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 (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 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; J aS 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

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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 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. The GPCR may 60 BAR labeled with monobromobimane at H271C at the cyto also be a hybrid GPCR that contains the IC3 loop of B2AR. plasmic end of TM6. Bimane fluorescence is quenched by Also provided is a method comprising reconstituting a W135 at the cytoplasmic end of TM3 upon agonist binding. GPCR in artificial phospholipid vesicles to make an antigen; The response to the full agonist isoproterenol, isoproterenol and immunizing an animal with the antigen. The animal may plus Fab 5, and Fab5 alone are shown. Fluorescence intensity be a rabbit, mouse or chicken, for example. 65 was corrected for background fluorescence from buffer and A method comprising screening a plurality of hybridoma ligands in all experiments. The data are the mean+S.E. of two lines obtained from an animal immunized with phospholipid independent experiments performed in triplicate. US 7,947,807 B2 3 4 FIG.4 Fluorescence images showing monoclonal antibody similar or equivalent to those described herein can be used in detection of flag tagged AR stably expressed in HEK-293 the practice or testing of the present invention, the preferred cells. HEK-293 cells stably expressing a flag tagged version methods and materials are described. of the BAR were cultured on coverslips and processed for All patents and publications, including all sequences dis immunocytochemistry. After fixation, with 4% paraformal closed within such patents and publications, referred to herein dehyde, the cells were washed and blocked with 2.5% goat are expressly incorporated by reference. serum in PBS alone (nonpermeabilized) or with 0.5% NP-40 Numeric ranges are inclusive of the numbers defining the in PBS (permeabilized). The cells were incubated with the range. Unless otherwise indicated, nucleic acids are written BAR specific antibody indicated on the left side of the figure. left to right in 5' to 3' orientation; amino acid sequences are After several washes, the BAR monoclonal antibodies were 10 written left to right in amino to carboxy orientation, respec detected with a Texas Red conjugated anti-mouse secondary tively. antibody. To demonstrate that all of the cells are expressing The headings provided herein are not limitations of the the receptor they were Subsequently stained with an Alexa various aspects or embodiments of the invention which can be 488 conjugated M1 antibody (M1 column), which recognizes had by reference to the specification as a whole. Accordingly, the amino terminal flag tag on the BAR. Antibodies 1, 3, 4 15 the terms defined immediately below are more fully defined and 8 stain cells in the absence of permeabilization and there by reference to the specification as a whole. fore bind an epitope on the extracellular face of the receptor. “G-protein coupled receptors’, or “GPCRs are polypep In contrast, antibodies 2, 5, 6, 7 and 9 only stain cells that have tides that share a common structural motif, having seven been permeabilized indicative of an intracellular epitope. The regions of between 22 to 24 hydrophobic amino acids that images shown are representative of at least 3 experiments. All form seven alpha helices, each of which spans a membrane. images were acquired using an Axioplan 2 microscope (Carl As illustrated in FIG.1, each span is identified by number, i.e., Zeiss MicroImaging), fitted with a camera (RTE/CCD-1300 transmembrane-1 (TM1), transmembrane-2 (TM2), etc. The Y/HS; Roper Scientific) controlled using IPLab software (BD transmembrane helices are joined by regions of amino acids Bioscience). between transmembrane-2 and transmembrane-3, transmem FIG.5 Affinity of Fab 5 for purified BAR determined by 25 brane-4 and transmembrane-5, and transmembrane-6 and isothermal titration calorimetry (ITC). ITC measurements transmembrane-7 on the exterior, or “extracellular side, of were performed at 25°C. using a VP-ITC calorimeter (Micro the cell membrane, referred to as “extracellular regions 1, 2 cal, Inc.). The ITC cell contained 11 uM detergent-solubi and 3 (EC1, EC2 and EC3), respectively. The transmembrane lized BAR or BAR buffer (blank titration), and the injection helices are also joined by regions of amino acids between syringe contained 70 uM Fab5. Titrations were initiated with 30 transmembrane-1 and transmembrane-2, transmembrane-3 a 3 ul injection, followed by a series of 8 ul injections (37 and transmembrane-4, and transmembrane-5 and transmem BAR and 31 for BAR buffer blank), with 240 s between brane-6 on the interior, or “intracellular side, of the cell injections. After Subtracting the blank titration, the measured membrane, referred to as “intracellular regions 1, 2 and 3 heat released upon binding was plotted against the Fab: BAR (IC1, IC2 and IC3), respectively. The “carboxy” (“C”) termi molar ratio, and the data were fitted to a single-site binding 35 nus of the receptor lies in the intracellular space within the model to obtain the association constant Ka, enthalpy change cell, and the “amino” (“N) terminus of the receptor lies in the AH, and stoichiometry n using the Origin software package' extracellular space outside of the cell. GPCR structure and (Microcal, Inc.) classification is generally well known in the art, and further FIG. 6 Image of Fab 5-3AR-TMR crystals. BAR was discussion of GPCRs may be found in Probst, DNA Cell Biol. labeled with tetramethylrhodamine (BAR-TMR) and mixed 40 1992 11:1-20; Marchese et al Genomics 23: 609-618, 1994; with an excess of Fab 5. The complex was purified by size and the following books: Jürgen Wess (Ed) Structure-Func exclusion chromatography. Crystals were grown in bicelles tion Analysis of G Protein-Coupled Receptors published by and ammonium sulfate (Rasmussen etal manuscript). Images Wiley-Liss (1st edition; Oct. 15, 1999); Kevin R. Lynch (Ed) were obtained by light (panel A) and fluorescence (panel B) Identification and Expression of G Protein-Coupled Recep microscopy. The fluorescence confirms the presence of 45 tors published by John Wiley & Sons (March 1998) and BAR-TMR in the crystals. Bar represents 50 m. All images Tatsuya Haga (Ed), G Protein-Coupled Receptors, published were acquired using an Axioplan 2 microscope (Carl Zeiss by CRC Press (Sep. 24, 1999); and Steve Watson (Ed) G-Pro MicroImaging), fitted with a camera (RTE/CCD-1300-Y/HS: tein Linked Receptor Factsbook, published by Academic Roper Scientific) controlled using IPLab software (BD Bio Press (1st edition: 1994). A schematic representation of an Science). 50 exemplary GPCR is shown in FIG.1. A GPCR may be natu FIG. 7 Figure illustrates packing of B2AR-Fab complex in rally occurring or non-naturally occurring (i.e., altered my a crystal. man). FIG. 8 Figure illustrates the B2AR-Fab complex. As The term “naturally-occurring in reference to a GPCR shown, the Fab5 antibody binds a three dimensional epitope means a GPCR that is naturally produced (for example and that contains amino acids from both ends of the IC3 loop. 55 not limitation, by a mammal or by a human). Such GPCRs are found in nature. The term “non-naturally occurring in refer DEFINITIONS ence to a GPCR means a GPCR that is not naturally-occur ring. Wild-type GPCRs that have been made constitutively Unless defined otherwise herein, all technical and scien active through mutation, and variants of naturally-occurring tific terms used herein have the same meaning as commonly 60 GPCRs are examples of non-naturally occurring GPCRs. understood by one of ordinary skill in the art to which this Non-naturally occurring GPCR may have an amino acid invention belongs. Singleton, et al., DICTIONARY OF MICROBIOL sequence that is at least 80% identical to, e.g., at least 90% OGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, identical to, at least 95% identical to or at lest 99% identical New York (1994), and Hale & Marham, THE HARPER COLLINS to, a naturally-occurring GPCR. DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide 65 The term “ligand’ means a molecule that specifically binds one of skill with general dictionaries of many of the terms to a GPCR. A ligand may be, for example a polypeptide, a used in this disclosure. Although any methods and materials lipid, a small molecule, an antibody. A "native ligand is a US 7,947,807 B2 5 6 ligand that is an endogenous, natural ligand for a native sequences, with or without N-terminal methionine residues; GPCR. A ligand may be a GPCR “antagonist”, “agonist'. immunologically tagged proteins; fusion proteins with “partial agonist' or “inverse agonist', or the like. detectable fusion partners, e.g., fusion proteins including as a A "modulator” is a ligand that increases or decreases a fusion partner a fluorescent protein, B-galactosidase, GPCR intracellular response when it is in contact with, e.g., luciferase, etc.; and the like. binds, to a GPCR that is expressed in a cell. This term includes The terms “nucleic acid molecule' and “polynucleotide' agonists, including partial agonists and inverse agonists, and are used interchangeably and refer to a polymeric form of antagonists. nucleotides of any length, either deoxyribonucleotides or A "deletion' is defined as a change in either amino acid or ribonucleotides, or analogs thereof. Polynucleotides may nucleotide sequence in which one or more amino acid or 10 nucleotide residues, respectively, are absent as compared to have any three-dimensional structure, and may perform any an amino acid sequence or nucleotide sequence of a parental function, known or unknown. Non-limiting examples of GPCR polypeptide or nucleic acid. In the context of a GPCR polynucleotides include a gene, a gene fragment, exons, or a fragment thereof, a deletion can involve deletion of about introns, messenger RNA (mRNA), transfer RNA, ribosomal 2, about 5, about 10, up to about 20, up to about 30 or up to 15 RNA, ribozymes, cDNA, recombinant polynucleotides, about 50 or more amino acids. A GPCR or a fragment thereof branched polynucleotides, plasmids, vectors, isolated DNA may contain more than one deletion. of any sequence, control regions, isolated RNA of any An “insertion' or “addition' is that change in an amino acid sequence, nucleic acid probes, and primers. The nucleic acid or nucleotide sequence which has resulted in the addition of molecule may be linear or circular. one or more amino acid or nucleotide residues, respectively, As used herein the term "isolated, when used in the con as compared to an amino acid sequence or nucleotide text of an isolated compound, refers to a compound of interest sequence of a parental GPCR. “Insertion' generally refers to that is in an environment different from that in which the addition to one or more amino acid residues within an amino compound naturally occurs. “Isolated' is meant to include acid sequence of a polypeptide, while “addition' can be an compounds that are within Samples that are substantially insertion or refer to amino acid residues added at an N- or 25 enriched for the compound of interest and/or in which the C-terminus, or both termini. In the context of a GPCR or compound of interest is partially or substantially purified. fragment thereof, an insertion or addition is usually of about As used herein, the term “substantially pure” refers to a 1, about 3, about 5, about 10, up to about 20, up to about 30 or compound that is removed from its natural environment and is up to about 50 or more amino acids. A GPCR or fragment at least 60% free, at least 75% free, or at least 90% free from thereof may contain more than one insertion. 30 other components with which it is naturally associated. A “substitution” results from the replacement of one or A “coding sequence' or a sequence that "encodes a more amino acids or nucleotides by different amino acids or selected polypeptide, is a nucleic acid molecule which can be nucleotides, respectively as compared to an amino acid transcribed (in the case of DNA) and translated (in the case of sequence or nucleotide sequence of a parental GPCR or a mRNA) into a polypeptide, for example, in a host cell when fragment thereof. It is understood that a GPCR or a fragment 35 placed under the control of appropriate regulatory sequences thereof may have conservative amino acid substitutions (or “control elements'). The boundaries of the coding which have substantially no effect on GPCR activity. By sequence are typically determined by a start codon at the 5' conservative substitutions is intended combinations such as (amino) terminus and a translation stop codon at the 3' (car gly, ala; Val, ile, leu; asp. glu; asn, gln, ser, thr; lys, arg; and boxy) terminus. A coding sequence can include, but is not phe, tyr. 40 limited to, cDNA from viral, procaryotic or eucaryotic The term “biologically active', with respect to a GPCR, mRNA, genomic DNA sequences from viral or prokaryotic refers to a GPCR having a biochemical function (e.g., a bind DNA, and synthetic DNA sequences. A transcription termi ing function, a signal transduction function, or an ability to nation sequence may be located 3' to the coding sequence. change conformation as a result of ligand binding) of a natu Other “control elements’ may also be associated with a cod rally occurring GPCR. 45 ing sequence. A DNA sequence encoding a polypeptide can As used herein, the terms “determining.” “measuring.” be optimized for expression in a selected cell by using the 'assessing.” and “assaying are used interchangeably and codons preferred by the selected cell to represent the DNA include both quantitative and qualitative determinations. Ref copy of the desired polypeptide coding sequence. erence to an “amount of a GPCR in these contexts is not “Operably linked’ refers to an arrangement of elements intended to require quantitative assessment, and may be 50 wherein the components so described are configured so as to either qualitative or quantitative, unless specifically indicated perform their usual function. In the case of a promoter, a otherwise. promoter that is operably linked to a coding sequence will The terms “polypeptide' and “protein', used interchange effect the expression of a coding sequence. The promoter or ably herein, refer to a polymeric form of amino acids of any other control elements need not be contiguous with the cod length, which can include coded and non-coded amino acids, 55 ing sequence, so long as they function to direct the expression chemically or biochemically modified or derivatized amino thereof. For example, intervening untranslated yet tran acids, and polypeptides having modified peptide backbones. scribed sequences can be present between the promoter The term “fusion protein’ or grammatical equivalents sequence and the coding sequence and the promoter sequence thereof is meanta protein composed of a plurality of polypep can still be considered “operably linked to the coding tide components, that while typically unjoined in their native 60 Sequence. state, are joined by their respective amino and carboxyl ter By “nucleic acid construct it is meant a nucleic acid mini through a peptide linkage to form a single continuous sequence that has been constructed to comprise one or more polypeptide. Fusion proteins may be a combination of two, functional units not found together in nature. Examples three or even four or more different proteins. The term include circular, linear, double-stranded, extrachromosomal polypeptide includes fusion proteins, including, but not lim 65 DNA molecules (plasmids), cosmids (plasmids containing ited to, fusion proteins with a heterologous amino acid COS sequences from lambda phage), viral genomes compris sequence, fusions with heterologous and homologous leader ing non-native nucleic acid sequences, and the like. US 7,947,807 B2 7 8 A “vector” is capable of transferring gene sequences to a producing the genetically coded antibody. Hybridomas and host cell. Typically, “vector construct.” “expression vector.” methodologies for making hybridomas may be found in U.S. and "gene transfer vector” mean any nucleic acid construct Pat. No. 6,420,140 and Harlow, (Antibodies: A Laboratory capable of directing the expression of a gene of interest and Manual, First Edition (1988) Cold Spring Harbor, N.Y.) for which can transfer gene sequences to host cells, which can be example. accomplished by genomic integration of all or a portion of the A “complex” between an antibody an antigen is character vector, or transient or inheritable maintenance of the vectoras ized by a K, (dissociation constant) of less than 10 M, less an extrachromosomal element. Thus, the term includes clon than 107M, less than 10 M, less than 10 M, or less than ing, and expression vehicles, as well as integrating vectors. 10 M. An 'expression cassette' comprises any nucleic acid con 10 The “IC3 loop' of a GPCR is the intracellular loop that is struct capable of directing the expression of a gene/coding between transmembrane region 5 (TM5) and transmembrane sequence of interest, which is operably linked to a promoter region 6 (TM6) of the GPCR. Such regions are readily iden of the expression cassette. Such cassettes can be constructed tifiable by analysis of the primary amino acid sequence of a into a “vector,” “vector construct.” “expression vector” or GPCR. “gene transfer vector” in order to transfer the expression 15 The terms “specifically binds' and “specific binding” refer cassette into a host cell. Thus, the term includes cloning and to the ability of an antibody to preferentially bind to a par expression vehicles, as well as viral vectors. ticular antigen that is present in a homogeneous mixture of A first polynucleotide is "derived from or “corresponds different antigens. In certain embodiments, a specific binding to a second polynucleotide if it has the same or substantially interaction will discriminate between desirable and undesir the same nucleotide sequence as a region of the second poly able antigens in a sample, in some embodiments more than nucleotide, its cDNA, complements thereof, or if it displays about 10 to 100-fold or more (e.g., more than about 1000- or sequence identity as described above. 10,000-fold). A first polypeptide is "derived from or “corresponds to a An antibody that specifically binds to a “three dimen second polypeptide if it is (i) encoded by a first polynucle sional epitope or “conformational epitope is an antibody otide derived from a second polynucleotide, or (ii) displays 25 that specifically binds to a tertiary (i.e., three dimensional) sequence identity to the second polypeptides as described structure a folded protein. Such an antibody binds at much above. reduced (i.e., by a factor of at least 2, 5, 10, 50 or 100) affinity The terms “antibodies” and “immunoglobulin' include to the linear (i.e., unfolded, denatured) form of the protein. antibodies or immunoglobulins of any isotype, fragments of The structure to which Such an antibody binds contains amino antibodies which retain specific binding to antigen, including, 30 acids that are dis-contiguous in the protein. In other words, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric binding of Such an antibody to a polypeptide is dependent antibodies, humanized antibodies, single-chain antibodies, upon the polypeptide being folded into a particular three and fusion proteins comprising an antigen-binding portion of dimensional conformation. an antibody and a non-antibody protein. The antibodies may be detectably labeled, e.g., with a radioisotope, an enzyme 35 DETAILED DESCRIPTION OF EXEMPLARY which generates a detectable product, a fluorescent protein, EMBODIMENTS and the like. The antibodies may be further conjugated to other moieties, such as members of specific binding pairs, As noted above, various compositions are provided, e.g., biotin (member of biotin-avidin specific binding pair), including an antibody that specifically binds to a three dimen and the like. The antibodies may also be bound to a solid 40 sional epitope of the IC3 loop of a GPCR, a complex contain Support, including, but not limited to, polystyrene plates or ing a monovalent fragment of the antibody and a GPCR, and beads, and the like. Also encompassed by the terms are Fab', a crystal containing the complex. Various crystallization Fv, F(ab'), and or other antibody fragments that retain spe methods are also provided. In particular embodiments, the cific binding to antigen. GPCR to be complexed with the antibody may contain the Antibodies may exist in a variety of other forms including, 45 B2AR IC3 loop, and, in certain embodiments, may be a for example, Fv, Fab, and (Fab'), as well as bi-functional (i.e. hybrid GPCR that contains the IC3 loop of B2AR. Methods of bi-specific) hybrid antibodies (e.g., Lanzavecchia et al., Eur. preparing an antigen, and screening for an antibody are also J. Immunol. 17, 105 (1987)) and in single chains (e.g., Huston provided. These embodiments are described in greater detail et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) below. and Bird et al., Science, 242, 423-426 (1988), which are 50 Antibodies incorporated herein by reference). (See, generally, Hood et A monoclonal antibody that that specifically binds to a al., “Immunology’. Benjamin, N.Y., 2nd ed. (1984), and three dimensional epitope of the IC3 loop of a GPCR is Hunkapiller and Hood, Nature, 323, 15-16 (1986).). This provided. Such antibodies may be made, in general terms, by term also encompasses so-called “phage display' antibodies. reconstituting an active GPCR in phospholipid vesicles, A "monovalent antibody is an antibody that has a single 55 immunizing a suitable animal with the phospholipid vesicles, antigen binding region. Fab fragments, scFv antibodies, and and screening antibody-producing cells of the animal (or phage display antibodies are types of monovalent antibodies, hybridomas thereof) for the antibody. although others are known. A “Fab' fragment of an antibody A GPCR may be produced and purified using conventional has a single binding region, and may be made by papain methods that may employ expressing a recombinant form of digestion of a full length monoclonal antibody. A single chain 60 the GPCR in a host cell, and purifying the GPCR using variable (or “scFv') fragment of an antibody is an antibody affinity chromatography and/or antibody-based methods. In fragment containing the variable regions of the heavy and particular embodiments, the bactulovirus/Sf-9 system may be light chains of immunoglobulins, linked together with a short employed for expression, although other expression systems flexible linker. (e.g., bacterial, yeast or mammalian cell Systems) may also be A “hybridoma' is a cell that is a hybrid of a spleen cell or 65 used. Exemplary methods for expressing and purifying other antibody producing cell and an immortal cell (e.g., a GCPRs are described in, for example, Kobilka (Anal Bio myeloma cell). Hybridomas are both immortal and capable of chem 1995 231, 269-71), Eroglu et al (EMBO 20023: 491 US 7,947,807 B2 10 496), Chelikani et al (Protein Sci. 2006 15:1433-40) and the is incorporated by reference) and, as such, at least 277 GPCRs book “Identification and Expression of G Protein-Coupled are suitable for the subject methods. A more recent disclosure Receptors' (Kevin R. Lynch (Editor), Wiley-Liss (March of the sequences and phylogenetic relationships between 367 1998)), among many others. human and 392 mouse GPCRs is provided in Vassilatis et al. Likewise, methods for reconstituting an active GPCR in (Proc Natl Acad Sci 2003 100:4903-8 and www.primalinc phospholipid vesicles are known, and are described in: Luca .com, each of which is hereby incorporated by reference in its etal (Proc. Natl. Acad. Sci. 2003 100:10706-11); Mansoor et entirety) and, as such, at least 367 human and at least 392 al (Proc. Natl. Acad. Sci. 2006 103: 3060-3065); Niu et al. mouse GPCRs are suitable for the subject methods. GPCR (Biophys J. 2005 89: 1833-1840); Shimada et al (J. Biol. families are also described in Fredriksson et al (Mol. Phar Chem. 2002 277:31774-80); and Eroglu et al. (Proc. Natl. 10 macol. 2003 63, 1256-72). Since the amino acid sequences of Acad. Sci. 2003 100: 10219-10224), among others. In certain many GPCRs are available and their code sequences are cases, the GPCR and phospholipids may be reconstituted at known, the subject antibody may be made for any GPCR. For high density (e.g., 1 mg receptor per mg of phospholipid). In example, a human, mouse, rat, insect or plant GPCR may be particular embodiments, the phospholipids vesicles may be used in the subject method. tested to confirm that the GPCR is active. In many cases, a 15 The methods may be used, by way of exemplification, for GPCR may be present in the phospholipid vesicle in both purinergic receptors, vitamin receptors, lipid receptors, pep orientations (in the normal orientation, and in the "upside tide hormone receptors, non-hormone peptide receptors, non down” orientation in which the intracellular loops are on the peptide hormone receptors, polypeptide receptors, protease outside of the vesicle). receptors, receptors for sensory signal mediator, and biogenic Any suitable animal, e.g., a warm-blooded animal, in par amine receptors not including 32-. In cer ticular a mammal Such as a rabbit, mouse, rat, camel, sheep, tain embodiments, said receptor does not cow or pig or a bird Such as a chicken or turkey, may be include an adrenoreceptor. C-type adrenoreceptors (e.g. C., immunized with the reconstituted GPCR using any of the C. or Cadrenoreceptors) and f-type adrenoreceptors (e.g. techniques well known in the art Suitable for generating an B, B, or B adrenoreceptors) are discussed in Singh et al., J. immune response. Procedures for immunizing animals are 25 Cell Phys. 189:257-265, 2001. well known in the art, and are described in Harlow (Antibod It is recognized that both naturally occurring and altered ies: A Laboratory Manual, First Edition (1988) Cold Spring Harbor, N.Y.) and Weir (Handbook of Experimental Immu native (non-naturally occurring) GPCRs may be used in the nology Vol 4, Blackwell Scientific Publishers, Oxford, Subject methods. In certain embodiments, therefore, an England, 1986). As will be appreciated by one of ordinary altered native GPCR (e.g. a native GPCR that is altered by an skill in the art, the immunogen may be admixed with an 30 amino acid Substitution, deletion and/or insertion) such that it adjuvant or hapten in order to increase the immune response binds the same ligand as a corresponding native GPCR may (for example, complete or incomplete Freund's or lipid A be used in the subject methods. adjuvant), or with a carrier Such as keyhole limpet hemocya As such, the following GPCRs (native or altered) find nin (KLH). particular use as parental GPCRs in the subject methods: Once a suitable animal has been immunized and an 35 cholinergic receptor, muscarinic 3; melanin-concentrating immune response against the antigen has been established by hormone receptor 2; cholinergic receptor, muscarinic 4: nia the animal, antibody producing cells from the animal are cin receptor; histamine 4 receptor; ghrelin receptor, CXCR3 screened to identify cells that produce antibodies having a , ; 5-hydroxytryptamine desired activity. In many embodiments, these methods may employ hybridoma technology in which cells from the spleen (serotonin) receptor 2A, 5-hydroxytryptamine (serotonin) of the immunized animal are fused with a suitable immortal 40 receptor 2B: 5-hydroxytryptamine (serotonin) receptor 2C: cell to produce hybridoma cells. Supernatants from these D3; , dopamine hybridoma cells may be screened, and positive clones are receptor D1; H2; histamine receptor H3; expanded according to standard procedures (Harlow et al. 1; Y1; angiotensin Antibodies: A Laboratory Manual, First Edition (1988) Cold II receptor 1; 1; ; spring Harbor, N.Y.; and Spieker-Polet et al., supra). 45 glucagon-like peptide 1 receptor, ; can The antibodies may be screened for binding to the GPCR nabinoid receptor 1; and melanin-concentrating hormone folded into a native conformation by, e.g., cell staining to receptor 1. identify those antibodies that bind a three dimensional In certain embodiments, the antibody is a monovalent anti epitope in the GPCR. In certain cases, the antibodies may also body that contains a single antigenbinding region comprising be screened for binding to denatured GPCR. Antibodies that 50 variable heavy and variable light domains. Such antibodies, bind to the IC3 loop of the polypeptide may be identified including Fab antibodies and schv antibodies, may be pro using any of a variety of different means, depending on the duced from a monoclonal antibody after it is identified. In one GPCR. For example, the antibody can be tested to determine embodiment, the monovalent antibody is an antibody con if it can block interactions with a G-protein, or it can be tested taining the heavy and light variable regions from antibody 5, on a GPCR containing amino acid substitutions in the IC3 55 which is described in the examples section of this disclosure. loop. Other methods are described in the examples section of Such antibodies include Fab fragment of antibody 5 (i.e., the this disclosure. Fab5 antibody) or a scFv antibody comprising the same vari In alternative embodiments, a phage display antibody may able domains as the Fab5 antibody, where the heavy and light be employed, methods for the production of which are well variable domains of the Fab5 antibody have the following known (see, e.g., Scott et al. Science 1990 249: 386; Devlin et 60 amino acid sequence: al., Science 1990 249:404; U.S. Pat. Nos. 5,223,409, 5,733, 731, 5,498,530, 5,432,018, 5,338,665, and 5,922,545, for example). Fab5 Light Chain: (SEQ ID NO: 1) Any known GPCR is suitable for use in the subject meth DIKMTOSPSSMYASLGERVTITCKASODINSYLSWFOOKPGKSPKTLIYR ods. A disclosure of the sequences and phylogenetic relation 65 ships between 277 GPCRs is provided in Joostetal. (Genome ANRLVDGVPSRFIGTGSGODYSLTISSLDYEDMGIYYCLOYDEFPYTFGG Biol. 2002 3:RESEARCHO063, the entire contents of which US 7,947,807 B2 11 12 - Continued Also provided is a method of determining a crystal struc ture. This method may comprise receiving an above described GTKLEIKRADAAPTVSIFPPSSEOLTSGGASVWCFLNNFYPKDINVKWKI complex, crystallizing the complex to produce a crystal, and obtaining atomic coordinates of the GPCR from the crystal. DGSERONGVLNSWTDODSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKT The complex may be received from a remote location (e.g., a STSPIWKSFNRNEC different laboratory in the same building or campus, or from Fab 5 Heavy chain: a different campus or city), and, in certain embodiments, the (SEQ ID NO: 2) method may also comprise transmitting the atomic coordi EVOLOOSGAELARPGASVKLSCKASGYIFTDYYINWVRORTGOGFEWIGE nates, e.g., by mail, e-mail or, using the internet, to the remote 10 location or to a third party. IYPGSGNIDYNERFKDKATLTADKSSSTAYMOLSSLTSEDSAVYFCVRGF In other embodiments, the method may comprise forward ing a complex to a remote location, where the complex may GYWGOGTTLTVSSAKTTPPSVYPLAPGSAAOTNSMVTLGCLVKGYFPEPV be crystallized and analyzed, and receiving the atomic coor TWTWNSGSLSSGVHTFPAVLOSDLYTLSSSVTVPSSTWPSETVTCNVAHP dinates of the GPCR. 15 In certain cases, the GPCR to be crystallized may be a ASSTKWDKKIWPRDCGC GPCR that has been modified to contain a three dimensional In certain cases, a Subjectantibody may contain: a) a heavy epitope from the IC3 loop from a different GPCR (e.g., the B2 chain containing the CDR1, CDR2 and CDR3 regions of the GPCR). In these embodiments, once a suitable antibody has heavy chain of an above-described antibody, separated by been identified for a first GPCR, the antigen binding region framework sequence and b) a light chain containing the for that antibody can be swapped from the first GPCR into a CDR1, CDR2 and CDR3 regions of the light chain of that second GPCR to make a chimeric GPCR. The chimeric antibody, separated by framework sequence. GPCR can then be bound to a monovalent antibody, and Also provided is a complex containing the GPCR and a crystallized using a method described herein. In one embodi single antigenbinding region of the antibody. Such a complex ment, the entire IC3 loop, including the three dimensional 25 epitope contained in the loop, may be grafted from one GPCR may be formed under antibody binding conditions described to another to produce an active GPCR. Such methods have in Harlow and Weir, supra. A crystal of the complex is also been successfully performed by others, e.g., Kim et al (Bio provided, and methods of making of which are described in chemistry 2005 44:2284-92): Yamashita etal (J. Biol. Chem. greater detail below. 2000 275:34272-9); Geiser et al (Protein Sci. 2006 15:1679 Crystallization Methods 30 90); Tumova et al (J. Biol. Chem. 2003 278:8146-53); Wong A crystallization method is also provided. The method etal (J. Biol. Chem. 1990 265:6219–24) and Wessetal (FEBS includes contacting a GPCR with a monovalent antibody that Lett. 1989 258:133-6). specifically binds to a three dimensional epitope of the IC3 The IC3 region of a GPCR lies in between transmembrane loop of a GPCR under binding conditions to form a complex: regions TM5 and TM6 and, may be about 12 amino acids and crystallizing the complex. 35 (CXCR3 and GPR40) to about 235 amino acids (cholinergic A Subject complex may be crystallized using any of a receptor, muscarinic 3) in length, for example. The TM5, IC3, variety of crystallization methods, many of which are and TM6 regions are readily discernable by one of skill in the reviewed in Caffrey Membrane protein crystallization. J art using, for example, a program for identifying transmem Struct. Biol. 2003 142: 108-32. In general terms, the methods brane regions; once transmembrane regions TM5 and TM6 are lipid-based methods that include adding lipid to the fusion 40 regions are identified, the IC3 region will be apparent. The protein prior to crystallization. Such methods have previously TM5, IC3, and TM6 regions may also be identified using such been used to crystallize other membrane proteins. Many of methods as pairwise or multiple sequence alignment (e.g. these methods including the exploit the spontaneous self using the GAP or BESTFIT of the University of Wisconsin's assembling properties of lipids and detergent as vesicles GCG program, or CLUSTAL alignment programs, Higgins et 45 al., Gene. 1988 73:237-44), using a target GPCR and, for (vesicle-fusion method), discoidal micelles (bicelle method), example, GPCRs of known structure. and liquid crystals or mesophases (in meso or cubic-phase Suitable programs for identifying transmembrane regions method). Lipidic cubic phases crystallization methods are include those described by Moller et al., (Bioinformatics, described in, for example: Landau et al. Lipidic cubic phases. 17:646-653, 2001). A particularly suitable program is called a novel concept for the crystallization of membrane proteins. 50 “TMHMM Krogh et al., (Journal of Molecular Biology, Proc. Natl. Acad. Sci. 1996 93:14532-5; Gouaux. It’s not just 305:567-580, 2001). To use these programs via a user inter a phase: crystallization and X-ray structure determination of face, a sequence corresponding to a GPCR or a fragment bacteriorhodopsin in lipidic cubic phases. Structure. 1998 thereof is entered into the user interface and the program run. 6:5-10; Rummel et al. Lipidic Cubic Phases. New Matrices Such programs are currently available over the world wide for the Three-Dimensional Crystallization of Membrane Pro 55 web, for example at the website of the Center for Biological teins.J. Struct. Biol. 1998 121:82–91; and Nollert etal Lipidic Sequence Analysis at cbs.dtu.dk/services/. The output of cubic phases as matrices for membrane protein crystalliza these programs may be variable in terms its format, however tion Methods. 2004 34:348-53, which publications are incor they usually indicate transmembrane regions of a GPCR porated by reference for disclosure of those methods. Bicelle using amino acid coordinates of a GPCR. crystallization methods are described in, for example: Faham 60 When TM regions of a GPCR polypeptide are determined etal Crystallization of bacteriorhodopsin from bicelle formu using TMHMM, the prototypical GPCR profile is usually lations at room temperature. Protein Sci. 2005 14:836-40. obtained: an N-terminus that is extracellular, followed by a 2005 and Faham et al. Bicelle Crystallization: a new method segment comprising seven TM regions, and further followed for crystallizing membrane proteins yields a monomeric bac by a C-terminus that is intracellular. TM numbering for this teriorhodopsin structure. J Mol Biol. 2002 Feb. 8; 316(1):1- 65 prototypical GPCR profile begins with the most N-terminally 6, each of which are incorporated by reference for disclosure disposed TM region (TM1) and concludes with the most of those methods. C-terminally disposed TM region (TM7). US 7,947,807 B2 13 14 Accordingly, in certain embodiments, the amino acid coor the On-line Mendelian Inheritance in Man database found at dinates of the TM5, IC-3, and TM6 regions of a GPCR are the world wide website of the NCBI. identified by a suitable method such as TMHMM. In certain embodiments as noted above, the antibody may In certain cases, once the TM5-IC3-TM6 segment is iden bind to the B2-adrenoreceptor, in which case it may be tified for a GPCR, a suitable region of amino acids is chosen employed in the treatment of a condition requiring relaxation for Substitution with the amino acid sequence of an IC3 region of Smooth muscle of the uterus or vascular system. Such an antibody may be thus used for the prevention oralleviation of of another GPCR, e.g. the B2 GPCR. In certain embodiments, premature labour pains in pregnancy, or in the treatment of the Substituted region may be identified using conserved or chronic or acute asthma, urticaria, psoriasis, rhinitis, allergic semi-conserved amino acids in the TM5 and TM6 transmem conjunctivitis, actinitis, hay fever, or mastocytosis, which brane regions. 10 conditions have been linked to the B2-adrenoreceptor. In For GPCRs that contain no conserved proline residues in these embodiments, the antibody may be employed as co TM5 and TM6, positions for inserting a different IC3 loop therapeutic agents for use in combination with other drug may be based on two considerations: a) alignment of the Substances such as anti-inflammatory, bronchodilatory or sequence of the GPCR with receptor members of the same antihistamine drug Substances, particularly in the treatment subfamily (which contained conserved proline residues in 15 of obstructive or inflammatory airways diseases such as those TM5 or TM6; b) by identifying the juxtaposition to the TM5/ mentioned hereinbefore, for example as potentiators of thera TM6 regions by hydrophobicity analysis. peutic activity of Such drugs or as a means of reducing required dosaging or potential side effects of Such drugs. A In addition to substituting IC3 region of a GPCR with a subject antibody may be mixed with the other drug substance stable, folded protein insertion, as described above, in certain in a fixed pharmaceutical composition or it may be adminis cases, the C-terminal region of the GPCR (which is C-termi tered separately, before, simultaneously with or after the nal to the cysteine palmitoylation site that is approximately 10 other drug Substance. Such anti-inflammatory drugs include to 25 amino acid residues downstream of a conserved steroids, in particular glucocorticosteroids Such as budes NPXXY motif), may be deleted. In certain cases, the 20-30 onide, beclamethasone, fluticasone, ciclesonide or mometa amino acids immediately C-terminal to the cysteine palmi sone or steroids described in WO 0200679 especially those of toylation site are not deleted. 25 Examples 3, 11, 14, 17, 19, 26, 34, 37, 39, 51, 60, 67, 72,73, In one embodiment, a region containing the binding region 90, 99 and 101, LTB4 antagonists such as those described in of antibody 5 described below (i.e., a region containing nine U.S. Pat. No. 5,451,700, LTD4 antagonists such as mon amino acids at the amino-terminal end of the B2ARIC3 loop telukast and Zafirlukast, and PDE4 inhibitors such as ArifloR) (I233-V242), having the amino acid sequence IDKSEGR (GlaxoSmith Kline), Roflumilast (Byk Gulden), V-11294A (Napp), BAY19-8004 (Bayer), SCH-351591 (Schering FHV: SEQ ID NO:3 and also containing L266 and/or K270 30 Plough), Arofylline (Almirall Prodesfarma), PD189659 from the C-terminal end of the B2AR IC3 loop) may be (Parke-Davis), AWD-12-281 (Asta Medica), CDC-801 (Cel substituted into a different (non-R2AR) GPCR at the same gene) and KW-4490 (Kyowa Hakko Kogyo) and A2a agonists position, to produce a chimeric GPCR. That chimeric GPCR such as those described in EP 1052264, EP 1241176, WO may be complexed with a monovalent version of antibody 5 0023457, WO0077018, WO 0123399, WO 0160835, WO (e.g., a Fab fragment of antibody 5 or a schv antibody) to 35 0194368, WO 0200676, WO 0222630, WO 0296462, WO make a complex that can be crystallized. In certain embodi 0127130, WO 0127131, WO 9602543, WO 9602553, WO ments, the antibody 5 binding site sequences may be trans 98.28319, WO 9924449, WO 9924.450, WO9924.451, WO ferred along with amino acids that flank the binding site (e.g., 9938877, WO994.1267, WO 9967263, WO 9967264, WO up to 2, 3, 4, or 5 amino acids from each side). In particular 9967265, WO 9967266, WO 94.17090, EP 409595A2 and embodiments, the entire IC3 region of the non-f2AR GPCR 40 WO 0078774 and A2b antagonists such as those described in WO 02/42298. Such bronchodilatory drugs include anticho may be substituted with the IC3 region of B2AR. In certain linergic or antimuscarinic agents, in particular ipratropium cases, the transferred may contain I233 to K270 of B2AR bromide, oxitropium bromide and tiotropium bromide. In GPCR, and may contain amino acids that flank that sequence general terms, these protocols involve administering to an (e.g., up to 2, 3, 4, or 5 amino acids from each side). In other individual suffering from a GPCR-related disorder an effec cases, two regions may be transferred, one containing the 45 tive amount of an antibody that modulates a GPCR to modu amino acid sequence of I233 to V242, and the other contain late the GPCR in the host and treat the individual for the ing the amino acid sequence of L266 to K270. These disorder. sequences can be transferred to the N-terminus and the C-ter In some embodiments, where a reduction in activity of a minus of the recipient GPCR's IC3, respectively. certain GPCR is desired, one or more compounds that Thus, a chimeric GPCR used in the method may containa) 50 decrease the activity of the GPCR may be administered, an IC3 loop of one GPCR (e.g., the B2 GPCR), and b) TM1 whereas when an increase in activity of a certain GPCR is TM7, IC1 and IC2 regions of another GPCR. desired, one or more compounds that increase the activity of Methods of Treatment the GPCR activity may be administered. Certain of the above-described antibodies may have thera A variety of individuals are treatable according to the sub peutic utility and may be administered to a subject having a 55 ject methods. Generally such individuals are mammals or condition in order to treat the subject for the condition. The mammalian, where these terms are used broadly to describe therapeutic utility for an antibody may be determined by the organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, GPCR to which the antibody binds in that signaling via that guinea pigs, and rats), and primates (e.g., humans, chimpan GPCR is linked to the condition. In certain cases, the GPCR Zees, and monkeys). In many embodiments, the individuals may be activated in the condition via by binding by a ligand. 60 will be humans. Subject treatment methods are typically per In other embodiments, the GPCR may be mutated to make it formed on individuals with such disorders or on individuals constitutively active, for example. A Subject antibody may be with a desire to avoid contracting Such disorders. employed for the treatment of a GPCR-mediated condition A variety of publications describe treatment regimens Such as Schizophrenia, migraine headache, reflux, asthma, using the antibodies including, for example, published patent bronchospasm, prostatic hypertrophy, ulcers, epilepsy, 65 applications 20080159981, 20080102069, 20070142346, angina, allergy, rhinitis, cancer e.g. prostate cancer, glaucoma 20060018899, 200501.48607 and 20030228663, as well as and stroke. Further exemplary GPCR-related conditions at U.S. Pat. Nos. 7,306,801, 6,399,063, 6,387,371 and 6,165, US 7,947,807 B2 15 16 464, which are each incorporated by reference in their Cell Culture entirety. In general terms, these documents described meth Myeloma cells were propagated in Dulbecco's Modified ods including administering a therapeutic monoclonal anti Eagle's Medium (DMEM-high glucose) supplemented with body to a Subject, singly or in combination with another 10% Fetal Bovine Serum (FBS), 0.15g/L oxaloacetate, 0.05 agent. The Subject may then be monitored for a clinically g/L pyruvate, 0.0082 g/L bovine insulin (OPI supplement), beneficial response, where a beneficial response to the anti 10% NCTC 109 medium and 4 mM L-glutamine. This formu body can be assessed according to whether an individual lation is referred to the SDMEM formulation. Newly formed patient experiences a desirable change in disease status. hybridoma were selected in SDMEM supplemented with In particular embodiments, the antibody may have a trans HAT mixture (1.0x10 M Hypoxanthine, 40x10 MAmi location sequence (e.g., an HIV TAT. AntP, or VP22 trasloca 10 nopterin, 1.6x10M thymidine).5% hybridoma cloning fac tion sequence, or other short sequence rich in basic amino tor, gentamicin (50 ug/ml) and B-mercaptoethanol at 0.055 acids) which facilitates translocation of the antibody across mM. This formulation is referred as SDMEM-HAT. After the plasma membrane into the cytoplasm of a target cell. Such selection, the newly formed hybridomas were propagated in translocation sequences and their use are known and SDMEM supplemented with 2% hybridoma cloning factor reviewed in, for example, Gupta (Drug Deliv. Rev. 2005 HT mixture (1.0x10 M hypoxanthine, 1.6x10M thymi 15 dine). This formulation is referred as DMEM-HT. For in vitro 57:637-51), Steffen (Methods Mol. Biol. 2001 161:141-8) antibody production, the hybridomas were propagated in and Hansen (Scientific World Journal. 2005 5:782-8). SDMEM supplemented with 1% hybridoma cloning factor In order to further illustrate the present invention, the fol and 5-10% low-IgG FBS. lowing specific examples are given with the understanding Cell Fusion Protocol that they are being offered to illustrate the present invention Balb/c mice were immunized weekly with 30 ug of BAR and should not be construed in any way as limiting its scope. in liposomes. The mice were injected intraperitoneally with 15ug of antigen and Subcutaneously at 2 sites with 7.5 mg of EXAMPLE1 antigen. Final boosts of 10 ug offAR were given intraperi toneally and intravenously on day 3 and 4 before fusion. Antigen Production 25 Spleens were harvested aseptically, placed in cold DMEM, diced and homogenized. The homogenate was centrifuged at Antigen was prepared by reconstituting purified, func 1000 rpm for 5 minutes. The cell pellet was incubated with 5 tional BAR at high density into phospholipid vesicles (1 mg ml of Red cell lysis buffer (Sigma R7757) for 3-5 mins. receptor per mg of phospholipids) using the following DMEM (20 ml) was added and the cells were centrifuged at method. 30 1000 rpm for 5 minutes. The pellet was rinsed twice in 20 ml BAR having an amino terminal Flag epitope tag and a DMEM. The final pellet was resuspended in 30 mL DMEM. carboxylhexahistidine tag was expressed in Sf9 insects cells Spleen and myeloma cells were combined at a 3:1 ratio, and purified by sequential M1 antibody and alprenolol affin centrifuged (1000 rpm) and resuspend in DMEM. The cen ity chromatography as previously described (Kobilka Anal trifugation was repeated and 1 mL PEG-1500 (50% solution) Biochem 1995 231,269-71). Purified BAR was immobilized 35 was added to the cell pellet over 3 min with gentle mixing. 4 on a Nicolumn and equilibrated with 10 column volumes of mL of SDMEM was slowly added to cells over 2 min. After 1 a mixture of 5 mg/ml DOPC (Avanti Polar Lipids) and 0.5 min incubation, 20 mL of SDMEM was added over 1 min. mg/ml Lipid A (Sigma) in 1% (w/v) octylglucoside The suspension was centrifuge (1000 rpm) and the pellet was (Anatrace), 100 mM. NaCl, 20 mM Hepes pH 7.5 and 1 uM carazolol (a faRantagonist). The faR was then eluted in resuspended in a total of 30 mL HAT-SDMEM and incubate the same buffer containing 200 mM imidazole. The concen 40 at 37° C. for 2 hours. HAT-SDMEM media was added to tration of the eluted BAR was adjusted to 5 mg/ml. This achieve a cell density of 5x10" cells/well and dispensed into usually involved diluting the protein with the same buffer, but 96-well plates at 200 uL per well. Cells were incubated for 11 occasionally required concentrating the protein up to two days before screening media for antibody production. fold with an Amicon ultrafiltration cell (100 kDa pore). The Fusions from two mice yielded seventeen hybridoma protein was then dialyzed against phosphate buffered saline 45 clones that produced antibody against AR as determined by containing 1 uM carazolol at 4°C. to remove detergent. The an ELISA assay on immobilized phospholipid vesicles con reconstituted protein was stored at -80° C. prior to use for taining purified BAR. Nine of these hybridomas produced immunization. sufficient quantities of BAR-specific antibody for further The phospholipid environment ensures the functional integrity of the protein after injection into mice. To provide characterization. The antibodies were characterized as bind additional stability, the BAR was liganded with carazolol, a 50 ing the intracellular or extracellular surface of the receptor in high affinity inverse agonist. To facilitate the immune immunofluorescence experiments with HEK-293 cells stably response, phospholipid vesicles consisted of a 10:1 mixture expressing an N-terminally FLAG-tagged version of the (by weight) of DOPC and the adjuvant Lipid A. The generated BAR. Antibodies that recognize an intracellular epitope only vesicles contained randomly oriented fAR, so that both stain permeabilized cells. Staining of cells that were either cytoplasmic and extracellular domains are presented to 55 fixed or fixed and permeabilized indicated that five of the immune cells. antibodies bound to the intracellular face and four bound to the extracellular surface (FIG. 4). EXAMPLE 2 EXAMPLE 3 Antibody Production 60 Antibody Screening Monoclonal antibodies (MABs) were generated using a conventional fusion protocol, as follows: Antibodies were screened for binding to a three dimen Myeloma Cell Lines sional epitope on the three-dimensional surface on the IC3 of The P3x63Ag8.653 myeloma cell line (ATCC # CRL 65 the native BAR, rather than a flexible linear epitope. Nine 1580; Lot No. 1131010) was used for the fusion experiment MABs were screened, along with positive (M1 antibody) and performed with splenocytes from immunized mice. negative (9E10) controls, for binding to BAR denatured with US 7,947,807 B2 17 18 sodium dodecyl sulfate (SDS) and spotted on nitrocellulose TABLE 1 (FIG. 2A). Antibody 5 and antibody 9 showed the weakest Antagonist and agonist binding properties of the 32AR with and binding to denatured protein, even though they showed without prebound Fab 5. Saturation and competition binding assays immunostaining comparable to M1 on binding to native were performed on purified BAR reconstituted in phospholipid receptor in fixed cells (see FIG. 4). The reduced binding of 5 vesicles in the absence and presence of prebound Fab 5. Values antibody 5 and antibody 9 to SDS denatured AR showed are from three independent experiments performed in triplicate. that these antibodies bind to a three-dimensional epitope. Hidihydroalprenolol Isoproterenol Antibodies 2, 5, 6, 7 and 9 all reacted with intracellular Kit S.E. K. S.E. interval epitopes. To select for antibodies that may interact with IC3, (nM) (nM) 10 we examined the effect of antibody binding on the fluores B2AR 1.02 0.09 290 (269-311 cence of BAR labeled at C265 (at the cytoplasmic end of B2AR-Fab5 1.08 O.OS 329 (310-348) TM6) with tetramethylrhodamine. Tetramethylrhodamine The IC50 values used for calculations of Kvalues were obtained from means of plC50 values bound to C265 is predicted to lie at the interface between TM5 determined by nonlinear regression analysis and the S.E. interval from pC50+ S.E. and TM6. Previous studies have shown that tetramethyl 15 rhodamine binding to C265 is sensitive to ligand-induced IC3 is known to be important for G protein activation. Fab three dimensional changes in the BAR (Swaminath et al., J. 5 prevented coupling of purified BAR to purified G protein Biol. Chem. 2004 279: 686-691). As such, antibodies binding (data not shown), most likely due to steric competition. How to IC3 may stabilize a specific conformation of TM5 relative ever, it is possible that Fab 5 restricts the conformational to TM6 that would be detected by a change in tetramethyl changes associated with receptor activation. The effect of Fab rhodamine fluorescence. It can be seen that antibody 5 binding on agonist-induced conformational changes was induced the largest fluorescence response and had the highest determined using a fluorescence-based assay (Yao et al. Nat. affinity for the BAR (FIG. 2B). The fluorescence change Chem. Biol. 2006 2, 417-422). induced by antibody 5 was significantly greater than the Purified receptor was reacted with 1:1 equivalent of mono response to antibody 9, the other intracellular binder that 25 bromobimane (mBBr, Invitrogen) in buffer A (100 mMNaCl, reacted weakly to SDS denatured receptor. It is notable that 20 mM HEPES, pH 7.5, 0.1% dodecyl maltoside) and incu there was a response to antibody 8 and antibody 4, two anti bated overnight on ice in the dark. The fluorophore-labeled bodies that bind to an extracellular epitope. This suggests that receptor was purified right before use by gel filtration on a binding of these antibodies to the extracellular surface of the desalting column equilibrated with buffer B. Fluorescence BAR influences the structure around the cytoplasmic end of 30 spectroscopy experiments were performed on a Spex Fluoro TM6. Max-3 spectrofluorometer (Jobin Yvon Inc, NJ) with photon Based on the results of assays shown in FIGS. 2A and 2B counting mode by using an excitation and emission bandpass antibody 5 was chosen for crystallography experiments. Anti of 4 nm. All experiments were performed at 25°C. For emis body 5 bound to an intracellular epitope, it exhibited rela 35 sion scans, excitation was set at 370 nm and emission was tively high affinity for native f3AR (FIG. 2B), but bound measured from 430-530 nm with an integration time of 1 weakly to SDS denatured protein (FIG. 2A). Antibody 5 also s/nm. To determine the effect of Fab5 antibody and drug, induced the largest fluorescence response in BAR labeled at three individual labeled protein samples were incubated with C265 with tetramethylrhodamine. Based on these experi antibody (1 uM Fab5) or 100LM Isoproterenol or both. Emis ments, antibody 5 binds to a three dimensional epitope on the 40 sion spectra of the samples were taken after 15 minutes incu native third intracellular loop. bation. Fluorescence intensity was corrected for background fluorescence from buffer and ligands in all experiments. The EXAMPLE 4 data are the meantS.E. of two independent experiments per formed in triplicate. Antibody Characterization 45 Agonist binding induces a change that brings the fluoro Fab 5 fragments were generated from purified antibody 5 phore monobromobimane bound to C271 at the cytoplasmic by papain cleavage and purified by ion exchange chromatog end of TM6 closer to W 135 at the cytoplasmic end of TM3 raphy. The dissociation constant for Fab 5 binding to purified resulting in a decrease in bimane fluorescence. This fluores BAR was determined to be 150 nM by isothermal titration 50 cence change was not affected by binding of antibody 5 (FIG. calorimetry (FIG.5). As expected, this value is higher than the 3B). In conclusion, while Fab 5 required a native f3AR to EC50 (23 nM) observed for the intact antibody in the fluo bind, it did not restrict the movement of transmembrane seg rescence experiments (FIG. 2B). ments involved in ligand binding and agonist activation. To localize the intracellular epitope of the BAR that inter To more easily identify crystals of the Fab 5-3AR com acts with Fab 5, limited tryptic digestions of purified AR 55 plex, we labeled purified BAR at C265 with tetramethyl were performed. Cleavage of purified AR with trypsin rhodamine (BAR-TMR). The Fab 5-BAR-TMR complex yielded two bands with molecular weights of approximately was formed by mixing BAR-TMR with a stoichiometric 27 and 29 kDa on a Western blot probed with the M1 anti excess of Fab 5 and isolating the complex by size exclusion FLAG antibody (FIG. 3A). The sizes of these fragments chromatography. Small fluorescent crystals formed by vapor indicate trypsin digestion at two often potential sites in IC3 60 phase diffusion using ammonium Sulfate as the precipitant (FIG. 3A). When preincubated with Fab 5, the 27 kDa band (FIG. 6). Importantly, no crystals formed from BAR alone or disappears Suggesting that the antibody binds to one of the from Fab 5 alone under these conditions, showing that the amino-terminal trypsin sites in IC3 (FIG. 3A). additional protein interactions and the stabilizing effects of The effect of Fab binding on ligand binding properties and the antibody were critical for successful crystallization of the on agonist-induced three dimensional changes was deter 65 BAR. Further refinement of the crystallography conditions mined. Fab 5 had no significant effect on antagonistoragonist has since produced diffraction quality crystals and a 3.4 A binding affinity (Table 1 below). structure of the Fab 5-fAR complex. US 7,947,807 B2 19 20 EXAMPLE 5 beamline ID23-2 of the ESRF, were used for the final fAR365-Fab5 data set (Table 2). The BAR24/365-Fab5 Preparation of Fab Fragments data set was obtained from 225° of data measured on beam line 23ID-B of the APS (Table 2). Monoclonal mouse immunoglobulins against the 32AR reconstituted in liposomes were raised, as described above. TABLE 2 Mab5 IgGs from mouse hybridoma cell culture supernatant X-ray data collection and refinement statistics were purified using a Protein G column (Pierce). Fab5 frag ments were generated by papain proteolysis (Worthington) BAR365-Fab5 BAR24/365-Fab5 and purified by Mono Q chromatography. The fragments 10 were concentrated to ~100 mg ml with a Millipore concen Data collection trator (5 kDa molecular weight cut off) in a solution of 10 mM Space group HEPES pH 7.5 and 100 mM NaCl. Cell dimensions

15 a, b, c (A) 338.4, 48.5, 89.4 338.4, 48.5, 89.4 EXAMPLE 6 Cl, f, Y () 90., 104.6, 90. 90., 104.6, 90. Resolution (A) 86.4-3.4 (3.49-3.40)* 50-3.4 (3.52-3.40)* Preparation of B2AR365-Fab5 Complexes Rmerge 0.117 (0.407) 0.120 (0.456) IoI 9.9 (2.3) 7.8 (2.6) Completeness (%) 98.9 (94.9) 99.4 (98.4) The BAR365 was expressed in Sf-9 insect cells infected Multiplicity 3.3 (2.9) 4.1 (3.4) with BAR365 baculovirus, and solubilized according to pre Refinement viously described methods. Functional protein was obtained Resolution (A) 20.-3.4 20.-3.4 by M1 FLAG affinity chromatography (Sigma) prior to and No. reflections 17658,1902 17458,1886 following alprenolol-Sepharose chromatography. Receptor work? free bound alprenolol was exchanged for carazolol on the second Ryor Rfree 0.216.0.269 O.226.O.279 M1 resin. N-linked glycolsylations were removed by treat 25 No. atoms 490S 4887 ment with PNGaseF (NEB), and the FLAG epitope was Average B values removed by treatment with AcTEV protease (Invitrogen). (A2) Protein was concentrated to ~50 mg/ml with a 100 kDa Receptor 156. 187. molecular weight cut off Vivaspin concentrator (Vivascience) Fab5 67. 91. and mixed in stoichiometric excess of Fab5. The complex was 30 Overall anisotropic purified on a Superdex-200 (10/300GL) column in a solution B (A2) of 10 mM HEPES pH 7.5, 100 mM NaCl, 0.1% dodecylmal B11/B2/B33/B13 -27.1/31.3, -4.24.4 -16.8. 20.4-3.5.12.4 toside, and 10 uM carazolol. The purified BAR365-Fab5 R.m. S deviations complexes were concentrated to ~60 mg ml using a 35 Bond lengths (A) O.007 O.OO8 Vivaspin concentrator. Bond angles () 1.4 1.5 Ramachandran EXAMPLE 7

Crystallization % most favored 76.3, 71.5 75.8, 71.8 40 allowed 22.1,27.2 22.1.26.6 The BAR365-Fab5 complexes were mixed with bicelles generously 1.61.3 2.1.1.6 (10% w/v 3:1 DMPC:CHAPSO in 10 mM HEPES pH 7.5, allowed 100 mM NaCl) at a 1:5 (protein: bicelle) ratio, and crystals disallowed 0.00.0 0.00.0 were grown in sitting- and hanging-drop formats at 22°C. *Highest resolution shell is shown in parenthesis. using equal Volumes of protein mixture and reservoir solu 45 As defined in PROCHECK tions. Initial crystallization leads were identified using mul ESRF data were processed with MOSFLM and SCALA tiple 96-well sitting-drop screens from Nextal (Qiagen). After (Collaborative Computational Project, N. Acta Cryst. 1994 extensive optimization, crystals for data collection were D50, 760-763) and data measured at the APS were processed grown in hanging-drop format over a reservoir solution of with HKL2000 (Otwinowski et al, Methods Enzymol. 1997 1.85-2.0 M ammonium sulfate, 180 mM sodium acetate, 5 50 276, 307-326). In many cases it was necessary to reindex the mM EDTA, 100 mM MES or HEPES pH 6.5-7.5. Crystals crystal after moving to a new position on the crystal, which grew to full size within 7 to 10 days. Crystals were flash may have been due to bending of the frozen crystals such that frozen and stored in liquid nitrogen, with reservoir Solution the indexing matrix from the previous Volume could not accu plus 20% glycerol as cryoprotectant. rately predict the diffraction pattern from a new volume. This 55 problem precluded global postrefinement of the unit cell EXAMPLE 8 parameters. The unit cell parameters used for Subsequent analysis (Table 1) were obtained from initial indexing and Microcrystallography Data Collection and refinement from one wedge of the ESRF data, and were Processing Subsequently found to be sufficient for processing the remain 60 ing data without unit cell constant refinement. Using a partial Microbeams were employed for data collection. The shape specific volume of 1.21 A/Da for protein, the unit cell would of the crystals permitted complete data to be measured from have 66% lipid, detergent and aqueous solvent for one BAR a single crystal. A small wedge of data, typically 5-10°, (1 Fab5 complex in the asymmetric unit. The structure of the per frame) could be measured before significant radiation BAR365-Fab5 complex was solved by molecular replace damage was observed. The crystal was then translated to a 65 ment, by searching with separate constant and variable new, undamaged position to collect the next wedge of data. A domain models against a low resolution (4.1 A) data set total of 182° of data collected in this manner, measured at measured at ESRF beamline ID-13. US 7,947,807 B2 21 22 The crystal structure confirms that Fab 5 binds to a three K270), but at a different region of the antibody. FIG. 7 illus dimensional epitope consisting of nine amino acids at the trating packing of B2AR-Fab complex in a crystal, and FIG.8 amino-terminal end of IC3 (I233-V242). The antibody also schematically illustrates the three-dimensional epitope to binds two amino acids at the carboxyl-terminal end (L266 and which the Fab 5 antibody binds.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 3

<21 Os SEQ ID NO 1 &211s LENGTH: 214 212s. TYPE: PRT <213> ORGANISM: mus musculis 22 Os. FEATURE: <221s NAME/KEY: UNSURE <222s. LOCATION: (0) . . . (O) <223> OTHER INFORMATION: Fab5 light chain <4 OOs SEQUENCE: 1 Asp Ile Llys Met Thr Glin Ser Pro Ser Ser Met Tyr Ala Ser Lieu. Gly 1. 5 1O 15 Glu Arg Val Thir Ile Thr Cys Lys Ala Ser Glin Asp Ile Asn. Ser Tyr 2O 25 3 O Leu Ser Trp Phe Glin Gln Llys Pro Gly Lys Ser Pro Llys Thr Lieu. Ile 35 4 O 45 Tyr Arg Ala Asn Arg Lieu Val Asp Gly Val Pro Ser Arg Phe Ile Gly SO 55 60 Thr Gly Ser Gly Glin Asp Tyr Ser Lieu. Thir Ile Ser Ser Lieu. Asp Tyr 65 70 7s 8O Glu Asp Met Gly Ile Tyr Tyr Cys Lieu. Glin Tyr Asp Glu Phe Pro Tyr 85 90 95 Thr Phe Gly Gly Gly. Thir Lys Lieu. Glu Ile Lys Arg Ala Asp Ala Ala 1OO 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Lieu. Thir Ser Gly 115 12O 125 Gly Ala Ser Val Val Cys Phe Lieu. Asn. Asn. Phe Tyr Pro Lys Asp Ile 13 O 135 14 O Asn Val Llys Trip Lys Ile Asp Gly Ser Glu Arg Glin Asn Gly Val Lieu. 145 15 O 155 16 O Asn Ser Trp Thr Asp Glin Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 1.65 17 O 17s Ser Thr Lieu. Thir Lieu. Thir Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 18O 185 190 Thr Cys Glu Ala Thr His Llys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 2 OO 2O5 Phe Asn Arg Asn. Glu. Cys 210

<21 Os SEQ ID NO 2 &211s LENGTH: 217 212s. TYPE: PRT <213> ORGANISM: mus musculis 22 Os. FEATURE: <221s NAME/KEY: UNSURE <222s. LOCATION: (0) . . . (O) <223> OTHER INFORMATION: Fab 5 Heavy chain

<4 OOs SEQUENCE: 2 Glu Val Glin Lieu. Glin Glin Ser Gly Ala Glu Lieu Ala Arg Pro Gly Ala 1. 5 1O 15

Ser Val Lys Lieu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asp Tyr US 7,947,807 B2 23 24 - Continued

2O 25

Ile Asn Trp Wall Arg Glin Arg Thir Gly Glin Gly Phe Glu Trp Ile 35 4 O 45

Gly Glu Ile Pro Gly Ser Gly ASn Ile Asp Tyr Asn Glu Arg Phe SO 55 6 O

Lys Asp Ala Thr Lell Thir Ala Asp Ser Ser Ser Thir Ala Tyr 65 70

Met Glin Luell Ser Ser Lieu. Thir Ser Glu Asp Ser Ala Wall Phe 85 90 95

Wall Arg Gly Phe Gly Tyr Trp Gly Glin Gly. Thir Thr Lieu. Thir Wall Ser 1OO 105 11 O

Ser Ala Lys Th Thir Pro Pro Ser Wall Pro Leu. Ala Pro Gly Ser 115 12 O 125

Ala Ala Gn. Thir Asn. Ser Met Val Thir Lieu. Gly Cys Lieu. Wall Lys Gly 13 O 135 14 O

Tyr Phe Pro Glu Pro Wall Thir Wall. Thir Trp ASn Ser Gly Ser Luell Ser 145 150 155 160

Ser Gly Wall His Thir Phe Pro Ala Wall Lieu. Glin Ser Asp Luell Tyr Thr 1.65 17O 17s

Leul Ser Ser Ser Wall. Thir Wall Pro Ser Ser Thr Trp Pro Ser Glu Thir 18O 185 19 O

Wall. Thir Cys Asn Wall Ala His Pro Ala Ser Ser Thr Lys Wall Asp 195 2O5

Ile Val Pro Arg Asp Cys Gly 21 O 215

SEQ ID NO 3 LENGTH: 10 TYPE PRT ORGANISM: homo sapien

<4 OOs, SEQUENCE: 3 Ile Asp Llys Ser Glu Gly Arg Phe His Val 1. 5 1O

What is claimed is: 3. The method of claim 1, wherein said antibody/GPCR 1. A method for obtaining crystals of a G protein-coupled complex is crystallized using a bicelle crystallization method receptor (GPCR), comprising: 45 or a cubic-phase crystallization method. a) contacting said GPCR with a monovalent antibody that 4. The method of claim 1, wherein said GPCR comprises the IC3 loop of the B2 adrenoreceptor (B2AR). i. specifically binds to a three dimensional epitope of the 5. The method of claim 1, wherein said GPCR is a chimeric intracellular region 3 (IC3) loop of said GPCR and GPCR that contains the IC3 loop of B2AR. ii. stabilizes the relative conformations of the transmem 50 6. The method of claim 1, wherein said GPCR is a chimeric brane region 5 (TM5) and transmembrane region 6 GPCR that comprises amino acid residues I223 to K270 of (TM6) of said GPCR, |B2AR. under conditions that provide for binding of said antibody 7. The method of claim 6, wherein said antibody comprises to said GPCR produce an antibody/GPCR complex: a light chain comprising the amino acid sequence of SEQID b) removing unbound antibody from the product of step a); 55 NO:1 and a heavy chain comprising the amino acid sequence and of SEQID NO:2. c) maintaining the product of step b) under conditions 8. The method of claim 1, wherein the antibody is made by: suitable for crystallizing said antibody/GPCR complex reconsitituting a GPCR in artificial phospholipid vesicles thereby producing a crystalline form of said antibody/ to make an antigen; GPCR complex. 60 immunizing an animal with said antigen; and 2. The method of claim 1, wherein said method comprises screening a plurality of hybridomalines obtained from said combining said antibody/GPCR complex with lipids prior to animal for a hybridoma that produces said antibody. crystallizing said complex. k k k k k UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. ; 7,947,807 B2 Page 1 of 1 APPLICATIONNO. : 12/284245 DATED : May 24, 2011 INVENTOR(S) : Brian Kobilka It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

O In column 1 lines 10-12: Please replace the paragraph beginning with “This work to and ending “in this invention. with the follow: -- This invention was made with Government support under contract NS028471 awarded by the National Institutes of Health. The Government has certain rights in this invention. --

Signed and Sealed this Twenty-fifth Day of October, 2011

David J. Kappos Director of the United States Patent and Trademark Office