CB-1 receptor antigen-binding proteins and uses thereof

Abstract

To provide CB1 receptor antigen-binding proteins, e.g., antibodies, and methods of using the CB1 receptor antibodies.SOLUTION: The CB1 receptor antibodies may include an antagonistic antibody to CB1 receptors and may be used to treat various health conditions. The health conditions can include obesity or diabetes, or any disease that benefits from antagonism of the CB1 receptor. Embodiment 1 is an antigen-binding protein consisting essentially of SEQ ID NO: 4 and SEQ ID NO: 5 that specifically binds to SEQ ID NO: 1.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 61 / 839,458, filed June 26, 2013. is incorporated herein by reference.

[0002] Sequence Listing Reference This application is submitted with a sequence listing in electronic format. It was created on June 25, 2014 under the file name 1835-WO-PCT_SEQ.txt and is 99,680 bytes in size. The electronic format of the sequence listing information is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to CB1 receptor antigen binding proteins, such as antibodies, and methods of using CB1 receptor antibodies. CB1 receptor antibodies may include antibodies that antagonize the signaling of the cannabinoid receptor CB1. [Background technology]

[0004] The CB1 receptor (cannabinoid receptor 1, gene name Cnr1) is a Gi-coupled G protein receptor that is widely expressed in the CNS and peripheral nervous system. Agonist stimulation of the CB1 receptor inhibits adenyly cyclase activity and activates mitogen-activated protein (MAP) kinase. CB1 receptors are highly conserved in humans, mice, and rats.

[0005] CB1 receptors are one of the most abundant and widely distributed G protein-coupled receptors in the mammalian brain. These receptors are also found in peripheral tissues such as adipose tissue, liver, muscle, and the gastrointestinal tract.

[0006] Endogenous agonists of the CB1 receptor include anandamide and 2-arachidonoylglycerol. Exogenous agonists include Δ 9 -Contains tetrahydrocannabinol Small molecule antagonists or inverse agonists (used interchangeably), such as rimonabant or taranabant, have been shown to reduce body weight and result in improved metabolic parameters, including lower plasma glucose and insulin levels.

[0007] Unfortunately, these small molecule antagonists have also been shown to have adverse CNS effects. For example, rimonabant, a small molecule CB1 receptor antagonist / inverse agonist that binds to the CB1 receptor, has been reported to increase the incidence of anxiety, depression, and suicidal ideation in multiple clinical trials (NDA 21-888, FDA Briefing Document 1). ), June 13, 2007) Small molecule antagonists with poor brain penetration (e.g., AM6545 and JD5037) have been reported to reduce food intake and weight gain and improve several metabolic parameters in mice (Tam et al., J. Clin. Invest. 120:2953-66, 2010; Tam et al., Cell Metab 16:1-13, 2012). Another small molecule with peripheral restriction has been described (US2011 / 0144157). The positive metabolic effects of CB1 antagonists may be mediated by peripheral receptors, and therefore peripherally acting macromolecules may be more effective and safer than unsuccessful small molecule treatments. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] US Patent Application Publication No. 2011 / 0144157 [Non-patent literature]

[0009] [Non-Patent Document 1] Tam et al,J.Clin.Invest.120:2953~66,2010 [Non-patent document 2] Tam et al Cell Metab 16:1~13,2012 Summary of the Invention [Means for solving the problem]

[0010] The CB1 antagonist antibodies disclosed herein are believed to be the only large molecule antagonists identified to date. Although small molecule antagonists have been described by various groups, CB1 antagonist antibodies differ from small molecule antagonists in that they have different pharmacokinetic properties, such as lack of CNS penetration and reduced clearance, which allows for less frequent administration.

[0011] The present invention relates to CB1 receptor antigen binding proteins and fragments thereof. CB1 receptor antigen binding proteins and fragments thereof that bind to CB1 receptors may be antagonistic CB1 receptor antigen binding proteins. In various embodiments, the antigen binding protein is an antibody. Uses of the antigen binding proteins described herein are also provided.

[0012] This disclosure provides various embodiments of the present invention.

[0013] Embodiment 1 is an antigen binding protein that specifically binds to SEQ ID NO: 1 and consists essentially of SEQ ID NO: 4 and SEQ ID NO: 5. Another related embodiment may consist of or comprise the amino acid sequence of the antigen binding protein.

[0014] Embodiment 2 is a method for determining whether binding to SEQ ID NO: 1 is achieved by a GTP-Eu assay, an aequorin assay or or cAMP assay.

[0015] Embodiment 3 is the antigen binding protein of embodiment 1, wherein said antigen binding protein is a monoclonal antibody or a fragment thereof.

[0016] Embodiment 4 is the antigen binding protein of embodiment 1, wherein said antigen binding protein is a murine antibody, a humanized antibody, a human antibody, a chimeric antibody, or a multispecific antibody.

[0017] Embodiment 5 is an antigen binding protein of embodiment 1, wherein the antigen binding protein binds to the EC2 domain region of the human CB1 receptor as shown in Figure 1. The exact EC2 domain may be represented in various embodiments with more or fewer amino acids.

[0018] Embodiment 6 is the antigen binding protein of embodiment 5, wherein the antigen binding protein specifically binds to amino acid NCEKLQSVCSDIFPHIDE of SEQ ID NO:1.

[0019] Embodiment 7 is the antigen binding protein of embodiment 6, wherein the antigen binding protein binds to at least 15 amino acids of the amino acid sequence NCEKLQSVCSDIFPHIDE.

[0020] Embodiment 8 is the antigen binding protein of embodiment 7, wherein the antigen binding protein binds to at least 10 amino acids of the amino acid sequence NCEKLQSVCSDIFPHIDE.

[0021] Embodiment 9 is a method for treating an inflammatory bowel disease, comprising administering to a subject the antigen binding protein a therapeutically effective amount of an IgG1 inhibitor, a IgG2 inhibitor, or a IgG2 inhibitor having an EC20 activity in a cAMP assay. 50 2. The antigen binding protein of embodiment 1, wherein the .times.

[0022] Embodiment 10 is a method for treating an inflammatory bowel disease, comprising administering to a subject the antigen binding protein a therapeutically effective amount of an IgG1 inhibitor, the EC200 / ... 50In various embodiments, the antigen binding protein has an EC 50 may be in the picomolar range.

[0023] Embodiment 11 is an antigen binding protein that is at least 99% identical to SEQ ID NO:4 and SEQ ID NO:5.

[0024] Embodiment 12 is an antigen binding protein that specifically binds to SEQ ID NO: 1, including SEQ ID NOs: 17-22, SEQ ID NOs: 36-37, or SEQ ID NOs: 36 and 38. Further related embodiments may either consist of, or consist essentially of, the amino acid sequence of the antigen binding sequence.

[0025] Embodiment 13 is a method for determining whether binding to SEQ ID NO: 1 is achieved by a GTP-Eu assay, an aequorin assay, or ... or the antigen binding protein of embodiment 12, which antagonizes G protein signaling as measured by a cAMP assay.

[0026] Embodiment 14 is the antigen binding protein of embodiment 12, wherein said antigen binding protein is a monoclonal antibody or a fragment thereof.

[0027] Embodiment 15 is the antigen-binding protein of embodiment 14, wherein said antigen-binding protein is a murine antibody, a humanized antibody, a human antibody, a chimeric antibody, or a multispecific antibody.

[0028] Embodiment 16 is the antigen binding protein of embodiment 12, wherein the antigen binding protein binds to the EC2 domain region of the human CB1 receptor as shown in Figure 1. The exact EC2 domain may be represented by a greater or lesser number of amino acids.

[0029] Embodiment 17 is the antigen binding protein of embodiment 16, wherein the antigen binding protein specifically binds to amino acid NCEKLQSVCSDIFPHIDE of SEQ ID NO:1.

[0030] Embodiment 18 is the antigen binding protein of embodiment 17, wherein the antigen binding protein binds to at least 15 amino acids of the amino acid sequence NCEKLQSVCSDIFPHIDE.

[0031] Embodiment 19 is the antigen binding protein of embodiment 18, wherein the antigen binding protein binds to at least 10 amino acids of the amino acid sequence NCEKLQSVCSDIFPHIDE.

[0032] Embodiment 20 is a method for treating an inflammatory bowel disease, comprising administering to a subject the antigen binding protein of any one of the following antibodies: 50 13. The antigen binding protein of embodiment 12, wherein the .times.

[0033] Embodiment 21 is a method for determining whether the antigen binding protein has an EC 300 or EC 300 antibody binding activity in a cAMP assay. 50 In various embodiments, the antigen binding protein has an EC 50 may be in the picomolar range.

[0034] Embodiment 22 is an antigen-binding protein having at least 99% identity to SEQ ID NOs: 17-22.

[0035] Embodiment 22 is an antigen binding protein that specifically binds to SEQ ID NO: 1, including SEQ ID NO: 7 and 8, SEQ ID NO: 7 and 10, SEQ ID NO: 7 and 12, SEQ ID NO: 6 and 16, SEQ ID NO: 4 and 14, SEQ ID NO: 36 and 37, or SEQ ID NO: 36 and 38. Further related embodiments may either consist of or consist essentially of the amino acid sequence of the antigen binding sequence.

[0036] Embodiment 23 is a method for determining whether binding to SEQ ID NO: 1 is achieved by a GTP-Eu assay, an aequorin assay, or ... or the antigen binding protein of embodiment 22, which antagonizes G protein signaling as measured by a cAMP assay.

[0037] Embodiment 24 is the antigen binding protein of embodiment 22, wherein said antigen binding protein is a monoclonal antibody or fragment thereof.

[0038] Embodiment 25 is the antigen-binding protein of claim 24, wherein the antigen-binding protein is a murine antibody, a humanized antibody, a human antibody, a chimeric antibody, or a multispecific antibody.

[0039] Embodiment 26 is the antigen binding protein of embodiment 22, wherein the antigen binding protein binds to the EC2 domain region of the human CB1 receptor as shown in Figure 4A. The exact EC2 domain may be represented by a greater or lesser number of amino acids.

[0040] Embodiment 27 is the antigen binding protein of embodiment 26, wherein the antigen binding protein binds to amino acid NCEKLQSVCSDIFPHIDE of SEQ ID NO:1.

[0041] Embodiment 28 is the antigen binding protein of embodiment 27, wherein the antigen binding protein binds to at least 15 amino acids of the amino acid sequence NCEKLQSVCSDIFPHIDE.

[0042] Embodiment 29 is the antigen binding protein of embodiment 28, wherein the antigen binding protein binds to at least 10 amino acids of the amino acid sequence NCEKLQSVCSDIFPHIDE.

[0043] Embodiment 30 is a method for determining the EC 50 23. The antigen binding protein of embodiment 22, wherein the .times.

[0044] Embodiment 31 is directed to an EC 300 / 300 antibody in a cAMP assay. 50 In various embodiments, the antigen binding protein has an EC 50 may be in the picomolar range.

[0045] Embodiment 32 is an antigen binding protein that is at least 99% identical to one of the antigen binding proteins of embodiment 22.

[0046] Embodiment 33 is a nucleic acid encoding the antigen-binding protein of any one of embodiments 1, 12, or 22.

[0047] Embodiment 34 is an expression vector comprising the nucleic acid of embodiment 33.

[0048] Embodiment 35 is a host cell comprising the vector of embodiment 34.

[0049] Embodiment 36 is the host cell of embodiment 35, wherein the cell is a eukaryotic or prokaryotic cell.

[0050] Embodiment 37 is the host cell of embodiment 36, wherein the eukaryotic cell is a mammalian cell.

[0051] Embodiment 38 is a method for producing an antigen binding protein, comprising culturing the host cell of embodiment 37 under suitable conditions such that the nucleic acid is expressed to produce the antigen binding protein.

[0052] Embodiment 39 is the method of embodiment 38, further comprising recovering the antibody from the host cell culture.

[0053] Embodiment 40 is a composition comprising an antigen binding protein of any one of embodiments 1, 12, 22 or 44, and a pharmaceutically acceptable carrier, diluent or excipient.

[0054] Embodiment 41 is a method of treating a patient in need of antagonism of the CB1 receptor, comprising administering a composition of embodiment 40.

[0055] Embodiment 42 is the method of embodiment 32, wherein treating the patient results in weight loss or improved metabolic parameters.

[0056] Embodiment 43 is the method of embodiment 42, wherein the improved metabolic parameter is reduced plasma glucose levels, reduced insulin levels, reduced triglyceride levels, reduced HbA1c, reduced intraperitoneal and liver fat, reduced blood pressure, increased adiponectin, increased HDL, cholesterol, or increased energy expenditure.

[0057] Embodiment 44 is a method for specifically binding to SEQ ID NO: 1, using a GTP-Eu assay, an aequorin assay, It is an antigen-binding protein that antagonizes G protein signaling as measured by the cAMP assay or cAMP assay.

[0058] Embodiment 45 is the antigen binding protein of embodiment 44, wherein said antigen binding protein is a monoclonal antibody or a fragment thereof.

[0059] Embodiment 46 is the antigen binding protein of embodiment 45, wherein said antigen binding protein is a murine antibody, a humanized antibody, a human antibody, a chimeric antibody or a multispecific antibody.

[0060] Embodiment 47 is the antigen binding protein of embodiment 44, wherein the antigen binding protein binds to the EC2 domain region of the human CB1 receptor. The exact EC2 domain may be represented by a greater or lesser number of amino acids.

[0061] Embodiment 48 is the antigen binding protein of embodiment 47, wherein the antigen binding protein specifically binds to amino acid NCEKLQSVCSDIFPHIDE of SEQ ID NO:1.

[0062] Embodiment 49 is the antigen binding protein of claim 48, wherein the antigen binding protein binds to at least 15 amino acids of the amino acid sequence NCEKLQSVCSDIFPHIDE.

[0063] Embodiment 50 is the antigen binding protein of claim 48, wherein said antigen binding protein binds to at least 10 amino acids of the amino acid sequence NCEKLQSVCSDIFPHIDE.

[0064] Embodiment 51 is directed to an EC 200 in a cAMP assay. 50 45. The antigen binding protein of embodiment 44, wherein the .DELTA.I.

[0065] Embodiment 52 is directed to an EC 200 in a cAMP assay. 50 In various embodiments, the antigen binding protein has an EC 50 may be in the picomolar range.

[0066] Other embodiments are described herein that will be appreciated by those skilled in the art. While various embodiments have been described above, those skilled in the art will appreciate that the examples and tests detailed herein are merely illustrative. It should be understood that various modifications are possible without departing from the spirit of the invention. In an embodiment of the present invention, for example, the following items are provided: (Item 1) An antigen binding protein that specifically binds to SEQ ID NO:1, consisting essentially of SEQ ID NO:4 and SEQ ID NO:5. (Item 2) The binding to SEQ ID NO: 1 is determined by a GTP-Eu assay, an aequorin assay or a cAM assay. 2. The antigen-binding protein of item 1, which antagonizes G protein signaling as measured in a P assay. (Item 3) 2. The antigen-binding protein of item 1, wherein the antigen-binding protein is a monoclonal antibody or a fragment thereof. (Item 4) 2. The antigen-binding protein of item 1, wherein the antigen-binding protein is a murine antibody, a humanized antibody, a human antibody, a chimeric antibody, or a multispecific antibody. (Item 5) 2. The antigen-binding protein of item 1, wherein the antigen-binding protein binds to the EC2 domain region of the human CB1 receptor. (Item 6) 6. The antigen-binding protein of item 5, wherein the antigen-binding protein specifically binds to the amino acid NCEKLQSVCSDIFPHIDE of SEQ ID NO: 1. (Item 7) 7. The antigen-binding protein of item 6, wherein the antigen-binding protein binds to at least 15 amino acids of the amino acid sequence NCEKLQSVCSDIFPHIDE. (Item 8) 8. The antigen-binding protein of item 7, wherein the antigen-binding protein binds to at least 10 amino acids of the amino acid sequence NCEKLQSVCSDIFPHIDE. (Item 9) the antigen binding protein has an EC 50 2. The antigen-binding protein of item 1, having the following structure: (Item 10) the antigen binding protein has an EC 50 10. The antigen-binding protein of item 9, having the following structure: (Item 11) An antigen binding protein having at least 99% identity to SEQ ID NO:4 and SEQ ID NO:5. (Item 12) An antigen-binding protein that specifically binds to SEQ ID NO: 1, including SEQ ID NOs: 17 to 22, SEQ ID NOs: 36 to 37, or 36 and 38. (Item 13) The binding to SEQ ID NO: 1 is determined by a GTP-Eu assay, an aequorin assay or a cAM assay. 13. The antigen-binding protein of item 12, which antagonizes G protein signaling as measured by a P assay. (Item 14) 13. The antigen-binding protein of item 12, wherein the antigen-binding protein is a monoclonal antibody or a fragment thereof. (Item 15) 15. The antigen-binding protein of item 14, wherein the antigen-binding protein is a murine antibody, a humanized antibody, a human antibody, a chimeric antibody, or a multispecific antibody. (Item 16) 13. The antigen-binding protein of item 12, wherein the antigen-binding protein binds to the EC2 domain region of the human CB1 receptor. (Item 17) 17. The antigen-binding protein of item 16, wherein the antigen-binding protein specifically binds to the amino acid NCEKLQSVCSDIFPHIDE of SEQ ID NO: 1. (Item 18) 18. The antigen-binding protein of item 17, wherein the antigen-binding protein binds to at least 15 amino acids of the amino acid sequence NCEKLQSVCSDIFPHIDE. (Item 19) 19. The antigen-binding protein of item 18, wherein the antigen-binding protein binds to at least 10 amino acids of the amino acid sequence NCEKLQSVCSDIFPHIDE. (Item 20) the antigen-binding protein has an EC 50 13. The antigen-binding protein of item 12, having the following structure: (Item 21) the antigen binding protein has an EC 50 21. The antigen-binding protein of item 20, having the following structure: (Item 22) An antigen-binding protein that specifically binds to SEQ ID NO: 1, which has at least 99% identity to SEQ ID NOs: 17-22. (Item 23) An antigen-binding protein comprising SEQ ID NOs: 7 and 8, SEQ ID NOs: 7 and 10, SEQ ID NOs: 7 and 12, SEQ ID NOs: 6 and 16, SEQ ID NOs: 4 and 14, SEQ ID NOs: 36 and 37, or SEQ ID NOs: 36 and 38. (Item 24) The binding to SEQ ID NO: 1 is determined by a GTP-Eu assay, an aequorin assay or a cAM assay. 13. The antigen-binding protein of item 12, which antagonizes G protein signaling as measured by a P assay. (Item 25) 24. The antigen-binding protein of item 23, wherein the antigen-binding protein is a monoclonal antibody or a fragment thereof. (Item 26) 26. The antigen-binding protein of item 25, wherein the antigen-binding protein is a murine antibody, a humanized antibody, a human antibody, a chimeric antibody, or a multispecific antibody. (Item 27) 24. The antigen-binding protein of item 23, wherein the antigen-binding protein binds to the EC2 domain region of the human CB1 receptor. (Item 28) 28. The antigen binding protein of item 27, wherein the antigen binding protein binds to amino acid NCEKLQSVCSDIFPHIDE of SEQ ID NO: 1. (Item 29) 29. The antigen-binding protein of item 28, wherein the antigen-binding protein binds to at least 15 amino acids of the amino acid sequence NCEKLQSVCSDIFPHIDE. (Item 30) 30. The antigen binding protein of item 29, wherein the antigen binding protein binds to at least 10 amino acids of the amino acid sequence NCEKLQSVCSDIFPHIDE. (Item 31) the antigen-binding protein has an EC 50 24. The antigen-binding protein of item 23, having the following structure: (Item 32) the antigen binding protein has an EC 50 32. The antigen-binding protein of item 31, having the following structure: (Item 33) 24. An antigen-binding protein having at least 99% identity to one of the antigen-binding proteins of item 23. (Item 34) 23. A nucleic acid encoding the antigen-binding protein of any one of items 1, 12 or 22. (Item 35) 35. An expression vector comprising the nucleic acid according to Item 34. (Item 36) A host cell comprising the vector described in Item 35. (Item 37) 37. The host cell of item 36, wherein the cell is a eukaryotic or prokaryotic cell. (Item 38) 38. The host cell according to item 37, wherein the eukaryotic cell is a mammalian cell. (Item 39) 38. A method for producing an antigen-binding protein, comprising culturing the host cell of Item 37 under suitable conditions such that the nucleic acid is expressed to produce the antigen-binding protein. (Item 40) 40. The method of claim 39, further comprising recovering the antigen binding protein from the host cell culture. (Item 41) 46. ​​A composition comprising the antigen-binding protein of any one of items 1, 12, 22 or 45 and a pharmacologically acceptable carrier, diluent or excipient. (Item 42) 42. A method for treating a patient in need of CB1 receptor antagonism, comprising administering any of the compositions of claim 41. (Item 43) 43. The method of claim 42, wherein said treatment of the patient results in weight loss or improvement of metabolic parameters. (Item 44) Item 44. The method of item 43, wherein the improved metabolic parameter is reduced plasma glucose concentration, reduced insulin concentration, reduced triglyceride concentration, reduced HbA1c, reduced intraperitoneal liver fat, reduced blood pressure, increased adiponectin, increased HDL, increased cholesterol, or increased energy expenditure. (Item 45) Specific binding to SEQ ID NO: 1, GTP-Eu assay, aequorin assay or cA An antigen-binding protein that antagonizes G protein signaling as measured by the MP assay. (Item 46) 46. ​​The antigen-binding protein of item 45, wherein the antigen-binding protein is a monoclonal antibody or a fragment thereof. (Item 47) 46. ​​The antigen-binding protein of item 45, wherein the antigen-binding protein is a murine antibody, a humanized antibody, a human antibody, a chimeric antibody, or a multispecific antibody. (Item 48) 46. ​​The antigen-binding protein of item 45, wherein the antigen-binding protein binds to the EC2 domain region of the human CB1 receptor. (Item 49) 49. The antigen binding protein of item 48, wherein the antigen binding protein specifically binds to amino acid NCEKLQSVCSDIFPHIDE of SEQ ID NO: 1. (Item 50) 50. The antigen-binding protein of item 49, wherein the antigen-binding protein binds to at least 15 amino acids of the amino acid sequence NCEKLQSVCSDIFPHIDE. (Item 51) 51. The antigen binding protein of item 50, wherein the antigen binding protein binds to at least 10 amino acids of the amino acid sequence NCEKLQSVCSDIFPHIDE. (Item 52) The antigen binding protein has an EC 50 46. ​​The antigen-binding protein of item 45, wherein the .alpha.-I.sub.2 binding activity is less than 250 nM. (Item 53) the antigen binding protein has an EC 50 53. The antigen-binding protein of item 52, having the following structure: [Brief explanation of the drawings]

[0067] [Figure 1]Figure 1 shows an alignment of the human (SEQ ID NO: 1), mouse (SEQ ID NO: 2), and rat (SEQ ID NO: 3) CB1 protein sequences, with the approximate locations of extracellular loops 1 and 2 (EC1 and EC2; also referred to as the EC domain region) indicated by solid lines on the sequences. Residues used in the epitope mapping studies shown in Figure 4 are indicated in Figure 1 by black shading, with positions in EC1 (R186P—between the human and mouse / rat sequences) and EC2 (E258K—between the human and mouse / rat sequences) and (H270L—between the human and mouse / rat sequences). It should be noted that the position numbers above the sequences are provided for convenience and do not necessarily align exactly as they appear. Additional differences between the human and rodent sequences are indicated by arrows.

[0068] [Figure 2A] Figures 2A and 2B compare the heavy and light chain protein sequences of various anti-CB1 antibodies, showing amino acid differences between several clones in the framework and CDR regions of the heavy and light chain protein sequences. [Figure 2B] Figures 2A and 2B compare the heavy and light chain protein sequences of various anti-CB1 antibodies, showing amino acid differences between several clones in the framework and CDR regions of the heavy and light chain protein sequences.

[0069] [Figure 3] FIG. 3 shows further characterization of wild-type and mutant forms of the CB1 antagonist antibody 10D10.

[0070] [Figure 4A] Figures 4A-4C show mutations made to the CB1 receptor useful for providing antibody binding sites: Figure 4A is a pictorial representation of the mutants, Figure 4B shows FACS binding data, and Figure 4C shows aequorin signaling data. [Figure 4B]Figures 4A-4C show mutations made to the CB1 receptor useful for providing antibody binding sites: Figure 4A is a pictorial representation of the mutants, Figure 4B shows FACS binding data, and Figure 4C shows aequorin signaling data. [Figure 4C] Figures 4A-4C show mutations made to the CB1 receptor useful for providing antibody binding sites: Figure 4A is a pictorial representation of the mutants, Figure 4B shows FACS binding data, and Figure 4C shows aequorin signaling data.

[0071] [Figure 5A] 5A-5C show the activity of CB1 antagonist antibodies in a cAMP (FIG. 5A) signaling assay, an aequorin (FIG. 5B) signaling assay, and a KinExA (FIG. 5C) binding assay. [Figure 5B] 5A-5C show the activity of CB1 antagonist antibodies in a cAMP (FIG. 5A) signaling assay, an aequorin (FIG. 5B) signaling assay, and a KinExA (FIG. 5C) binding assay. [Figure 5C] 5A-5C show the activity of CB1 antagonist antibodies in a cAMP (FIG. 5A) signaling assay, an aequorin (FIG. 5B) signaling assay, and a KinExA (FIG. 5C) binding assay.

[0072] [Figure 6A] FIG. 6A shows the activity of CB1 antagonist antibodies compared to anandamide in the GTP-Eu assay. [Figure 6B] FIG. 6B shows the effect of varying concentrations of 10D10 on GTP-Eu.

[0073] [Figure 7] FIG. 7 shows the various mutans and CB1 activity of CB1 antagonist antibodies. DETAILED DESCRIPTION OF THE INVENTION

[0074] Disclosed herein are CB1 receptor antigen binding proteins (e.g., antibodies and functional binding fragments thereof) that bind to the CB1 receptor. Such antigen binding proteins bind to the CB1 receptor and prevent the CB1 receptor from performing various functions, e.g., antagonizing receptor activity. The CB1 receptor binding proteins bind to the CB1 receptor and inhibit signal transduction as measured, for example, by a cAMP assay, an aequorin assay, or a GTP-Eu assay. Prevent it from happening.

[0075] The above summary is not intended to define all aspects or embodiments of the present invention, and additional aspects may be described in other sections. It is intended that this entire document be linked as a unified disclosure, and thus it should be understood that any combination of features described herein is contemplated, even if that combination of features is not simultaneously described within the same sentence, paragraph, or section of this document.

[0076] In addition to the above, as an additional aspect, all embodiments narrower in scope than the variations defined by a particular paragraph herein may be included in the present disclosure. For example, when an aspect refers to a genus, it should be understood that all members of that genus can individually and separately be an embodiment. Also, when an aspect is described as being selected from a genus or members of a genus, it should be understood to encompass combinations of two or more members of that genus. While various embodiments may be presented herein using the term "comprising" in various contexts, it should also be understood that related embodiments may be described using the terms "consisting of" or "consisting essentially of."

[0077] It will be understood that the descriptions in this specification are merely illustrative and explanatory and do not limit the claims of the present invention. In this specification, the use of the singular includes the plural unless specifically stated otherwise. In this specification, the use of "or" means "and / or" unless specifically stated otherwise. Furthermore, when used, terms such as "including" and other forms of "comprises" and "included" are used, they are open-ended. Furthermore, terms such as "element" or "component" include both elements and components containing one unit and elements and components containing one or more subunits, unless specifically stated otherwise. Furthermore, the use of the term "portion" can include a portion of a moiety or the entire moiety.

[0078] It should also be understood that when a range of values ​​is described, the described characteristic is considered to be one value within that range. For example, "a pH of about pH 4 to about pH 6" contemplates any value within that range, including, but not limited to, pH 4, 4.2, 4.6, 5.1, 5.5, etc. Furthermore, "a pH of about pH 4 to about pH 6" should not be construed to mean that the pH in question varies from pH 4 to pH 6 by two pH units; rather, the pH value of a solution may be selected within a range of two pH values.

[0079] In some embodiments, when the term "about" is used, it means that 5%, 10%, 15% or more of the stated numerical value is added to or subtracted from the stated numerical value. The actual variable intended can be determined from the context.

[0080] The section titles used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All materials, or portions of materials, including but not limited to patents, patent applications, articles, books, and treatises cited herein are expressly incorporated herein in their entirety by reference thereto for any purpose. As used in accordance with this disclosure, the following terms shall be understood to have the following meanings unless otherwise indicated:

[0081] As used herein, the term "CB1" refers to cannabinoid receptor 1, a Gi-coupled G protein receptor widely expressed in the CNS and peripheral nervous system. Stimulation of the CB1 receptor is known to inhibit adenyly cyclase and activate mitogen-activated protein (MAP) kinase. The CB1 protein sequence is highly conserved among humans, mice, and rats. The CB1 receptor is one of the most abundant and widely distributed G protein-coupled receptors in the mammalian brain. These receptors are also found in other tissues, such as adipose tissue, liver, muscle, and the gastrointestinal tract.

[0082] As used herein, an antibody or antigen-binding fragment can be an agonist or an antagonist.

[0083] "Agonist" refers to a substance that binds to a polypeptide (such as a receptor) or polynucleotide and, in particular, stimulates, increases, activates, promotes, enhances activation, sensitizes, or upregulates the activity or expression of the polypeptide or polynucleotide.

[0084] "Antagonist" refers to a substance that binds to a polypeptide (such as a receptor) or polynucleotide and, in particular, partially or completely blocks stimulation, reduces, prevents, delays activation, inactivates, desensitizes, or downregulates the activity of the bound polypeptide or polynucleotide. Antagonists may also be inverse agonists, particularly those that can reduce constitutive signal transduction of the receptor.

[0085] "Antigen binding protein" ("ABP") refers to any protein that binds a specific target antigen. As used herein, the specific target antigen may be the CB1 receptor or a fragment or region thereof. "Antigen binding protein" includes, but is not limited to, antibodies and binding portions thereof, such as immunologically functional fragments. Peptibodies are also an example of an antigen binding protein.

[0086] A "CB1 receptor antigen binding protein" refers to a protein capable of binding to the CB1 receptor. The "CB1 receptor antigen binding protein" may be a "CB1 receptor antagonist antibody" that binds the receptor. The CB1 receptor antibody can block CB1 receptor signaling. Blocking signaling can result in both cellular and physiological responses, such as weight loss and improved metabolic parameters, e.g., reduced plasma glucose and insulin levels.

[0087] The "extracellular (EC) binding region" of the CB1 protein is shown in FIG. 1 and is also referred to as the "extracellular loop" or "extracellular domain region." For example, the EC2 domain region is believed to have the sequence NCEKLQSVCSDIFPHIDE of SEQ ID NO: 1. In various embodiments, the EC2 domain region may further comprise an additional 1, 2, 3, 4, or 5 amino acids at either the amino or carboxyl terminus. In other embodiments, the EC2 domain region may have 1, 2, 3, 4, or 5 fewer amino acids at either the amino or carboxyl terminus of the sequence. The antibodies disclosed herein may bind to any of the EC2 domains described above, or to the sequence NCEKLQSVCSDIFPHIDE of SEQ ID NO: 1, or at least 10 or at least 15 amino acids of this sequence. As shown in FIG. 1, various antibodies may alternatively bind to the EC1 domain region.

[0088] The terms "polynucleotide" or "nucleic acid" include both single-stranded and double-stranded nucleotide polymers. The nucleotides comprising a polynucleotide can be ribonucleotides or deoxyribonucleotides or modified forms of these nucleotides. Such modifications include base modifications such as bromouridine derivatives and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoroaniladate, and phosphoroamidate.

[0089] The term "oligonucleotide" refers to a polynucleotide containing 200 or fewer nucleotides. In some embodiments, the oligonucleotide is about 10 to about 60 bases in length. In other embodiments, the oligonucleotide is about 12, 13, 14, 15, 16, 17, 18, 19, or 20 to about 40 nucleotides in length. Oligonucleotides can be single-stranded or double-stranded, for example, when used in constructing a mutant gene. Oligonucleotides can be sense or antisense oligonucleotides. Oligonucleotides can include a label, such as a radioactive label, fluorescent label, hapten, or antigenic label, for detection methods. Oligonucleotides can be used, for example, as PCR primers, cloning primers, or hybridization probes.

[0090] An "isolated nucleic acid molecule" refers to DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or some combination thereof, that is not associated with all or part of the polynucleotides that comprise the isolated polynucleotide as found in nature, or that is linked to polynucleotides with which it is not naturally linked. For purposes of this disclosure, it should be understood that a "nucleic acid molecule comprising" a particular nucleotide sequence does not encompass an entire chromosome. An isolated nucleic acid molecule "comprising" a particular nucleic acid sequence can, in addition to the specified sequence, include coding sequences encoding up to 10, or even up to 20, other proteins or portions thereof, or can include operably linked regulatory sequences that control expression of the coding region of the described nucleic acid sequence, and / or can include vector sequences. In various embodiments, a described nucleic acid can be an "isolated nuclear molecule."

[0091] Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence discussed herein is the 5'-end, and the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction. The 5' to 3' direction in which the nascent RNA transcript is added is referred to as the transcription direction; the region of the sequence on the DNA strand that corresponds 5' to the 5' end of the RNA transcript and has the same sequence as the RNA transcript is referred to as the "upstream sequence"; the region of the sequence on the DNA strand that corresponds 3' to the 3' end of the RNA transcript and has the same sequence as the RNA transcript is referred to as the "downstream sequence."

[0092] A nucleic acid can encode an antigen binding protein, e.g., a CB1 receptor antigen binding protein or an anti-CB1 receptor antibody, as disclosed in various embodiments herein. A nucleic acid is said to be "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it expresses the polypeptide as a precursor protein involved in the secretion of that polypeptide; a promoter or enhancer is operably linked if it is linked to a coding sequence and affects the transcription of the sequence; or a ribosome binding site is linked to a coding sequence and positioned so as to promote translation. Generally, "operably linked" means that the linked DNA sequences are contiguous, and, in the case of a secretory leader, the sequences are contiguous and in reading phase. However, enhancers need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites are absent, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

[0093] The term "amino acid" refers to natural and / or non-natural amino acids and includes the usual meaning in the art. Amino acids are also referred to as standard or non-standard amino acids.

[0094] The terms "polypeptide" or "protein" refer to a polymer having the amino acid sequence of a wild-type protein, i.e., a protein produced by a naturally occurring, non-recombinant cell; or to a protein that can be produced by a genetically engineered or recombinant cell, including molecules having the amino acid sequence of a wild-type protein or molecules having deletions, additions, and / or substitutions of one or more amino acids of the wild-type sequence. The terms also include amino acid polymers in which one or more amino acids are chemical analogs of the corresponding naturally occurring amino acids and polymers. The terms "polypeptide" and "protein" specifically encompass sequences having deletions, additions, and / or substitutions of one or more amino acids of a CB1 receptor antigen binding protein, antibody, or antigen binding protein. The term "polypeptide fragment" refers to a polypeptide having an amino-terminal deletion, a carboxyl-terminal deletion, and / or a deletion within the sequence compared to the full-length wild-type protein. Such fragments can also contain modified amino acids compared to the wild-type protein. In various embodiments, the length of a fragment can range from about 5 to about 500 amino acids. For example, the length of a fragment can be at least about 5, about 6, about 8, about 10, about 14, about 20, about 50, about 70, about 100, about 150, about 200, about 250, about 300, about 350, about 400, or about 450 amino acids. Useful polypeptide fragments include immunologically functional fragments of antibodies, including binding domains. In the case of CB1 receptor-binding antibodies, useful fragments include those of the CB1 receptor-binding domain, the CB1 receptor-binding domain, and the CB1 receptor-binding domain. Suitable fragments include, but are not limited to, CDR regions, heavy and / or light chain variable domains, portions of antibody chains, or only the variable regions thereof containing 1, 2, 3, 4, 5, or 6 CDRs.

[0095] The term "isolated protein" means that the protein of interest is (1) free from at least some other proteins with which it is normally found; (2) essentially free from other proteins from the same source, e.g., the same species; (3) expressed by cells of a different species; (4) at least about 50 percent separated from polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated; (5) functionally associated (by covalent or noncovalent interactions) with polypeptides with which it is not naturally associated; or (6) not naturally occurring. An isolated protein can be a CB1 antigen-binding protein or antibody. Typically, an "isolated protein" can represent at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or more of a sample. Synthetic, genomic DNA, cDNA, mRNA, or other RNA, or any combination thereof, can encode such an isolated protein. In various embodiments, an isolated protein is substantially free of proteins or polypeptides or other contaminants present in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic, research, or other use. An isolated "antigen binding protein" or an "isolated antibody" can be considered an "isolated protein." In various embodiments, the antigen binding protein can be an isolated antigen binding protein or an isolated antibody.

[0096] "Variants" or "mutants" of a polypeptide (e.g., an antigen-binding protein or antibody) include amino acid sequences that have insertions, deletions, and / or substitutions of one or more amino acid residues in the amino acid sequence compared to another polypeptide sequence. Variants include fusion proteins.

[0097] As used herein, the 20 common (standard or naturally occurring) amino acids and their abbreviations follow conventional usage. nd Ed., E.S. Golub & D.R. Gren, Eds., Sinauer Assoc., Sunderland, Mass. (1991), which is incorporated herein by reference for all purposes. Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids), α-, α-disubstituted amino acids, N-alkylamino acids, lactic acid, and other non-conventional amino acids may also be suitable components of the polypeptides of the various embodiments described herein. Examples of non-conventional amino acids include 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethylamino acid, γ-amino acid, ε-N,N,N-trimethyl ... Lysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylserine Examples of amino acids include methionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In annotating polypeptides used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0098] Conservative amino acid substitutions can include non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than synthesis in biological systems. These include peptidomimetics and other amino acid moiety reverses or inversions.

[0099] Naturally occurring residues can be divided into classes based on common properties of their side chains: · Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; · Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; · Acidic: Asp, Glu; · Basic: His, Lys, Arg; Residues that influence chain orientation: Gly, Pro; and · Aromatics: Trp, Tyr, Phe.

[0100] For example, non-conservative substitutions may involve exchanging a member of one of these classes for a member of another class, and such substituted residues may be introduced, for example, into regions of a human antibody that are homologous with a non-human antibody, or into non-homologous regions of the molecule.

[0101] When making modifications to an antigen binding protein (such as an antibody), according to certain embodiments, the hydropathic index of amino acids can be taken into consideration. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. These amino acids include isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine / cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9), and ribonucleotides (-0.9). ATP (-0.9), tyrosine (-1.3), proline (-1.6), histidine (-3.2), glutamate (-3.5), glutamine (-3.5), aspartic acid (-3.5), aspartic acid (-3.5), Paragine (-3.5), Lysine (-3.9); and Arginine (-4.5).

[0102] The importance of the hydropathic index of amino acids in determining the biological interaction function of a protein is understood in the art. Kyte et al., J. Mol. Biol., 157:105-131, (1982). It is known that certain amino acids can be substituted with other amino acids having a similar hydropathic index or score and still retain similar biological activity. When making changes based on the hydropathic index, some embodiments include substitution of amino acids with a hydropathic index within ±2. Some embodiments include amino acids within ±1, and some embodiments include those within ±0.5.

[0103] It is also understood in the art that similar amino acid substitutions can be made effectively on the basis of hydrophilicity, particularly when the resulting biologically functional proteins or peptides are intended for use in immunological embodiments. In certain embodiments, the local maximum average hydrophilicity value of a protein, which depends on the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., correlates with a biological property of the protein.

[0104] The hydrophilicity values ​​of these amino acid residues are as follows: arginine (+3.0), lysine (+3.0), aspartic acid (+3.0±1), glutamate (+3.0±1), serine (+0.3), asparagine (+0.2), glutamine (+0.2), glycine (0), threonine (-0.4), proline (-0.5±1), alanine (-0.5), histidine (-0.5), and threonine (-0.4). sine (-0.5), cysteine ​​(-1.0), methionine (-1.3), valine (-1.5), leucine (-1.8), isoleucine (-1.8), tyrosine (-2.3), phenylalanine The hydrophilicity values ​​of amino acids are: lanine (-2.5), and tryptophan (-3.4). When making modifications based on hydrophilicity values, some embodiments include substitution of amino acids with hydrophilicity values ​​within ±2, some embodiments include those within ±1, and some embodiments include those within ±0.5. It is also possible to identify epitopes based on hydrophilicity from the primary amino acid sequence. These regions are also referred to as "epitope core regions."

[0105] Exemplary amino acid substitutions are listed in Table 1. Table 1: Amino acid substitutions [Table 1-1] [Table 1-2]

[0106] The term "derivative" refers to a molecule that contains a chemical modification other than an amino acid (or nucleic acid) insertion, deletion, or substitution. In certain embodiments, a derivative includes a covalent modification, such as, but not limited to, chemical conjugation with a polymer, lipid, or other organic or inorganic moiety. In certain embodiments, a chemically modified antigen-binding protein may have a longer circulating half-life than an antigen-binding protein that is not chemically modified. In certain embodiments, a chemically modified antigen-binding protein may have improved targeting ability to desired cells, tissues, and / or organs. In some embodiments, a derivative of an antigen-binding protein is covalently modified to include the attachment of one or more water-soluble polymers, including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. See, e.g., U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192, and 4,179,337. In certain embodiments, the derivatives of the antigen binding proteins comprise one or more polymers, including, but not limited to, carbohydrate-based polymers such as monomethoxypolyethylene glycol, dextran, cellulose, poly(N-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polymers of oxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, and mixtures of such polymers.

[0107] In some embodiments, the derivatives are covalently modified with polyethylene glycol (PEG) subunits. In some embodiments, one or more water-soluble polymers are attached to one or more specific positions of the derivative, e.g., at the amino terminus. In some embodiments, one or more water-soluble polymers are randomly attached to one or more side chains of the derivative. In some embodiments, PEG is used to improve the therapeutic potential of antigen-binding proteins. In some embodiments, PEG is used to improve the therapeutic potential of humanized antibodies. Certain of these methods are discussed, for example, in U.S. Patent No. 6,133,426, which is incorporated herein by reference for any purpose.

[0108] Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties similar to those of the template peptide. These types of non-peptide compounds are called "peptidomimetics" or "peptidomimetics." Fauchere, J., Adv. Drug Res., 15:29, (1986); Veber & Freidinger, TINS, p. 392, (1985); and Evans et al., J. Med. Chem., 30:1229, (1987), which are incorporated herein by reference for all purposes. Such compounds are often generated by computer-assisted molecular modeling. Peptidomimetics, which are structurally similar to therapeutically useful peptides, can be used to similarly achieve therapeutic or prophylactic effects. Generally, peptidomimetics are structurally similar to a reference polypeptide (i.e., a polypeptide that has biochemical properties or pharmacological activity), such as a human antibody, but can be modified by methods well known in the art to include --CH2NH--, --CH2S--, --CH2-CH2--, --CH= CH-(cis & trans), --COCH2--, --CH(OH)CH2--, and --CH2S In one embodiment, one or more amino acids of the consensus sequence are replaced with a D-amino acid of the same kind (e.g., D-lysine in place of L-lysine) to generate a more stable peptide. In addition, constrained peptides containing consensus sequences or substantially identical variants of the consensus sequences can be generated by methods known in the art (Rizo & Gierasch, Ann. Rev. Biochem., 61:387, (1992), which is incorporated herein by reference for any purpose), such as by adding internal cysteine ​​residues that can form intramolecular disulfide bridges to cyclize the peptide.

[0109] The term "native" as used throughout this specification in connection with biological material such as polypeptides, nucleic acids, host cells, etc., refers to a material found in nature or a form of material found in nature.

[0110] The term "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, means that two or more sequences or subsequences are identical or have a specified percentage of identical amino acid residues or nucleotides, as determined using the BLAST or BLAST 2.0 sequence comparison algorithm with the default parameters described below, or by manual alignment and visual inspection (e.g., in various embodiments, within a comparison window for maximum correspondence). When compared and aligned over a specified region (window), the antigen binding protein may have about 60% identity, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more identity over a particular region to a sequence presented herein (see, e.g., the NCBI website http: / / www.ncbi.nlm.nih.gov / BLAST / ). Such sequences are also said to be "substantially identical." This definition may also refer to, or apply to, the complement sequence of a test sequence. This definition also includes sequences that have deletions and / or additions, as well as sequences that have substitutions. As described herein, each algorithm can take into account gaps, etc. In various embodiments, identity exists over a region that is at least about 25 amino acids, about 50 amino acids or nucleotides in length, or over a region that is 50-100 amino acids or nucleotides in length.

[0111] In the case of sequence comparison, typically one sequence is used as a reference sequence, and test sequences are compared to it. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence based on the program parameters.

[0112] The "comparison window" includes reference to a segment at any one of the desired consecutive position numbers. In some embodiments, the "comparison window" can be selected from the group consisting of about 50 to about 200, or about 100 to about 150, or 150 or more. If desired, the comparison sequence and the reference sequence at the same consecutive position number may be optimally aligned before comparing the two sequences. Methods for aligning sequences to be compared are well known in the art. Optimal alignment of the sequences to be compared can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math., 2:482, (1981); by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol., 48:443, (1970); by the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA, 85:2444, (1988); by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.); or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology (Ausubel et al., eds. 1995, supplement)).

[0113] Examples of algorithms suitable for determining percent sequence identity and sequence similarity include the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res., 25:3389-3402, (1977) and Altschul et al., J. Mol. Biol., 215:403-410, (1990), respectively. BLAST and BLAST 2.0 are used with the parameters described herein to determine percent sequence identity for nucleic acids and proteins of various embodiments. Software for performing BLAST analyses is available from the National Center for Biotechnology Information. The algorithm is publicly available at http: / / www.ncbi.nlm.nih.gov / (National Institute of Biotechnology Information) (http: / / www.ncbi.nlm.nih.gov / ). The algorithm identifies high-scoring sequence pairs (HSPs) by first identifying short words W in the query sequence that, when aligned with words of the same length in a database sequence, match or meet a positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These first found neighborhood words are used to initiate a search for longer HSPs containing them. The found words are extended toward both ends of each sequence, as far as the cumulative alignment score can be. Cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (mismatching residues; always < 0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the found word in each direction is stopped if the cumulative alignment score falls by an amount X from the maximum achieved; if the cumulative score falls below zero due to the accumulation of one or more negative residue alignment scores; or if the end of the sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. For the BLASTN program (for nucleotide sequences), the word length (W) is 11, the expectation (E) is 10, M=5, and N=-4. For amino acid sequences, the BLASTP program uses a default word length of 3 and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915, (1989)) uses a default alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and a default comparison of both strands.

[0114] The term "control sequences" refers to polynucleotide sequences that affect the expression and processing of coding sequences to which they are linked. The nature of such control sequences may vary depending on the host organism. In particular embodiments, prokaryotic control sequences may include a promoter, ribosomal binding site, and transcription termination sequence. For example, eukaryotic control sequences may include a promoter containing one or more recognition sites for transcription factors, transcription enhancer sequences, and transcription termination sequences. "Control sequences" may include leader sequences and / or fusion partner sequences.

[0115] The term "vector" refers to any molecule or entity (eg, nucleic acid, plasmid, bacteriophage, or virus) used to introduce protein-coding information into a host cell.

[0116] The term "expression vector" or "expression construct" refers to a vector suitable for transformation of a host cell and containing nucleic acid sequences that (in conjunction with the host cell) direct and / or control the expression of one or more heterologous coding regions operably linked thereto. Expression constructs can include, but are not limited to, sequences that affect or control transcription, translation, and, if present, introns, sequences that affect RNA splicing of the operably linked coding region. Expression vectors useful in the various embodiments described herein can contain at least one expression control sequence operably linked to the DNA sequence or fragment to be expressed. Control sequences are inserted into the vector to control and regulate expression of the cloned DNA sequence. Examples of useful expression control sequences include those from the lac system, trp system, tac system, trc system, lambda phage major operator and promoter region, the fd coat protein control region, yeast glycolytic promoters, e.g., the promoter for 3-phosphoglycerate kinase. promoters derived from polyoma, adenovirus, retrovirus, and simian virus, such as the early and late promoters or SV40, and other sequences known to control gene expression in prokaryotic or eukaryotic cells and viruses in these cells, or combinations thereof.

[0117] The term "host cell" refers to a cell that has been transformed, or is capable of being transformed, with a nucleic acid sequence and thereby expresses a gene of interest. The term includes the progeny of the parent cell, whether or not such progeny are identical in morphology or genetic make-up to the original parent cell, so long as the gene of interest is present.

[0118] The term "transfection" refers to the uptake of foreign or exogenous DNA by a cell; a cell is said to be "transfected" when the exogenous DNA is introduced inside the cell membrane. Numerous transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into a suitable host cell. Transfection can be transient.

[0119] The term "transformation" refers to a change in the genetic characteristics of a cell; a cell is said to be transformed if it has been modified to contain new DNA or RNA. For example, a cell has been transformed if it has been genetically modified from its wild-type state by the introduction of new genetic material via transfection, transduction, or other techniques. After transfection or transduction, the transforming DNA can either be physically integrated into the cell's chromosome and recombine with the cell's DNA, or it can be maintained transiently as an episomal element without replication, or it can be replicated separately as a plasmid. A cell is considered "stably transformed" if the transforming DNA is replicated with cell division.

[0120] The term "immunologically functional fragment" (or simply "fragment") of an antigen-binding protein of an antibody or immunoglobulin chain (heavy or light chain), as used herein, refers to a species of antigen-binding protein that contains a portion of an antibody (regardless of how that portion is obtained or synthesized) that is missing at least some amino acids present in the full-length chain but that does not lose its ability to specifically bind to the antigen.

[0121] "Specific binding," "specifically binds," or "binds specifically" should be understood to mean that the antigen-binding protein selectively binds to CB1. However, this does not necessarily exclude the antigen-binding protein from binding to proteins other than CB1. In various embodiments, binding to other proteins accounts for less than about 5%, less than about 10%, less than about 15%, less than about 20%, or less than about 25% of the total protein bound. A CB1 antigen-binding protein that "specifically binds" primarily binds to CB1 or a specific sequence of CB1, e.g., the CB1 extracellular domain. "Specific binding" or "binds specifically" should not be construed to exclude binding to targets other than the stated target(s) or specific sequences, but the primary binding activity should be for the specific target(s) or amino acid sequence.

[0122] Fragments of antigen-binding proteins are biologically active, in that they bind to a target antigen and can compete with other antigen-binding proteins, such as intact antibodies, to bind to a given epitope or antigen. In some embodiments, the fragments are neutralizing fragments. In some embodiments, the fragments can block or inhibit the potential interaction of CB1 with its ligand(s). In certain aspects, such fragments retain at least one CDR present in a full-length light or heavy chain, and in some embodiments, include a heavy and / or light chain or a portion thereof. These biologically active fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of antigen-binding proteins, such as intact antibodies. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, diabodies (in which the heavy chain variable domain is connected on the same polypeptide with the light chain variable domain via a short peptide linker that is too short to pair the two domains on the same chain), Fab', F(ab')2, Fv, domain antibodies, and single-chain antibodies, and such fragments may be derived from any mammalian source, including, but not limited to, human, mouse, rat, camelid, or rabbit. It is further contemplated that functional portions of the antigen-binding proteins disclosed herein, e.g., one or more CDRs, can be covalently linked to a second protein or small molecule to create therapeutic agents that are directed to specific targets in the body, have dual therapeutic properties, or have long serum half-lives. As will be understood by those of skill in the art, antigen-binding proteins can include non-proteinaceous components.

[0123] Certain antigen-binding proteins described herein are antibodies or are derived from antibodies. In certain embodiments, the polypeptide structure of the antigen-binding protein is based on an antibody, including, but not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "pseudo-antibodies"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments of each. In some embodiments, the antigen-binding protein comprises or consists of avimers (tightly binding peptides).

[0124] The "Fc" region is the C H 1 and C H The heavy chain fragments each contain two domains. The two heavy chain fragments are separated by two or more disulfide bonds and a C H The three domains are held together by hydrophobic interactions.

[0125] A "Fab fragment" is a fragment consisting of one light chain and one heavy chain. H 1 and variable regions. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0126] "Fab' fragment" consists of one light chain and one V H Domain and C H In addition to 1 domain, C H 1 and C H It contains a portion of one heavy chain that also contains the region between the two domains, allowing disulfide bonds to form between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule.

[0127] "F(ab')2 fragment" is a fragment consisting of two light chains and C H 1 and C H It contains two heavy chains containing a portion of the constant region between the two domains, allowing disulfide bonds to form between the two heavy chains. Thus, an F(ab')2 fragment is composed of two Fab' fragments linked by disulfide bonds between the two heavy chains.

[0128] The "Fv region" comprises the variable regions of each of the heavy and light chains, but lacks the constant regions.

[0129] A "single-chain antibody" is an Fv molecule in which the variable regions of the heavy and light chains are connected by a flexible linker to form a single polypeptide chain that forms the antigen-binding region. Single-chain antibodies are discussed in detail in International Patent Application Publication No. WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated by reference.

[0130] A "domain antibody" is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some cases, two or more V H The domains are covalently linked with a peptide linker to create a bivalent domain antibody. H The regions can target the same or different antigens.

[0131] A "bivalent antigen-binding protein" or "bivalent antibody" comprises two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. Bivalent antigen-binding proteins and bivalent antibodies may be bispecific, as defined herein. In certain embodiments, bivalent antibodies other than "multispecific" or "multifunctional" antibodies are typically understood to have each binding site identical.

[0132] A "multispecific antigen-binding protein" or "multispecific antibody" is one that targets more than one antigen or epitope.

[0133] "Bispecific," "CB1-specific," or "bifunctional" antigen-binding proteins or antibodies are hybrid antigen-binding proteins or antibodies, respectively, that possess two distinct antigen-binding sites. Bispecific antigen-binding proteins and antibodies are multispecific antigen-binding protein antibodies and can be produced in a variety of ways, including, but not limited to, fusion of hybridomas or linking of Fab' fragments. For example, Songsivilai See, for example, Kostelny et al., 1992, J. Immunol., 148:1547-1553. The two binding sites of a bispecific antigen-binding protein or antibody bind to two different epitopes, which may be on the same or different target proteins.

[0134] Each individual CB1 immunoglobulin chain is typically "composed of several immunoglobulin domains." These domains are the basic units that make up an antibody polypeptide. In humans, IgA and IgD isotypes contain four heavy chains and four light chains; IgG and IgE isotypes contain two heavy chains and two light chains; and IgM isotypes contain five heavy chains and five light chains. The heavy chain C region typically contains one or more domains that may be involved in effector functions. The number of constant region domains in a heavy chain depends on the isotype. An IgG heavy chain, for example, contains C H 1. C H 2 and C H The antibodies contain three C region domains known as isotypes and subtypes. 3. The antibodies provided may have any of these isotypes and subtypes.

[0135] An "antigen-binding region" refers to a protein or a portion of a protein (e.g., a paratope) that specifically binds a specific antigen. For example, a portion of an antigen-binding protein containing amino acid residues that interact with an antigen and confer its specificity and affinity for the antigen to the antigen-binding protein is called an "antigen-binding region." An antigen-binding region typically contains one or more complementary binding regions (CDRs). A particular antigen-binding region also contains one or more "framework" regions. A "CDR" is an amino acid sequence that contributes to the specificity and affinity of antigen binding. A "framework" region functions to maintain the proper conformation of the CDRs, thereby promoting binding between the antigen-binding region and the antigen. Structurally, a framework region can be located between the CDRs in an antibody.

[0136] In certain embodiments, recombinant antigen-binding proteins that bind the CB1 receptor are provided. In this context, a "recombinant antigen-binding protein" is a protein made using recombinant techniques, i.e., by expressing a recombinant nucleic acid as described herein. Recombinant protein methods and techniques are well known in the art.

[0137] The term "antibody" refers to an intact immunoglobulin of any isotype, or a fragment thereof, that can compete with a complete antibody for specific binding to a target antigen, including, for example, chimeric antibodies, humanized antibodies, fully human antibodies, and bispecific antibodies. An "antibody" is a species of antigen-binding protein. Complete antibodies generally contain at least two full-length heavy chains and two full-length light chains, but in some cases may contain fewer chains, such as naturally occurring antibodies from camelids, which may contain only heavy chains. Antibodies may be derived from a single source or, as described in more detail below, may be so-called "chimeric" antibodies, in which different portions of the antibody may be derived from two different antibodies. Antigen-binding proteins, antibodies, or binding fragments can be produced in hybridomas by recombinant DNA techniques or by enzymatic or chemical cleavage of complete antibodies. Unless otherwise specified, the term "antibody" includes antibodies containing two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and muteins thereof, examples of which are described below. Furthermore, unless expressly excluded, antibody includes monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "pseudo-antibodies"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments of each. In some embodiments, the term also encompasses peptibodies.

[0138] The structural unit of a natural antibody typically comprises a tetramer. Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one full-length "light" chain and one full-length "heavy" chain. The amino-terminal portion of each chain typically contains a variable region that is typically responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant region that may be involved in effector function. The variable regions of each light / heavy chain pair typically form the antigen-binding site.

[0139] Variable regions typically share the same general structure, consisting of three hypervariable regions, also called complementarity-determining regions or CDRs, connected by relatively conserved framework regions (FRs). The CDRs of the two chains of each pair are typically arranged between framework regions, enabling binding to a specific epitope. From the N-terminus to the C-terminus, both light-chain and heavy-chain variable regions typically contain the following domains: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid sequence of each domain typically follows the Kabat definition of Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., (1987 and 1991) or Chothia & Lesk, J. Mol. Biol., 196:901-917, (1987); Chothia et al., Nature, 342:878-883, (1989)).

[0140] In some embodiments, the antibody heavy chain binds to the antigen in the absence of the antibody light chain. In some embodiments, the antibody light chain binds to the antigen in the absence of the antibody heavy chain. In some embodiments, the antibody binding region binds to the antigen in the absence of the antibody light chain. In some embodiments, the antibody binding region binds to the antigen in the absence of the antibody heavy chain. In some embodiments, the variable region of indiviCB1 specifically binds to the antigen in the absence of other variable regions.

[0141] In one embodiment, the CDR profile and identification of residues comprising the binding site of the antibody are finally obtained by solving the structure of the antibody and / or by solving the structure of the antibody-ligand complex. In some embodiments, this can be accomplished by any of a variety of techniques known to those of skill in the art, such as X-ray crystallography. In some embodiments, various analytical methods can be used to identify or approximate CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the "AbM" definition, and the contact definition.

[0142] The Kabat definition is a standard for numbering each residue in an antibody and is typically used to identify CDR regions. See, e.g., Johnson & Wu, Nucleic Acids Res., 28:214-8, (2000). The definition of Chothia is K Similar to the Abbat definition, the Chothia definition takes into account the location of specific structural loop regions. See, e.g., Chothia et al., J. Mol. Biol., 196:901-17, (1986); Chothia et al., Nature, 342:877-83, (1989). The "AbM" definition uses an integrated package of computer programs for modeling antibody structure created by the Oxford Molecular Group. See, e.g., Martin et al., Proc. Natl. Acad. Sci. (USA), 86:9268-9272, (1989); "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd. The AbM definition is based on the AbM definition by Samudrala et al., "Ab The tertiary structure of antibodies is modeled from the primary sequence using a combination of knowledge databases and various ab initio methods, such as those described in "Initio Protein Structure Prediction Using a Combined Hierarchical Approach," PROTEINS, Structure, Function and Genetics, Suppl. 3:194-198, (1999). Contact definitions are based on crystal structure analyses of available complexes. See, e.g., MacCallum et al., J. Mol. Biol., 5:732-45, (1996).

[0143] By convention, heavy chain CDR regions are typically referred to as H1, H2, and H3, numbered sequentially from the amino terminus to the carboxy terminus, and light chain CDR regions are typically referred to as L1, L2, and L3, numbered sequentially from the amino terminus to the carboxy terminus.

[0144] The term "light chain" includes full-length light chains and fragments of full-length light chains containing sufficient variable region sequence to confer binding specificity. A full-length light chain contains the V L and C of the constant region domain L The variable region domain of the light chain is at the amino terminus of the polypeptide. Light chains include kappa chains and lambda chains.

[0145] The specificity of various embodiments of antibodies or fragments thereof for the CB1 receptor can be measured based on affinity and / or avidity. Affinity is expressed as the equilibrium dissociation constant (Kd) of the antibody-bound antigen and measures the binding strength between an antigenic determinant and an antibody-binding site. Avidity measures the binding strength between an antibody and its antigen. Avidity is related to both the affinity between an epitope and the antigen-binding site on the antibody for that epitope and the valency of the antibody, which refers to the number of antigen-binding sites specific for a particular epitope. The smaller the Kd value, the stronger the binding strength between an antigenic determinant and an antibody-binding site.

[0146] The term "heavy chain" includes full-length heavy chains and fragments of full-length heavy chains having sufficient variable region sequence to confer binding specificity. A full-length heavy chain includes the V H , and three constant region domains C H 1. C H 2, and C H Contains 3. V H The domain is located at the amino terminus of the polypeptide and at the C H The domain is located at the carboxyl terminus and is C H3 is closest to the carboxy terminus of the polypeptide. The heavy chain may be of any isotype, such as IgG (including the subtypes IgG1, IgG2, IgG3, and IgG4), IgA (including the subtypes IgA1 and IgA2), IgM, or IgE.

[0147] Bispecific or bifunctional antibodies are typically artificial hybrid antibodies in which the two pairs of heavy / light chains are different and the two binding sites are different. Bispecific antibodies can be produced in a variety of ways, including, but not limited to, fusion of hybridomas or linking of Fab' fragments. They can be produced by various methods. See, for example, Songsivilai et al., Clin. Exp. Immunol., 79:315-321, (1990); Kostelny et al., J. Immunol., 148:1547-1553, (1992).

[0148] Some mammalian species can also produce antibodies that have only one heavy chain.

[0149] Each individual CB1 immunoglobulin chain typically consists of several "immunoglobulin domains." These domains are the basic units that make up an antibody polypeptide. The heavy chain C region typically contains one or more domains that may be involved in effector function. The number of constant region domains in a heavy chain is determined by the isotype. The provided antibodies may have any isotype and subtype.

[0150] The term "variable region" or "variable domain" refers to a portion of an antibody's light and / or heavy chain. In certain embodiments, the variable regions of different antibodies, even those from the same species, differ significantly in amino acid sequence. The variable regions of an antibody typically determine the specificity of a particular antibody for a particular target.

[0151] The terms "neutralizing antigen-binding protein" or "neutralizing antibody" refer to an antigen-binding protein or antibody, respectively, that binds to a ligand and prevents or inhibits the binding of that ligand to its binding partner. This can be done, for example, by directly blocking the binding site on the ligand, or by binding to the ligand and altering the ligand's binding ability by indirect means, such as altering the structure or energy of the ligand. In some embodiments, the terms can also refer to an antigen-binding protein that prevents the protein to which it is bound from performing a biological function. In assessing the binding and / or specificity of an antigen-binding protein, e.g., an antibody or immunologically functional fragment thereof, an antibody or fragment can substantially inhibit binding of a ligand to its binding partner if the amount of binding partner bound to the ligand by an excess of antibody is reduced by at least about 1-20%, about 20-30%, about 30-40%, about 40-50%, about 50-60%, about 60-70%, about 70-80%, about 80-85%, about 85-90%, about 90-95%, about 95-97%, about 97-98%, about 98-99% or more (as measured in an in vitro competitive binding assay). In some embodiments, in the case of a CB1 receptor antigen-binding protein, such a neutralizing molecule can reduce the binding ability of the receptor. In some embodiments, neutralizing ability is determined and / or described via a competitive assay. In some embodiments, neutralizing ability is measured using an IC 50 Value or EC 50 In some embodiments, the antigen binding protein may be a non-neutralizing antigen binding protein.

[0152] The term "target" refers to a molecule or portion of a molecule that can be bound by an antigen-binding protein. In some embodiments, a target can have one or more epitopes. In some embodiments, a target is an antigen. The use of "antigen" in the phrase "antigen-binding protein" simply means that a protein sequence that includes such an antigen can be bound by an antibody. In this context, it is not necessary for the protein to be foreign or capable of inducing an immune response.

[0153] The term "compete," as used in the context of antigen-binding proteins (e.g., neutralizing antigen-binding proteins or antibodies) competing for the same epitope, means that the antigen-binding proteins compete with each other, and as measured in an assay, the test antigen-binding protein (e.g., an antibody or immunologically functional fragment thereof) prevents or inhibits (e.g., inhibits) the specific binding of a reference antigen-binding protein (e.g., a ligand or reference antibody) to a common antigen (e.g., CB1 or a fragment thereof). Many types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another, including, for example, solid-phase radioimmunoassays (RIAs) direct or indirect, solid-phase enzyme immunoassays (EIAs) direct or indirect, sandwich competition assays (see, e.g., Stahl et al., 1983, Methods in Enzymology, 9:242-253), and biotin-avidin EIA solid-phase direct. direct labeling (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619); solid-phase direct labeling; solid-phase direct labeling sandwich method (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); I-125 solid-phase direct label RIA using a label (see, e.g., Morel et al., 1988, Molec. Immunol., 25:7-15); solid-phase direct biotin-avidin EIA method (See, e.g., Cheung et al., 1990, Virology, 176:546-552); and direct labeling RIA (Moldenhauer et al., 1990, Scand. J. Immunol., 32:77-82). Typically, such assays use purified antigen bound to a solid surface or cells bearing an unlabeled test antigen-binding protein and a labeled reference antigen-binding protein. Competitive inhibition is determined by measuring the amount of label bound to the solid surface or cells in the presence of the test antigen-binding protein. Typically, the test antigen-binding protein is present in excess. Antigen-binding proteins identified in competitive assays (competing antigen-binding proteins) include antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein and antigen-binding proteins that, due to steric hindrance, bind to an epitope that is sufficiently close and adjacent to the epitope bound by the reference antigen-binding protein. Further details regarding methods for measuring competitive binding are provided in the Examples herein. Typically, when a competing antigen-binding protein is present in excess, specific binding of the reference antigen-binding protein to a common antigen is inhibited (e.g., suppressed) by at least about 40-45%, about 45-50%, about 50-55%, about 55-60%, about 60-65%, about 65-70%, about 70-75%, or about 75% or more. In some cases, binding is inhibited by at least about 80-85%, about 85-90%, about 90-95%, about 95-97%, or about 97% or more.

[0154] The term "antigen" refers to a molecule or portion of a molecule capable of being bound by a selective binding substance, such as an antigen-binding protein (including, for example, an antibody or an immunologically functional fragment thereof). In some embodiments, such antigens can be used in an animal to produce antibodies capable of binding to that antigen. An antigen can have one or more epitopes, each capable of interacting with a different antigen-binding protein, e.g., an antibody.

[0155] The term "epitope" includes any determinant capable of being bound by an antigen-binding protein such as an antibody or capable of binding to a T-cell receptor. An epitope is a region of an antigen to which an antigen-binding protein that targets that antigen binds, and, if the antigen is a protein, includes specific amino acids that make direct contact with the antigen-binding protein. Most often, epitopes are found on proteins, but they can also be found on other molecular species, such as nucleic acids. Epitopes include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural features and / or specific charge characteristics. Generally, antibodies specific for a particular target antigen selectively recognize epitopes on complex mixtures of proteins and / or macromolecules.

[0156] As used herein, "substantially pure" means that the recited molecular species is the predominant molecular species present, i.e., it is more abundant, on a molar basis, than any other IndiviCB1 species in the same mixture. In certain embodiments, a substantially pure molecule is a composition in which the target species constitutes at least about 50% (on a molar basis) of all macromolecular species present therein. In other embodiments, a substantially pure composition constitutes at least about 80%, about 85%, about 90%, about 95%, or about 99% of all macromolecular species present in the composition. In other embodiments, the target species is purified to essentially homogeneity, where contaminating species in the composition are not detectable by conventional detection methods, and thus the composition is composed of a single detectable macromolecule. In various embodiments, the antigen binding protein can be a purified antigen binding protein or a purified antibody.

[0157] The term "biological sample," as used herein, includes, but is not limited to, any quantity of material derived from a living or formerly living organism. Such organisms include, but are not limited to, animals such as humans, mice, monkeys, rats, rabbits, etc. Such material includes, but is not limited to, blood, serum, urine, cells, organs, tissues, bone, bone marrow, lymph nodes, and skin.

[0158] As used herein, the term "pharmaceutical composition" (or agent or drug) refers to a chemical compound, composition, agent or drug that, when properly administered to a patient, is capable of inducing a desired therapeutic effect, although this does not necessarily require one or more components.

[0159] The terms "therapeutically effective amount" and "therapeutically effective dose" refer to an amount of CB1 receptor antigen-binding protein determined to confer a therapeutic response in a mammal. Such a therapeutically effective amount can be ascertained by one skilled in the art. The exact dose and formulation will depend on the therapeutic purpose and can be ascertained by one skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (Vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro, Editor (2003), and Pickar, Dosage Calculations (1999).

[0160] The term "pharmacologically acceptable salts" or "pharmacologically acceptable carriers" is intended to include salts of the active compounds prepared with relatively non-toxic acids or bases, depending on the particular substituents of the compounds described herein.

[0161] The term "modulator," as used herein, refers to a compound that changes or alters the activity or function of a molecule. For example, a modulator can increase or decrease the magnitude of a particular activity or function of a molecule compared to the degree of the activity or function observed in the absence of the modulator. In certain embodiments, a modulator is an inhibitor, decreasing the magnitude of at least one activity or function of a molecule. Certain exemplary activities and functions of a molecule include, but are not limited to, binding affinity, enzymatic activity, and signal transduction. Certain exemplary inhibitors include, but are not limited to, proteins, peptides, antigen-binding fragments, antibodies, peptibodies, carbohydrates, or small organic molecules. Antibodies can be generated against the CB1 receptor. Peptibodies are described, for example, in US Patent No. 6,660,843 (corresponding to PCT Application No. WO 01 / 83525).

[0162] The terms "patient" and "subject" are used interchangeably and include human and non-human animal subjects who have previously been diagnosed with a disorder, who have previously had a disorder but are currently free of the disorder, who are undergoing medical treatment, who are at risk for developing a disorder, etc.

[0163] The terms "treat" and "treatment" include therapeutic treatment, prophylactic, and applications in which a subject reduces the risk of developing a disorder or other risk factors. Treatment does not require a complete cure of a disorder, but encompasses embodiments in which symptoms or underlying risk factors are reduced.

[0164] The term "prevent" does not require 100% elimination of the likelihood of an event, but rather means that the likelihood of the event occurring is reduced in the presence of the compound or method.

[0165] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications, as common in the art, or as described herein. The techniques and procedures described above can generally be performed according to conventional methods known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference for all purposes. Unless otherwise defined, the terminology and laboratory procedures and techniques used in connection with analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein are those well known and commonly used in the art. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0166] Provided herein are antigen binding proteins that bind the CB1 receptor. In some embodiments, the provided antigen binding proteins are polypeptides comprising one or more complementarity determining regions (CDRs) as described herein. In some antigen binding proteins, the CDRs are embedded in "framework" regions, which orient the CDR(s) so that the appropriate antigen binding properties of the CDR(s) are achieved. In some embodiments, the antigen binding proteins provided herein can interfere with, block, inhibit, or modulate the CB1 receptor.

[0167] In some embodiments, the antigen binding proteins provided herein are capable of inhibiting CB1-mediated activity, e.g., ligand binding. In other embodiments, antigen binding proteins that bind to a CB1 receptor epitope are capable of inhibiting a physiological effect mediated by the CB1 receptor. In some embodiments, the antigen binding protein is chimeric, such as a human / mouse chimera.

[0168] The antigen binding proteins can be used in a variety of therapeutic applications, for example to reduce body weight or to improve metabolic parameters such as plasma glucose, insulin, HDL cholesterol, triglycerides, adiponectin, and HbA1c levels, abdominal and liver fat, energy expenditure, and blood pressure.

[0169] In some embodiments, the antigen binding proteins provided comprise one or more CDRs (e.g., 1, 2, 3, 4, 5, or 6 CDRs). In other embodiments, the antigen binding proteins comprise (a) a polypeptide structure and (b) one or more CDRs inserted into and / or attached to the polypeptide structure. The polypeptide structure can take a wide variety of forms, for example, it can be or comprise a naturally occurring antibody framework, or a fragment or variant thereof, or it can be entirely synthetic.

[0170] In certain embodiments, the polypeptide structure of the antigen-binding protein is or is derived from an antibody, including, but not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "pseudo-antibodies"), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and portions or fragments of each. In some cases, the antigen-binding protein is an immunological fragment of an antibody (e.g., a Fab fragment, a Fab' fragment, etc.). , F(ab')2 fragment, Fv fragment, diabody, or scFv (single-chain antibody molecules such as IgG1, IgG2, IgG3, IgG4, IgG5,

[0171] In embodiments where the antigen binding protein is used for therapeutic purposes, the antigen binding protein can inhibit, interfere with, or modulate one or more biological activities of CB1. In certain embodiments, the antigen binding protein specifically binds to the CB1 receptor and / or substantially inhibits human CB1 receptor binding or signaling by at least about 20%-40%, about 40-60%, or more. %, about 60-80%, about 80-85%, or more (e.g., as measured in an in vitro assay).

[0172] Some antigen binding proteins provided herein are antibodies. In some embodiments, the antigen binding protein is a K d is about 10 -7 , about 10 -8 , about 10 -9 , about 10 -10 , about 10 -11 , about 10 -12 , about 10 -13 Smaller than M (stronger bond). In embodiments, the antigen binding protein exhibits an IC of CB1 receptor binding or signaling blockade. 50 is less than about 1 μM, about 1000 nM to about 100 nM, about 100 nM to about 10 nM, about 10 nM to about 1 nM, about 1000 pM to about 500 pM, about 500 pM to about 200 pM, less than about 200 pM, about 200 pM to about 150 pM, about 200 pM to about 100 pM, about 100 pM to about 10 pM, or about 10 pM to about 1 pM.

[0173] In some embodiments, the antigen binding protein binds to a specific conformational state of the CB1 receptor and prevents receptor activity.

[0174] The antigen-binding proteins against the CB1 receptor described herein can include humanized antibodies and / or portions thereof, and a practical application of such methods is the "humanization" of the mouse humoral immune system.

[0175] In some embodiments, a humanized antibody is substantially non-immunogenic in humans. In some embodiments, the affinity of a humanized antibody for a target is substantially the same as the affinity of an antibody from another species from which the humanized antibody is derived. See, e.g., US Patent No. 5,530,101; US ​​Patent No. 5,693,761; US ​​Patent No. 5,693,762; and US Patent No. 5,585,089.

[0176] In certain embodiments, amino acids in antibody variable domains are identified that can be modified to reduce the immunogenicity of the antigen-binding domain without reducing its native affinity (see, e.g., U.S. Patent Nos. 5,766,886 and 5,869,619).

[0177] In some embodiments, antibody modification by methods known in the art is typically designed to achieve high binding affinity for the target and / or reduce the immunogenicity of the recipient antibody. In some embodiments, humanized antibodies can be modified to eliminate glycosylation sites to increase the affinity of the antibody for its cognate antigen. See, e.g., Co et al., Mol. Immunol., 30:1361-1367, (1993). In some embodiments, techniques such as "reshaping," "hyperchimerization," or "veneering / resurfacing" are used to generate humanized antibodies. See, e.g., Vaswami et al., Annals of Allergy, Asthma, & Immunol., 81:105, (1998); Roguska et al., Prot. Engineer., 9:895-904, (1996); and U.S. Patent No. 6,072,035. In certain such embodiments, such techniques typically reduce the immunogenicity of antibodies by reducing the number of foreign residues, but do not prevent anti-idiotypic and anti-allotypic responses after repeated administration of the antibody. Other specific methods of reducing immunogenicity are described, for example, in Gilliland et al., J. Immunol., 62(6):3663-71, (1999).

[0178] In some cases, humanization of an antibody can result in a loss of antigen-binding ability. A humanized antibody can then be "backmutated." In such embodiments, the humanized antibody can be mutated to introduce one or more amino acid residues found in the donor antibody. See, e.g., Saldanha et al., Mol. Immunol., 36:709-19, (1999).

[0179] In some embodiments, the complementarity-determining regions (CDRs) of the light and heavy chain variable regions of an antibody against the CB1 receptor can be grafted onto framework regions (FRs) derived from the same or another species. In some embodiments, the CDRs of the light and heavy chain variable regions of an antibody against the CB1 receptor can be grafted onto consensus human FRs. To create consensus human FRs, in some embodiments, FRs derived from several amino acid sequences of human heavy or light chains are aligned to identify a consensus amino acid sequence. In some embodiments, FRs of the heavy or light chain of an antibody against the CB1 receptor are replaced with FRs from another heavy or light chain. In some embodiments, rare amino acids in the FRs of the heavy and light chains of an antibody against the CB1 receptor are not replaced, while other FR amino acids are replaced. Rare amino acids are specific amino acids located at positions not normally found in FRs. In some embodiments, a grafted variable region from an antibody against the CB1 receptor can be used with a constant region different from the constant region of the antibody against the CB1 receptor. In some embodiments, the grafted variable region is part of a single-chain Fv antibody. CDR grafting is described, for example, in U.S. Patent Nos. 6,180,370; 6,054,297; 5,693,762; 5,859,205; 5,693,761; 5,565,332; 5,585,089; and 5,530,101, as well as Jones, et al., Nature, 321:522-525, (1986); Riechmann et al., Nature, 332:323-327, (1988); Verhoeyen, et al., Science, 239:1534-1536, (1988); Winter, FEBS Letts., 430:92-94, (1998), which are incorporated herein by reference for any purpose.

[0180] In some embodiments, antigen-binding proteins (such as antibodies) are produced by immunization with an antigen (e.g., the CB1 receptor or a fragment thereof). The antibodies may be produced by immunization with the full-length receptor, a soluble form of the receptor, the catalytic domain alone, the mature form of the CB1 receptor, a splice variant of the receptor, or a fragment thereof. In some embodiments, the antibodies may be polyclonal or monoclonal, and / or may be recombinant.

[0181] In certain embodiments, various methods can be used to manipulate the inherent properties of an antibody, such as its affinity for its target. Such methods include, but are not limited to, the use of site-directed or random mutagenesis of a polynucleotide molecule encoding an antibody to generate antibody variants. In certain embodiments, antibody variants are then screened for those that exhibit the desired change, e.g., improved or decreased affinity.

[0182] In some embodiments, the target amino acid residues for mutagenesis are those within the CDRs. In other embodiments, the target amino acid residues may be those within the framework regions of the variable domains. Such framework regions have been shown to contribute to the target binding properties of certain antibodies. See, e.g., Hudson, Curr. Opin. Biotech., 9:395-402, (1999), and references therein.

[0183] In certain embodiments, smaller, more efficiently screened antibody variant libraries can be generated by restricting random or site-directed mutagenesis to hypermutation sites within the CDRs, i.e., sites corresponding to regions susceptible to mutation during somatic affinity maturation. See, e.g., Chowdhury & Pastan, Nature Biotech., 17:568-572, (1999) and references therein. In certain embodiments, specific types of DNA elements can be used to identify hypermutation sites, including, but not limited to, specific direct and inverted repeats, specific consensus sequences, specific secondary structures, and specific palindromes. For example, such DNA elements that can be used to identify hypermutation sites include four-base sequences containing a purine (A or G) followed by a guanine (G), followed by a pyrimidine (C or T), followed by either adenosine or thymidine (A or T) (i.e., A / GGC / TA / T). Another example of a DNA element that can be used to identify hypermutation sites is the AGC / T codon for serine.

[0184] Many techniques known in the art can be used to prepare suitable antibodies of various embodiments, e.g., recombinant antibodies, monoclonal antibodies, or polyclonal antibodies (see, e.g., Kohler & Milstein, Nature, 256:495-497, (1975); Kozbor et al., Immunology Today, 4:72, (1983); Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986)). Genes encoding the heavy and light chains of a subject antibody can be cloned from cells; for example, genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody. Gene libraries encoding the heavy and light chains of monoclonal antibodies can also be generated from hybridomas or plasma cells. Random combination of heavy and light chain gene products can generate a large pool of antibodies with different antigen specificities (see, for example, Kuby, Immunol., (3 rded. 1997). Techniques for producing single chain antibodies or recombinant antibodies (US Patent No. 4,946,778; US Patent No. 4,816,567) can be adapted to produce antibodies to the polypeptides of various embodiments. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized or human antibodies (see, e.g., U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; Marks et al., Bio / Technology, 10:779-783, (1992); Lonberg et al., Nature, 368:856-859, (1994); Morrison, Nature, 368:812-13, (1994); Fishwild et al., Nature Biotechnology, 14:845-51, (1996); Neuberger, Nature Biotechnology, 14:826, (1996); and Lonberg & Huszar, Intern. Rev. Immunol., 13:65-93, (1995). Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to a selected antigen (see, e.g., McCafferty et al., Nature, 348:552-554, (1990); Marks et al., Biotechnology, 10:779-783, (1992)). Antibodies can be engineered to be bispecific, i.e., capable of recognizing two different antigens (see, e.g., WO 93 / 08829; Traunecker et al., EMBO J., 10:3655-3659, (1991); and Suresh et al., Methods in Enzymology, 121:210, (1986)). The antibody can also be a heteroconjugate, eg, two covalently linked antibodies, or an immunotoxin (see, e.g., US Patent No. 4,676,980, WO91 / 00360; WO92 / 200373; and EP03089).

[0185] Methods for humanizing or primatizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues, often referred to as imported residues, are typically taken from an imported variable domain. Humanization can be performed essentially according to the method of Winter and coworkers (see, e.g., Jones et al., Nature, 321:522-525, (1986); Riechmann et al., Nature, 332:323-327, (1988); Verhoeyen et al., Science, 239:1534-1536, (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596, (1992)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) in which substantially less than an entire human variable domain has been substituted by the corresponding non-human species sequence. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0186] Another alternative, such as that used by GenPharm International, Inc., is the "minilocus" approach. In the minilocus approach, an exogenous Ig locus is mimicked by including a piece from the Ig locus (the indiviCB1 gene). Thus, one or more V H Gene, one or more D H Gene, one or more J HThe gene, the mu constant region, and usually a second constant region (eg, a gamma constant region) are formed into a construct that is inserted into the animal. This technique is disclosed in U.S. Patent No. 5,545,807 (Surani, et al.) and U.S. Patent Nos. 5,545,806; 5,625,825; 5,625,126; 5,633,425; 5,661,016; 5,770,429; 5,789,650; 5,814,318; 5,877,397; 5,874,299; and 6,255,458 (Lonberg & Kay, respectively), U.S. Patent Nos. 5,591,669 and 6,023,010 (Krimpenfort & Berns), U.S. Patent Nos. 5,612,205, 5,721,367, and 5,789,215 (Berns et al.), and U.S. Patent No. No. 5,643,763 (Choi & Dunn), and U.S. Patent Application Serial Nos.: 07 / 574,748; 07 / 575,962; 07 / 810,279; 07 / 853,408; 07 / 904,068; 07 / 990,860; 08 / 053,131; 08 / 096,762; 08 / 155,301; 08 / 161,739; 08 / 165,699; 08 / 209,741 to GenPharm International, the disclosures of which are incorporated herein by reference. See also European Patent No. 0546073B1, International Patent Application Nos. WO92 / 03918; WO92 / 22645; WO92 / 22647; WO92 / 22670; WO93 / 12227; WO94 / 00569; WO94 / 25585; WO96 / 14436; WO97 / 13852; and WO98 / 24884, and U.S. Patent No. 5,981,175, the disclosures of which are incorporated herein by reference in their entireties. See also Taylor et al., 1992; Chen et al., 1993; Tuaillon et al., 1993; Choi et al., 1993; Lonberg et al. (1994); Taylor et al., (1994), and Tuaillon et al., (1995), Fishwild et al., (1996), the disclosures of which are incorporated herein by reference.

[0187] In certain embodiments, the antibody is conjugated to an "effector" moiety, which can be any number of molecules, including a labeling moiety such as a radioactive or fluorescent label, or can be a therapeutic moiety.

[0188] The antibody may be fused to additional amino acid residues, perhaps a peptide tag to facilitate isolation. Other amino acid residues for homing the antibody to a particular organ or tissue are also contemplated.

[0189] In certain embodiments, the antibodies described herein or the antigen-binding region of any monoclonal antibody described herein can be used to treat cancer or retinopathies.

[0190] As will be appreciated, antibodies can be expressed in cell lines other than hybridoma cell lines. A sequence encoding a particular antibody can be used to transform a suitable mammalian host cell. Transformation can be by any known method for introducing a polynucleotide into a host cell, such as packaging the polynucleotide into a virus (or viral vector) and transducing the host cell with such a virus (or vector), or by transfection procedures known in the art, such as those exemplified in U.S. Patent Nos. 4,399,216; 4,912,040; 4,740,461; and 4,959,455 (hereby incorporated by reference). The transformation procedure used will depend on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, liposome encapsulation of the polynucleotide(s), and direct DNA microinjection into the nucleus.

[0191] Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, Chinese hamster ovary (CHO) cells, Hela cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., HEPG2), human epithelial kidney 293 cells, and many other cell lines. Selection of a particular desirable cell line is accomplished by assaying for cell lines that highly express the antibody of interest.

[0192] In some embodiments, the antigen binding protein can comprise at least one immunoglobulin molecule of an IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, or IgM isotype. In some embodiments, the antigen binding protein comprises a human kappa light chain and / or a human heavy chain. In some embodiments, the heavy chain is an IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, or IgM isotype. In some embodiments, the antigen binding protein has been cloned for expression in mammalian cells. In some embodiments, the antigen binding protein comprises a constant region that is not one of the constant regions of an IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, or IgM isotype.

[0193] In certain embodiments, substantially altering the functional and / or chemical properties of antibodies against the CB1 receptor can be achieved by selecting substitutions in the heavy and light chain amino acid sequences that differ significantly in their ability to maintain (a) the molecular backbone structure of the substituted region, e.g., a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the side chains.

[0194] For example, "conservative amino acid substitution" may be used to replace a wild-type amino acid residue with a non-wild-type residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Additionally, any wild-type residue in a polypeptide can be substituted with alanine, as described above for "alanine scanning mutagenesis."

[0195] Desired amino acid substitutions (whether conservative or non-conservative) can be determined by one of skill in the art at the time such substitutions are desired. In certain embodiments, amino acid substitutions can be used to identify key residues of an antibody to the CB1 receptor or to improve or decrease the affinity of an antibody to the CB1 receptor described herein.

[0196] In certain embodiments, antibodies or antigen-binding proteins can be expressed in cell lines other than hybridoma cell lines. Sequences encoding a particular antibody can be used to transform a suitable mammalian host cell. According to certain embodiments, transformation can be achieved by packaging the polynucleotide into a virus (or viral vector) and transducing the host cell with such a virus (or vector), or by any known method for introducing polynucleotides into a host cell, such as transfection procedures known in the art, such as those exemplified in U.S. Patent Nos. 4,399,216; 4,912,040; 4,740,461; and 4,959,455 (hereby incorporated by reference for all purposes). In certain embodiments, the transformation procedure used can depend on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, liposome encapsulation of the polynucleotide(s), and direct DNA microinjection into the nucleus.

[0197] Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, the many immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HEPG2), and many other cell lines. In certain embodiments, cell lines are selected through determination of cell lines with high expression levels and cell lines that produce antibodies with constitutive HGF binding properties. Expression vectors suitable for mammalian host cells are well known.

[0198] In certain embodiments, the antigen binding protein comprises one or more polypeptides. Any of a variety of expression vector / host systems can be used to express polynucleotide molecules encoding polypeptides comprising one or more antigen binding protein components or the antigen binding protein itself. Such systems include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transfected with viral expression vectors (e.g., cauliflower mosaic virus (CaMV), tobacco mosaic virus (TMV)) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

[0199] In some embodiments, a polypeptide comprising one or more antigen binding protein components or the antigen binding protein itself is recombinantly expressed in yeast. In certain such embodiments, commercially available expression systems, such as the Pichia Expression System (Invitrogen, San Diego, CA), are used according to the manufacturer's instructions. In some embodiments, such systems rely on the pre-pro-alpha sequence for direct secretion. In some embodiments, transcription of the insert is driven by the alcohol oxidase (AOX1) promoter upon induction with methanol.

[0200] In one embodiment, secreted polypeptides comprising one or more antigen binding protein components or the antigen binding protein itself are purified from the yeast growth medium, hi one embodiment, the methods used to purify the yeast growth medium are the same as those used to purify polypeptides from bacterial and mammalian cell supernatants.

[0201] In one embodiment, a nucleic acid encoding a polypeptide comprising one or more antigen binding protein components or the antigen binding protein itself is cloned into a baculovirus expression vector such as pVL1393 (PharMingen, San Diego, CA). In one embodiment, such a vector is used according to the manufacturer's instructions (PharMingen) to infect Spodoptera frugiperda cells in sF9 protein-free medium to produce the recombinant polypeptide. In one embodiment, the polypeptide is purified from the medium and separated by heparin-Sepharose. The concentrate is concentrated using a cellulose column (Pharmacia).

[0202] In some embodiments, a polypeptide comprising one or more antigen-binding protein components or the antigen-binding protein itself is expressed in an insect system. Specific insect systems for polypeptide expression are well known to those of skill in the art. In one such system, the Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or Trichoplusia larvae. In some embodiments, a nucleic acid molecule encoding a polypeptide is inserted into a nonessential gene of the virus, e.g., the polyhedrin gene, and placed under the control of that gene's promoter. In some embodiments, successful insertion of the nucleic acid molecule renders the nonessential gene inactive. In some embodiments, such inactivation results in a detectable characteristic. For example, inactivation of the polyhedrin gene results in a virus lacking coat protein.

[0203] In certain embodiments, the recombinant viruses can be used to infect S. frugiperda cells or Trichoplusia larvae. See, e.g., Smith et al., J. Virol., 46:584, (1983); Engelhard et al., Proc. Nat. Acad. Sci. (USA), 91:3224-7, (1994).

[0204] In one embodiment, polypeptides comprising one or more antigen binding protein components or the antigen binding protein itself produced within bacterial cells are produced within the bacteria as insoluble inclusion bodies. Host cells containing such inclusion bodies are centrifuged and resuspended in 0.15M NaCl, 10 mM NaCl, After washing with Tris, pH 8, and 1 mM EDTA, the cells are harvested by treatment with 0.1 mg / ml lysozyme (Sigma, St. Louis, MO) for 15 minutes at room temperature. In one embodiment, the lysate is clarified by sonication and centrifuged at 12,000×g for 10 minutes to pellet the cell debris. In one embodiment, the polypeptide-containing pellet is resuspended in 50 mM Tris, pH 8, and 10 mM EDTA, layered on 50% glycerol, and centrifuged at 6000×g for 30 minutes. In one embodiment, the resulting pellet is purified by Mg ++ and Ca ++ The polypeptide can be resuspended in standard phosphate-buffered saline (PBS) lacking SDS. In certain embodiments, the resuspended pellet is further purified by fractionating it on a denaturing SDS-polyacrylamide gel (see, e.g., Sambrook et al., supra). In certain embodiments, such a gel can be soaked in 0.4 M KCl to visualize the protein, which can be excised and electroeluted in a gel-running buffer lacking SDS. For example, glutathione S-transferase (GST) fusion proteins are expressed as soluble proteins in cells. In one embodiment, such GST fusion proteins are purified using the GST Purification Module (Pharmacia).

[0205] In some embodiments, it is desirable to "refold" a particular polypeptide, e.g., a polypeptide comprising one or more antigen-binding protein components or the antigen-binding protein itself. In some embodiments, such polypeptides are produced using certain recombinant systems discussed herein. In some embodiments, the polypeptide is "refolded" and / or oxidized to form a desired tertiary structure and / or generate disulfide bonds. In some embodiments, such structures and / or bonds are related to the particular biological activity of the polypeptide. In some embodiments, refolding is achieved using any of a number of procedures known in the art. Exemplary methods include, but are not limited to, exposing the solubilized polypeptide material to a pH typically greater than 7 in the presence of a chaotropic agent. An exemplary chaotropic agent is guanidine. In some embodiments, the refolding / oxidation solution also contains a reducing agent and an oxidized form of the reducing agent. In some embodiments, the reducing agent and the oxidized form are present in a ratio that generates a particular redox potential that allows disulfide bond shuffling to occur. In some embodiments, such shuffling allows cysteine ​​bridge formation. Exemplary redox couples include cysteine / cystamine, glutathione / dithiobisGSH, cupric chloride, dithiothreitol DTT / dithiane DTT, and 2-mercaptoethanol (bME). In some embodiments, the co-solvent may be, but is not limited to, methyl-2-methyl-2-propanol / dithio-bME. Exemplary co-solvents include, but are not limited to, glycerol, polyethylene glycols of various molecular weights, and arginine.

[0206] In some embodiments, a polypeptide comprising one or more antigen-binding protein components or the antigen-binding protein itself is substantially purified. Specific protein purification techniques are known to those of skill in the art. In some embodiments, a crude polypeptide fraction separated from non-polypeptide fractions is used to purify the protein. In some embodiments, the polypeptide is purified using chromatographic and / or electrophoretic methods. Exemplary purification methods include, but are not limited to, ammonium sulfate precipitation; PEG precipitation; immunoprecipitation; heat denaturation followed by centrifugation; chromatography, including, but not limited to, affinity chromatography (e.g., protein A Sepharose), ion exchange chromatography, exclusion chromatography, and reverse-phase chromatography; gel filtration; hydroxyapatite chromatography; isoelectric focusing; polyacrylamide gel electrophoresis; and various combination techniques. In some embodiments, the polypeptide is purified by fast protein liquid chromatography or high performance liquid chromatography (HPLC). In some embodiments, purification steps can be interchanged or certain steps can be omitted and still be suitable methods for preparing substantially purified polypeptides.

[0207] In some embodiments, the degree of purification of a polypeptide preparation is quantified. Specific methods for quantifying the degree of purification are known to those of skill in the art. Examples of specific methods include, but are not limited to, measuring the specific binding activity of the preparation and assessing the amount of polypeptide in the preparation by SDS / PAGE analysis. Specific examples of methods for assessing the degree of purification of a polypeptide preparation include calculating the binding activity of the preparation and comparing it to the binding activity of the initial extract. In some embodiments, the result of such calculations is expressed as "fold purified?" The units used to express the amount of binding activity are determined by the particular assay being performed.

[0208] In some embodiments, a polypeptide comprising one or more antigen-binding protein components or the antigen-binding protein itself is partially purified. Partial purification can be achieved by using fewer purification steps or different forms of the same general purification scheme. For example, in some embodiments, cation exchange column chromatography performed using an HPLC instrument generally results in a greater "fold purification" than the same technique using a low-pressure chromatography system. In some embodiments, less purified methods offer advantages in terms of total polypeptide recovery or retention of the binding activity of the polypeptide.

[0209] In some cases, the electrophoretic migration of polypeptides varies depending on the conditions of SDS / PAGE, sometimes quite significantly. See, e.g., Capaldi, et al., Biochem. Biophys. Res. Comm., 76:425, (1977). It will be recognized that the apparent molecular weight of purified or partially purified polypeptides may vary under different electrophoretic conditions.

[0210] In various embodiments described herein, the antibodies can be used in vivo and in vitro for research or diagnostic methods, which are well known in the art. Such diagnostic methods include kits containing the antibodies of various embodiments. In other embodiments, the antibodies described herein can be used therapeutically.

[0211] When CB1-receptor antibodies are used in mammals for prophylactic or therapeutic purposes, such antibodies It is understood that the compound can be administered in the form of a composition that can further contain a pharmacologically acceptable carrier. Suitable pharmacologically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof.

[0212] Pharmaceutically acceptable carriers may further contain minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antigen-binding protein. Injectable compositions can be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to a mammal, as is well known in the art.

[0213] Pharmaceutical formulations, particularly formulations of antibodies for use as described herein, can be prepared by mixing an antibody having the desired purity with any pharmacologically acceptable carrier, excipient, or stabilizer. Such formulations can be lyophilized or aqueous solutions. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations used. Acceptable carriers, excipients, or stabilizers include organic acids such as acetate, phosphate, and citrate; antioxidants (e.g., ascorbic acid); preservatives; low molecular weight polypeptides; proteins such as serum albumin or gelatin, or hydrophilic polymers such as polyvinylpyllolidone; and amino acids, monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents; and ionic and nonionic surfactants (e.g., polysorbates); salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants. The antibody may be formulated at a concentration of 0.5 to 200 mg / ml.

[0214] In therapeutic applications, the compositions are administered in a "therapeutically effective dose" to a patient suffering from a disease (e.g., a muscle-wasting disease). The effective amount for this application depends on the severity of the disease and the patient's general health. The compositions may be administered in single or multiple doses, depending on the dosage and frequency required and tolerated by the patient. As used herein, "patient" or "subject" can include both humans and other animals, particularly mammals. Thus, such methods are applicable to both human treatment and veterinary applications. In various embodiments, the patient is a mammal. The mammal can be a primate, or even a human.

[0215] The routes of administration of pharmaceutical compositions are consistent with known methods, including oral administration, intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, intralesional, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, or intraperitoneal injection; and administration via nasal, enteral, topical, sublingual, urethral, ​​vaginal, or rectal sustained-release systems or implanted devices. If desired, compositions may be administered via bolus injection or continuous infusion, or via an implanted device. Alternatively or additionally, compositions may be administered locally via a membrane, sponge, or other suitable material into which the desired molecule has been absorbed or encapsulated. When an implanted device is used, such a device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may occur via diffusion, sustained-release bolus, or continuous administration.

[0216] In some embodiments, the formulation components are present in concentrations that are acceptable to the site of administration. In some embodiments, a buffer is used to maintain the composition at or slightly below physiological pH, typically within the range of about 5 to about 8 pH.

[0217] In certain embodiments, when parenteral administration is contemplated, the therapeutic composition may be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired CB1 receptor antigen binding protein against CB1, with or without additional therapeutic agents, in a pharmacologically acceptable vehicle. In certain embodiments, the injection vehicle is sterile distilled water, in which the CB1 receptor antigen binding protein against CB1 receptor, with or without one or more additional therapeutic agents, is formulated as a properly preserved, sterile, isotonic solution. In certain embodiments, the desired molecule may be formulated using substances such as injectable microparticles, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, which allow for controlled or sustained release of the formulation and can be delivered via depot injection. In certain embodiments, hyaluronic acid may also be used, which may have the effect of promoting sustained release in the circulation. In certain embodiments, the desired molecule may be introduced using an implantable drug delivery device.

[0218] Uses of CB1 receptor antigen-binding compositions The present invention provides methods and pharmaceutical compositions for inhibiting, reducing or neutralizing the amount or activity of CB1 receptors.

[0219] In one aspect, the present invention provides methods and reagents for treating a CB1-related disorder in a subject in need of such treatment by administering to the subject an effective dose of a CB1 receptor antigen binding protein composition. As used herein, the term "subject" refers to any animal, such as a mammal, including a human.

[0220] Disorders treatable with CB1 receptor antigen binding protein compositions include, but are not limited to, various metabolic disorders such as diabetes and related disorders.

[0221] Administration of the antigen-binding proteins described herein may improve plasma glucose levels or lipid concentrations. Thus, administration of the compositions disclosed herein may improve diabetes, obesity, or hyperglycemic conditions in suitable subjects. Furthermore, compositions containing antigen-binding proteins may reduce an individual's food intake.

[0222] Other aspects of the present invention will be apparent to those skilled in the art and are described herein. Various embodiments of the present invention are described herein, including the following examples, but those skilled in the art will appreciate that the specific examples and tests detailed herein are for illustrative purposes only. It should be understood that various modifications are possible without departing from the spirit of the present invention. [Example]

[0223] The following sequences are relevant to the present application: Table 2 [Table 2-1] [Table 2-2]

[0224] Example 1 antibody generation Multiple antibody campaigns were conducted. In the first campaign, 41 CB1 binders were identified, including the human CB1 antagonist 10D10. In the second campaign, 507 CB1 binders were obtained, none of which were CB1 antagonists. In the third campaign, 58 CB1 binders were identified, all of which were human CB1 antagonists. None of the CB1-binding antibodies were antagonists of mouse CB1.

[0225] The hCB1 antibody 10D10 was generated by immunizing Xenomouse strains XMG2 / K and XMG4 / KL with transiently transfected HEK293 cells expressing PADRE-hCB1 and E3K-hCB1. Immune tissue from mice showing positive antibody titers was collected, pooled, and used for hybridoma generation. Hybridoma supertants containing hCB1-binding antibodies were identified by FACS analysis using hCB1-expressing CHO cells, and antagonist activity was assessed using the hCB1 cAMP assay.

[0226] Antibody 10D10 was obtained and sequenced. The sequences of the heavy chain, light chain, CDR regions and framework regions are provided below. 10D10 HC (SEQ ID NO: 4) QVQLQESGPGLVKPSQTLSLTCTVSGGSIRRGGDYWSWIRQHPGKGLEWIGYIYYSGSTNYNPSLKSRATISVDTSKNQFSLKLSSVTAADTAVYYCARDYDILTGYSYYYYG MDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVE RKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE KTISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 10D10 LC (SEQ ID NO: 5) DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKIRRVEAEEDVGVYYCMQALQTPRTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0227] The sequences of the CDR and framework regions of 10D10 are as follows: CDR sequences HC-CDR1 RGGDYWS (SEQ ID NO: 17) HC-CDR2 YIYYSGSTNYNPSLKS (SEQ ID NO: 18) HC-CDR3 DYDILTGYSYYYYGMDV (SEQ ID NO: 19) LC-CDR1 RSSQSLLHSNGYNYLD (SEQ ID NO: 20) LC-CDR2 LGSNRAS (SEQ ID NO: 21) LC-CDR3 MQALQTPRT (SEQ ID NO: 22) Framework Array HC-FR1 QVQLQESGPGLVKPSQTLSLTCTVSGGSIR (sequence Number 23) HC-FR2 WIRQHPGKGLEWIG (SEQ ID NO: 24) HC-FR3 RATISVDTSKNQFSLKLSSVTAADTAVYYCAR( SEQ ID NO: 27) LC-FR1 DIVMTQSPLSLPVTPGEPASISC (SEQ ID NO: 28) LC-FR2 WYLQKPGQSPQLLIY (SEQ ID NO: 29) LC-FR3 GVPDRFSGSGSGTDFTLKIRRVEAEDVGYYC (distribution Column number 30)

[0228] The HC and LC framework and CDR regions of several additional antibodies are provided in Figures 2A-2B. These are HC FR1 (SEQ ID NO:23), HC FR2 (SEQ ID NO:24), and HC FR3 (SEQ ID NO:27). The associated HC CDR1 (SEQ ID NO:17), HC CDR2 (SEQ ID NO:26-yiyysgstYynpslks), and HC CDR3 (SEQ ID NO:19) sequences are also provided in the figures. The associated LC CDR1 (SEQ ID NO:20), LC CDR2 (SEQ ID NO:21), and LC CDR3 (SEQ ID NO:22) sequences are also shown. One skilled in the art, reading the figures, will understand that starting with the sequence of 1A11 and making the indicated amino acid changes will result in the sequences of the additional antibodies described.

[0229] Mutagenesis of the CDR regions of the heavy and light chains of the antibody was performed to obtain additional antigen-binding proteins. Random mutagenesis (NNK codons) of single amino acid residues (N = A, T, G, or C; K = T or G) was performed on all residues in all three HC-CDR regions and all three LC-CDR regions (SEQ ID NOS: 17 to 22).

[0230] Mutagenic primers were prepared by splitting the 5-prime of the 24 wild-type nucleotides and the 24 wild-type nucleotides. The NNK was designed by flanking the target position with a 3-prime of leutidine. Forty positions in the HC-CDR and 32 positions in the LC-CDR were mutated, resulting in a total of 1368 mutants.

[0231] Plasmid DNA containing the 10D10 heavy chain and the 10D10 light chain in the pTT5 vector was used as a template for mutagenesis reactions. CB1 mutants were identified by sequencing and isolation. Single-residue mutants were created by pairing all light chain mutants with the wild-type heavy chain and all heavy chain mutants with the wild-type light chain. Conditioned medium (CM) was collected on day 7 posttransfection and used in a cell-based ELISA to assess binding.

[0232] Beneficial mutations 2-4 in the CDRs were combined using specific mutagenesis primers to generate site-directed combination mutants (CSDMs). CSDMs with further improved affinity were identified by FACS at an antibody concentration of 0.1 μg / mL. Pairing of CSDM mutant LC with CSDM mutant HC further improved affinity. To improve the biochemical properties of the CSDM mutants, the N35Y mutation was added to CSDM mutant LC using specific mutagenesis primers.

[0233] The following LC and HC mutants based on 10D10 were obtained, the sequences of which are shown below:

[0234] 10D10-D83K HC (SEQ ID NO: 6) QVQLQESGPGLVKPSQTLSLTCTVSGGSIRRGGDYWSWIRQHPGKGLEWIGYIYYSGSTNYNPSLKSRATISVKTSKNQFSLKLSSVTAADTAVYYCARDYDILTGYSYYYYG MDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVE RKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE KTISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0235] 10D10-31Y HC, 10D10-41Y HC, 10D10-43Y HC(H 2-1) (SEQ ID NO: 7) QVQLQESGPGLVKPSQTLSLTCTVSGGSIRRGGDYWSWIRQHPGKGLEWIGYIYYSGSTLYNPRLKSRATISVDTSKNQFSLKLSSVTAADTAVYYCARDYDILTGYSYYYYG MDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVE RKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE KTISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0236] 10D10-31Y LC(L1-8Y) (SEQ ID NO: 8) DIVMTQSPLSLPVTPGEPASISCRSSQSLYHSYGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKIRRVEAEDVGVYYCMQALQTPRTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0237] 10D10-31Y R94S LC (L1-8Y R94S) (SEQ ID NO: 9) DIVMTQSPLSLPVTPGEPASISCRSSQSLYHSYGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0238] 10D10-41Y LC(L2-1Y) DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSYGYNYLDWYLQKPGQSPQLLIYLGYKKASGVPDRFSGSGSGTDFTLKIRRVEAEDVGVYYCMQALQTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0239] 10D10-41Y R94S LC(L2-1Y R94S) DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSYGYNYLDWYLQKPGQSPQLLIYLGYKKASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0240] 10D10-43Y LC(L3-2Y) DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSYGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKIRRVEAEDVGVYYCMQARGTVRTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0241] 10D10-43Y R94S LC (L3-2Y R94S) (SEQ ID NO: 13) DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSYGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARGTVRTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0242] 10D10-LYYY-5 LC (L1YYY-5) (SEQ ID NO: 14) DIVMTQSPLSLPVTPGEPASISCRSSQSLLHYYGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKIRRVEAEDVGVYYCMQALQTPRTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0243] 10D10-LYYY-5 R94S LC(L1YYY-5 R94S) (SEQ ID NO: 1 5) DIVMTQSPLSLPVTPGEPASISCRSSQSLLHYYGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0244] 10D10-N35Y LC(N35Y) (SEQ ID NO: 16) DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSYGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKIRRVEAEDVGVYYCMQALQTPRTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0245] Various combinations of mutated HC and mutated LC were made and are listed in Table 3. Table 3 [Table 3]

[0246] The characteristics of high affinity matured antibodies are shown in Figure 3.

[0247] Figure 2 provides information about binders from the first campaign. The HC and LC framework and CDR sequences are shown. It will be noted that there are no significant differences in the sequences between the various antibodies. In reviewing Figures 2A and 2B, one skilled in the art will recognize that substitutions of 1-8 amino acids in the presented sequence of 1A11 are possible to obtain additional antibodies disclosed in the figures. Any of these antibodies may be encompassed by various embodiments of the present disclosure.

[0248] Analysis of covariance suggests further modifications to the antibody, and these suggested modifications are shown in Tables 4 and 5 below. Table 4. Covariance analysis suggests 31Y, 41Y, 43Y, and LYYY-5 variants Changes [Table 4] Table 5. Further covariance analysis suggests N35Y / D83K and D83R variant changes [Table 5]

[0249] Example 2 Antibody properties Several different antibodies were characterized using various assays to study the properties of CB1 receptor antibodies. The exemplary antibody 10D10 is a xenomouse antibody (i.e., a human antibody) that exhibits cAMP, aequorin, and GTP-Eu assays. The results of these assays demonstrate that the antibody antagonizes a synthetic agonist (CP55,940) and an endogenous (anandamide) agonist, and binds to an epitope presented by the EC2 domain region of Figure 1.

[0250] The binding results of 10D10 and another non-antagonist antibody, 3A4, to both wild-type and mutant CB1 receptor sequences are summarized in Figure 4A. The different mutant CB1 receptors used in the analysis are depicted graphically, including wild-type human CB1 (hCB1), wild-type mouse CB1 (mCB1), human CB1 lacking the extracellular N-terminus (ΔNT), human CB1 receptors containing single amino acid substitutions in EC1 (R186P) and EC2 (E258K and H270L), and human CB2, which contains three extracellular loops of human CB1 (hCB2 / hCB1).

[0251] We found that 10D10 does not bind or antagonize CB1 receptors with mutations in the EC2 domain (E258K and H270L) or with the murine CB1 sequence, but still antagonizes when mutated in the EC1 domain (R186P). 10D10 will also bind and antagonize mutant CB1 receptors that do not contain the extracellular N-terminus. In contrast, the 3A4 antibody still binds and does not antagonize mutant CB1 receptors that do not contain the extracellular N-terminus, despite mutations in either the EC1 or EC2 domains of CB1.

[0252] Figure 4B shows the binding of CB1 antibodies to cells transfected with various receptor constructs in a FACS assay. Receptor expression was recorded using antibodies raised against an epitope tag (e.g., V5 or E3K) at the N-terminus of each receptor. The receptors used for the study were wild-type mouse CB1 (V5-mCB1), wild-type human CB1 (V5-hCB1), human CB1 lacking the extracellular N-terminus (E3K-hCB1Del.NT), and human CB1. Human CB2 (V5-hCB2 / hCB1 ECL1-3), which contains three extracellular loops of CB1, and three human CB1 receptors containing single amino acid substitutions in EC1 (V5-hCB1 R186P) and EC2 (V5-hCB1 E258K and V5-hCB1 H270L). The cells were treated with no antibody (Un), control antibody (IgG-PE), anti-V5 antibody (V5), anti- The cells were probed with either the E3K antibody (E3K) and various anti-CB1 antibodies (3A4, 3H7, 10B4, 10D2, 10D10, 1A11, 1E9, and 5G4). Highlighted columns indicate positive signals in the assay. It should be noted that 10B4 and 10D2 do not appear to demonstrate binding. This may be due to low Ab concentrations or loss of binding activity as the clones were expanded.

[0253] Figure 4C shows that 10D10 requires binding to CB1 for antagonist activity. Data shown are from an aequorin assay. The positive control small molecule antagonist SR141716A inhibited wild-type CB1, hCB1 R186P, and hCB1 10D10 exhibits antagonist activity only with wild-type CB1 and hCB1 R186P, which are binding partners, but does not exhibit antagonist activity with hCB1 H270L and hCB1 E258K, which are not binding partners.

[0254] The assays used to characterize the antibodies are described below.

[0255] GTP-Eu assay PerkinElmer DELFIA GTP-Eu Reagents, D GTP-binding buffers and hCB1 membranes were used to detect GTP. P-Eu assay was performed. Antagonists were added to the membrane at 4.5 μg / well, 50 μg / well. Saponin, 150 mM NaCl, 10 mM MgCl2, 10 nM GTP-E Antagonist activity was measured by preincubating 96-well Pall ArcoWell plates with 5 μM GDP, 0.1% BSA, and 50 mM HEPES for 15 minutes, followed by the addition of 600 nM (Figure 6A) or a dose-response of the CB1 agonist anandamide (Figure 6B) for 45 minutes. Plates were washed twice with GTP wash buffer using a Millipore manifold and read on a Victor reader. cAMP assay CHO cells stably expressing hCB1 (Euroscreen) were cultured in 10% FBS, 1% Pen / Strep / L-glutamine, 25 mM Hepes, 0.1 mM NEA. A, 1 mM sodium pyruvate, and 400 μg / ml G418 were grown in DMEM containing 0.5% FBS, 1% Pen / Strep / L-glutamine, 25 mM Hepes, 0.1 mM NEAA, 1 mM pyruvate, and 400 μg / ml G418. To measure antagonist activity, Cells were seeded into 96-well plates at a density of 10,000 cells per well in 80 μl of DMEM containing sodium bicarbonate and 400 μg / ml G418. After overnight incubation, the medium was replaced with 5 μl of fresh medium, and then 5 μl of forskolin and CP55-940 were added to the medium, followed by 40 μl of antibody. The final concentrations of forskolin and CP55,940 were 15 μM and 250 pM, respectively. The antibody was diluted in 10 mM NaAcetate, 150 mM NaCl, pH 5.0. The forskolin / CP55,940 / antibody mixture was left on the cells for 30 minutes at 37°C, then removed, and cAMP levels were measured using the DiscoverX XS+ cAMP Assay Kit according to the manufacturer's instructions. Plates were read for 30 seconds on a PerkinElmer ViewLux Microplate Imager. Aequorin assay CHOK1 cells grown in DMEM / F12 containing 10% FBS were harvested in 10 cm dishes at a density of 5 x 10 cells per dish and then transiently transfected into Opti-Mem using Lipofectamine 2000 with a plasmid encoding CB1, Gal6, and aequorin at a ratio of 2:1:10. After overnight incubation, the cells were incubated for 1 hour. Afterward, cells were trypsinized and resuspended in 10 ml of aequorin buffer containing HBSS, 20 mM HEPES, 0.01% fatty acid-free BSA, and 10 ul coelenterazine (1 ug / ul), transferred to a foil-covered beaker, and gently agitated for 2 hours at room temperature. Cells were dispensed using a Microlumat into pre-warmed (37°C) assay plates containing 2x the final concentration of antibody and 60 nM CP55,940. Typically, 100 ul of cells were added to 100 ul of test article. Kinetic measurements were performed for 20 s and dose-response curves were constructed using the area under the curve from 2 to 20 s. KinExA assay 1x10 CHO35 cells expressing huCB1 in DMEM / F12 containing 1% FBS and 0.05% sodium azide 6 ,3x10 6 , and 9x10 6 100 pM antibody was incubated with cells / ml and the sample was rocked for 4 hours at room temperature. Unbound free antibody was separated from whole cells and antibody-cell complexes using a Beckman GS-6R centrifuge at approximately 220 x g for 5 minutes. The supernatant was filtered through a 0.22 μM filter. The beads were then passed through goat anti-huFc coated UltraLink Biosupport resin. The amount of Ab bound to the beads was quantified using a fluorescently labeled anti-huIgG (H+L) antibody. The binding signal is proportional to the free Ab concentration in solution at each cell concentration. A relative binding signal of 100% represents 100 pM antibody alone. A decrease in signal indicates antibody binding to the cells.

[0256] The 10D10 antibody does not recognize mouse CB1 (SEQ ID NO: 2) and has an affinity for hCB1 of less than 200 nM. Subsequent in vitro maturation efforts yielded additional antibodies that were more potent (approximately 4-6 fold) and also antagonized human CB1 receptor signaling. Examples of these antibodies are N35Y / D83K, 41Y, 43Y, and LYYY-5.

[0257] N35W is an affinity-matured, antagonistic CB1 antibody based on 10D10, and is approximately 4-fold more potent than 10D10. This is shown in Figures 5A-5C. In Figure 5A, the cAMP receptor N35W EC by Issey 50 is 6.973 x 10 -8 whereas 10D10 is 2.826×10 -7 In addition, in both 10D10 and N35W, the concentration increased In the aequorin assay (Fig. 5B), the EC 50 is 3.878 x 10 -7 whereas 10D10 is 1.520 x 1 0 -6 In both figures, the area under the curve decreases as the amount of antibody increases. CB1 inhibition associated with

[0258] KinExA binding analysis (Fig. 5c) showed that more N35W bound to CB1 expressing CHO cells than 10D10 (fewer free antibodies after incubation), implying that N35W has a higher binding affinity than 10D10.

[0259] FIG. 6A shows the results of the CB1 antibodies 10D10, 1E9, and This indicates that 3H7 antagonizes the endogenous CB1 agonist anandamide. In this assay, the small molecule antagonist SR141716A was used as a control, and the IC values ​​of all four antagonists were 50 The values ​​are shown in Table 6. Figure 6B shows the results of the GTP-Eu assay. , indicating that increasing concentrations of 10D10 shift the anandamide dose-response curve to the right, further demonstrating the antagonist activity of 10D10. Table 6 [Table 6]

[0260] Example 3 Several 10D10 variants were prepared. The antibody example tested was Y54143, which has the 10D10-Y54143 LC (SEQ ID NO: 36) paired with the 10D10-LYYY5.002 HC (SEQ ID NO: 37), containing a T144L mutation within the HC. Y54143-B also has the 10D10-Y54143 LC (SEQ ID NO: 36), which is paired with the 10D10-LYYY5.002 HC (SEQ ID NO: 37). It is paired with 31Y.002 HC (SEQ ID NO: 38). The sequence is shown below:

[0261] 10D10-3141 LC (SEQ ID NO: 31) DIVMTQSPLSLPVTPGEPASISCRSSQSLYHSYGYNYLDWYLQKPGQSPQLLIYLGYKKASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0262] 10D10-3143 LC (SEQ ID NO: 32) DIVMTQSPLSLPVTPGEPASISCRSSQSLYHSYGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARGTVRTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0263] 10D10-314143 LC (SEQ ID NO: 33) DIVMTQSPLSLPVTPGEPASISCRSSQSLYHSYGYNYLDWYLQKPGQSPQLLIYLGYKKASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARGTVRTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0264] 10D10-Y541 LC (SEQ ID NO: 34) DIVMTQSPLSLPVTPGEPASISCRSSQSLLHYYGYNYLDWYLQKPGQSPQLLIYLGYKKASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0265] 10D10-Y543 LC (SEQ ID NO: 35) DIVMTQSPLSLPVTPGEPASISCRSSQSLLHYYGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARGTVRTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0266] 10D10-Y54143 LC (SEQ ID NO: 36) DIVMTQSPLSLPVTPGEPASISCRSSQSLLHYYGYNYLDWYLQKPGQSPQLLIYLGYKKASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARGTVRTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0267] 10D10-LYYY5.002 HC (SEQ ID NO: 37) QVQLQESGPGLVKPSQTLSLTCTVSGGSIRRGGDYWSWIRQHPGKGLEWIGYIYYSGSTNYNPSLKSRATISVDTSKNQFSLKLSSVTAADTAVYYCARDYDILTGYSYYYGMDVWGQGTLVTSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0268] 10D10-31Y.002HC QVQLQESGPGLVKPSQTLSLTCTVSGGSIRRGGDYWSWIRQHPGKGLEWIGYIYYSGSTLYNPRLKSRATISVDTSKNQFSLKLSSVTAADTAVYCARDYDILTGYYYYYGMDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVE RKCCVECPPCPAPPVAGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPPSREEMT

[0269] LC and HC of 10D10 mutants with proline substitutions 10D10-Y54143-LP1 LC (SEQ ID NO: 39) DIVMTQSPLSLPVTPGEPASISCRSSQSLLHYYGYNYLDWYLQKPGQSPQLLIYLGYKKASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARGTVPTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0270] 10D10-Y54143-LP2 LC (SEQ ID NO: 40) DIVMTQSPLSLPVTPGEPASISCRSSQSLLHYYGYNYLDWYLQKPGQSPQLLIYLGYKKASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYYCMQPRGTVRTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0271] 10D10-Y54143-LP3 LC (SEQ ID NO: 41) DIVMTQSPLSLPVTPGEPASISCRSSQSLLHYYGYNYLDWYLQKPGQSPQLLIYLGYKKASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYYCMQPRGTVPTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0272] 10D10-Y54143-HP1 HC (SEQ ID NO: 42) QVQLQESGPGLVKPSQTLSLTCTVSGGSIRRGGDYWSWIRQHPGKGLEWIGYIYYSGSTLYNPRLKSRATISVDTSKNQFSLKLSSVTAADTAVYYCARDYDPLTGYSYYYYGMDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0273] 10D10-Y54143-HP2 HC (SEQ ID NO: 43) QVQLQESGPGLVKPSQTLSLTCTVSGGSIRRGGDYWSWIRQHPGKGLEWIGYIYYSGSTLYNPRLKSRATISVDTSKNQFSLKLSSVTAADTAVYYCARDYDIPTGYSYYYYGMDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0274] 10D10-Y54143-HP3 HC (SEQ ID NO: 44) QVQLQESGPGLVKPSQTLSLTCTVSGGSIRRGGDYWSWIRQHPGKGLEWIGYIYYSGSTLYNPRLKSRATISVDTSKNQFSLKLSSVTAADTAVYYCARDYDILTGYSYYYYG MDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVE RKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE KTISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0275] 10D10-Y54143-HP4 HC (SEQ ID NO: 45) QVQLQESGPGLVKPSQTLSLTCTVSGGSIRRGGDYWSWIRQHPGKGLEWIGYIYYSGSTLYNPRLKSRATISVDTSKNQFSLKLSSVTAADTAVYYCARDYDPPTGYSYYYYG MDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVE RKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE KTISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0276] 10D10-Y54143-HP5 HC (SEQ ID NO: 46) QVQLQESGPGLVKPSQTLSLTCTVSGGSIRRGGDYWSWIRQHPGKGLEWIGYIYYSGSTLYNPRLKSRATISVDTSKNQFSLKLSSVTAADTAVYYCARDYDPLTGYSYYYYYG MDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVE RKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE KTISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0277] 10D10-Y54143-HP6 HC (SEQ ID NO: 47) QVQLQESGPGLVKPSQTLSLTCTVSGGSIRRGGDYWSWIRQHPGKGLEWIGYIYYSGSTLYNPRLKSRATISVDTSKNQFSLKLSSVTAADTAVYYCARDYDIPTGYSYYYYG MDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVE RKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE KTISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0278] 10D10-Y54143-HP7 HC (SEQ ID NO: 48) QVQLQESGPGLVKPSQTLSLTCTVSGGSIRRGGDYWSWIRQHPGKGLEWIGYIYYSGSTLYNPRLKSRATISVDTSKNQFSLKLSSVTAADTAVYYCARDYDPPTGYSYYYYG MDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVE RKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE KTISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0279] Figure 7 shows the results for some of these mutants. Some mutants are more potent than 10D10, as evidenced by the shift of the curves to the left. It should also be noted that some of the tested 10D10 antigen binding proteins with proline substitutions did not exhibit greater potency than Y54143.

[0280] Various publications, patents, and patent applications are referenced throughout this specification. The disclosures of these documents are incorporated herein by reference in their entireties. However, the citation of such documents should not be construed as an admission that such documents are prior art to the present specification. Furthermore, the mere incorporation of a document by reference does not necessarily mean that applicants agree entirely with the contents of the document.

Claims

[Claim 1] The method described in the specification.

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