US 7,947,807 B2 1. 2 METHOD FOR OBTAINING G vesicles comprising a GPCR for a hybridoma that produces PROTEIN-COUPLED RECEPTOR (GPCR) an antibody that bind to a three dimensional epitope of the IC3 DIFFRACTION-QUALITY CRYSTALS loop of said GPCR is also provided. This method may further EMPLOYING AMONOCLONAL ANTIBODY comprise isolating a hybridoma line that produces the anti THAT BINDS TO THE THIRD body. INTRACELLULAR LOOP (IL3) A method of making GPCR-containing vesicular antigens is also provided, as is a method of screening hybridomas for GOVERNMENT RIGHTS the production of antibodies that bind to GPCR-containing vesicles. This work was supported in part by federal grant number 10 A method ofusing the antibody as a treatment for a GPCR NS28471 from the National Institutes of Health. The federal mediated disorder is also provided. government has certain rights in this invention. BRIEF DESCRIPTION OF THE FIGURES BACKGROUND 15 G protein-coupled receptor (GPCR) signaling plays a vital The patent or application file contains at least one drawing role in a number of physiological contexts including, but not executed in color. Copies of this patent or patent application limited to, metabolism, inflammation, neuronal function, and publication with color drawing(s) will be provided by the cardiovascular function. For instance, GPCRs include recep Office upon request and payment of the necessary fee. tors for biogenic amines, e.g., dopamine, epinephrine, hista FIG. 1 is a schematic illustration of a GPCR, showing the mine, glutamate, acetylcholine, and serotonin; for purines canonical transmembrane regions (TM1, TM2, TM3, TM4. such as ADP and ATP; for the vitamin niacin; for lipid media TM5, TM6, and TM7), intracellular regions (IC1, IC2, and tors of inflammation Such as prostaglandins, lipoxins, platelet IC3), and extracellular regions (EC1, EC2, and EC3). activating factor, and leukotrienes; for peptide hormones Such FIGS. 2A and 2B. Binding characteristics of BAR-spe as calcitonin, follicle stimulating hormone, gonadotropin 25 cific antibodies. 2A, Dot-blots showing binding of nine AR releasing hormone, ghrelin, motilin, neurokinin, and oxyto specific antibodies to denatured receptor. Equal amounts of cin; for non-hormone peptides such as beta-endorphin, BAR denatured with SDS and B-mercaptoethanol were spot dynorphin A. Leu-enkephalin, and Met-enkephalin; for the ted in triplicate on nitrocellulose strips. The strips were non-peptide hormone melatonin; for polypeptides such as blocked with 5% non-fat dry milk in PBS-Tween (0.05% C5a anaphylatoxin and chemokines; for proteases such as 30 Tween-20) and then probed with 1 mg/ml of the indicated thrombin, trypsin, and factor Xa, and for sensory signal antibodies diluted in blocking buffer. Binding of the primary mediators, e.g., retinal photopigments and olfactory stimula antibody to the denatured BAR was detected with an Alexa tory molecules. GPCRs are of immense interest for drug 688 labeled anti-mouse secondary antibody. The top panel is development. a graphical representation of the average dot intensity from Efforts to crystallize GPCRs have been frustrated by intrin 35 three independent experiments. The lower panel is a repre sic characteristics of integral membrane proteins. Bovine sentative experiment. The binding of all 9 antibodies to dena rhodopsin is the only GPCR for which a high-resolution tured AR is reduced compared to M1 binding to the linear structure has been determined by X-ray crystallography; and Flag epitope. 2B, Dose-responsecurves showing the effect on this is in part due to its natural abundance and atypical stabil increasing amounts of antibodies 4-9 on the fluorescence of ity. The seven hydrophobic transmembrane helices of GPCRs 40 f3AR labeled at C265 with tetramethylrhodamine (BAR make poor Surfaces for crystal contacts, and the extracellular TMR). BAR-TMR was diluted to 4 nM in 500 uL of buffer and intracellular domains are often relatively short and/or consisting of 0.1% dodecylmaltoside, 100 mM sodium chlo poorly structured. ride, and 20 mM HEPES buffer (pH 7.5). Data represent ann of 3. SUMMARY OF THE INVENTION 45 FIGS. 3A and 3B. Fab 5 binds IC3 (labeled as IL3) of the BAR but does not effect structural changes associated with G An antibody that specifically binds a three dimensional protein activation. 3A, Western blot analysis of the BAR epitope of the IC3 loop of a GPCR is provided. In certain digested with trypsin in the absence and presence of Fab 5. embodiments, the antibody may specifically bind to the IC3 The fragmented BAR was visualized using an Alexa-680 loop of the B2 adrenoreceptor. Also provided is a complex 50 labeled M1 antibody against the N-terminal Flag epitope. In comprising a GPCR and a monovalent antibody that binds to the absence of Fab 5, two fragments at approximately 27 and a three dimensional epitope of the IC3 loop of that GPCR. The 29 kDa appear corresponding to cleavage in the third intrac complex may be in a crystalline form. ellular loop. In contrast, the presence of Fab 5 protects the A method is also provided. In general terms, the method N-terminal end of the loop thus leaving the larger of the two comprises: a) contacting a GPCR with a monovalent antibody 55 fragments. The diagram shows the third intracellular loop that specifically binds to a three dimensional epitope of the connecting TM5 and TM6. The loop is marked with MW IC3 loop of a GPCR under binding conditions to form a indicators corresponding to N-terminal fragments containing complex; and b) crystallizing the complex. In one embodi the Flag epitope. Residues sensitive to trypsin digest are ment, the GPCR comprises the B2ARIC3 loop, and the Fab shown in red. 3B. The change in the bimane fluorescence of fragment may bind to the B2AR IC3 loop.
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