US 2011 0166330A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0166330 A1 Kobilka et al. (43) Pub. Date: Jul. 7, 2011 (54) GPCR CRYSTALIZATION METHOD USING (60) Provisional application No. 60/980,122, filed on Oct. AN ANTIBODY 15, 2007. (76) Inventors: Brian Kobilka, Palo Alto, CA Publication Classification (US); Daniel Rohrer, Los Gatos, CA (US); Peter Brams, (51) Int. Cl. Sacramento, CA (US); Asna C07K 6/00 (2006.01) Masood, Saratoga, CA (US) (52) U.S. Cl. ................................... 530/387.3; 530/388.1 (21) Appl. No.: 12/931,528 (57) ABSTRACT (22) Filed: Feb. 2, 2011 An antibody that specifically binds a three dimensional Related U.S. Application Data epitope on the IC3 loop of a GPCR is provided. The antibody AV may be employed in a method that comprises: contacting a (62) Division of application No. 12/284.245, filed on Sep. GPCR with a monovalent version of the antibody binding 19, 2008, now Pat. No. 7,947,807. conditions to form a complex; and crystallizing the complex. Patent Application Publication Jul. 7, 2011 Sheet 1 of 8 US 2011/O166330 A1 Patent Application Publication Jul. 7, 2011 Sheet 2 of 8 US 2011/O166330 A1 8 : & is 8 & k x x X x : ·xxx x8: ; &š83 : 8888 X Patent Application Publication Jul. 7, 2011 Sheet 3 of 8 US 2011/O166330 A1 *& Patent Application Publication Jul. 7, 2011 Sheet 4 of 8 US 2011/O166330 A1 *iopetreatized termeabized 38-88 &: 88: 83-88 A: Patent Application Publication Jul. 7, 2011 Sheet 5 of 8 US 2011/O166330 A1 38 33: s: 8: 3. 8: wporogopogos 33-3-3-3-3-3-3-3-3-3-3-3-3-3 3. *:::::::::::: Patent Application Publication Jul. 7, 2011 Sheet 6 of 8 US 2011/O166330 A1 Patent Application Publication Jul. 7, 2011 Sheet 7 of 8 US 2011/O166330 A1 Patent Application Publication Jul. 7, 2011 Sheet 8 of 8 US 2011/O166330 A1 US 2011/0166330 A1 Jul. 7, 2011 GPCR CRYSTALIZATION METHOD USING of the IC3 loop of said GPCR is also provided. This method AN ANTIBODY may further comprise isolating a hybridoma line that pro duces the antibody. GOVERNMENT RIGHTS 0008. A method of making GPCR-containing vesicular antigens is also provided, as is a method of Screening hybri 0001. This work was supported in part by federal grant domas for the production of antibodies that bind to GPCR number NS28471 from the National Institutes of Health. The containing vesicles. federal government has certain rights in this invention. 0009. A method of using the antibody as a treatment for a GPCR-mediated disorder is also provided. BACKGROUND 0002 G protein-coupled receptor (GPCR) signaling plays BRIEF DESCRIPTION OF THE FIGURES a vital role in a number of physiological contexts including, 0010. The patent or application file contains at least one but not limited to, metabolism, inflammation, neuronal func drawing executed in color. Copies of this patent or patent tion, and cardiovascular function. For instance, GPCRs application publication with color drawing(s) will be pro include receptors for biogenic amines, e.g., dopamine, epi vided by the Office upon request and payment of the neces nephrine, histamine, glutamate, acetylcholine, and serotonin; sary fee. for purines such as ADP and ATP; for the vitamin niacin; for 0011 FIG. 1 is a schematic illustration of a GPCR, show lipid mediators of inflammation is such as prostaglandins, ing the canonical transmembrane regions (TM1, TM2, TM3. lipoxins, platelet activating factor, and leukotrienes; for pep TM4, TM5, TM6, and TM7), intracellular regions (IC1, IC2, tide hormones Such as calcitonin, follicle stimulating hor and IC3), and extracellular regions (EC1, EC2, and EC3). mone, gonadotropin releasing hormone, ghrelin, motilin, 0012 FIGS. 2A and 2B. Binding characteristics of BAR neurokinin, and oxytocin, for non-hormone peptides such as specific antibodies. 2A, Dot-blots showing binding of nine beta-endorphin, dynorphin A, Leu-enkephalin, and Met-en BAR specific antibodies to denatured receptor. Equal kephalin; for the non-peptide hormone melatonin; for amounts of BAR denatured with SDS and B-mercaptoetha polypeptides such as C5a anaphylatoxin and chemokines; for nol were spotted in triplicate on nitrocellulose strips. The proteases such as thrombin, trypsin, and factor Xa; and for strips were blocked with 5% non-fat dry milk in PBS-Tween sensory signal mediators, e.g., retinal photopigments and (0.05% Tween-20) and then probed with 1 mg/ml of the olfactory stimulatory molecules. GPCRs are of immense indicated antibodies diluted in blocking buffer. Binding of the interest for drug development. primary antibody to the denatured BAR was detected with an 0003. Efforts to crystallize GPCRs have been frustrated by Alexa-688 labeled anti-mouse secondary antibody. The top intrinsic characteristics of integral membrane proteins. panel is a graphical representation of the average dot intensity Bovine rhodopsin is the only GPCR for which a high-resolu from three independent experiments. The lower panel is a tion structure has been determined by X-ray crystallography; representative experiment. The binding of all 9 antibodies to and this is in part due to its natural abundance and atypical denatured fAR is reduced compared to M1 binding to the stability. The seven hydrophobic transmembrane helices of linear Flag epitope. 2B, Dose-response curves showing the GPCRs make poor surfaces for crystal contacts, and the extra effect on increasing amounts of is antibodies 4-9 on the fluo cellular and intracellular domains are often relatively short rescence of BAR labeled at C265 with tetramethyl and/or poorly structured. rhodamine (BAR-TMR). BAR-TMR was diluted to 4 nM in 500 uL of buffer consisting of 0.1% dodecylmaltoside, 100 SUMMARY OF THE INVENTION mM sodium chloride, and 20 mM HEPES buffer (pH 7.5). Data represent an in of 3. 0004 An antibody that specifically binds a three dimen (0013 FIGS. 3A and 3B. Fab 5 binds IC3 (labeled as IL3) sional epitope of the IC3 loop of a GPCR is provided. In of the BAR but does not effect structural changes associated certain embodiments, the antibody may specifically bind to with G protein activation. 3A, Western blot analysis of the the IC3 loop of the B2 adrenoreceptor. Also provided is a BAR digested with trypsin in the absence and presence of complex comprising a GPCR and a monovalent antibody that Fab 5. The fragmented fAR was visualized using an Alexa binds to a three dimensional epitope of the IC3 loop of that 680 labeled M1 antibody against the N-terminal Flag epitope. GPCR. The complex may be in a crystalline form. In the absence of Fab 5, two fragments at approximately 27 0005. A method is also provided. In general terms, the and 29 kDa appear corresponding to cleavage in the third method comprises: a) contacting a GPCR with a monovalent intracellular loop. In contrast, the presence of Fab 5 protects antibody that specifically binds to a three dimensional epitope the N-terminal end of the loop thus leaving the larger of the of the IC3 loop of a GPCR under binding conditions to form two fragments. The diagram shows the third intracellular loop a complex; and b) crystallizing the complex. In one embodi connecting TM5 and TM6. The loop is marked with MW ment, the GPCR comprises the B2ARIC3 loop, and the Fab indicators corresponding to N-terminal fragments containing fragment may bind to the B2AR IC3 loop. The GPCR may the Flag epitope. Residues sensitive to trypsin digest are also be a hybrid GPCR that contains the IC3 loop of B2AR. shown in red. 3B. The change in the bimane fluorescence of 0006. Also provided is a method comprising reconstitut BAR labeled with monobromobimane at H271C at the cyto ing a GPCR in artificial phospholipid vesicles to make an plasmic end of TM6. Bimane fluorescence is quenched by antigen; and immunizing an animal with the antigen. The W135 at the cytoplasmic end of TM3 upon agonist binding. animal may be a rabbit, mouse or chicken, for example. The response to the full agonist isoproterenol, isoproterenol 0007. A method comprising screening a plurality of hybri plus Fab 5, and Fab5 alone are shown. Fluorescence intensity doma lines obtained from an animal immunized with phos was corrected for background fluorescence from buffer and pholipid vesicles comprising a GPCR for a hybridoma that ligands in all experiments. The data are the mean+S.E. of two produces an antibody that bind to a three dimensional epitope independent experiments performed in triplicate. US 2011/0166330 A1 Jul. 7, 2011 0014 FIG. 4 Fluorescence images showing monoclonal nial, NY (1991) provide one of skill with general dictionaries antibody detection of flag tagged BAR stably expressed in of many of the terms used in this disclosure. Although any HEK-293 cells. HEK-293 cells stably expressing a flag methods and materials similar or equivalent to those tagged version of the BAR were cultured on coverslips and described herein can be used in the practice or testing of the processed for immunocytochemistry. After fixation, with 4% present invention, the preferred methods and materials are paraformaldehyde, the cells were washed and blocked with described. 2.5% goat serum in PBS alone (nonpermeabilized) or with 0020 All patents and publications, including all 0.5% NP-40 in PBS (permeabilized). The cells were incu sequences disclosed within Such patents and publications, bated with the BAR specific antibody indicated on the left referred to herein are expressly incorporated by reference. side of the figure. After several washes, the BAR monoclonal 0021 Numeric ranges are inclusive of the numbers defin antibodies were detected with a Texas Red conjugated anti ing the range.