Anti corticotropin-releasing hormone antibodies and polycystic ovarian syndrome

Abstract

Disclosed herein are methods of treating polycystic ovarian syndrome (PCOS) and related conditions using antibodies and antigen-binding fragments thereof that specifically bind to corticotropin releasing hormone (CRH). Also disclosed herein are kits comprising antibodies and antigen-binding fragments thereof that specifically bind to CRH and instructions for using the antibodies and antigen-binding fragments thereof in such methods.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 370,541, filed August 5, 2022, and U.S. Provisional Application No. 63 / 491,151, filed March 20, 2023, the general references of which are incorporated herein by reference in their entirety.

[0003] Reference to sequence listings submitted electronically

[0004] The entire contents of the electronically submitted sequence list (name: 4857_0020001_SequenceListing_ST26; size: 686,196 bytes; creation date: March 9, 2023) are incorporated herein by reference. Technical Field

[0005] This disclosure relates to methods for treating polycystic ovary syndrome (PCOS) and related conditions using antibodies that specifically bind to corticotropin-releasing hormone (CRH) and their antigen-binding fragments. This disclosure also relates to kits containing such antibodies and antigen-binding fragments, as well as instructions for using such antibodies and antigen-binding fragments in such methods. Background Technology

[0006] Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among women of reproductive age, affecting 4% to 50% of women worldwide. Deswal et al., J. Hum. Reprod. Sci., 13(4):261-271 (2020). Women with PCOS may experience (i) infrequent, irregular, or prolonged menstrual cycles; (ii) excessive androgen levels, which can lead to hypertrichosis (hirsutism), acne, hair loss, or skin abnormalities; and (iii) polycystic ovaries that may lead to infertility. Azziz, Obstet. Gynecol., 132(2):321-336 (2018). Key features of PCOS include hyperandrogenism, anovulation, insulin resistance, and neuroendocrine disorders. Crespo et al., Arch. Endocrin. Metab., 62(3):352-361 (2018).

[0007] Due to this complexity, treating PCOS remains a significant challenge. Treatment is often based on the symptoms experienced, which can include androgen-related symptoms, infertility associated with ovulation dysfunction, and menstrual irregularities. (Badawy et al., Int. J. Womens Health, 3:25-35 (2011)). There is evidence that both clinicians and consumers are dissatisfied with current treatments. (Hoeger et al., J. Clin. Endocrinol. Metab., 106(3):e1071-e1083 (2021)). Therefore, there is a need for improved therapies for PCOS and related conditions. Summary of the Invention

[0008] This article provides a method for treating polycystic ovary syndrome (PCOS) in a subject in need, comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to corticotropin-releasing hormone (CRH).

[0009] This article also provides a method for treating PCOS in a subject in need, the method comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to CRH, wherein the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable domain (VH) CDR1 comprising an amino acid sequence of any one of SEQ ID NO:22-35, a VH CDR2 comprising an amino acid sequence of any one of SEQ ID NO:53-78, and a VH CDR3 comprising an amino acid sequence of any one of SEQ ID NO:113-122 or an amino acid sequence of PDV or GID; and (b) a light chain variable domain (VL) CDR1 comprising an amino acid sequence of any one of SEQ ID NO:152-174, a VL CDR2 comprising an amino acid sequence of any one of SEQ ID NO:193-198, and a VL CDR3 comprising an amino acid sequence of any one of SEQ ID NO:223-232.

[0010] In one aspect, the antibody or its antigen-binding fragment comprises (a) VH, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NO:240-295, 459, and 461; and / or (b) VL, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NO:296-347 and 462.

[0011] In another aspect, the antibody or its antigen-binding fragment comprises (a) a heavy chain having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NO:348-403, 460, and 463; and / or (b) a light chain having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NO:404-455 and 464.

[0012] This article also provides a method for reducing androgen levels in subjects with PCOS, which involves administering to the subject an antibody that specifically binds to CRH or an antigen-binding fragment thereof.

[0013] This article also provides a method for reducing androgen levels in a subject with PCOS, the method comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to CRH, wherein the antibody or antigen-binding fragment thereof comprises: (a) a VH CDR1 comprising an amino acid sequence of any one of SEQ ID NO:22-35, a VH CDR2 comprising an amino acid sequence of any one of SEQ ID NO:53-78, and a VH CDR3 comprising an amino acid sequence of any one of SEQ ID NO:113-122 or an amino acid sequence of PDV or GID; and (b) a VLCDR1 comprising an amino acid sequence of any one of SEQ ID NO:152-174, a VL CDR2 comprising an amino acid sequence of any one of SEQ ID NO:193-198, and a VL CDR3 comprising an amino acid sequence of any one of SEQ ID NO:223-232.

[0014] In one aspect, the antibody or its antigen-binding fragment comprises (a) VH, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NO:240-295, 459, and 461; and / or (b) VL, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NO:296-347 and 462.

[0015] In another aspect, the antibody or its antigen-binding fragment comprises (a) a heavy chain having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NO:348-403, 460, and 463; and / or (b) a light chain having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NO:404-455 and 464.

[0016] In another respect, androgens are testosterone, free testosterone, androstenedione, 11-oxyandrogens, or combinations thereof. In yet another respect, 11-oxyandrogens are 11-hydroxyandrostenedione (11OHA4), 11-hydroxytestosterone (11OHT), 11-ketoandrostenedione (11KA4), 11-ketotestosterone (11KT), or combinations thereof.

[0017] This article also provides a method for reducing the severity of one or more symptoms or signs in a subject with PCOS, the method comprising administering to the subject an antibody that specifically binds to CRH or an antigen-binding fragment thereof.

[0018] This article also provides a method for reducing the severity of one or more symptoms or signs in a subject with PCOS, the method comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to CRH, wherein the antibody or antigen-binding fragment thereof comprises: (a) a VH CDR1 comprising an amino acid sequence of any one of SEQ ID NO:22-35, a VH CDR2 comprising an amino acid sequence of any one of SEQ ID NO:53-78, and a VH CDR3 comprising an amino acid sequence of any one of SEQ ID NO:113-122 or an amino acid sequence of PDV or GID; and (b) a VL CDR1 comprising an amino acid sequence of any one of SEQ ID NO:152-174, a VL CDR2 comprising an amino acid sequence of any one of SEQ ID NO:193-198, and a VL CDR3 comprising an amino acid sequence of any one of SEQ ID NO:223-232.

[0019] In one aspect, the antibody or its antigen-binding fragment comprises (a) VH, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NO:240-295, 459, and 461; and / or (b) VL, which comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NO:296-347 and 462.

[0020] In one aspect, the antibody or its antigen-binding fragment comprises (a) a heavy chain having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NO:348-403, 460, and 463; and / or (b) a light chain having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NO:404-455 and 464.

[0021] In another aspect, one or more symptoms or signs include: anovulation; irregular or absent menstruation; hyperandrogenism; elevated levels of testosterone, free testosterone, androstenedione, 11-oxyandrogens, or combinations thereof; abnormal uterine bleeding; enlarged ovaries with multiple follicles; infertility; obesity; insulin resistance; hyperinsulinemia; hypertension; hyperlipidemia; type 2 diabetes; excessive facial hair growth; hirsutism; alopecia; or acne. In another aspect, 11-oxyandrogens are 11-hydroxyandrostenedione (11OHA4), 11-hydroxytestosterone (11OHT), 11-ketoandrostenedione (11KA4), 11-ketotestosterone (11KT), or combinations thereof.

[0022] On the other hand, antibodies can be polyclonal, monoclonal, human, humanized, or chimeric.

[0023] In another respect, the antigen-binding fragments are Fab, scFab, Fab', F(ab')2, Fv, scFv, biantibody, or triantibody.

[0024] In another aspect, the antibody or its antigen-binding fragment is administered in a pharmaceutical composition comprising the antibody or its antigen-binding fragment and a pharmaceutically acceptable carrier.

[0025] In another aspect, the antibody or its antigen-binding fragment is administered in combination with one or more PCOS therapies. In another aspect, one or more PCOS therapies are selected from combined birth control, progestin therapy, clomiphene, tamoxifen, aromatase inhibitors, gonadotropins, ovarian drilling, spironolactone, efornithine, electrolytes, and antidiabetic drugs. In another aspect, combined birth control is an oral contraceptive containing both estrogen and progestin. In another aspect, the antidiabetic drug is metformin. In another aspect, the aromatase inhibitor is letrozole.

[0026] In another aspect, one or more PCOS treatments are selected from spironolactone, metformin, and combined birth control. In yet another aspect, combined birth control is an oral contraceptive containing both estrogen and progestin.

[0027] In another respect, the subjects were human beings.

[0028] This article also provides a kit comprising (i) an antibody that specifically binds to CRH or an antigen-binding fragment thereof; and (ii) instructions for use in treating PCOS in subjects in need, for reducing androgen levels in subjects with PCOS, or for reducing the severity of one or more symptoms or signs in subjects with PCOS.

[0029] This document also provides a kit comprising (i) a pharmaceutical composition comprising an antibody that specifically binds to CRH or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier; and (ii) instructions for use in treating PCOS in a subject in need, for reducing androgen levels in a subject with PCOS, or for reducing the severity of one or more symptoms or signs in a subject with PCOS. Attached Figure Description

[0030] This document describes some aspects of the invention by way of example only, with reference to the accompanying drawings. Now, specific details are referred to in the drawings, with emphasis on the fact that the details shown are by way of example and are intended to illustrate various aspects of the invention.

[0031] Figure 1 The open reading frame of the coding sequence of mouse CRH (GenBank NM_205769) is shown, as well as the CRISPR-targeting gRNA and PCR primers used in Example 1.

[0032] Figures 2A to 2B The identification of CRH knockout founder mice by PCR is shown. The expected PCR DNA fragment size from wild-type mice was 146 base pairs (bp) and 103 bp. PCR fragments smaller than these expected sizes indicate deletion in founder mice. PCR primers CRHbp70F and CRHbp215R, flanking gRNA1, were used to extract from wild-type H2L2 Harbour mice. The expected size of the amplified 146bp ( Figure 2A (Left figure). PCR primers CRHbp208F and CRHbp310R were used to PCR extract wild-type H2L2 Harbour The expected size of the amplified 103bp ( Figure 2A (middle image). PCR primers CRHbp70F and CRHbp310R produce a PCR fragment of 241 bp in size. Figure 2A (See right figure). PCR products smaller than these expected sizes indicate a mutation. Founder mice 1, 2, 4, and 5 were identified as carrying the mutation. (Source: [Insert source here]) Figure 2BThe founder mouse 5 of the nest 12198 carried an 11bp deletion (5'-CAGCCGGTTCT-3') (SEQ ID NO:465), which was used for subsequent breeding.

[0033] Figure 3 shows the location of an 11 bp deletion (5'-CAGCCGGTTCT-3') (SEQ ID NO:465) in the coding region 157-167 of the CRH gene, which causes downstream reading frame shift and premature stop codon. This defective CRH open reading frame cannot synthesize the CRH peptide. Builders with this deletion are selected for further breeding to achieve homozygosity, rescue, and immunization.

[0034] Figures 4A to 4N The CRH ELISA binding activity of the antibodies shown is indicated by PR designations. Figure 4A , Figure 4B , Figure 4D , Figure 4E , Figure 4G , Figure 4H , Figure 4J , Figure 4K , Figure 4L and Figure 4M ), and demonstrated the reporter gene blocking activity of human CRHR1 (hCRHR1) luciferase with anti-CRH monoclonal antibody (mAb). Figure 4C , Figure 4F , Figure 4I and Figure 4N Unrelated human IgG1 was used as a negative control.

[0035] Figures 5A to 5L The CRH ELISA binding activity of the series 1 mAb PR004290 and its ΔPTM variants is shown. Figures 5A to 5D ) and the gene blocking activity of human CRHR1 (hCRHR1), human CRHR2 (hCRHR2), mouse CRHR1 (mCRHR1) and mouse CRHR2 (mCRHR2) luciferase reporter genes ( Figures 5E to 5L ).

[0036] Figures 6A to 6D The gene blocking activities of the series 2mAb PR006669 and its ΔPTM variants for human CRHR1, human CRHR2, mouse CRHR1 and mouse CRHR2 luciferase reporter genes are shown.

[0037] Figures 7A to 7L The CRH ELISA binding activity of a series of 5mAbs PR301777 and their humanized variants was demonstrated. Figures 7A to 7D ) and the gene blocking activity of human CRHR1, human CRHR2, mouse CRHR1 and mouse CRHR2 luciferase reporter genes ( Figures 7E to 7L).

[0038] Figures 8A to 8D The human CRHR1 luciferase reporter gene blocking activity of the series 7mAb PR302309 and its humanized variants is shown.

[0039] Figures 9A to 9F The gene blocking activities of the series 9mAb PR302334 and its humanized variants for human CRHR1, human CRHR2, mouse CRHR1 and mouse CRHR2 luciferase reporter genes are shown.

[0040] Figures 10A to 10G The gene blocking activities of the series of 10mAb PR302341 and its humanized variants for human CRHR1, human CRHR2, mouse CRHR1 and mouse CRHR2 luciferase reporter genes are shown.

[0041] Figures 11A to 11L The gene blocking activities of a series of 10 humanized mAbs PR302341-10, PR302341-20 and PR302341-23 and their ΔPTM variants for human CRHR1, human CRHR2, mouse CRHR1 and mouse CRHR2 luciferase reporter genes are shown.

[0042] Figures 12A to 12B This indicates that some anti-CRH mAb binds to human UCN1 by ELISA but not to human UCN2 or human UCN3.

[0043] Figures 13A to 13D The selected mAb was shown to bind to CRH and human UCN1 in an ELISA.

[0044] Figures 14A to 14H The inhibition of human UCN1-mediated human CRHR1 reporter gene activity and human CRHR2 reporter gene activity is shown.

[0045] Figures 15A to 15D The binding of anti-CRH mAb to the N-terminal region of the CRH peptide was demonstrated.

[0046] Figure 16 Is using A schematic diagram of the epitope binding experiment on the platform.

[0047] Figure 17 The results indicate that anti-CRH mAb PR302050 reduced the concentrations of restraint stress-induced ACTH and corticosterone in wild-type mice.

[0048] Figure 18 This indicates that anti-CRH mAb PR302050 reduced constitutive high ACTH in Mrap1 knockout mice.

[0049] Figures 19A to 19C This indicates that anti-CRH mAb reduces constitutive high ACTH in Mrap1 knockout mice.

[0050] Figures 20A to 20B Another anti-CRH mAb was shown, which reduced constitutive high ACTH in Mrap1 knockout mice.

[0051] Figures 21A to 21B The results showed that anti-CRH mAb PR302038, PR005660 and PR302050 reduced plasma ACTH concentration in Mrap1 KO mice in a dose-dependent manner at antibody doses of 20 mg / kg and 5 mg / kg. Figure 21A The results are shown on day 2 after mAb administration. Figure 21B The results are shown on day 16 after mAb administration.

[0052] Figures 22A to 22B The results showed that anti-CRH mAbs PR302334-24 and PR005660 significantly reduced plasma ACTH concentrations in Mrap1 KO mice at 20 mg / kg, while PR301429 (an anti-CRH mAb with considerable binding affinity, but a non-blocker in vitro) did not inhibit ACTH production. PR303394, an analogue of CTRND05, was used as a comparative. Figure 22A The results are shown on day 2 after mAb administration. Figure 22B The results are shown on day 16 after mAb administration.

[0053] Figures 23A to 23B This indicates that anti-CRH mAb PR302064 inhibits constraint-induced ACTH ( Figure 23A ) and constraint-induced corticosterone ( Figure 23B It is more effective than SSR125543A in terms of release.

[0054] Figures 24A to 24B This indicates that anti-CRH mAbs PR302064 and PR302334-24 are superior to SSR125543A in the constitutive high ACTH model of Mrap1 KO mice. The results show the efficacy on day 2 after mAb administration. Figure 24A ) and the 14th day ( Figure 24B ACTH levels.

[0055] Figures 25A to 25B This indicates that the anti-CRH mAb PR302334-24 is superior to SSR125543A in a wild-type mouse model of high ACTH and corticosterone induced by constraint stress. The ACTH levels induced after mAb administration are shown. Figure 25A ) and corticosterone ( Figure 25BPlasma levels.

[0056] Figure 26A This is a schematic diagram of a method for testing the pharmacokinetics of anti-CRH mAb. Specifically, anti-CRH mAb in human IgG1-WT-Fc present in mouse serum is captured by a goat anti-human Fc polyclonal antibody and then detected using a goat anti-human (H+L) secondary antibody conjugated with HRP.

[0057] Figures 26B to 26D The mean serum concentration-time curve of anti-CRH mAb administered at a single intravenous dose of 5 mg / kg in female C57BL / 6 wild-type mice is shown. Detailed Implementation

[0058] Various terms used throughout the specification and claims are relevant to various aspects of this disclosure. Unless otherwise stated, such terms shall be given their ordinary meaning in the art. Other specifically defined terms will be interpreted in a manner consistent with the definitions provided herein.

[0059] 1. Definition

[0060] The term "antibody" (Ab) refers to, but is not limited to, glycoprotein immunoglobulins that specifically bind to antigens and comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain contains a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region contains three constant domains CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated as VL) and a light chain constant region. This light chain constant region contains one constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The heavy chain may or may not have a C-terminal lysine. Unless otherwise stated herein, amino acids in the variable region are numbered using the Kabat numbering system, and amino acids in the constant region are numbered using the EU system.

[0061] As used herein, the term "monoclonal antibody" refers to an antibody produced by a single clone of a B cell and binding to the same epitope. Conversely, the term "polyclonal antibody" refers to a group of antibodies produced by different B cells and binding to different epitopes of the same antigen. The term "antibody" includes, for example, monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human or non-human antibodies, fully synthetic antibodies, and single-chain antibodies. Non-human antibodies can be humanized through recombinant methods to reduce their immunogenicity in humans.

[0062] Antibodies can be altered (e.g., through mutation, deletion, substitution, or conjugation to non-antibody parts). For example, an antibody may include one or more variant amino acids that alter the properties (e.g., functional properties) of the antibody compared to naturally occurring antibodies. Many such alterations are known in the art, affecting, for example, half-life, effector function, and / or the patient's immune response to the antibody. The term "antibody" also includes artificial polypeptide constructs that contain at least one antibody-derived antigen-binding site.

[0063] An antibody's "antigen-binding fragment" refers to one or more fragments or portions of an antibody that retain the ability to specifically bind antigens bound by the whole antibody. Studies have shown that the antigen-binding function of an antibody can be achieved through fragments or portions of a full-length antibody. Examples of binding fragments encompassed within the terms "antigen-binding portion" or "antigen-binding fragment" of antibodies disclosed herein include: the term "antibody fragment" refers to a portion of a complete antibody. "Antigen-binding fragment," "antigen-binding domain," or "antigen-binding region" refers to a portion of a complete antibody that binds an antigen. An antigen-binding fragment may contain an antigen-determining region (e.g., a complementarity-determining region (CDR)) of the complete antibody. Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single-chain antibodies. Antigen-binding fragments of antibodies can be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans, or can be artificially generated.

[0064] Furthermore, although the two domains (VL and VH) of the Fv fragment are encoded by separate genes, these domains can still be linked via synthetic linkers using recombinant methods, allowing these domains to be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv)). See, for example, Bird et al., Science, 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988). Such single-chain antibodies are also intended to be encompassed within the terminology of antibodies, which refers to "antigen-binding moieties" or "antigen-binding fragments." These antibody fragments are obtained using conventional techniques known to those skilled in the art and are screened for use in the same manner as intact antibodies. Antigen-binding moieties can be produced via recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. In some respects, antibodies are antigen-binding fragments.

[0065] As used herein, the terms “variable region” and “variable domain” are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a portion of the light or heavy chain, typically 110 to 120 or 110 to 125 amino acids at the amino terminus in the mature heavy chain and approximately 90 to 115 amino acids in the mature light chain. These amino acids vary considerably in sequence between antibodies and are responsible for the binding and specificity of a particular antibody to its specific antigen. Sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while more highly conserved regions within a variable domain are called frame regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of both the light and heavy chains are primarily responsible for antibody-antigen interactions and specificity. In some respects, variable regions are mammalian variable regions, such as human or rabbit variable regions. In some respects, variable regions comprise rodent or mouse CDRs and human frame regions (FRs). In some respects, variable regions are primate (e.g., non-human primate) variable regions. In some respects, the variable region includes the rodent or mouse CDR and the primate (e.g., non-human primate) frame region (FR).

[0066] As used herein, the term "complementarity-determining region" or "CDR" refers to each region of the variable structural domains of an antibody that is highly variable in sequence and / or forms a structurally defined loop (hypervariant loop) and / or contains antigen contact residues. An antibody may contain six CDRs, for example, three in the VH and three in the VL.

[0067] The terms “VL” and “VL domain” are used interchangeably to refer to the variable region of the light chain of an antibody.

[0068] The terms “VH” and “VH domain” are used interchangeably to refer to the variable region of the heavy chain of an antibody.

[0069] The term "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues in the variable regions of the heavy and light chains of an antibody or its antigen-binding fragment. In some respects, CDRs can be determined according to the Kabat numbering system. See, for example, Kabat et al., Ann. NY Acad. Sci., 190:382-391 (1971) and Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991). Using the Kabat numbering system, CDRs within the antibody heavy chain molecule are typically located at amino acid positions 31 to 35, which may optionally include one or two additional amino acids following 35 (referred to as 35A and 35B in the Kabat numbering scheme) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs within antibody light chain molecules are typically located at amino acid positions 24 to 34 (CDR1), 50 to 56 (CDR2), and 89 to 97 (CDR3).

[0070] Chothia refers to the location of the structural loop. Chothia et al., J.Mol.Biol., 196:901-917 (1987). When using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop terminates at 32; if only 35A is present, the loop terminates at 33; if both 35A and 35B are present, the loop terminates at 34). The AbM hypervariable region represents a compromise between the Kabat CDR and the Chothia structural loop and is used by the AbM antibody modeling software of Oxford Molecular.

[0071] As used herein, the terms “constant region” and “constant domain” are used interchangeably and have their common meaning in the art. A constant region is an antibody portion, such as the carboxyl-terminal portion of the light chain and / or heavy chain, that does not directly participate in antibody-antigen binding but can exhibit various effector functions, such as interaction with Fc receptors. The constant regions of immunoglobulin molecules typically have a more conserved amino acid sequence than the variable domains of immunoglobulins. In some respects, antibody or antigen-binding fragments contain a constant region or a portion thereof sufficient for antibody-dependent cell-mediated cytotoxicity (ADCC).

[0072] As used herein, the term "heavy chain," when used to refer to antibodies, can refer to any different type of amino acid sequence based on a constant structural domain, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which produce IgA, IgD, IgE, IgG, and IgM antibodies, including subclasses of IgG, such as IgG1, IgG2, IgG3, and IgG4. The amino acid sequence of the heavy chain is well known in the art. In some respects, the heavy chain is the human heavy chain.

[0073] As used herein, the term "light chain" when referring to antibodies can refer to any different type, such as kappa(κ) or lambda(λ) based on the amino acid sequence of a constant structural domain. The amino acid sequence of a light chain is well known in the art. In some respects, the light chain is the human light chain.

[0074] The term "chimeric" antibody or its antigen-binding fragment refers to an antibody or its antigen-binding fragment whose amino acid sequence originates from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of an antibody or its antigen-binding fragment derived from one mammal (e.g., mouse, rat, rabbit, etc.) that possesses the desired specificity, affinity, and ability, while the constant regions are homologous to sequences in an antibody or its antigen-binding fragment derived from another (typically human) to avoid evoking an immune response in that species.

[0075] The term "humanized" antibody or its antigen-binding fragment refers to a non-human (e.g., mouse) antibody or antigen-binding fragment that is a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof containing a minimal non-human (e.g., mouse) sequence. Typically, humanized antibodies or their antigen-binding fragments are human immunoglobulins in which residues from the complementarity-determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) with the desired specificity, affinity, and capability ("CDR transplantation") (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). In some cases, Fv framework region (FR) residues of human immunoglobulins are replaced by corresponding residues from antibodies or fragments of non-human species with the desired specificity, affinity, and capability. Humanized antibodies or their antigen-binding fragments can be further modified by substituting additional residues within the Fv framework region and / or within the substituted non-human residues to improve and optimize the specificity, affinity, and / or capability of the antibody or its antigen-binding fragment. Generally, humanized antibodies or their antigen-binding fragments will contain substantially all of at least one, typically two or three, variable domains that contain all or substantially all of the CDR regions corresponding to non-human immunoglobulins, and all or substantially all of the FR regions that are those common sequences of human immunoglobulins. Humanized antibodies or their antigen-binding fragments may also contain at least a portion of the immunoglobulin constant region or domain (Fc), typically the constant region or domain of a human immunoglobulin. Examples of methods for generating humanized antibodies are described in U.S. Patent No. 5,225,539, Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994) and Roguska et al., Protein Eng., 9(10):895-904 (1996). In some respects, "humanized antibodies" are surface remodeling antibodies.

[0076] The term "human" antibody or antigen-binding fragment thereof means an antibody or antigen-binding fragment thereof having an amino acid sequence derived from a human immunoglobulin locus, wherein such antibody or antigen-binding fragment is prepared using any technique known in the art. This definition of human antibody or antigen-binding fragment thereof includes complete or full-length antibodies and fragments thereof.

[0077] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or its antigen-binding fragment) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody or its antigen-binding fragment and an antigen). The affinity of molecule X for its partner Y can generally be determined by the dissociation constant (K). D Affinity can be measured and / or expressed in many ways known in the art, including but not limited to the equilibrium dissociation constant (K). D ) and equilibrium binding constant (K) A K D By k off / k on The quotient calculation, and K A By k on / k off Calculate the quotient of k. on This refers to, for example, the rate constant of binding between an antibody or its antigen-binding fragment and an antigen, and k off This refers to, for example, the dissociation of an antibody or its antigen-binding fragment from an antigen. on and k off It can be determined using techniques known to those skilled in the art, such as BLI Or KinExA.

[0078] As used herein, "epitope" is a term in the art and refers to a localized region of an antigen to which an antibody or its antigen-binding fragment can specifically bind. An epitope can be, for example, a continuous amino acid sequence of a polypeptide (linear or continuous epitope), or an epitope can be, for example, derived from two or more discontinuous regions of a polypeptide (conformal, nonlinear, discontinuous, or non-continuous epitopes). In some aspects, epitopes to which antibodies or their antigen-binding fragments bind can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA assays, hydrogen / deuterium exchange coupled to mass spectrometry (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be performed using any method known in the art. See, for example, Giegé et al., Acta Crystallogr DBiol. Crystallogr., 50(Pt 4):339-350 (1994); McPherson, Eur. J. Biochem., 189:1-23 (1990); Chayen, Structure 5:1269-1274 (1997); and McPherson, J. Biol. Chem. 251:6300-6303 (1976). Antibody / its antigen-binding fragment: Antigen crystals can be studied using well-known X-ray diffraction techniques and can be optimized using computer software such as X-PLOR (Yale University, 1992, published by Molecular Simulations, Inc.; see, for example, Meth. Enzymol. (1985), vols. 114 and 115, edited by Wyckoff et al.; US Publication No. 2004 / 0014194) and BUSTER (Bricogne, Acta Crystallogr. D Biol. Crystallogr., 49(Pt 1):37-60 (1993); Bricogne, Meth. Enzymol., 276A:361-423, edited by Carter CW (1997); Roversi et al., Acta Crystallogr D Biol. Crystallogr., 56(Pt10):1316-1323 (2000)). Mutagenic mapping studies can be performed using any method known to those skilled in the art. See, for example, Champe et al., J. Biol. Chem., 270:1388-1394 (1995) and Cunningham et al., Science, 244:1081-1085 (1989) for descriptions of mutagenic techniques, including alanine scanning mutagenesis.

[0079] "Isolated" peptides, antibodies, polynucleotides, carriers, cells, or compositions are peptides, antibodies, polynucleotides, carriers, cells, or compositions in forms not found in nature. Isolated peptides, antibodies, polynucleotides, carriers, cells, or compositions include those that have been purified to the point that they are no longer in their naturally occurring forms. In some respects, isolated antibodies, polynucleotides, carriers, cells, or compositions are substantially pure. As used herein, "substantially pure" means material that is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0080] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. Polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. The term also covers amino acid polymers that have been modified naturally or through intervention; for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other operation or modification, such as conjugation with a labeled component. For example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids) and other modifications known in the art are also included within this definition. It should be understood that because the polypeptides of the present invention are antibody-based, in some aspects, the polypeptides may exist as single chains or associated chains.

[0081] As used herein, the term "host cell" can refer to any type of cell, such as primary cells, cultured cells, or cells derived from a cell line. In some respects, the term "host cell" refers to cells transfected with nucleic acid molecules and their offspring or potential offspring. For example, the offspring of such cells may differ from the parent cells transfected with nucleic acid molecules due to mutations or environmental influences that may occur in subsequent generations or the integration of nucleic acid molecules into the host cell genome.

[0082] The term “pharmaceutically acceptable carrier” includes any and all solvents, co-solvents, complexing agents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents, etc., which are biologically or otherwise desirable. The use of such media and reagents for pharmaceutically active substances is well known in the art. Unless any conventional media or reagent is incompatible with the active ingredient, its use in therapeutic formulations is considered. Complementary active ingredients may also be incorporated into formulations. Furthermore, a variety of excipients, such as those commonly used in the art, may be included. These pharmaceutically acceptable carriers and other such compounds are described in references, e.g., Merck Index, Merck & Company, Rahway, NJ. Considerations for including various components in pharmaceutical compositions are described, e.g., Gilman et al. (ed.) (2010); Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 12th edition, The McGraw-Hill Companies.

[0083] As used herein, the term "subject" means human or non-human mammal (e.g., dog, cat, mouse, rat, cow, sheep, pig, goat), non-human primate or bird, such as chicken, and any other vertebrate or invertebrate. In some respects, the subject is human. In some respects, the subject is female. In some respects, the woman is a woman of reproductive age. In some respects, the subject has experienced and / or exhibited at least one symptom of PCOS. In some respects, the subject has been identified or diagnosed with PCOS. In some respects, the subject is suspected of having PCOS.

[0084] As used herein, the term “corticotropin-releasing hormone” is used interchangeably with the terms “CRH,” “corticotropin-releasing factor,” “CRF,” and “CRH1.” CRH is a neuropeptide hormone that activates the synthesis and release of adrenocorticotropic hormone (ACTH) from the pituitary gland. CRH regulates various neuroendocrine, sympathetic, and behavioral functions, including key roles in controlling stress responses, anxiety and depression, wakefulness, feeding behavior, energy metabolism, and digestive and cardiovascular functions. CRH is a 41-amino acid peptide derived from a 196-amino acid prohormone via enzymatic cleavage and secreted from the paraventricular nucleus (PVN) of the hypothalamus. The amino acid sequence of human and mouse CRH (UniProtKB-P06850; Genbank accession number EAW86897.1) is: SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII (SEQ ID NO:466).

[0085] CRH functions via two distinct G protein-coupled receptors, CRHR1 and CRHR2. CRHR1 expression is widespread in brain regions responsible for sensory and motor control, such as the cortical mantle, olfactory bulb, hippocampus, amygdala, basal ganglia, medial and lateral hypothalamic nuclei, and cerebellum. Conversely, CRHR2 is primarily found in subcortical brain regions, including the lateral septum, stria terminalis bed nucleus, ventromedial hypothalamic nucleus, and the medial and cortical nuclei of the amygdala. In the anterior pituitary gland, CRHR1 responds to CRH-mediated ACTH release.

[0086] In response to stress, the hypothalamus releases CRH, which triggers the release of ACTH from the anterior pituitary gland into the circulation. Subsequently, ACTH binds to receptors on its adrenal cortex and triggers the release of stress hormones such as cortisol. This entire system is known as the hypothalamic-pituitary-adrenal (HPA) axis, which plays a crucial role in regulating the fight-or-flight response to stress.

[0087] As used herein, the term “polycystic ovary syndrome” is used interchangeably with the terms “polycystic ovary syndrome,” “PCOS,” “polycystic ovary disease,” “polycystic ovary disorder,” and “Stein-Leventhal syndrome.” Women diagnosed with PCOS exhibit one or more of the following symptoms: anovulation (e.g., irregular or absent menstruation); hyperandrogenism (e.g., elevated levels of testosterone, free testosterone, androstenedione, 11-oxyandrogens, or combinations thereof); abnormal uterine bleeding; enlarged ovaries with multiple follicles; infertility; obesity; insulin resistance; hyperinsulinemia; hypertension; hyperlipidemia; type 2 diabetes; hirsutism; excessive facial hair growth; hair loss; and acne. In some respects, the diagnosis of PCOS is based on biochemical records of hyperandrogenism (e.g., two or more standard deviations above the control mean for testosterone, free testosterone, androstenedione, 11-oxyandrogens, or combinations thereof), chronic anovulation (e.g., <6 menstrual cycles / year or dysfunctional uterine bleeding), and / or vaginal ovarian ultrasound showing the presence of polycystic ovaries.

[0088] As used herein, the terms “polycystic ovary syndrome therapy” or “PCOS therapy” refer to any treatment for PCOS. PCOS therapy includes, but is not limited to, combined contraceptive therapy (e.g., oral contraceptives containing estrogen and progestin) or progestin therapy, such as for regulating the menstrual cycle. PCOS therapy also includes, but is not limited to, clomiphene, tamoxifen, aromatase inhibitors (e.g., letrozole), metformin, gonadotropins, or ovarian drilling, such as to promote ovulation. PCOS therapy also includes, but is not limited to, combined contraceptive therapy (e.g., oral contraceptives containing estrogen and progestin), spironolactone, efornithine, or electrolysis, such as to reduce excessive hair growth. PCOS therapy also includes, but is not limited to, antidiabetic drugs (e.g., metformin), such as to lower insulin and / or blood glucose levels.

[0089] As used herein, the term “hyperandrogenism” refers to a condition in which a subject exhibits elevated levels of androgens (e.g., testosterone, free testosterone, androstenedione, 11-oxyandrogens, or combinations thereof) in the serum.

[0090] As used herein, the term "11-oxyandrogen" refers to a 19-carbon steroid that is primarily derived from the adrenal glands and shares an oxygen atom on carbon 11. Examples of 11-oxyandrogens include, but are not limited to, 11-hydroxyandrostenedione (11OHA4), 11-hydroxytestosterone (11OHT), 11-ketoandrostenedione (11KA4), and 11-ketotestosterone (11KT).

[0091] As used in this article, the term "hirsutism" refers to a condition in which subjects exhibit abnormally excessive hair growth.

[0092] As used in this article, the term "hyperinsulinemia" refers to a condition in which subjects exhibit elevated plasma insulin levels.

[0093] As used in this article, the term “hyperlipidemia” refers to a condition in which a subject exhibits elevated concentrations of any or all lipids in the plasma.

[0094] As used in this article, the term "hypertension" refers to a condition in which a subject experiences persistent high blood pressure (i.e., systolic blood pressure equal to or greater than 140 mmHg and diastolic blood pressure equal to or greater than 90 mmHg).

[0095] As used in this article, the term "type 2 diabetes" refers to a condition also known as non-insulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes mellitus (AODM), in which a subject has elevated blood glucose levels.

[0096] As used herein, the term “insulin resistance” describes a subnormal biological response to a given concentration of insulin (e.g., reduced glucose transport across the cell membrane in response to insulin).

[0097] As used herein, the term "treat" refers to a therapeutic or palliative measure. Beneficial or desired clinical outcomes include, but are not limited to: complete or partial relief of symptoms associated with the disease or condition, reduction of disease severity, stabilization (i.e., no worsening) of the disease state, delay or slowing of disease progression, improvement or reduction of the disease state (e.g., one or more symptoms of the disease), and relief (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean extended survival compared to expected survival without treatment.

[0098] As used herein, the term "therapeutic effective dose" is the amount of the anti-CRH antibody or its antigen-binding fragment disclosed herein required to reduce or alleviate PCOS or any of its symptoms (e.g., insulin resistance, hyperinsulinemia, type 2 diabetes, obesity, hypertension, hyperlipidemia, anovulation or irregular ovulation, infertility, hyperandrogenism, hirsutism, hair loss, acne, polyfollicular enlargement of the ovaries, and the severity of abnormal uterine bleeding lasting for a period of time). Therapeutic effective dose also means the amount required to improve the clinical symptoms of a subject in need.

[0099] The use of alternatives (e.g., "or") should be understood to mean one, both, or any combination of the alternatives. As used herein, the indefinite article "a" or "one" should be understood to mean "one or more" of any of the enumerated or listed components.

[0100] As used herein, the term “and / or” should be considered to be a specific disclosure of each of two specified features or components with or without the other. Therefore, the term “and / or” as used herein in phrases such as “A and / or B” is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0101] It should be understood that the various aspects disclosed herein are "included" anywhere in the language, and similar aspects described in the terms "consisting of" and / or "substantially composed of" are also provided.

[0102] The term "about" refers to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within a standard deviation of 1 or greater than 1 based on every practice in the art. Alternatively, "about" may mean a range of up to 10% or 20% (i.e., ±10% or ±20%). For example, about 3 mg may include any value between 2.7 mg and 3.3 mg (at 10%) or 2.4 mg and 3.6 mg (at 20%). Furthermore, particularly for biological systems or processes, the term may mean a value up to an order of magnitude or up to 5 times higher. When a particular value or composition is provided in this application and claims, unless otherwise stated, the meaning of "about" should be assumed to be within an acceptable margin of error for that particular value or composition.

[0103] As disclosed herein, unless otherwise stated, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within the range, and, where appropriate, to include fractions thereof (such as one-tenth and one-hundredth of an integer).

[0104] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art relating to this disclosure. For example, The Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 5th edition, 2013, Academic Press; and The Oxford Dictionary of Biochemistry and Molecular Biology, 2006, Oxford University Press, provide a general dictionary for those skilled in the art of the use of many of the terms used in this disclosure.

[0105] Units, prefixes, and symbols are represented in their form accepted by the Système International de Unites (SI). Numerical ranges include the numbers that define the range. The headings provided herein are not intended to limit the various aspects of this disclosure, which can be obtained by referring to the specification as a whole. Therefore, the terms defined below are defined more fully by reference to the specification as a whole.

[0106] Each aspect is described in further detail in the following sections.

[0107] 2. Anti-CRH antibody or its antigen-binding fragment

[0108] The methods and kits disclosed herein contain antibodies or antigen-binding fragments thereof that specifically bind to corticotropin-releasing hormone (CRH) (i.e., anti-CRH antibodies or antigen-binding fragments thereof).

[0109] Examples of anti-CRH antibodies and their antigen-binding fragments are known and described, for example, in International Publication No. WO2019 / 241127; International Publication No. WO 2020 / 115555; CN107043752; Futch et al., J. Exp. Med., 216(11):2479-2491 (2019); Kawahito et al., Gut, 37(4):544-551 (1995); Kageyama et al., Neuroimmunomod., 2(3):137-140 (1995); Carnes et al., Life Sci., 45(12):1049-1056 (1989); Okamoto et al., Brain Res., 855(1):192-197 (2000); Kawahito et al., Gastroenterol., 106(4):859-865 (1994); Miyazaki et al., Mod.Rheumatol., 12(3):206-212 (2002); Perkins et al., Placenta, 16(3):233-243 (1995); Yamada et al., Intern. Med., 41(7):549-554 (2002); and Sasaki et al., Neuroimmunomod., 2(3):134-136 (1995). The entire contents of these patents and documents are incorporated herein by reference.

[0110] Other examples of anti-CRH antibodies and their antigen-binding fragments are described, for example, in U.S. Provisional Application No. 63 / 234,130, the entire contents of which are incorporated herein by reference.

[0111] In some aspects, the anti-CRH antibody or its antigen-binding fragment comprises: a heavy chain variable domain (VH)CDR1 containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with any of SEQ ID NO:22-35; VH CDR2 containing an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with any of SEQ ID NO:53-78; and VH CDR3 containing an amino acid sequence or PDV or GID having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with any of SEQ ID NO:113-122; and / or a light chain variable domain (VL)CDR1 containing an amino acid sequence ... VL CDR2, which contains an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of SEQ ID NO: 152-174, and VL CDR3, which contains an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of SEQ ID NO: 193-198, and VL CDR3, which contains an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of SEQ ID NO: 223-232.

[0112] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:22, VH CDR2 contains the amino acid sequence of SEQ ID NO:53, VH CDR3 contains the amino acid sequence of SEQ ID NO:113, VL CDR1 contains the amino acid sequence of SEQ ID NO:152, VL CDR2 contains the amino acid sequence of SEQ ID NO:193, and VL CDR3 contains the amino acid sequence of SEQ ID NO:223.

[0113] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:22, VH CDR2 contains the amino acid sequence of SEQ ID NO:54, VH CDR3 contains the amino acid sequence of SEQ ID NO:113, VL CDR1 contains the amino acid sequence of SEQ ID NO:152, VL CDR2 contains the amino acid sequence of SEQ ID NO:193, and VL CDR3 contains the amino acid sequence of SEQ ID NO:223.

[0114] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:22, VH CDR2 contains the amino acid sequence of SEQ ID NO:55, VH CDR3 contains the amino acid sequence of SEQ ID NO:113, VL CDR1 contains the amino acid sequence of SEQ ID NO:152, VL CDR2 contains the amino acid sequence of SEQ ID NO:193, and VL CDR3 contains the amino acid sequence of SEQ ID NO:223.

[0115] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:22, VH CDR2 contains the amino acid sequence of SEQ ID NO:53, VH CDR3 contains the amino acid sequence of SEQ ID NO:113, VL CDR1 contains the amino acid sequence of SEQ ID NO:153, VL CDR2 contains the amino acid sequence of SEQ ID NO:193, and VL CDR3 contains the amino acid sequence of SEQ ID NO:223.

[0116] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:22, VH CDR2 contains the amino acid sequence of SEQ ID NO:54, VH CDR3 contains the amino acid sequence of SEQ ID NO:113, VL CDR1 contains the amino acid sequence of SEQ ID NO:153, VL CDR2 contains the amino acid sequence of SEQ ID NO:193, and VL CDR3 contains the amino acid sequence of SEQ ID NO:223.

[0117] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:22, VH CDR2 contains the amino acid sequence of SEQ ID NO:55, VH CDR3 contains the amino acid sequence of SEQ ID NO:113, VL CDR1 contains the amino acid sequence of SEQ ID NO:153, VL CDR2 contains the amino acid sequence of SEQ ID NO:193, and VL CDR3 contains the amino acid sequence of SEQ ID NO:223.

[0118] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:22, VH CDR2 contains the amino acid sequence of SEQ ID NO:54, VH CDR3 contains the amino acid sequence of SEQ ID NO:113, VL CDR1 contains the amino acid sequence of SEQ ID NO:154, VL CDR2 contains the amino acid sequence of SEQ ID NO:193, and VL CDR3 contains the amino acid sequence of SEQ ID NO:223.

[0119] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:22, VH CDR2 contains the amino acid sequence of SEQ ID NO:54, VH CDR3 contains the amino acid sequence of SEQ ID NO:113, VL CDR1 contains the amino acid sequence of SEQ ID NO:155, VL CDR2 contains the amino acid sequence of SEQ ID NO:193, and VL CDR3 contains the amino acid sequence of SEQ ID NO:223.

[0120] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:22, VH CDR2 contains the amino acid sequence of SEQ ID NO:54, VH CDR3 contains the amino acid sequence of SEQ ID NO:113, VL CDR1 contains the amino acid sequence of SEQ ID NO:156, VL CDR2 contains the amino acid sequence of SEQ ID NO:193, and VL CDR3 contains the amino acid sequence of SEQ ID NO:223.

[0121] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:22, VH CDR2 contains the amino acid sequence of SEQ ID NO:54, VH CDR3 contains the amino acid sequence of SEQ ID NO:113, VL CDR1 contains the amino acid sequence of SEQ ID NO:157, VL CDR2 contains the amino acid sequence of SEQ ID NO:193, and VL CDR3 contains the amino acid sequence of SEQ ID NO:223.

[0122] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:22, VH CDR2 contains the amino acid sequence of SEQ ID NO:55, VH CDR3 contains the amino acid sequence of SEQ ID NO:113, VL CDR1 contains the amino acid sequence of SEQ ID NO:154, VL CDR2 contains the amino acid sequence of SEQ ID NO:193, and VL CDR3 contains the amino acid sequence of SEQ ID NO:223.

[0123] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:22, VH CDR2 contains the amino acid sequence of SEQ ID NO:55, VH CDR3 contains the amino acid sequence of SEQ ID NO:113, VL CDR1 contains the amino acid sequence of SEQ ID NO:155, VL CDR2 contains the amino acid sequence of SEQ ID NO:193, and VL CDR3 contains the amino acid sequence of SEQ ID NO:223.

[0124] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:22, VH CDR2 contains the amino acid sequence of SEQ ID NO:55, VH CDR3 contains the amino acid sequence of SEQ ID NO:113, VL CDR1 contains the amino acid sequence of SEQ ID NO:156, VL CDR2 contains the amino acid sequence of SEQ ID NO:193, and VL CDR3 contains the amino acid sequence of SEQ ID NO:223.

[0125] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:22, VH CDR2 contains the amino acid sequence of SEQ ID NO:55, VH CDR3 contains the amino acid sequence of SEQ ID NO:113, VL CDR1 contains the amino acid sequence of SEQ ID NO:157, VL CDR2 contains the amino acid sequence of SEQ ID NO:193, and VL CDR3 contains the amino acid sequence of SEQ ID NO:223.

[0126] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:22, VH CDR2 contains the amino acid sequence of SEQ ID NO:56, VH CDR3 contains the amino acid sequence of SEQ ID NO:113, VL CDR1 contains the amino acid sequence of SEQ ID NO:154, VL CDR2 contains the amino acid sequence of SEQ ID NO:193, and VL CDR3 contains the amino acid sequence of SEQ ID NO:223.

[0127] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:22, VH CDR2 contains the amino acid sequence of SEQ ID NO:56, VH CDR3 contains the amino acid sequence of SEQ ID NO:113, VL CDR1 contains the amino acid sequence of SEQ ID NO:155, VL CDR2 contains the amino acid sequence of SEQ ID NO:193, and VL CDR3 contains the amino acid sequence of SEQ ID NO:223.

[0128] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:22, VH CDR2 contains the amino acid sequence of SEQ ID NO:56, VH CDR3 contains the amino acid sequence of SEQ ID NO:113, VL CDR1 contains the amino acid sequence of SEQ ID NO:156, VL CDR2 contains the amino acid sequence of SEQ ID NO:193, and VL CDR3 contains the amino acid sequence of SEQ ID NO:223.

[0129] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:22, VH CDR2 contains the amino acid sequence of SEQ ID NO:56, VH CDR3 contains the amino acid sequence of SEQ ID NO:113, VL CDR1 contains the amino acid sequence of SEQ ID NO:157, VL CDR2 contains the amino acid sequence of SEQ ID NO:193, and VL CDR3 contains the amino acid sequence of SEQ ID NO:223.

[0130] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:23, VH CDR2 contains the amino acid sequence of SEQ ID NO:57, VH CDR3 contains the amino acid sequence of SEQ ID NO:114, VL CDR1 contains the amino acid sequence of SEQ ID NO:158, VL CDR2 contains the amino acid sequence of SEQ ID NO:193, and VL CDR3 contains the amino acid sequence of SEQ ID NO:224.

[0131] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:23, VH CDR2 contains the amino acid sequence of SEQ ID NO:58, VH CDR3 contains the amino acid sequence of SEQ ID NO:114, VL CDR1 contains the amino acid sequence of SEQ ID NO:158, VL CDR2 contains the amino acid sequence of SEQ ID NO:193, and VL CDR3 contains the amino acid sequence of SEQ ID NO:224.

[0132] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:23, VH CDR2 contains the amino acid sequence of SEQ ID NO:59, VH CDR3 contains the amino acid sequence of SEQ ID NO:114, VL CDR1 contains the amino acid sequence of SEQ ID NO:158, VL CDR2 contains the amino acid sequence of SEQ ID NO:193, and VL CDR3 contains the amino acid sequence of SEQ ID NO:224.

[0133] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:23, VH CDR2 contains the amino acid sequence of SEQ ID NO:60, VH CDR3 contains the amino acid sequence of SEQ ID NO:114, VL CDR1 contains the amino acid sequence of SEQ ID NO:158, VL CDR2 contains the amino acid sequence of SEQ ID NO:193, and VL CDR3 contains the amino acid sequence of SEQ ID NO:224.

[0134] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:24, VH CDR2 contains the amino acid sequence of SEQ ID NO:61, VH CDR3 contains the amino acid sequence of SEQ ID NO:115, VL CDR1 contains the amino acid sequence of SEQ ID NO:159, VL CDR2 contains the amino acid sequence of SEQ ID NO:194, and VL CDR3 contains the amino acid sequence of SEQ ID NO:225.

[0135] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:25, VH CDR2 contains the amino acid sequence of SEQ ID NO:62, VH CDR3 contains the amino acid sequence of PDV, VL CDR1 contains the amino acid sequence of SEQ ID NO:160, VL CDR2 contains the amino acid sequence of SEQ ID NO:195, and VL CDR3 contains the amino acid sequence of SEQ ID NO:226.

[0136] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:24, VH CDR2 contains the amino acid sequence of SEQ ID NO:63, VH CDR3 contains the amino acid sequence of SEQ ID NO:115, VL CDR1 contains the amino acid sequence of SEQ ID NO:161, VL CDR2 contains the amino acid sequence of SEQ ID NO:196, and VL CDR3 contains the amino acid sequence of SEQ ID NO:225.

[0137] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:26, VH CDR2 contains the amino acid sequence of SEQ ID NO:64, VH CDR3 contains the amino acid sequence of SEQ ID NO:116, VL CDR1 contains the amino acid sequence of SEQ ID NO:162, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:227.

[0138] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:24, VH CDR2 contains the amino acid sequence of SEQ ID NO:63, VH CDR3 contains the amino acid sequence of SEQ ID NO:115, VL CDR1 contains the amino acid sequence of SEQ ID NO:163, VL CDR2 contains the amino acid sequence of SEQ ID NO:196, and VL CDR3 contains the amino acid sequence of SEQ ID NO:225.

[0139] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:24, VH CDR2 contains the amino acid sequence of SEQ ID NO:65, VH CDR3 contains the amino acid sequence of SEQ ID NO:115, VL CDR1 contains the amino acid sequence of SEQ ID NO:161, VL CDR2 contains the amino acid sequence of SEQ ID NO:196, and VL CDR3 contains the amino acid sequence of SEQ ID NO:225.

[0140] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:27, VH CDR2 contains the amino acid sequence of SEQ ID NO:66, VH CDR3 contains the amino acid sequence of SEQ ID NO:117, VL CDR1 contains the amino acid sequence of SEQ ID NO:164, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:228.

[0141] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:28, VH CDR2 contains the amino acid sequence of SEQ ID NO:67, VH CDR3 contains the amino acid sequence of SEQ ID NO:118, VL CDR1 contains the amino acid sequence of SEQ ID NO:165, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:227.

[0142] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:29, VH CDR2 contains the amino acid sequence of SEQ ID NO:68, VH CDR3 contains the amino acid sequence of SEQ ID NO:119, VL CDR1 contains the amino acid sequence of SEQ ID NO:166, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:227.

[0143] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:30, VH CDR2 contains the amino acid sequence of SEQ ID NO:69, VH CDR3 contains the amino acid sequence of SEQ ID NO:120, VL CDR1 contains the amino acid sequence of SEQ ID NO:167, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:229.

[0144] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:29, VH CDR2 contains the amino acid sequence of SEQ ID NO:70, VH CDR3 contains the amino acid sequence of SEQ ID NO:121, VL CDR1 contains the amino acid sequence of SEQ ID NO:166, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:230.

[0145] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:31, VH CDR2 contains the amino acid sequence of SEQ ID NO:71, VH CDR3 contains the amino acid sequence of GID, VL CDR1 contains the amino acid sequence of SEQ ID NO:168, VL CDR2 contains the amino acid sequence of SEQ ID NO:198, and VL CDR3 contains the amino acid sequence of SEQ ID NO:231.

[0146] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:32, VH CDR2 contains the amino acid sequence of SEQ ID NO:72, VH CDR3 contains the amino acid sequence of SEQ ID NO:118, VL CDR1 contains the amino acid sequence of SEQ ID NO:166, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:227.

[0147] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:33, VH CDR2 contains the amino acid sequence of SEQ ID NO:73, VH CDR3 contains the amino acid sequence of SEQ ID NO:118, VL CDR1 contains the amino acid sequence of SEQ ID NO:169, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:227.

[0148] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:34, VH CDR2 contains the amino acid sequence of SEQ ID NO:71, VH CDR3 contains the amino acid sequence of GID, VL CDR1 contains the amino acid sequence of SEQ ID NO:168, VL CDR2 contains the amino acid sequence of SEQ ID NO:198, and VL CDR3 contains the amino acid sequence of SEQ ID NO:231.

[0149] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:30, VH CDR2 contains the amino acid sequence of SEQ ID NO:74, VH CDR3 contains the amino acid sequence of SEQ ID NO:122, VL CDR1 contains the amino acid sequence of SEQ ID NO:170, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:232.

[0150] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:35, VH CDR2 contains the amino acid sequence of SEQ ID NO:71, VH CDR3 contains the amino acid sequence of GID, VL CDR1 contains the amino acid sequence of SEQ ID NO:168, VL CDR2 contains the amino acid sequence of SEQ ID NO:198, and VL CDR3 contains the amino acid sequence of SEQ ID NO:231.

[0151] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:29, VH CDR2 contains the amino acid sequence of SEQ ID NO:75, VH CDR3 contains the amino acid sequence of SEQ ID NO:121, VL CDR1 contains the amino acid sequence of SEQ ID NO:171, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:230.

[0152] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:29, VH CDR2 contains the amino acid sequence of SEQ ID NO:76, VH CDR3 contains the amino acid sequence of SEQ ID NO:121, VL CDR1 contains the amino acid sequence of SEQ ID NO:171, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:230.

[0153] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:29, VH CDR2 contains the amino acid sequence of SEQ ID NO:77, VH CDR3 contains the amino acid sequence of SEQ ID NO:121, VL CDR1 contains the amino acid sequence of SEQ ID NO:171, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:230.

[0154] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:29, VH CDR2 contains the amino acid sequence of SEQ ID NO:75, VH CDR3 contains the amino acid sequence of SEQ ID NO:121, VL CDR1 contains the amino acid sequence of SEQ ID NO:172, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:230.

[0155] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:29, VH CDR2 contains the amino acid sequence of SEQ ID NO:75, VH CDR3 contains the amino acid sequence of SEQ ID NO:121, VL CDR1 contains the amino acid sequence of SEQ ID NO:173, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:230.

[0156] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:29, VH CDR2 contains the amino acid sequence of SEQ ID NO:76, VH CDR3 contains the amino acid sequence of SEQ ID NO:121, VL CDR1 contains the amino acid sequence of SEQ ID NO:172, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:230.

[0157] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:29, VH CDR2 contains the amino acid sequence of SEQ ID NO:77, VH CDR3 contains the amino acid sequence of SEQ ID NO:121, VL CDR1 contains the amino acid sequence of SEQ ID NO:173, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:230.

[0158] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:29, VH CDR2 contains the amino acid sequence of SEQ ID NO:76, VH CDR3 contains the amino acid sequence of SEQ ID NO:121, VL CDR1 contains the amino acid sequence of SEQ ID NO:173, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:230.

[0159] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:29, VH CDR2 contains the amino acid sequence of SEQ ID NO:77, VH CDR3 contains the amino acid sequence of SEQ ID NO:121, VL CDR1 contains the amino acid sequence of SEQ ID NO:172, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:230.

[0160] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:29, VH CDR2 contains the amino acid sequence of SEQ ID NO:78, VH CDR3 contains the amino acid sequence of SEQ ID NO:121, VL CDR1 contains the amino acid sequence of SEQ ID NO:172, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:230.

[0161] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:29, VH CDR2 contains the amino acid sequence of SEQ ID NO:78, VH CDR3 contains the amino acid sequence of SEQ ID NO:121, VL CDR1 contains the amino acid sequence of SEQ ID NO:173, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:230.

[0162] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:29, VH CDR2 contains the amino acid sequence of SEQ ID NO:76, VH CDR3 contains the amino acid sequence of SEQ ID NO:121, VL CDR1 contains the amino acid sequence of SEQ ID NO:174, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:230.

[0163] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:29, VH CDR2 contains the amino acid sequence of SEQ ID NO:77, VH CDR3 contains the amino acid sequence of SEQ ID NO:121, VL CDR1 contains the amino acid sequence of SEQ ID NO:174, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:230.

[0164] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:29, VH CDR2 contains the amino acid sequence of SEQ ID NO:78, VH CDR3 contains the amino acid sequence of SEQ ID NO:121, VL CDR1 contains the amino acid sequence of SEQ ID NO:174, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:230.

[0165] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:29, VH CDR2 contains the amino acid sequence of SEQ ID NO:78, VH CDR3 contains the amino acid sequence of SEQ ID NO:121, VL CDR1 contains the amino acid sequence of SEQ ID NO:171, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:230.

[0166] In some respects, VH CDR1 contains the amino acid sequence of SEQ ID NO:29, VH CDR2 contains the amino acid sequence of SEQ ID NO:75, VH CDR3 contains the amino acid sequence of SEQ ID NO:121, VL CDR1 contains the amino acid sequence of SEQ ID NO:174, VL CDR2 contains the amino acid sequence of SEQ ID NO:197, and VL CDR3 contains the amino acid sequence of SEQ ID NO:230; or VH CDR1 contains the amino acid sequence of SEQ ID NO:22, VH CDR2 contains the amino acid sequence of SEQ ID NO:53, VH CDR3 contains the amino acid sequence of SEQ ID NO:113, VL CDR1 contains the amino acid sequence of SEQ ID NO:152, VL CDR2 contains the amino acid sequence of SEQ ID NO:193, and VL CDR3 contains the amino acid sequence of SEQ ID NO:223.

[0167] In some aspects, the anti-CRH antibody or its antigen-binding fragment comprises VH, which contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with any one of SEQ ID NO:240-295, 459, and 461; and / or VL, which contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with any one of SEQ ID NO:296-347 and 462. In some aspects, the antibody or antigen-binding fragment comprises VH, which contains an amino acid sequence of any one of SEQ ID NO:240-295, 459, and 461; and / or VL, which contains an amino acid sequence of any one of SEQ ID NO:296-347 and 462.

[0168] In some respects, VH contains the amino acid sequence of SEQ ID NO:240, and / or VL contains the amino acid sequence of SEQ ID NO:296.

[0169] In some respects, VH contains the amino acid sequence of SEQ ID NO:241, and / or VL contains the amino acid sequence of SEQ ID NO:296.

[0170] In some respects, VH contains the amino acid sequence of SEQ ID NO:242, and / or VL contains the amino acid sequence of SEQ ID NO:296.

[0171] In some respects, VH contains the amino acid sequence of SEQ ID NO:240, and / or VL contains the amino acid sequence of SEQ ID NO:297.

[0172] In some respects, VH contains the amino acid sequence of SEQ ID NO:241, and / or VL contains the amino acid sequence of SEQ ID NO:297.

[0173] In some respects, VH contains the amino acid sequence of SEQ ID NO:242, and / or VL contains the amino acid sequence of SEQ ID NO:297.

[0174] In some respects, VH contains the amino acid sequence of SEQ ID NO:241, and / or VL contains the amino acid sequence of SEQ ID NO:298.

[0175] In some respects, VH contains the amino acid sequence of SEQ ID NO:241, and VL contains the amino acid sequence of SEQ ID NO:299.

[0176] In some respects, VH contains the amino acid sequence of SEQ ID NO:241, and VL contains the amino acid sequence of SEQ ID NO:300.

[0177] In some respects, VH contains the amino acid sequence of SEQ ID NO:241, and / or VL contains the amino acid sequence of SEQ ID NO:301.

[0178] In some respects, VH contains the amino acid sequence of SEQ ID NO:242, and / or VL contains the amino acid sequence of SEQ ID NO:298.

[0179] In some respects, VH contains the amino acid sequence of SEQ ID NO:242, and / or VL contains the amino acid sequence of SEQ ID NO:299.

[0180] In some respects, VH contains the amino acid sequence of SEQ ID NO:242, and / or VL contains the amino acid sequence of SEQ ID NO:300.

[0181] In some respects, VH contains the amino acid sequence of SEQ ID NO:242, and / or VL contains the amino acid sequence of SEQ ID NO:301.

[0182] In some respects, VH contains the amino acid sequence of SEQ ID NO:243, and / or VL contains the amino acid sequence of SEQ ID NO:298.

[0183] In some respects, VH contains the amino acid sequence of SEQ ID NO:243, and / or VL contains the amino acid sequence of SEQ ID NO:299.

[0184] In some respects, VH contains the amino acid sequence of SEQ ID NO:243, and / or VL contains the amino acid sequence of SEQ ID NO:300.

[0185] In some respects, VH contains the amino acid sequence of SEQ ID NO:243, and / or VL contains the amino acid sequence of SEQ ID NO:301.

[0186] In some respects, VH contains the amino acid sequence of SEQ ID NO:244, and / or VL contains the amino acid sequence of SEQ ID NO:302.

[0187] In some respects, VH contains the amino acid sequence of SEQ ID NO:245, and / or VL contains the amino acid sequence of SEQ ID NO:302.

[0188] In some respects, VH contains the amino acid sequence of SEQ ID NO:246, and / or VL contains the amino acid sequence of SEQ ID NO:302.

[0189] In some respects, VH contains the amino acid sequence of SEQ ID NO:247, and / or VL contains the amino acid sequence of SEQ ID NO:302.

[0190] In some respects, VH contains the amino acid sequence of SEQ ID NO:248, and / or VL contains the amino acid sequence of SEQ ID NO:303.

[0191] In some respects, VH contains the amino acid sequence of SEQ ID NO:249, and / or VL contains the amino acid sequence of SEQ ID NO:304.

[0192] In some respects, VH contains the amino acid sequence of SEQ ID NO:250, and / or VL contains the amino acid sequence of SEQ ID NO:305.

[0193] In some respects, VH contains the amino acid sequence of SEQ ID NO:251, and / or VL contains the amino acid sequence of SEQ ID NO:306.

[0194] In some respects, VH contains the amino acid sequence of SEQ ID NO:252, and / or VL contains the amino acid sequence of SEQ ID NO:305.

[0195] In some respects, VH contains the amino acid sequence of SEQ ID NO:252, and / or VL contains the amino acid sequence of SEQ ID NO:307.

[0196] In some respects, VH contains the amino acid sequence of SEQ ID NO:253, and / or VL contains the amino acid sequence of SEQ ID NO:308.

[0197] In some respects, VH contains the amino acid sequence of SEQ ID NO:254, and / or VL contains the amino acid sequence of SEQ ID NO:309.

[0198] In some respects, VH contains the amino acid sequence of SEQ ID NO:255, and / or VL contains the amino acid sequence of SEQ ID NO:310.

[0199] In some respects, VH contains the amino acid sequence of SEQ ID NO:255, and / or VL contains the amino acid sequence of SEQ ID NO:311.

[0200] In some respects, VH contains the amino acid sequence of SEQ ID NO:255, and / or VL contains the amino acid sequence of SEQ ID NO:312.

[0201] In some respects, VH contains the amino acid sequence of SEQ ID NO:256, and / or VL contains the amino acid sequence of SEQ ID NO:310.

[0202] In some respects, VH contains the amino acid sequence of SEQ ID NO:256, and / or VL contains the amino acid sequence of SEQ ID NO:311.

[0203] In some respects, VH contains the amino acid sequence of SEQ ID NO:256, and / or VL contains the amino acid sequence of SEQ ID NO:312.

[0204] In some respects, VH contains the amino acid sequence of SEQ ID NO:257, and / or VL contains the amino acid sequence of SEQ ID NO:310.

[0205] In some respects, VH contains the amino acid sequence of SEQ ID NO:257, and / or VL contains the amino acid sequence of SEQ ID NO:311.

[0206] In some respects, VH contains the amino acid sequence of SEQ ID NO:257, and / or VL contains the amino acid sequence of SEQ ID NO:312.

[0207] In some respects, VH contains the amino acid sequence of SEQ ID NO:258, and / or VL contains the amino acid sequence of SEQ ID NO:310.

[0208] In some respects, VH contains the amino acid sequence of SEQ ID NO:258, and / or VL contains the amino acid sequence of SEQ ID NO:311.

[0209] In some respects, VH contains the amino acid sequence of SEQ ID NO:258, and / or VL contains the amino acid sequence of SEQ ID NO:312.

[0210] In some respects, VH contains the amino acid sequence of SEQ ID NO:259, and / or VL contains the amino acid sequence of SEQ ID NO:310.

[0211] In some respects, VH contains the amino acid sequence of SEQ ID NO:259, and / or VL contains the amino acid sequence of SEQ ID NO:311.

[0212] In some respects, VH contains the amino acid sequence of SEQ ID NO:259, and / or VL contains the amino acid sequence of SEQ ID NO:312.

[0213] In some respects, VH contains the amino acid sequence of SEQ ID NO:260, and / or VL contains the amino acid sequence of SEQ ID NO:317.

[0214] In some respects, VH contains the amino acid sequence of SEQ ID NO:260, and / or VL contains the amino acid sequence of SEQ ID NO:313.

[0215] In some respects, VH contains the amino acid sequence of SEQ ID NO:261, and / or VL contains the amino acid sequence of SEQ ID NO:314.

[0216] In some respects, VH contains the amino acid sequence of SEQ ID NO:261, and / or VL contains the amino acid sequence of SEQ ID NO:315.

[0217] In some respects, VH contains the amino acid sequence of SEQ ID NO:261, and / or VL contains the amino acid sequence of SEQ ID NO:316.

[0218] In some respects, VH contains the amino acid sequence of SEQ ID NO:262, and / or VL contains the amino acid sequence of SEQ ID NO:317.

[0219] In some respects, VH contains the amino acid sequence of SEQ ID NO:261, and / or VL contains the amino acid sequence of SEQ ID NO:317.

[0220] In some respects, VH contains the amino acid sequence of SEQ ID NO:263, and / or VL contains the amino acid sequence of SEQ ID NO:317.

[0221] In some respects, VH contains the amino acid sequence of SEQ ID NO:264, and / or VL contains the amino acid sequence of SEQ ID NO:317.

[0222] In some respects, VH contains the amino acid sequence of SEQ ID NO:262, and / or VL contains the amino acid sequence of SEQ ID NO:318.

[0223] In some respects, VH contains the amino acid sequence of SEQ ID NO:263, and / or VL contains the amino acid sequence of SEQ ID NO:318.

[0224] In some respects, VH contains the amino acid sequence of SEQ ID NO:264, and / or VL contains the amino acid sequence of SEQ ID NO:318.

[0225] In some respects, VH contains the amino acid sequence of SEQ ID NO:260, and / or VL contains the amino acid sequence of SEQ ID NO:318.

[0226] In some respects, VH contains the amino acid sequence of SEQ ID NO:264, and / or VL contains the amino acid sequence of SEQ ID NO:314.

[0227] In some respects, VH contains the amino acid sequence of SEQ ID NO:264, and / or VL contains the amino acid sequence of SEQ ID NO:316.

[0228] In some respects, VH contains the amino acid sequence of SEQ ID NO:260, and / or VL contains the amino acid sequence of SEQ ID NO:319.

[0229] In some respects, VH contains the amino acid sequence of SEQ ID NO:260, and / or VL contains the amino acid sequence of SEQ ID NO:314.

[0230] In some respects, VH contains the amino acid sequence of SEQ ID NO:260, and / or VL contains the amino acid sequence of SEQ ID NO:315.

[0231] In some respects, VH contains the amino acid sequence of SEQ ID NO:260, and / or VL contains the amino acid sequence of SEQ ID NO:316.

[0232] In some respects, VH contains the amino acid sequence of SEQ ID NO:261, and / or VL contains the amino acid sequence of SEQ ID NO:313.

[0233] In some respects, VH contains the amino acid sequence of SEQ ID NO:261, and / or VL contains the amino acid sequence of SEQ ID NO:318.

[0234] In some respects, VH contains the amino acid sequence of SEQ ID NO:261, and / or VL contains the amino acid sequence of SEQ ID NO:319.

[0235] In some respects, VH contains the amino acid sequence of SEQ ID NO:265, and / or VL contains the amino acid sequence of SEQ ID NO:320.

[0236] In some respects, VH contains the amino acid sequence of SEQ ID NO:266, and / or VL contains the amino acid sequence of SEQ ID NO:321.

[0237] In some respects, VH contains the amino acid sequence of SEQ ID NO:267, and / or VL contains the amino acid sequence of SEQ ID NO:322.

[0238] In some respects, VH contains the amino acid sequence of SEQ ID NO:268, and / or VL contains the amino acid sequence of SEQ ID NO:323.

[0239] In some respects, VH contains the amino acid sequence of SEQ ID NO:269, and / or VL contains the amino acid sequence of SEQ ID NO:324.

[0240] In some respects, VH contains the amino acid sequence of SEQ ID NO:270, and / or VL contains the amino acid sequence of SEQ ID NO:325.

[0241] In some respects, VH contains the amino acid sequence of SEQ ID NO:271, and / or VL contains the amino acid sequence of SEQ ID NO:326.

[0242] In some respects, VH contains the amino acid sequence of SEQ ID NO:272, and / or VL contains the amino acid sequence of SEQ ID NO:331.

[0243] In some respects, VH contains the amino acid sequence of SEQ ID NO:272, and / or VL contains the amino acid sequence of SEQ ID NO:327.

[0244] In some respects, VH contains the amino acid sequence of SEQ ID NO:273, and / or VL contains the amino acid sequence of SEQ ID NO:328.

[0245] In some respects, VH contains the amino acid sequence of SEQ ID NO:273, and / or VL contains the amino acid sequence of SEQ ID NO:329.

[0246] In some respects, VH contains the amino acid sequence of SEQ ID NO:273, and / or VL contains the amino acid sequence of SEQ ID NO:330.

[0247] In some respects, VH contains the amino acid sequence of SEQ ID NO:273, and / or VL contains the amino acid sequence of SEQ ID NO:331.

[0248] In some respects, VH contains the amino acid sequence of SEQ ID NO:274, and / or VL contains the amino acid sequence of SEQ ID NO:331.

[0249] In some respects, VH contains the amino acid sequence of SEQ ID NO:275, and / or VL contains the amino acid sequence of SEQ ID NO:331.

[0250] In some respects, VH contains the amino acid sequence of SEQ ID NO:276, and / or VL contains the amino acid sequence of SEQ ID NO:331.

[0251] In some respects, VH contains the amino acid sequence of SEQ ID NO:277, and / or VL contains the amino acid sequence of SEQ ID NO:331.

[0252] In some respects, VH contains the amino acid sequence of SEQ ID NO:272, and / or VL contains the amino acid sequence of SEQ ID NO:332.

[0253] In some respects, VH contains the amino acid sequence of SEQ ID NO:276, and / or VL contains the amino acid sequence of SEQ ID NO:332.

[0254] In some respects, VH contains the amino acid sequence of SEQ ID NO:275, and / or VL contains the amino acid sequence of SEQ ID NO:328.

[0255] In some respects, VH contains the amino acid sequence of SEQ ID NO:275, and / or VL contains the amino acid sequence of SEQ ID NO:330.

[0256] In some respects, VH contains the amino acid sequence of SEQ ID NO:277, and / or VL contains the amino acid sequence of SEQ ID NO:328.

[0257] In some respects, VH contains the amino acid sequence of SEQ ID NO:277, and / or VL contains the amino acid sequence of SEQ ID NO:330.

[0258] In some respects, VH contains the amino acid sequence of SEQ ID NO:272, and / or VL contains the amino acid sequence of SEQ ID NO:333.

[0259] In some respects, VH contains the amino acid sequence of SEQ ID NO:272, and / or VL contains the amino acid sequence of SEQ ID NO:328.

[0260] In some respects, VH contains the amino acid sequence of SEQ ID NO:272, and / or VL contains the amino acid sequence of SEQ ID NO:329.

[0261] In some respects, VH contains the amino acid sequence of SEQ ID NO:272, and / or VL contains the amino acid sequence of SEQ ID NO:330.

[0262] In some respects, VH contains the amino acid sequence of SEQ ID NO:273, and / or VL contains the amino acid sequence of SEQ ID NO:327.

[0263] In some respects, VH contains the amino acid sequence of SEQ ID NO:273, and / or VL contains the amino acid sequence of SEQ ID NO:332.

[0264] In some respects, VH contains the amino acid sequence of SEQ ID NO:273, and / or VL contains the amino acid sequence of SEQ ID NO:333.

[0265] In some respects, VH contains the amino acid sequence of SEQ ID NO:278, and / or VL contains the amino acid sequence of SEQ ID NO:334.

[0266] In some respects, VH contains the amino acid sequence of SEQ ID NO:279, and / or VL contains the amino acid sequence of SEQ ID NO:323.

[0267] In some respects, VH contains the amino acid sequence of SEQ ID NO:280, and / or VL contains the amino acid sequence of SEQ ID NO:339.

[0268] In some respects, VH contains the amino acid sequence of SEQ ID NO:280, and / or VL contains the amino acid sequence of SEQ ID NO:335.

[0269] In some respects, VH contains the amino acid sequence of SEQ ID NO:281, and / or VL contains the amino acid sequence of SEQ ID NO:336.

[0270] In some respects, VH contains the amino acid sequence of SEQ ID NO:281, and / or VL contains the amino acid sequence of SEQ ID NO:337.

[0271] In some respects, VH contains the amino acid sequence of SEQ ID NO:281, and / or VL contains the amino acid sequence of SEQ ID NO:338.

[0272] In some respects, VH contains the amino acid sequence of SEQ ID NO:282, and / or VL contains the amino acid sequence of SEQ ID NO:339.

[0273] In some respects, VH contains the amino acid sequence of SEQ ID NO:281, and / or VL contains the amino acid sequence of SEQ ID NO:339.

[0274] In some respects, VH contains the amino acid sequence of SEQ ID NO:283, and / or VL contains the amino acid sequence of SEQ ID NO:339.

[0275] In some respects, VH contains the amino acid sequence of SEQ ID NO:284, and / or VL contains the amino acid sequence of SEQ ID NO:339.

[0276] In some respects, VH contains the amino acid sequence of SEQ ID NO:285, and / or VL contains the amino acid sequence of SEQ ID NO:339.

[0277] In some respects, VH contains the amino acid sequence of SEQ ID NO:286, and / or VL contains the amino acid sequence of SEQ ID NO:339.

[0278] In some respects, VH contains the amino acid sequence of SEQ ID NO:282, and / or VL contains the amino acid sequence of SEQ ID NO:340.

[0279] In some respects, VH contains the amino acid sequence of SEQ ID NO:280, and / or VL contains the amino acid sequence of SEQ ID NO:340.

[0280] In some respects, VH contains the amino acid sequence of SEQ ID NO:283, and / or VL contains the amino acid sequence of SEQ ID NO:340.

[0281] In some respects, VH contains the amino acid sequence of SEQ ID NO:284, and / or VL contains the amino acid sequence of SEQ ID NO:340.

[0282] In some respects, VH contains the amino acid sequence of SEQ ID NO:285, and / or VL contains the amino acid sequence of SEQ ID NO:340.

[0283] In some respects, VH contains the amino acid sequence of SEQ ID NO:286, and / or VL contains the amino acid sequence of SEQ ID NO:340.

[0284] In some respects, VH contains the amino acid sequence of SEQ ID NO:284, and / or VL contains the amino acid sequence of SEQ ID NO:336.

[0285] In some respects, VH contains the amino acid sequence of SEQ ID NO:284, and / or VL contains the amino acid sequence of SEQ ID NO:338.

[0286] In some respects, VH contains the amino acid sequence of SEQ ID NO:286, and / or VL contains the amino acid sequence of SEQ ID NO:336.

[0287] In some respects, VH contains the amino acid sequence of SEQ ID NO:286, and / or VL contains the amino acid sequence of SEQ ID NO:338.

[0288] In some respects, VH contains the amino acid sequence of SEQ ID NO:287, and / or VL contains the amino acid sequence of SEQ ID NO:340.

[0289] In some respects, VH contains the amino acid sequence of SEQ ID NO:288, and / or VL contains the amino acid sequence of SEQ ID NO:340.

[0290] In some respects, VH contains the amino acid sequence of SEQ ID NO:280, and / or VL contains the amino acid sequence of SEQ ID NO:341.

[0291] In some respects, VH contains the amino acid sequence of SEQ ID NO:286, and / or VL contains the amino acid sequence of SEQ ID NO:342.

[0292] In some respects, VH contains the amino acid sequence of SEQ ID NO:286, and / or VL contains the amino acid sequence of SEQ ID NO:343.

[0293] In some respects, VH contains the amino acid sequence of SEQ ID NO:287, and / or VL contains the amino acid sequence of SEQ ID NO:342.

[0294] In some respects, VH contains the amino acid sequence of SEQ ID NO:288, and / or VL contains the amino acid sequence of SEQ ID NO:343.

[0295] In some respects, VH contains the amino acid sequence of SEQ ID NO:287, and / or VL contains the amino acid sequence of SEQ ID NO:343.

[0296] In some respects, VH contains the amino acid sequence of SEQ ID NO:288, and / or VL contains the amino acid sequence of SEQ ID NO:342.

[0297] In some respects, VH contains the amino acid sequence of SEQ ID NO:289, and / or VL contains the amino acid sequence of SEQ ID NO:342.

[0298] In some respects, VH contains the amino acid sequence of SEQ ID NO:289, and / or VL contains the amino acid sequence of SEQ ID NO:343.

[0299] In some respects, VH contains the amino acid sequence of SEQ ID NO:287, and / or VL contains the amino acid sequence of SEQ ID NO:344.

[0300] In some respects, VH contains the amino acid sequence of SEQ ID NO:288, and / or VL contains the amino acid sequence of SEQ ID NO:344.

[0301] In some respects, VH contains the amino acid sequence of SEQ ID NO:280, and / or VL contains the amino acid sequence of SEQ ID NO:336.

[0302] In some respects, VH contains the amino acid sequence of SEQ ID NO:289, and / or VL contains the amino acid sequence of SEQ ID NO:344.

[0303] In some respects, VH contains the amino acid sequence of SEQ ID NO:289, and / or VL contains the amino acid sequence of SEQ ID NO:340.

[0304] In some respects, VH contains the amino acid sequence of SEQ ID NO:286, and / or VL contains the amino acid sequence of SEQ ID NO:344.

[0305] In some respects, VH contains the amino acid sequence of SEQ ID NO:290, and / or VL contains the amino acid sequence of SEQ ID NO:336.

[0306] In some respects, VH contains the amino acid sequence of SEQ ID NO:291, and / or VL contains the amino acid sequence of SEQ ID NO:336.

[0307] In some respects, VH contains the amino acid sequence of SEQ ID NO:292, and / or VL contains the amino acid sequence of SEQ ID NO:336.

[0308] In some respects, VH contains the amino acid sequence of SEQ ID NO:290, and / or VL contains the amino acid sequence of SEQ ID NO:345.

[0309] In some respects, VH contains the amino acid sequence of SEQ ID NO:291, and / or VL contains the amino acid sequence of SEQ ID NO:345.

[0310] In some respects, VH contains the amino acid sequence of SEQ ID NO:292, and / or VL contains the amino acid sequence of SEQ ID NO:345.

[0311] In some respects, VH contains the amino acid sequence of SEQ ID NO:290, and / or VL contains the amino acid sequence of SEQ ID NO:346.

[0312] In some respects, VH contains the amino acid sequence of SEQ ID NO:280, and / or VL contains the amino acid sequence of SEQ ID NO:337.

[0313] In some respects, VH contains the amino acid sequence of SEQ ID NO:291, and / or VL contains the amino acid sequence of SEQ ID NO:346.

[0314] In some respects, VH contains the amino acid sequence of SEQ ID NO:292, and / or VL contains the amino acid sequence of SEQ ID NO:346.

[0315] In some respects, VH contains the amino acid sequence of SEQ ID NO:290, and / or VL contains the amino acid sequence of SEQ ID NO:347.

[0316] In some respects, VH contains the amino acid sequence of SEQ ID NO:291, and / or VL contains the amino acid sequence of SEQ ID NO:347.

[0317] In some respects, VH contains the amino acid sequence of SEQ ID NO:292, and / or VL contains the amino acid sequence of SEQ ID NO:347.

[0318] In some respects, VH contains the amino acid sequence of SEQ ID NO:293, and / or VL contains the amino acid sequence of SEQ ID NO:340.

[0319] In some respects, VH contains the amino acid sequence of SEQ ID NO:294, and / or VL contains the amino acid sequence of SEQ ID NO:340.

[0320] In some respects, VH contains the amino acid sequence of SEQ ID NO:295, and / or VL contains the amino acid sequence of SEQ ID NO:340.

[0321] In some respects, VH contains the amino acid sequence of SEQ ID NO:293, and / or VL contains the amino acid sequence of SEQ ID NO:343.

[0322] In some respects, VH contains the amino acid sequence of SEQ ID NO:294, and / or VL contains the amino acid sequence of SEQ ID NO:343.

[0323] In some respects, VH contains the amino acid sequence of SEQ ID NO:280, and / or VL contains the amino acid sequence of SEQ ID NO:338.

[0324] In some respects, VH contains the amino acid sequence of SEQ ID NO:295, and / or VL contains the amino acid sequence of SEQ ID NO:343.

[0325] In some respects, VH contains the amino acid sequence of SEQ ID NO:293, and / or VL contains the amino acid sequence of SEQ ID NO:342.

[0326] In some respects, VH contains the amino acid sequence of SEQ ID NO:294, and / or VL contains the amino acid sequence of SEQ ID NO:342.

[0327] In some respects, VH contains the amino acid sequence of SEQ ID NO:295, and / or VL contains the amino acid sequence of SEQ ID NO:342.

[0328] In some respects, VH contains the amino acid sequence of SEQ ID NO:293, and / or VL contains the amino acid sequence of SEQ ID NO:344.

[0329] In some respects, VH contains the amino acid sequence of SEQ ID NO:294, and / or VL contains the amino acid sequence of SEQ ID NO:344.

[0330] In some respects, VH contains the amino acid sequence of SEQ ID NO:295, and / or VL contains the amino acid sequence of SEQ ID NO:344.

[0331] In some respects, VH contains the amino acid sequence of SEQ ID NO:281, and / or VL contains the amino acid sequence of SEQ ID NO:345.

[0332] In some respects, VH contains the amino acid sequence of SEQ ID NO:281, and / or VL contains the amino acid sequence of SEQ ID NO:346.

[0333] In some respects, VH contains the amino acid sequence of SEQ ID NO:281, and / or VL contains the amino acid sequence of SEQ ID NO:347.

[0334] In some respects, VH contains the amino acid sequence of SEQ ID NO:281, and / or VL contains the amino acid sequence of SEQ ID NO:335.

[0335] In some respects, VH contains the amino acid sequence of SEQ ID NO:283, and / or VL contains the amino acid sequence of SEQ ID NO:343.

[0336] In some respects, VH contains the amino acid sequence of SEQ ID NO:283, and / or VL contains the amino acid sequence of SEQ ID NO:342.

[0337] In some respects, VH contains the amino acid sequence of SEQ ID NO:283, and / or VL contains the amino acid sequence of SEQ ID NO:344.

[0338] In some respects, VH contains the amino acid sequence of SEQ ID NO:281, and / or VL contains the amino acid sequence of SEQ ID NO:340.

[0339] In some respects, VH contains the amino acid sequence of SEQ ID NO:281, and / or VL contains the amino acid sequence of SEQ ID NO:341, VH contains the amino acid sequence of SEQ ID NO:461, and / or VL contains the amino acid sequence of SEQ ID NO:462, or VH contains the amino acid sequence of SEQ ID NO:459, and / or VL contains the amino acid sequence of SEQ ID NO:296.

[0340] In some aspects, the anti-CRH antibody or its antigen-binding fragment comprises a heavy chain having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with any one of SEQ ID NO:348-403, 460, and 463; and / or a light chain having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with any one of SEQ ID NO:404-455 and 464. In some aspects, the antibody or its antigen-binding fragment comprises a heavy chain having an amino acid sequence having any one of SEQ ID NO:348-403, 460, and 463; and / or a light chain having an amino acid sequence having any one of SEQ ID NO:404-455 and 464.

[0341] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:348, and / or the light chain contains the amino acid sequence of SEQ ID NO:404.

[0342] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:349, and / or the light chain contains the amino acid sequence of SEQ ID NO:404.

[0343] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:350, and / or the light chain contains the amino acid sequence of SEQ ID NO:404.

[0344] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:348, and / or the light chain contains the amino acid sequence of SEQ ID NO:405.

[0345] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:349, and / or the light chain contains the amino acid sequence of SEQ ID NO:405.

[0346] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:350, and / or the light chain contains the amino acid sequence of SEQ ID NO:405.

[0347] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:349, and / or the light chain contains the amino acid sequence of SEQ ID NO:406.

[0348] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:349, and / or the light chain contains the amino acid sequence of SEQ ID NO:407.

[0349] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:349, and / or the light chain contains the amino acid sequence of SEQ ID NO:408.

[0350] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:349, and / or the light chain contains the amino acid sequence of SEQ ID NO:409.

[0351] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:350, and / or the light chain contains the amino acid sequence of SEQ ID NO:406.

[0352] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:350, and / or the light chain contains the amino acid sequence of SEQ ID NO:407.

[0353] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:350, and / or the light chain contains the amino acid sequence of SEQ ID NO:408.

[0354] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:350, and / or the light chain contains the amino acid sequence of SEQ ID NO:409.

[0355] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:351, and / or the light chain contains the amino acid sequence of SEQ ID NO:406.

[0356] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:351, and / or the light chain contains the amino acid sequence of SEQ ID NO:407.

[0357] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:351, and / or the light chain contains the amino acid sequence of SEQ ID NO:408.

[0358] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:351, and / or the light chain contains the amino acid sequence of SEQ ID NO:409.

[0359] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:352, and / or the light chain contains the amino acid sequence of SEQ ID NO:410.

[0360] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:353, and / or the light chain contains the amino acid sequence of SEQ ID NO:410.

[0361] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:354, and / or the light chain contains the amino acid sequence of SEQ ID NO:410.

[0362] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:355, and / or the light chain contains the amino acid sequence of SEQ ID NO:410.

[0363] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:356, and / or the light chain contains the amino acid sequence of SEQ ID NO:411.

[0364] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:357, and / or the light chain contains the amino acid sequence of SEQ ID NO:412.

[0365] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:358, and / or the light chain contains the amino acid sequence of SEQ ID NO:413.

[0366] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:359, and / or the light chain contains the amino acid sequence of SEQ ID NO:414.

[0367] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:360, and / or the light chain contains the amino acid sequence of SEQ ID NO:413.

[0368] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:360, and / or the light chain contains the amino acid sequence of SEQ ID NO:415.

[0369] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:361, and / or the light chain contains the amino acid sequence of SEQ ID NO:416.

[0370] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:362, and / or the light chain contains the amino acid sequence of SEQ ID NO:417.

[0371] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:363, and / or the light chain contains the amino acid sequence of SEQ ID NO:418.

[0372] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:363, and / or the light chain contains the amino acid sequence of SEQ ID NO:419.

[0373] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:363, and / or the light chain contains the amino acid sequence of SEQ ID NO:420.

[0374] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:364, and / or the light chain contains the amino acid sequence of SEQ ID NO:418.

[0375] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:364, and / or the light chain contains the amino acid sequence of SEQ ID NO:419.

[0376] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:364, and / or the light chain contains the amino acid sequence of SEQ ID NO:420.

[0377] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:365, and / or the light chain contains the amino acid sequence of SEQ ID NO:418.

[0378] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:365, and / or the light chain contains the amino acid sequence of SEQ ID NO:419.

[0379] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:365, and / or the light chain contains the amino acid sequence of SEQ ID NO:420.

[0380] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:366, and / or the light chain contains the amino acid sequence of SEQ ID NO:418.

[0381] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:366, and / or the light chain contains the amino acid sequence of SEQ ID NO:419.

[0382] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:366, and / or the light chain contains the amino acid sequence of SEQ ID NO:420.

[0383] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:367, and / or the light chain contains the amino acid sequence of SEQ ID NO:418.

[0384] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:367, and / or the light chain contains the amino acid sequence of SEQ ID NO:419.

[0385] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:367, and / or the light chain contains the amino acid sequence of SEQ ID NO:420.

[0386] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:368, and / or the light chain contains the amino acid sequence of SEQ ID NO:425.

[0387] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:368, and / or the light chain contains the amino acid sequence of SEQ ID NO:421.

[0388] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:369, and / or the light chain contains the amino acid sequence of SEQ ID NO:422.

[0389] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:369, and / or the light chain contains the amino acid sequence of SEQ ID NO:423.

[0390] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:369, and / or the light chain contains the amino acid sequence of SEQ ID NO:424.

[0391] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:370, and / or the light chain contains the amino acid sequence of SEQ ID NO:425.

[0392] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:369, and / or the light chain contains the amino acid sequence of SEQ ID NO:425.

[0393] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:371, and / or the light chain contains the amino acid sequence of SEQ ID NO:425.

[0394] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:372, and / or the light chain contains the amino acid sequence of SEQ ID NO:425.

[0395] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:370, and / or the light chain contains the amino acid sequence of SEQ ID NO:426.

[0396] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:371, and / or the light chain contains the amino acid sequence of SEQ ID NO:426.

[0397] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:372, and / or the light chain contains the amino acid sequence of SEQ ID NO:426.

[0398] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:368, and / or the light chain contains the amino acid sequence of SEQ ID NO:426.

[0399] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:372, and / or the light chain contains the amino acid sequence of SEQ ID NO:422.

[0400] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:372, and / or the light chain contains the amino acid sequence of SEQ ID NO:424.

[0401] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:368, and / or the light chain contains the amino acid sequence of SEQ ID NO:427.

[0402] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:368, and / or the light chain contains the amino acid sequence of SEQ ID NO:422.

[0403] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:368, and / or the light chain contains the amino acid sequence of SEQ ID NO:423.

[0404] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:368, and / or the light chain contains the amino acid sequence of SEQ ID NO:424.

[0405] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:369, and / or the light chain contains the amino acid sequence of SEQ ID NO:421.

[0406] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:369, and / or the light chain contains the amino acid sequence of SEQ ID NO:426.

[0407] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:369, and / or the light chain contains the amino acid sequence of SEQ ID NO:427.

[0408] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:373, and / or the light chain contains the amino acid sequence of SEQ ID NO:428.

[0409] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:374, and / or the light chain contains the amino acid sequence of SEQ ID NO:429.

[0410] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:375, and / or the light chain contains the amino acid sequence of SEQ ID NO:430.

[0411] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:376, and / or the light chain contains the amino acid sequence of SEQ ID NO:431.

[0412] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:377, and / or the light chain contains the amino acid sequence of SEQ ID NO:432.

[0413] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:378, and / or the light chain contains the amino acid sequence of SEQ ID NO:433.

[0414] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:379, and / or the light chain contains the amino acid sequence of SEQ ID NO:434.

[0415] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:380, and / or the light chain contains the amino acid sequence of SEQ ID NO:439.

[0416] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:380, and / or the light chain contains the amino acid sequence of SEQ ID NO:435.

[0417] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:381, and / or the light chain contains the amino acid sequence of SEQ ID NO:436.

[0418] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:381, and / or the light chain contains the amino acid sequence of SEQ ID NO:437.

[0419] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:381, and / or the light chain contains the amino acid sequence of SEQ ID NO:438.

[0420] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:381, and / or the light chain contains the amino acid sequence of SEQ ID NO:439.

[0421] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:382, and / or the light chain contains the amino acid sequence of SEQ ID NO:439.

[0422] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:383, and / or the light chain contains the amino acid sequence of SEQ ID NO:439.

[0423] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:384, and / or the light chain contains the amino acid sequence of SEQ ID NO:439.

[0424] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:385, and / or the light chain contains the amino acid sequence of SEQ ID NO:439.

[0425] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:380, and / or the light chain contains the amino acid sequence of SEQ ID NO:440.

[0426] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:384, and / or the light chain contains the amino acid sequence of SEQ ID NO:440.

[0427] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:383, and / or the light chain contains the amino acid sequence of SEQ ID NO:436.

[0428] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:383, and / or the light chain contains the amino acid sequence of SEQ ID NO:438.

[0429] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:385, and / or the light chain contains the amino acid sequence of SEQ ID NO:436.

[0430] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:385, and / or the light chain contains the amino acid sequence of SEQ ID NO:438.

[0431] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:380, and / or the light chain contains the amino acid sequence of SEQ ID NO:441.

[0432] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:380, and / or the light chain contains the amino acid sequence of SEQ ID NO:436.

[0433] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:380, and / or the light chain contains the amino acid sequence of SEQ ID NO:437.

[0434] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:380, and / or the light chain contains the amino acid sequence of SEQ ID NO:438.

[0435] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:381, and / or the light chain contains the amino acid sequence of SEQ ID NO:435.

[0436] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:381, and / or the light chain contains the amino acid sequence of SEQ ID NO:440.

[0437] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:381, and / or the light chain contains the amino acid sequence of SEQ ID NO:441.

[0438] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:386, and / or the light chain contains the amino acid sequence of SEQ ID NO:442.

[0439] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:387, and / or the light chain contains the amino acid sequence of SEQ ID NO:431.

[0440] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:388, and / or the light chain contains the amino acid sequence of SEQ ID NO:447.

[0441] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:388, and / or the light chain contains the amino acid sequence of SEQ ID NO:443.

[0442] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:389, and / or the light chain contains the amino acid sequence of SEQ ID NO:444.

[0443] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:389, and / or the light chain contains the amino acid sequence of SEQ ID NO:445.

[0444] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:389, and / or the light chain contains the amino acid sequence of SEQ ID NO:446.

[0445] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:390, and / or the light chain contains the amino acid sequence of SEQ ID NO:447.

[0446] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:389, and / or the light chain contains the amino acid sequence of SEQ ID NO:447.

[0447] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:391, and / or the light chain contains the amino acid sequence of SEQ ID NO:447.

[0448] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:392, and / or the light chain contains the amino acid sequence of SEQ ID NO:447.

[0449] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:393, and / or the light chain contains the amino acid sequence of SEQ ID NO:447.

[0450] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:394, and / or the light chain contains the amino acid sequence of SEQ ID NO:447.

[0451] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:390, and / or the light chain contains the amino acid sequence of SEQ ID NO:448.

[0452] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:388, and / or the light chain contains the amino acid sequence of SEQ ID NO:448.

[0453] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:391, and / or the light chain contains the amino acid sequence of SEQ ID NO:448.

[0454] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:392, and / or the light chain contains the amino acid sequence of SEQ ID NO:448.

[0455] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:393, and / or the light chain contains the amino acid sequence of SEQ ID NO:448.

[0456] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:394, and / or the light chain contains the amino acid sequence of SEQ ID NO:448.

[0457] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:392, and / or the light chain contains the amino acid sequence of SEQ ID NO:444.

[0458] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:392, and / or the light chain contains the amino acid sequence of SEQ ID NO:446.

[0459] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:394, and / or the light chain contains the amino acid sequence of SEQ ID NO:444.

[0460] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:394, and / or the light chain contains the amino acid sequence of SEQ ID NO:446.

[0461] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:395, and / or the light chain contains the amino acid sequence of SEQ ID NO:448.

[0462] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:396, and / or the light chain contains the amino acid sequence of SEQ ID NO:448.

[0463] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:388, and / or the light chain contains the amino acid sequence of SEQ ID NO:449.

[0464] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:394, and / or the light chain contains the amino acid sequence of SEQ ID NO:450.

[0465] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:394, and / or the light chain contains the amino acid sequence of SEQ ID NO:451.

[0466] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:395, and / or the light chain contains the amino acid sequence of SEQ ID NO:450.

[0467] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:396, and / or the light chain contains the amino acid sequence of SEQ ID NO:451.

[0468] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:395, and / or the light chain contains the amino acid sequence of SEQ ID NO:451.

[0469] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:396, and / or the light chain contains the amino acid sequence of SEQ ID NO:450.

[0470] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:397, and / or the light chain contains the amino acid sequence of SEQ ID NO:450.

[0471] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:397, and / or the light chain contains the amino acid sequence of SEQ ID NO:451.

[0472] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:395, and / or the light chain contains the amino acid sequence of SEQ ID NO:452.

[0473] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:396, and / or the light chain contains the amino acid sequence of SEQ ID NO:452.

[0474] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:388, and / or the light chain contains the amino acid sequence of SEQ ID NO:444.

[0475] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:397, and / or the light chain contains the amino acid sequence of SEQ ID NO:452.

[0476] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:397, and / or the light chain contains the amino acid sequence of SEQ ID NO:448.

[0477] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:394, and / or the light chain contains the amino acid sequence of SEQ ID NO:452.

[0478] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:398, and / or the light chain contains the amino acid sequence of SEQ ID NO:444.

[0479] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:399, and / or the light chain contains the amino acid sequence of SEQ ID NO:444.

[0480] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:400, and / or the light chain contains the amino acid sequence of SEQ ID NO:444.

[0481] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:398, and / or the light chain contains the amino acid sequence of SEQ ID NO:453.

[0482] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:399, and / or the light chain contains the amino acid sequence of SEQ ID NO:453.

[0483] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:400, and / or the light chain contains the amino acid sequence of SEQ ID NO:453.

[0484] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:398, and / or the light chain contains the amino acid sequence of SEQ ID NO:454.

[0485] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:388, and / or the light chain contains the amino acid sequence of SEQ ID NO:445.

[0486] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:399, and / or the light chain contains the amino acid sequence of SEQ ID NO:454.

[0487] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:400, and / or the light chain contains the amino acid sequence of SEQ ID NO:454.

[0488] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:398, and / or the light chain contains the amino acid sequence of SEQ ID NO:455.

[0489] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:399, and / or the light chain contains the amino acid sequence of SEQ ID NO:455.

[0490] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:400, and / or the light chain contains the amino acid sequence of SEQ ID NO:455.

[0491] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:401, and / or the light chain contains the amino acid sequence of SEQ ID NO:448.

[0492] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:402, and / or the light chain contains the amino acid sequence of SEQ ID NO:448.

[0493] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:403, and / or the light chain contains the amino acid sequence of SEQ ID NO:448.

[0494] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:401, and / or the light chain contains the amino acid sequence of SEQ ID NO:451.

[0495] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:402, and / or the light chain contains the amino acid sequence of SEQ ID NO:451.

[0496] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:388, and / or the light chain contains the amino acid sequence of SEQ ID NO:446.

[0497] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:403, and / or the light chain contains the amino acid sequence of SEQ ID NO:451.

[0498] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:401, and / or the light chain contains the amino acid sequence of SEQ ID NO:450.

[0499] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:402, and / or the light chain contains the amino acid sequence of SEQ ID NO:450.

[0500] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:403, and / or the light chain contains the amino acid sequence of SEQ ID NO:450.

[0501] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:401, and / or the light chain contains the amino acid sequence of SEQ ID NO:452.

[0502] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:402, and / or the light chain contains the amino acid sequence of SEQ ID NO:452.

[0503] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:403, and / or the light chain contains the amino acid sequence of SEQ ID NO:452.

[0504] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:389, and / or the light chain contains the amino acid sequence of SEQ ID NO:453.

[0505] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:389, and / or the light chain contains the amino acid sequence of SEQ ID NO:454.

[0506] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:389, and / or the light chain contains the amino acid sequence of SEQ ID NO:455.

[0507] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:389, and / or the light chain contains the amino acid sequence of SEQ ID NO:443.

[0508] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:391, and / or the light chain contains the amino acid sequence of SEQ ID NO:451.

[0509] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:391, and / or the light chain contains the amino acid sequence of SEQ ID NO:450.

[0510] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:391, and / or the light chain contains the amino acid sequence of SEQ ID NO:452.

[0511] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:389, and / or the light chain contains the amino acid sequence of SEQ ID NO:448.

[0512] In some respects, the heavy chain contains the amino acid sequence of SEQ ID NO:389, and / or the light chain contains the amino acid sequence of SEQ ID NO:449, the heavy chain contains the amino acid sequence of SEQ ID NO:463, and / or the light chain contains the amino acid sequence of SEQ ID NO:464, or the heavy chain contains the amino acid sequence of SEQ ID NO:460, and / or the light chain contains the amino acid sequence of SEQ ID NO:404.

[0513] In some respects, anti-CRH antibodies or antigen-binding fragments are the antibodies or antigen-binding fragments listed in Table 1.

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524]

[0525]

[0526]

[0527]

[0528]

[0529] In some aspects, the methods and kits disclosed herein comprise a pharmaceutical composition comprising an anti-CRH antibody or an antigen-binding fragment thereof disclosed herein and a pharmaceutically acceptable carrier.

[0530] In some respects, the anti-CRH antibodies used in the methods and kits disclosed herein are polyclonal, monoclonal, human, humanized, or chimeric.

[0531] In other respects, the anti-CRH antigen binding fragments used in the methods and kits disclosed herein are Fab, scFab, Fab', F(ab')2, Fv, scFv, biantibody, or triantibody.

[0532] Methods well known to those skilled in the art can be used to obtain antibodies or antigen-binding fragments thereof containing the CDR, VH, VL, heavy chain sequences and / or light chain sequences disclosed herein. These methods include, for example, constructing expression vectors containing coding sequences for such antibodies or antigen-binding fragments thereof or their domains (e.g., CDR, VH, VL, heavy chain and / or light chain) and appropriate transcription and translation control signals using in vitro recombinant DNA technology, synthetic techniques and in vivo genetic recombination.

[0533] Expression vectors can be transferred into cells (e.g., host cells) using conventional techniques, and the resulting cells can then be cultured using conventional techniques to produce antibodies or antigen-binding fragments thereof disclosed herein (e.g., antibodies or antigen-binding fragments thereof comprising the six CDRs, VH, VL, VH and VL, heavy chain, light chain, or heavy chain and light chain of the antibodies disclosed herein) or their domains (e.g., VH, VL, VH and VL, heavy chain, or light chain of the antibodies disclosed herein).

[0534] In some aspects, to express a double-stranded antibody or its antigen-binding fragment, a vector encoding both the heavy chain and the light chain, respectively, can be co-expressed in a host cell to express the complete immunoglobulin. In some aspects, the host cell contains a vector comprising polynucleotides encoding both the heavy chain and the light chain, or their domains, of the antibody disclosed herein. In some aspects, the host cell contains two different vectors: a first vector containing polynucleotides encoding the heavy chain or a heavy chain variable region of the antibody disclosed herein, and a second vector containing polynucleotides encoding the light chain or a light chain variable region of the antibody disclosed herein. In some aspects, a first host cell contains a first vector containing polynucleotides encoding the heavy chain or a heavy chain variable region of the antibody disclosed herein, and a second host cell contains a second vector containing polynucleotides encoding the light chain or a light chain variable region of the antibody disclosed herein. In some aspects, the heavy chain / heavy chain variable region expressed by the first cell associates with the light chain / light chain variable region of the second cell to form the antibody disclosed herein or its antigen-binding fragment. In some respects, this article provides a host cell population comprising such first host cells and such second host cells.

[0535] A variety of host expression vector systems can be used to express the antibodies and their antigen-binding fragments disclosed herein. These host expression systems represent vectors that can generate and subsequently purify the coding sequences of interest, and cells that can express the proteins or antibodies or their antigen-binding fragments disclosed herein in situ when transformed or transfected with appropriate nucleotide coding sequences. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant phage DNA, plasmid DNA, or copious DNA expression vectors containing antibody-encoding sequences (e.g., *Escherichia coli* and *Bacillus subtilis*); yeast transformed with recombinant yeast expression vectors containing antibody-encoding sequences (e.g., *Pichia pastoris*); insect cell systems infected with recombinant viral expression vectors containing antibody-encoding sequences (e.g., baculoviruses); plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus CaMV; tobacco mosaic virus TMV) or transfected with recombinant plasmid expression vectors containing antibody-encoding sequences (e.g., Ti plasmids) (e.g., green algae, such as *Chlamydomonas reinhardtii*); or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NSO, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH). Cells including 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 carried recombinant expression constructs containing promoters from mammalian cell genomes (e.g., metallothionein promoters) or from mammalian viruses (e.g., adenovirus late promoters; vaccinia virus 7.5K promoters). In some respects, the cells used to express the proteins or antibodies disclosed herein and their antigen-binding fragments are CHO cells, such as those from the CHO GS System. TM (Lonza) CHO cells. In some respects, the cells used to express the antibodies and their antigen-binding fragments disclosed herein are human cells, such as human cell lines. In some respects, the mammalian expression vector is pOptiVEC. TM Or pcDNA3.3. In some respects, bacterial cells such as *Escherichia coli* or eukaryotic cells (e.g., mammalian cells), especially those used to express complete recombinant antibody molecules, are used to express recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary (CHO) cells combined with vectors such as major intermediate early gene promoter elements from human cytomegalovirus are efficient antibody expression systems (Foecking et al., *Gene*, 45:101-105 (1986) and Cockett et al., *Biotechnology*, 8:662-667 (1990)). In some respects, the proteins or antibodies or their antigen-binding fragments disclosed herein are produced by HEK-293T cells.

[0536] Furthermore, host cell lines can be selectively regulated to modulate the expression of the inserted sequence or to modify and process the gene product in a desired specific manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can contribute to protein function. For this purpose, eukaryotic host cells with appropriate cellular machinery for the processing, glycosylation, and phosphorylation of the primary transcript of the gene product can be used. These mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, MDCK, HEK293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10 and HsS78Bst cells.

[0537] Once the disclosed antibody or its antigen-binding fragment has been generated through recombinant expression, it can be purified using any method known in the art for purifying proteins or immunoglobulin molecules, such as chromatography (e.g., ion exchange, affinity, particularly by affinity for a specific antigen after protein A, and size exclusion chromatography), centrifugation, differential dissolution, or any other standard technique for purifying proteins. Furthermore, the disclosed antibody or its antigen-binding fragment can be fused with the disclosed heterologous polypeptide sequence or other heterologous polypeptide sequences known in the art to facilitate purification.

[0538] In some respects, the methods and kits disclosed herein contain isolated or purified antibodies or antigen-binding fragments thereof. Typically, the isolated protein or antibody or antigen-binding fragment thereof is substantially free of other proteins or antibodies or antigen-binding fragments thereof having an antigen specificity different from that of the isolated antibody or antigen-binding fragment thereof. For example, in some respects, the antibodies or antigen-binding fragments thereof disclosed herein are substantially free of cellular material and / or chemical precursors.

[0539] 3. How to use

[0540] Women with polycystic ovary syndrome (PCOS) synthesize and secrete excess androgens from their adrenal glands and ovaries. Recent analyses of the androgen sources in PCOS patients have found that the majority of these increased androgens originate from the adrenal glands (O'Reilly et al., J Clin Endocrinol Metab 102:840, 2017). The synthesis and secretion of adrenal androgens in the human body require ACTH (Weber et al., Clin Endocrinol 46:431, 2003), while the synthesis and secretion of ACTH require CRH (Muglia et al., Journal of Clinical Investigation, 105:1269, 2000). Therefore, blocking the CRH effect in PCOS patients with anti-CRH antibodies should reduce CRH, ACTH, and adrenal androgen levels in these patients.

[0541] In some aspects, this disclosure provides methods for treating PCOS and related conditions. These methods include administering to a subject in need a therapeutically effective amount of the disclosed anti-CRH antibody or its antigen-binding fragment. In some aspects, the method includes administering to a subject a therapeutically effective amount of a pharmaceutical composition of this disclosure, the pharmaceutical composition containing the disclosed anti-CRH antibody or its antigen-binding fragment.

[0542] In some aspects, this disclosure provides methods for reducing androgen levels in subjects suffering from PCOS. These methods include administering to the subject a therapeutically effective amount of the disclosed anti-CRH antibody or its antigen-binding fragment. In some aspects, the method includes administering to the subject a therapeutically effective amount of a pharmaceutical composition of this disclosure containing the disclosed anti-CRH antibody or its antigen-binding fragment. In some aspects, the androgen is testosterone, free testosterone, androstenedione, 11-oxyandrogens, or combinations thereof. In another aspect, the 11-oxyandrogens are 11-hydroxyandrostenedione (11OHA4), 11-hydroxytestosterone (11OHT), 11-ketoandrostenedione (11KA4), 11-ketotestosterone (11KT), or combinations thereof.

[0543] In some aspects, this disclosure provides methods for reducing the severity of one or more symptoms or signs in a subject suffering from PCOS. These methods include administering to the subject suffering from PCOS a therapeutically effective amount of the disclosed anti-CRH antibody or its antigen-binding fragment. In some aspects, the method includes administering to the subject a therapeutically effective amount of a pharmaceutical composition of this disclosure, the pharmaceutical composition containing the disclosed anti-CRH antibody or its antigen-binding fragment.

[0544] In some respects, one or more symptoms or signs of PCOS include anovulation (e.g., irregular or absent menstruation); hyperandrogenism (e.g., elevated levels of testosterone, free testosterone, androstenedione, 11-oxyandrogens, or combinations thereof); abnormal uterine bleeding; enlarged ovaries with multiple follicles; infertility; obesity; insulin resistance; hyperinsulinemia; hypertension; hyperlipidemia; type 2 diabetes; excessive facial hair growth; hirsutism; alopecia; and acne. In other respects, 11-oxyandrogens are 11-hydroxyandrostenedione (11OHA4), 11-hydroxytestosterone (11OHT), 11-ketoandrostenedione (11KA4), 11-ketotestosterone (11KT), or combinations thereof.

[0545] In some respects, upon administration of the anti-CRH antibody or its antigen-binding fragment disclosed herein, subjects exhibited a reduction in the severity of one or more symptoms of PCOS or an improvement in one or more symptoms of PCOS. In some respects, one or more symptoms include anovulation (e.g., irregular or absent menstruation); hyperandrogenism (e.g., elevated testosterone, free testosterone, androstenedione, 11-oxyandrogens, or combinations thereof); abnormal uterine bleeding; enlarged ovaries with multiple follicles; infertility; obesity; insulin resistance; hyperinsulinemia; hypertension; hyperlipidemia; type 2 diabetes; excessive facial hair growth; hirsutism; alopecia; or acne. In another respects, 11-oxyandrogens are 11-hydroxyandrostenedione (11OHA4), 11-hydroxytestosterone (11OHT), 11-ketoandrostenedione (11KA4), 11-ketotestosterone (11KT), or combinations thereof.

[0546] In some respects, subjects showed a reduction in anovulation after administration of the anti-CRH antibody or its antigen-binding fragment disclosed herein compared to before administration of the antibody or its antigen-binding fragment. In some respects, subjects resumed ovulation after administration of the anti-CRH antibody or its antigen-binding fragment disclosed herein.

[0547] In some respects, subjects showed a reduction in menstrual irregularities after administration of the anti-CRH antibody or its antigen-binding fragment disclosed herein, compared to menstrual irregularities prior to administration of the antibody or its antigen-binding fragment. In some respects, subjects resumed menstrual cycles after administration of the anti-CRH antibody or its antigen-binding fragment disclosed herein. In some respects, subjects resumed regular menstrual cycles after administration of the anti-CRH antibody or its antigen-binding fragment disclosed herein.

[0548] In some respects, subjects showed remission of hyperandrogenemia after administration of the anti-CRH antibody or its antigen-binding fragment disclosed herein, compared to before administration of the antibody or its antigen-binding fragment. In some respects, subjects showed a reduction in testosterone, free testosterone, androstenedione, 11-oxyandrogens, or combinations thereof after administration of the anti-CRH antibody or its antigen-binding fragment disclosed herein, compared to before administration of the antibody or its antigen-binding fragment. In another respect, the 11-oxyandrogens are 11-hydroxyandrostenedione (11OHA4), 11-hydroxytestosterone (11OHT), 11-ketoandrostenedione (11KA4), 11-ketotestosterone (11KT), or combinations thereof.

[0549] In some respects, subjects showed a reduction in abnormal uterine bleeding after administration of the anti-CRH antibody or its antigen-binding fragment disclosed herein, compared to before administration of the antibody or its antigen-binding fragment.

[0550] In some respects, subjects showed a reduction in ovarian cysts and / or a reduction in multifollicular ovaries after administration of the anti-CRH antibody or its antigen-binding fragment disclosed herein, compared to before administration of the antibody or its antigen-binding fragment.

[0551] In some respects, subjects showed improved fertility after administration of the anti-CRH antibody or its antigen-binding fragment disclosed herein, compared with those before administration of the antibody or its antigen-binding fragment.

[0552] In some respects, subjects showed improved insulin resistance after administration of the anti-CRH antibody or its antigen-binding fragment disclosed herein, compared with those before administration of the antibody or its antigen-binding fragment.

[0553] In some respects, subjects showed improvement in hyperinsulinemia after administration of the anti-CRH antibody or its antigen-binding fragment disclosed herein, compared with those before administration of the antibody or its antigen-binding fragment.

[0554] In some respects, subjects showed improvement in hypertension after administration of the anti-CRH antibody or its antigen-binding fragment disclosed herein, compared with those before administration of the antibody or its antigen-binding fragment.

[0555] In some respects, subjects showed improvement in hyperlipidemia after administration of the anti-CRH antibody or its antigen-binding fragment disclosed herein compared to before administration of the antibody or its antigen-binding fragment.

[0556] In some respects, subjects showed excessive remission of hair growth after administration of the anti-CRH antibody or its antigen-binding fragment disclosed herein, compared with those before administration of the antibody or its antigen-binding fragment.

[0557] In some respects, subjects showed remission of hirsutism after administration of the anti-CRH antibody or its antigen-binding fragment disclosed herein, compared with those before administration of the antibody or its antigen-binding fragment.

[0558] In some respects, subjects showed a reduction in hair loss (e.g., male pattern baldness) after administration of the antiCRH antibody or its antigen-binding fragment disclosed herein compared to before administration of the antibody or its antigen-binding fragment.

[0559] In some respects, subjects showed a reduction in acne after administration of the anti-CRH antibody or its antigen-binding fragment disclosed herein, compared with those before administration of the antibody or its antigen-binding fragment.

[0560] In some aspects of any of the methods provided herein, the anti-CRH antibody or its antigen-binding fragment disclosed herein is administered in combination with one or more PCOS therapies. In some aspects, the one or more PCOS therapies are selected from combined contraception (e.g., oral contraceptives containing estrogen and progestin); progestin therapy; clomiphene; tamoxifen; aromatase inhibitors (e.g., letrozole); gonadotropins; ovarian drilling; spironolactone; efornithine; electrolytes; and antidiabetic drugs (e.g., metformin). In some aspects, the anti-CRH antibody or its antigen-binding fragment disclosed herein is administered in combination with one or more PCOS therapies selected from spironolactone, metformin, and combined contraception (e.g., oral contraceptives containing estrogen and progestin).

[0561] In some respects, the subjects were human beings. In some respects, the subjects were women. In some respects, the women were women of childbearing age.

[0562] Sometimes it is necessary to detect the presence of CRH or measure its amount in a sample (e.g., a biological sample). In some aspects, this disclosure provides a method for measuring the amount of CRH in a sample from a subject diagnosed with or suspected of having PCOS using an anti-CRH antibody or an antigen-binding fragment thereof disclosed herein. To determine the measurement of CRH, a biological sample from the subject is contacted with an anti-CRH antibody or an antigen-binding fragment thereof disclosed herein for a time sufficient to allow for immune complex formation. The immune complex formed between the antibody and CRH in the sample is then detected. Optionally, the amount of CRH in the biological sample is quantified by measuring the amount of the immune complex formed between the antibody and CRH. For example, if the antibody has a detectable label, the antibody can be quantified, or a secondary antibody can be used to quantify the immune complex.

[0563] In some respects, biological samples include tissue samples, cell samples, or biological fluid samples, such as blood, saliva, serum, or plasma.

[0564] The conditions used to incubate the antibody with the test sample vary. The incubation conditions depend on the form used in the assay, the detection method used, and the type and nature of the antibody used in the assay. Those skilled in the art will recognize that any of the commonly available forms of immunoassays can be readily adapted to use the antibodies (or fragments thereof) disclosed herein. Examples of such assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock et al., Techniques in Immunocytochemistry, Academic Press, Orlando, FL, Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, Practice and Theory of Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985). The test samples used in the above methods will vary depending on the form of measurement, the nature of the detection method, and the tissue, cells, or fluid used as the test sample.

[0565] Such methods can be used, for example, to evaluate the efficacy of specific treatment regimens in animal studies and clinical trials, or to monitor the treatment of individual patients.

[0566] In some aspects, a CRH or anti-CRH antibody or its antigen-binding fragment is attached to a solid support, and binding is detected by detecting the complex between the CRH and the anti-CRH antibody or its antigen-binding fragment on the solid support. The anti-CRH antibody or its antigen-binding fragment optionally contains a detectable label, and binding is detected by detecting the label in the CRH-antibody complex.

[0567] The detection of the presence or absence of a CRH-antibody complex can be performed using any method known in the art. For example, transcripts produced by a reporter gene transcription assay of a CRH peptide interacting with a target molecule (e.g., an antibody) typically encode directly or indirectly detectable products (e.g., β-galactosidase activity and luciferase activity). For cell-free binding assays, one of the components typically includes or is coupled to a detectable label. A variety of labels can be used, such as those providing direct detection (e.g., radioactivity, luminescence, optical, or electron density) or indirect detection (e.g., epitope labels such as FLAG epitopes, enzyme labels such as horseradish peroxidase). The label can bind to the antibody or be integrated into the antibody's structure.

[0568] Depending on the properties of the label and other analytes, a variety of methods can be used to detect the label. For example, the label can be detected either when bound to a solid matrix or after separation from the solid matrix. The label can be detected directly by optical or electron density, radioactive emission, nonradiative energy transfer, or indirectly by antibody conjugates or streptavidin-biotin conjugates. Methods for detecting the label are well known in the art.

[0569] 4. Reagent kit

[0570] In some respects, this disclosure provides a kit comprising the anti-CRH antibody or its antigen-binding fragment disclosed herein.

[0571] In some respects, the kit contains (i) the anti-CRH antibody or antigen-binding fragment disclosed herein, and (ii) instructions for treating PCOS in a subject in need of the anti-CRH antibody or antigen-binding fragment.

[0572] In some respects, the kit contains (i) an anti-CRH antibody or antigen-binding fragment disclosed herein, and (ii) instructions for using the anti-CRH antibody or antigen-binding fragment to reduce androgen concentrations (e.g., testosterone, free testosterone, androstenedione, 11-oxyandrogens, or combinations thereof) in subjects with PCOS. In another respect, the 11-oxyandrogens are 11-hydroxyandrostenedione (11OHA4), 11-hydroxytestosterone (11OHT), 11-ketoandrostenedione (11KA4), 11-ketotestosterone (11KT), or combinations thereof.

[0573] In some respects, the kit contains (i) an anti-CRH antibody or antigen-binding fragment disclosed herein, and (ii) instructions for using the anti-CRH antibody or antigen-binding fragment to reduce the severity of one or more symptoms or signs in a subject with PCOS.

[0574] In some respects, the anti-CRH antibody or antigen-binding fragment in the kit is present in the pharmaceutical composition disclosed herein.

[0575] In some aspects, anti-CRH antibodies, antigen-binding fragments, or pharmaceutical compositions are stored in containers, such as sterile vials, in the form of solutions, suspensions, gels, emulsions, solids, crystals, or dehydrated or lyophilized powders. Such aspects may be provided in ready-to-use form or in a form reconstituted before application (e.g., lyophilized). In some aspects, the kit includes a first container containing an anti-CRH antibody, antigen-binding fragment, or pharmaceutical composition in dehydrated or lyophilized powder, and a second container containing an aqueous formulation. In some aspects, the kit includes a pre-filled syringe containing an anti-CRH antibody, antigen-binding fragment, or pharmaceutical composition.

[0576] Example

[0577] The following embodiments are provided to further illustrate various aspects of this disclosure, and it is not intended to limit this disclosure to any particular application or operational theory.

[0578] Example 1

[0579] H2L2 Harbour Production of CRH gene knockout mice in the context

[0580] Using the standard transgenic method described in Nagy et al., Cold Spring Harbor Laboratory Press's laboratory manual "Manipulating the Mouse Embryo," in H2L2 Harbor... Against this background, CRH-releasing hormone (CRH) knockout (KO) mice were generated. The CRH KO H2L2 mice were generated in accordance with animal license ADV101002016512, animal protocol 16-512-11, which is a scientific procedure approved by the Central Netherlands Authority for use on animals in (CCD) and EDC animal facilities.

[0581] In short, superovulatory H2L2 Harbour Females and males were used for mating. Collected fertilized eggs were microinjected at 20 ng / µL into a microinjection buffer of 5 mM tris(hydroxymethyl)aminomethane (TRIS) and 0.1 mM ethylenediaminetetraacetic acid (EDTA) in ultrapure water adjusted to pH 7.4 with two guide RNAs (gRNAs): gRNA1 Mm.Cas9.CRH.1.AA and gRNA2 Mm.Cas9.CRH.1.AB, along with the CAS9 protein. The following gRNA1 sequence was used:

[0582] gRNA1=Sequence-Mm.Cas9CHR.1.AA:

[0583] 5'-AltR1 / rArArC rUrCrC rArCrG rCrCrC rCrUrC rArCrC rGrCrG rUrUrUrUrArG rArGrC rUrArU rGrCrU / altR2 / -3'

[0584] gRNA2=Mm.Cas9.CRH.1.AB:

[0585] 5'-AltR1 / rUrCrA rCrCrC rArUrG rCrGrG rArUrC rArGrArArCrG rUrUrUrUrArG rArGrC rUrArU rGrCrU / altR2 / -3'

[0586] Guide RNA and CAS9 were obtained from Integrated DNA Technologies (IDT).

[0587] Injected oocytes were transferred into B6CBAF1 mice. Knockout pups were identified from toeDNA by amplifying DNA across a region on the gRNA. Polymerase chain reaction (PCR) fragments smaller than these expected sizes indicated deletion in the founder mice. PCR primers CRHbp70F and CRHbp215R, flanking gRNA1, were used to extract oocytes from wild-type H2L2 Harbour mice. The expected size of the amplified 146bp ( Figure 2A (Left figure). PCR primers CRHbp208F and CRHbp310R were used to PCR extract wild-type H2L2 Harbour The expected size of the amplified 103bp ( Figure 2A (middle image). PCR primers CRHbp70F and CRHbp310R produce a PCR fragment of 241 bp in size. Figure 2A (See right figure). PCR products smaller than these expected sizes indicate a mutation. Founder mice 1, 2, 4, and 5 were identified as carrying the mutation. Further analysis of PCR products deviating from the wild-type expected size was performed by cutting the PCR amplification products from the agarose gel, isolating the DNA, and sequencing, using the same primers used for amplification. (Source: [Insert source here]) Figure 2B The founder mouse 5 of the nest 12198 carried an 11bp deletion (5'-CAGCCGGTTCT-3') (SEQ ID NO:465), which was used for subsequent breeding.

[0588] CRH knockout of H2L2 Harbour The rescue

[0589] Rescue and immunization of CRH knockout mice were performed according to Animal Permit ADV101002016512 and Animal Working Protocol 16-512-19. In short, homozygous CRH knockout H2L2 Harbour mice were... Males and females were placed in cages one-to-one. Females were removed from the cages and separated by observing the mating plug (0.5 dpc). Ten days later, pregnant females were randomly given cortisol (30 μg / ml) in drinking water (dark bottle). This resulted in the birth of live knockout offspring for immunization.

[0590] CRH knockout immunization H2L2 Harbour

[0591] On days 0, 14, 28, 42, and 50, ten homozygous CRH knockout H2L2 Harbour mice of approximately 8 weeks of age were immunized subcutaneously (SC) and intraperitoneally (IP) with a maximum volume of 100 μl of the CRH-KLH conjugate. .

[0592] Use the following conjugates:

[0593] CRH-KLH: SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII-KLH (C-terminal) (GenScript) (SEQ ID NO: 467)

[0594] CRH-KLH conjugates were mixed with the adjuvant Stimmune for the first injection and with the adjuvant Ribi for subsequent boosters. The final booster was an IP (injection). Blood samples were collected after the fourth injection and at the end of the successful immunization experiment. Three to five days after the final IP injection, mice were euthanized, and the spleen and lymph nodes were used for fusion experiments with myeloma fusion partners to generate hybridomas, according to a standard protocol. Serum anti-CRH antibody titers were screened by enzyme-linked immunosorbent assay (ELISA) using biotinylated CRH captured on streptavidin (SA)-coated plates, followed by hybridoma screening. The detailed protocol is as follows.

[0595] CRH ELISA

[0596] Coat ELISA plates overnight at 4°C with 5 μg / ml streptavidin in phosphate-buffered saline (PBS) (Sigma 189730-1 mg) or at room temperature (RT) for 2 hours. Use 50 μl / well for 96-well plates and a smaller amount for 384-well plates. After incubation, remove the streptavidin solution and add a biotinylated peptide (ThermoFisher, with biotin at the N-terminus). After incubating at RT for 30 minutes, wash the plates with 1X phosphate-buffered saline (PBS) / 0.1% Tween-20 and block with 1% milk / 1% bovine serum albumin (BSA) in the wash solution. After washing with 3X PBS / 0.1% Tween-20, add 50 μl of antibody to the blocking buffer and incubate at RT for 2 hours. The plates were then washed with 3X PBS / 0.1% Tween-20, and 50 μl of anti-rat IgG-HRP (horseradish peroxidase) diluted 1:2,000 was added. The plates were incubated at RT for 2 hours. After washing with 3X PBS / 0.1% Tween-20, 50 μl of peroxidase substrate (POD or equivalent) was added, and the absorbance was measured at 450 nm.

[0597] Immunity in Balb / c wild-type mice

[0598] Balb / c wild-type mice were immunized with CRH-KLH protein to generate anti-CRH antibodies. The CRH and KLH conjugates were either N-terminal conjugated CRH (peptide 1: KLH-CRH) or C-terminal conjugated CRH (peptide 2: CRH-KLH), via an additional cysteine ​​(C) at the N-terminus or C-terminus of the CRH, respectively. Each mouse underwent a first perfusion of 50 μg of protein via IP with complete Freund's adjuvant (CFA, Sigma, catalog number F5881) and subsequent boosters of 25 μg of protein via IP with Ribi (Sigma S6322). Boosters were administered every two weeks for a total of four times. At designated time points, mouse serum was sampled and titrated by indirect ELISA to test for binding to human CRH.

[0599] Mice with high serum titers and a specific immune response to human CRH were selected and injected with 25 μg of protein in PBS solution to enhance immunity. Three days later, the mice were sacrificed, and their spleen cells and lymph node cells were collected.

[0600] Preparation of CRH-KLH conjugates

[0601] Weigh the KLH carrier protein (Sigma, H7017-50MG) and dialyze it against 0.1M sodium phosphate buffer at pH 7.5. Add Sulfo-MBS (BBI, C100314-0050) solution to the KLH carrier protein solution at a ratio of 1:10. After incubating at room temperature for one hour, apply the reaction mixture to a G-50 desalting column (Sigma, G50150-10G) pre-equilibrated with conjugation buffer (0.1M sodium phosphate buffer, pH 6.5, containing 1mM EDTA). Collect the fraction corresponding to the first protein peak based on A280 nm. Mix the KLH carrier protein-MBS and CRH peptide with an additional cysteine ​​residue at the N-terminus or C-terminus and incubate at room temperature for 2 to 4 hours. Terminate the reaction by adding 2-mercaptoethanol (Sigma, M3148-25ML) to a final concentration of 10mM and incubate at room temperature for more than 30 minutes. The CRH-KLH conjugate was purified using a Sephadex G-25 desalting column to separate unconjugated peptides from the CRH-KLH conjugate. Fractions corresponding to the first protein peak were collected. The protein concentration of the CRH-KLH conjugate was determined using a NanoPhotometer.

[0602] The following peptides were used to immunize Balb / c wild-type mice:

[0603] Peptide 1 (KLH-CRH) (SEQ ID NO:468):

[0604] KLH-C-SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII-CONH2

[0605] Peptide 2 (CRH-KLH) (SEQ ID NO:469):

[0606] SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII-C-KLH

[0607] Furthermore, the following peptides are used in ELISA:

[0608] Peptide 3 (Biotin-CRH) (SEQ ID NO:470):

[0609] Biotin-K-SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII-CONH2

[0610] Peptide 4 (CRH-Biotin) (SEQ ID NO:471):

[0611] SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII-K-Biotin

[0612] Biotin-CRH (SEQ ID NO:472):

[0613] Biotin-SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII-COOH (ThermoFisher)

[0614] based on Optofluidic system for single B-cell screening

[0615] Use around Beacon TM The nanofluidic photoelectric B lymphocyte antibody screening technology (NanOBlast) was established, which is a commercially available integrated culture and imaging platform (Berkeley Lights).

[0616] The NanOBlast workflow begins with antigen-specific antibody-secreting cells (ASCs) generated via in vivo immunization. Selectively enriched, antigen-experienced mouse ASCs are harvested from the spleen and lymph nodes. ASCs are microfluidically introduced into the chip and isolated into individual nanopens for screening via opto-localization (OEP). ASCs secreting antigen-specific IgG are detected using a bead-based color fluorescence binding assay initiated by characteristic fluorescence. The selected single cells are then released using OEP and exported directly from the chip to 96-well plates containing cell lysis buffer. The antibody heavy chain variable domain (VH) and light chain variable domain (VL) sequences are recovered using rapid single-cell amplification (RACE) of cDNA ends, cloned, and recombinantly expressed as canonical antibodies using standard methods. The recombinant antibodies are used for binding confirmation and final validation in relevant downstream assays.

[0617] Antibody production and purification

[0618] Recombinant plasmids encoding the target antibody were transiently co-transfected into HEK293-6E or ExpiCHO cell cultures using polyethyleneimine (PEI) (Polyscience, 24885). Cells were seeded in Corning Erlenmeyer flasks the day before transfection. On the day of transfection, the recombinant plasmid encoding the target protein was mixed with the transfection reagent at the optimal ratio (heavy chain:light chain = 2:3) and added to the seeded flasks for transfection. Cell culture supernatant was collected on day 6 for purification. The cell culture medium was centrifuged and then filtered. The filtered cell culture supernatant was loaded onto an affinity purification column. After washing and elution, the eluted fractions were combined, and the buffer was replaced with stock buffer. The purified protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) to determine molecular weight and purity. Concentration was determined by absorbance at 280 nm.

[0619] Antibody engineering

[0620] The heavy chain variable domain (VH) and light chain variable domain (VL) sequences of the anti-CRH chimeric antibody were further optimized through humanization and post-translational modification (PTM) removal procedures. In the humanization process, the VH and VL sequences were first aligned to the nearest human germline sequence using algorithms such as NCBI / Ig-BLAST, and then positions within the frame region were restored to human germline sequences. All critical reversion mutation sites were scanned using Chothia numbering. If mismatches were found, they were replaced with mouse counterpart residues. The humanized antibody composed of sequence variants was then recombined using standard molecular biology techniques.

[0621] To remove PTM, VH and VL sequences are scanned to determine the presence of PTM motifs, such as isomerized motifs (e.g., DG). "Hotspot" residues (e.g., D or G in the DG motif) are mutated to their germline counterparts or other residues with similar biophysical properties. Antibodies containing the sequence variants after PTM removal are then recombined using standard molecular biology techniques.

[0622] Anti-CRH antibodies are bound to CRH or human UCN1 via ELISA.

[0623] Streptavidin (Thermo, catalog number: 21125) was diluted in PBS to a concentration of 2 μg / mL. 100 μl of the diluted streptavidin was added to each well of an ELISA microplate, and the plates were incubated overnight at 4°C. The plates were blocked with ELISA blocking buffer (containing 2% w / v BSA, 0.05% (v / v) Tween-20, pH 7.4 PBS buffer) at 37°C for 1 hour, and then incubated with 0.5 μg / mL CRH (NT Biotin), 0.5 μg / mL CRH (CT Biotin), 1 μg / mL human UCN1 (NT Biotin), or 1 μg / mL human UCN1 (CT Biotin) at 37°C for 1 hour. The plates were then washed and incubated with 15 μg / mL (100 nM, 10-fold dilution, 8 spots) of anti-CRH antibody (Ab) at 37°C for 1 hour. Subsequently, the plates were washed and incubated with HRP-conjugated goat anti-human IgG (H+L) antibody (Jackson, catalog number: 109-035-088) at 37°C for 1 hour. 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Biopanda, catalog number: TMB-S-003) was added, and the plates were incubated at room temperature for 15 minutes. 100 μL of ELISA stop solution (Solarbio, catalog number: C1058) was added to terminate the reaction, and the optical density (OD450nm) at 450 nm was measured using an ELISA reader (Molecular Devices, Spectra max 384plus).

[0624] Preparation of plasmids and cell lines

[0625] The CHO-K1 cell line (catalog number CCL-61) was obtained from the American Type Culture Collection (ATCC), and the pGL4.29 / luc2P / CRE / Hygro assay (catalog number E8471) and Bio-Glo luciferase assay system (catalog number G7940) were obtained from Promega. The following plasmids were synthesized: pcDNA3.1-human CRHR1 (clone ID: OHu27188C), pcDNA3.1-human CRHR2 (clone ID: OHu10803), pcDNA3.1-mouse CRHR1 (clone ID: OMu00753), and pcDNA3.1-mouse CRHR2 (clone ID: OMu23246), all of which were G418 resistant. The human CRH peptide was obtained from Peptide International (catalog number 4136-s). Fossiline (catalog number 1099) and the human CRHR1 antagonist small molecule (Antalarmin, catalog number 2778) were obtained from Tocris. The anti-human CRHR1 antibody (catalog number MAB3930) was obtained from R&D System. Electroporation was performed using an Electro Cell Manipulator (catalog number ECM630) and electroporation cuvettes plus (2 mm gap cuvettes, catalog number 45-0125) from BTX. Electroporation buffer (Ingenio Solution, catalog number MIR50111) was obtained from Mirus, and sheep anti-human CRH polyclonal antibody was obtained from Salk Institute.

[0626] CHO-CRE-luciferase-human CRHR1,-human CRHR2,-mouse CRHR1 and-mouse CRHR2 reporter gene cells The emergence of systems

[0627] To generate a stable CHO-CRE-luciferase reporter gene cell line, 2 million CHO-K1 cells were transferred to an electroporation cuvette with a mixture of 10 μg pGL4.29 / luc2P / CRE / Hygro plasmid in 100 μl Ingenio solution and pulsed once or twice using an Electro Cell Manipulator (950 uF and 120 V). The treated cells were then transferred to 6-well plates containing 2 mL of Dulbecco Modified Eagle Medium (DMEM) / F12 with 10% fetal bovine serum (FBS). After 24 hours, 500 μg / mL of the selective antibiotic hygromycin was added to the cells. This selection process took approximately 10 to 14 days. The CHO-CRE cell pool was subcloned into 96-well plates using matrix dilutions. Individual stable clones were harvested when the selected colonies became apparent. Positive clones were validated using a series of diluted forscoringin. Validated positive clones were frozen for future use.

[0628] To generate CHO-CRE cell lines reporting the hCRHR1, hCRHR2, mCRHR1, and mCRHR2 gene signatures, pcDNA3.1-hCRHR1, hCRHR2, mCRHR1, and mCRHR2 were introduced into the established CHO-CRE cells via electroporation as described above. Stable cell lines were characterized individually using 500 μg / ml of the selective antibiotic hygromycin and 1000 μg / ml of G418. Both were stimulated with CRH and forscorin to characterize the individual stable cell lines.

[0629] Development of luciferase reporter gene assay

[0630] Forscorin stimulates adenylate cyclase in cells and increases intracellular cyclic adenosine monoester (cAMP) levels. To verify whether cells respond to increased cAMP, CHO-CRE cells were stimulated with a series of concentrations of forscorin, and cAMP-dependent luciferase expression was measured using the Bio-Glo luciferase assay system. CHO-CRE cells responded to forscorin stimulation in a dose-dependent manner, increasing luciferase expression (data not shown). CRH binds to CRHR1 and CRHR2, which are Gαs-coupled G protein-coupled receptors (GPCRs).

[0631] Activated GPCR proteins increase intracellular cAMP production. Stable clone 11 of CHO-CRE-human CRHR1 was generated by introducing pcDNA3.1-human CRHR1 under G418 and hygromycin selection. Human CRHR1 expression in CHO-CRE-hCRHR1 clone 11 was confirmed by flow cytometry (data not shown). To further validate the cell line's functionality, CHO-CRE-hCRHR1 clone 11 cells were treated with forscolin and CRH peptide. CHO-CRE-hCRHR1 clone 11 cells showed a significant increase in luciferase expression after CRH treatment compared to the parental CHO-CRE, while both cell lines showed comparable levels of intracellular cAMP in response to forscolin treatment. Subsequently, CHO-CRE-hCRHR2 clone 32, -mCRHR1 clone 22, and -mCRHR2 clone 8 cell lines were established using a similar approach and validated with CRH treatment (data not shown).

[0632] If the CRH-CRHR1 system functions in CHO-CRE-hCRHR1 cells, blocking CRHR1 with small molecules or neutralizing CRH peptides can reduce luciferase expression. Antalarmin, a small molecule CRHR1 antagonist, blocked CRH-mediated luciferase expression in CHO-CRE-hCRHR1 clone 11 (data not shown). Furthermore, sheep anti-CRH polyclonal antibodies reduced CRH-mediated luciferase expression in these cells (data not shown). Similarly, sheep anti-CRH polyclonal antibodies blocked CRH-mediated luciferase expression in CHO-CRE-hCRHR2, CHO-CRE-mCRHR1, and CHO-CRE-mCRHR2 cells (data not shown).

[0633] use Determining affinity

[0634] Red 96e is used for affinity determination. The run buffer was prepared by diluting kinetic buffer 10X (Fortebio, catalog 18-1105) in PBS according to the manufacturer's instructions. Two SA sensors (Fortebio, catalog 18-5019) were hydrated in the run buffer for 10 min, one for ligand capture and the other for reference. The antibody was diluted 2-fold and added to 96-well plates. 30 nM biotinylated CRH (GL Biochem, catalog 784751) was captured by the first SA sensor for 20 s, while the reference sensor was immersed in the buffer wells. After a 60-second baseline step, the sensors were successively immersed in the analyte and buffer wells for binding and dissociation times of 180 s and 800 s, respectively. Before the next cycle, the sensors were regenerated in glycine pH 1.5 (Cytiva, catalog BR-1003-54). The equilibrium dissociation constant (KD) value was evaluated using data analysis software 11.0 and a 1:1 global fit model. Before data fitting, subtract the signals from the reference aperture and the reference sensor by selecting "Dual Reference".

[0635] CRH-induced signaling in reporter gene cell lines (CHO-CRE-hCRHR1, CHO-CRE-hCRHR2, CHO- Blocking activity of CRE-mCRHR1 and CHO-CRE-mCRHR2

[0636] Cells were digested with trypsin-EDTA (Life Technologies, catalog number: 12604-013), centrifuged at 1,000 rpm for 5 minutes, resuspended in growth medium, counted using a cell counter, and seeded at a concentration of 5,000 cells / 100 μl in 96-well plates and incubated overnight. 50 μl of serially diluted 4X antibody (8 spots, 1 / 4 diluted with growth medium) was added to a plate containing CHO-CRE-hCRHR1 cells, along with 50 μl of 4X CRH (4X at 2 nM, final concentration 0.5 nM). Similarly, 4X CRH (4X at 0.4 nM, final concentration 0.1 nM) was added to CHO-CRE-hCRHR2 and CHO-CRE-mCRHR1 cells, and 4X CRH (4X at 20 nM, final concentration 5 nM) was added to CHO-CRE-mCRHR2 cells. Next, incubate the plates in a CO2 incubator at 37°C for 4 to 6 hours. Remove 60 μl of culture medium, add 60 μl of Bio-Glo luciferase, and then incubate at RT for 10 minutes. Read the plates using an EnVision (PerkinElmer) luciferase reader.

[0637] Human UCN1, Human UCN2 and Human UCN3 ELISA

[0638] Streptavidin (Thermo, catalog number: 21125) was diluted in PBS to a concentration of 2 μg / mL. 100 μl of the diluted streptavidin was added to each well of an ELISA microplate, and the plates were incubated overnight at 4°C. The plates were blocked with ELISA blocking buffer (containing 2% w / v BSA, 0.05% (v / v) Tween-20, pH 7.4 PBS buffer) at 37°C for 1 hour, and then incubated with 1 μg / mL hUCN1 (NT biotin), hUCN1 (CT biotin), hUCN2 (NT biotin), hUCN2 (CT biotin), hUCN3 (NT biotin), or hUCN3 (CT biotin) at 37°C for 1 hour. The plates were then washed and incubated with 100 nM and 10 nM anti-CRH antibody at 37°C for 1 hour. The plates were then washed and incubated with HRP-conjugated goat anti-human IgG (H+L) antibody (Jackson, catalog number: 109-035-088) at 37°C for 1 hour. 100 μL of TMB substrate (Biopanda, catalog number: TMB-S-003) was added, and the plates were incubated at room temperature for 15 minutes. Then, 100 μL of ELISA stop solution (Solarbio, catalog number: C1058) was added to terminate the reaction, and the OD450 nm was measured using an ELISA reader (Molecular Devices, Spectra max 384plus).

[0639] Assay of human UCN1-induced luciferase reporter gene

[0640] CHO-CRE-hCRHR1 and CHO-CRE-hCRHR2 cells were harvested using trypsin-EDTA (Life Technologies, catalog number: 12604-013). Cells were centrifuged at 1,000 rpm for 5 minutes and resuspended in growth medium. Cells were counted using a cell counter and seeded into 96-well plates at 100 μL per well, and incubated overnight. 50 μL of serially diluted 4X antibody (8 spots, 1 / 3 diluted with growth medium) or CP 376395 hydrochloride (TOCRIS, catalog number: 3212) (8 spots, 1 / 10 diluted with growth medium) was added to the plates, followed by 50 μL of 4X hUCN1 (4X is 4 nM, final concentration is 1 nM) to either the CHO-CRE-hCRHR1 or CHO-CRE-hCRHR2 cells. The plates were then incubated in a CO2 incubator at 37°C for 4 to 6 hours. Then, remove 60 μl of culture medium, add 60 μl of Bio-Glo luciferase, and incubate at RT for 10 minutes. Then, read the plate using an EnVision (PerkinElmer) luciferase reader.

[0641] In vivo efficacy models - wild-type female C57BL / 6 mice and Mrap1 KO mice

[0642] Wild-type female C57BL / 6 mice aged 8 to 10 weeks were obtained from Charles River Laboratories. Mrap1 KO mice were obtained from Queen Mary University of London, UK. SSR125543A, a small molecule CRHR1 antagonist, was obtained from Axon Medchem (catalog number Axon 1799). Mouse corticosterone (55-Corms-E01) and adrenocorticotropic hormone (ACTH) ELISA kits (21-ACTHU-E01) were obtained from ALPCO, USA. All procedures were performed in accordance with the National Institutes of Health's guidelines for laboratory animal care and use. The protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Charles River Laboratories.

[0643] Mrap1 KO mouse breeding, ear markers, and genotyping

[0644] To generate Mrap1 KO mice, Mrap1- / - male mice were mated with Mrap1+ / - female mice. When the female mice became pregnant and their pregnancy was palpable (approximately day 10 of embryonic development), corticosteroid replacement was administered to their cages at a dose of 50 μg / ml of drinking water. The drinking water was changed weekly, and treatment was discontinued at weaning. At tail clipping, the mice were uniquely identified by ear tags to match the future genotype of each mouse. DNA was extracted from the tail tissue and used for mouse genotyping.

[0645] Genotyping of Mrap1- / - mice was performed using PCR and the following PCR primers:

[0646] For the wild-type allele of exon 1 of Mrap1:

[0647] Positive 5'-GCGTCTCTTAGTAGCCTTTGG (SEQ ID NO:473)

[0648] Inverse 5'-CTTGTTGGCTTTCAGCTTCT (SEQ ID NO:474)

[0649] For the mutant alleles of Mrap1:

[0650] Positive 5'-GAACTTGCCTGGTCAACTGTTAAAAGGAC (SEQ ID NO:475)

[0651] Reverse 5'-TCTTTAGGCTCACTTCCTCCACTGACC (SEQ ID NO:476)

[0652] PCR band size: wild-type allele is 345 bp, mutant allele is 459 bp.

[0653] Body restraint in wild-type mice

[0654] To determine the effects of non-painful physical restraint on stress responses, mice were restrained in a 50 ml polypropylene ventilated tube, the tip of which was cut off with a razor blade to allow the mice to breathe easily for 15 to 20 minutes. The mice were led to the open tube and climbed inside. A paper towel was placed behind the mouse to prevent it from exiting, and the cap was screwed on. The mice were then returned to their cages, placed belly-down on bedding. This stressful stimulus was not considered painful because the mice were confined to a limited space (within the ventilated restraint tube) but not in an unnatural body position. The researchers were present in the room throughout the process. At the end of the procedure, the mice were removed by their tails.

[0655] The role of CRH Ab in a constraint stress model in wild-type mice

[0656] On day 1, each mouse was intraperitoneally injected with 20 mg / kg anti-CRH mAb, hIgG1, or PBS (5 mice per group). On days 2 or 3 and day 14, each mouse was subjected to the above-described physical restraint stress for 15 or 20 minutes. Immediately after the restraint stress, 200 μl of blood was collected from the submandibular vein of each mouse into an EDTA tube. Plasma was separated using a refrigerated centrifuge (4°C, 500 x g, 10 min) and rapidly frozen on dry ice for subsequent ACTH and corticosterone ELISA measurements.

[0657] The role of anti-CRH Ab in the Mrap1 KO mouse model

[0658] On day 1, each mouse was injected (IP) with 20 mg / kg anti-CRH Ab or hIgG1 (n=5 or 6 per group). On days 2 or 3, 14, and 28, 200 μl of blood was collected from the submandibular vein of each mouse into EDTA tubes. Plasma was separated using a refrigerated centrifuge (4°C, 500 x g, 10 min) and rapidly frozen on dry ice for subsequent ACTH ELISA measurements.

[0659] Parallel comparison of anti-CRH mAb and SSR125543A in a wild-type mouse constraint stress model

[0660] On day 1, half of the mice were injected with 20 mg / kg anti-CRH Ab or hIgG1 via intraperitoneal injection (IP) (n=5 mice per group). On day 3, the other half of the mice were injected with 30 mg / kg SSR125543A or a solvent via IP (n=5 mice per group). Two hours later, all four groups of mice were subjected to restraint stress for 20 minutes as described above. Immediately after the restraint stress, 200 μl of blood was collected from the submandibular vein of each mouse in all four groups into EDTA tubes. Plasma was separated as described above and rapidly frozen in dry ice for subsequent ACTH and corticosterone ELISA measurements. On day 4, mice treated with SSR125543A or the solvent underwent a second round of restraint stress for 20 minutes, and plasma was collected. Two weeks later, mice treated with anti-CRH mAb or hIgG1 underwent somatic stress for 20 minutes, followed by plasma collection.

[0661] In vivo PK research

[0662] Application and blood collection

[0663] For each antibody test, six female C57BL / 6 mice, weighing 18 to 22 grams, were selected and administered the antibody intravenously in a single dose of 5 mg / kg. Whole blood was collected from three mice before administration and at 15 minutes, 24 hours (1 day), 4 days, and 10 days after administration. Whole blood was collected from the remaining three mice before administration and at 5 hours, 2 days, 7 days, and 14 days after administration. The whole blood was allowed to stand for 30 minutes to coagulate, and then centrifuged. The separated serum was then frozen at -80°C until analysis.

[0664] Analytical methods

[0665] ELISA was used to quantify drug concentrations in mouse serum. Test antibodies (containing human Fc) in mouse serum were captured by pre-coated goat anti-human Fc polyclonal antibody (Rockland, #609-101-017) on a 96-well plate. HRP-labeled goat anti-human (H+L) secondary antibody (Abcam, #ab97175) was then added to the wells. The plates were then sealed and incubated at 37°C for 30 minutes, followed by washing and the addition of TMB substrate and ELISA stop solution. The 96-well plates were read at 450 / 630 nm using a SpectraMax M2. Data were processed using SoftMax Pro.

[0666] Pharmacokinetic (PK) parameters were analyzed using a non-compartmental (NCA) model with Phoenix WinNonlin version 8.2.

[0667] The binding region between CRH and anti-CRH antibody was determined by ELISA.

[0668] Streptavidin (Thermo, catalog number: 21125) was diluted in PBS to a concentration of 2 μg / ml. 100 μl of the diluted streptavidin was added to each well of an ELISA microplate, and the plates were incubated overnight at 4°C. The plates were blocked with ELISA blocking buffer (containing 2% w / v BSA, 0.05% (v / v) Tween-20, pH 7.4 PBS buffer) at 37°C for 1 hour, then incubated at 37°C for 1 hour with 1 μg / mL CRH N-terminal region plus a central region (CT biotin), a central region only (NT biotin or CT biotin), or a central region plus a C-terminal region (NT biotin). The plates were then washed and incubated at 37°C for 1 hour with 15 μg / ml (100 nM, 10-fold dilution, 8 spots) of anti-CRH antibody (Ab). Subsequently, the plates were washed and incubated with HRP-conjugated goat anti-human IgG (H+L) antibody (Jackson, catalog number: 109-035-088) at 37°C for 1 hour. 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Biopanda, catalog number: TMB-S-003) was added, and the plates were incubated at room temperature for 15 minutes. 100 μL of ELISA stop solution (Solarbio, catalog number: C1058) was added to terminate the reaction, and the optical density (OD450nm) at 450 nm was measured using an ELISA reader (Molecular Devices, Spectra max 384plus).

[0669] Epitope binding via competitive ELISA

[0670] For epitope binding, a competitive ELISA was performed. Specifically, 1 μg / ml capture antibody (100 μl / well) was coated onto a 96-well plate overnight at 4°C. The plates were blocked for 1 hour at 37°C with ELISA blocking buffer (containing 2% w / v BSA, 0.05% (v / v) Tween-20, pH 7.4 PBS buffer). Then, a mixture of 50 μl / well of an appropriate concentration of competitive antibody (40 μg / ml) and 50 μl / well of biotin-labeled CRH was added to the 96-well plate, followed by incubation at 37°C for 1 hour. Subsequently, the plates were washed and incubated at 37°C for 1 hour with streptavidin-peroxidase (SIGMA, catalog number: S2438-250UG). Add 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Biopanda, catalog number: TMB-S-003) and incubate the plate at room temperature for 15 minutes. Add 100 μL of ELISA stop solution (Solarbio, catalog number: C1058) to terminate the reaction, and measure the optical density (OD450nm) at 450 nm using an ELISA reader (Molecular Devices, Spectra max 384plus).

[0671] use epitope binding

[0672] Epitope binding in The procedure was performed using Red 96e. The run buffer was prepared by diluting kinetic buffer 10x (Fortebio, catalog number 18-1105) in PBS according to the manufacturer's instructions.

[0673] The SA sensor (Fortebio, catalog number 18-5019) was hydrated in run buffer for 10 minutes. The tandem epitope binding assay consisted of a four-step binding cycle: 1) a setup regeneration step of 30 seconds; 2) CRH capture at 30 nM with a loading threshold of 0.12 nm; 3) loading with 60 nM antibody (the first Ab) to saturate the immobilized peptide for 200 seconds; 4) binding of the mixture of the first Ab and the competing antibody from step 3 for 200 seconds.

[0674] The data was processed using Fortebio data analysis software 11.0. After the output signal, the inhibition rate was calculated as follows:

[0675] Inhibition rate (%) = (ab) / a * 100%

[0676] Taking antibody Abn as an example

[0677] Variable a: The signal of Abn binding to the antigen alone (also refers to the 100% signal of Abn).

[0678] Variable b: When Abn is the second Ab, the signal indicating its binding to the antigen.

[0679] Example 2

[0680] H2L2 Harbour Generation of CRH knockout mice in the context

[0681] Using the above materials and methods, in H2L2 Harbour CRH knockout mice were generated in this context. Figure 1 The open reading frame of the coding sequence of mouse CRH (GenBank NM_205769) is shown, along with the CRISPR-targeted gRNA and PCR primers used. Specifically, genotyping, i.e., amplification of genomic DNA, was performed using the following primers: Figure 1 As shown:

[0682] CRHbp70F: 5'-GCC CTG CTC AGC AGG GGA TCC GT-3' (SEQ ID NO: 477)

[0683] CRHbp215R: 5'-CTG TTG AGA TTC CCC AGG CGG AG-3' (SEQ ID NO: 478)

[0684] CRHbp208F: 5'-CTC AAC AGA AGT CCC GCT CGG-3' (SEQ ID NO: 479)

[0685] CRHbp310R: 5'-GGA AAA AGT TAG CCG CAG CCT GG-3' (SEQ ID NO: 480)

[0686] The expected PCR DNA fragment sizes from wild-type mice were 146 bp and 103 bp, respectively. PCR fragments smaller than these expected sizes indicate deletions in the founder mice. Figures 2A to 2B The method was used to identify CRH knockout establisher mice.

[0687] Figure 3 shows the location of an 11 bp deletion (5'-CAGCCGGTTCT-3') (SEQ ID NO:465) in the coding region 157-167 of the CRH gene, which results in a downstream reading frame shift and a premature stop codon. This defective CRH open reading frame cannot synthesize the CRH peptide. Builders with this deletion are selected for further breeding to achieve homozygosity, rescue, and immunization.

[0688] Example 3

[0689] H2L2 Harbour Identification of anti-CRH mAb from CRH knockout mice in this context

[0690] In H2L2 Harbour Against this background, numerous positive hybridoma clones were identified from multiple rounds of hybridoma fusion by immunization with CRH peptides in CRH knockout mice. In ELISA, purified monoclonal antibodies (mAbs) from the hybridoma supernatant bound CRH with high affinity. However, in a human CRHR1 luciferase reporter gene assay, three functional monoclonal antibody clones, namely hybridoma clones 16g2, 28b3, and 52g7, were identified as having blocking activity. These three clones were recombinantly expressed with human IgG1 wild-type Fc as PR004289, PR004290, and PR006669, respectively. PR004289 (from hybridoma clone 16g2), PR004290 (from hybridoma clone 28b3), and PR006669 (from hybridoma clone 52g7) share the same germline sequences IGHV3-7 and IGKV2-28. PR004289 and PR004290 differ by one residue at position 28 of the IGHV, namely I28 in PR004289 and T28 in PR004290. Both PR004289 and PR004290 have a different complementarity-determining region (CDR) than PR006669. These three mAbs are designated as Series 1 (PR004289 and PR004290) and Series 2 (PR006669) mAbs. See Table 2.

[0691] Table 2. Identification of CRH mAb resistance

[0692]

[0693]

[0694] Example 4

[0695] Identification of anti-CRH mAb from wild-type mice

[0696] Using the materials and methods described above, over 200,000 single B cells from wild-type mice immunized with CRH-KLH conjugates were screened using the Beacon system. Over 500 monoclonal antibodies (mAbs) with binding activity were identified in the bead binding assay. Over 300 of these mAbs were recombinantly expressed with human IgG1 wild-type Fc. Many of these recombinantly expressed mAbs bound to CRH with high affinity in ELISA. Using the materials and methods described above, eight series of functional mAbs with blocking activity in the human CRHR1 luciferase reporter gene assay were identified. mAbs from different series had different germline sequences and different CDRs. mAbs within each series shared the same germline sequence but had different CDRs.

[0697] These mAbs are named as follows: Series 3 to Series 10.

[0698] Series 3 includes PR301306.

[0699] Series 4 includes PR301612.

[0700] Series 5 includes PR301777, PR301788, PR301790 and PR301803.

[0701] Series 6 includes PR301806.

[0702] Series 7 includes PR302309, PR302332 and PR302333.

[0703] Series 8 includes PR302315 and PR302335.

[0704] Series 9 includes PR302328, PR302331, PR302334 and PR302339.

[0705] Series 10 includes PR302312, PR302324 and PR302341.

[0706] See also Table 2 and Figures 4A to 4N .

[0707] Example 5

[0708] Antibody engineering

[0709] PTM removal of series 1mAb PR004290

[0710] PR004290 contains two potential post-translational modifications (PTMs): DG in the heavy chain CDR2 (HCDR2) and NS in the light chain CDR1 (LCDR1). PR006669 has one potential PTM with DG in HCDR2. These potential PTMs are mutated either alone in HCDR2, alone in LCDR1, or in various combinations of mutations, as detailed in Table 3. Their binding activity in CRH ELISA and their functional blocking activity in human CRHR1 (hCRHR1), human CRHR2 (hCRHR2), mouse CRHR1 (mCRHR1), and mouse CRHR2 (mCRHR2) luciferase reporter gene assays are shown in Table 3 and Figures 5A to 5L Most ΔPTM variants retain functional activity. In hCRHR1 reporter gene assays, the 50% inhibitory concentration (IC50) of PR005660 was approximately 10 nM, while that of the parental PR004290 was approximately 5 nM.

[0711] Table 3. CRH ELISA binding and functional blocking activities of series 1 mAb PR004290 and its ΔPTM variants

[0712]

[0713]

[0714] PTM removal of series 2 mAb PR006669

[0715] PR006745 and PR006746 are two ΔPTM variants of PR006669, exhibiting slightly weaker ELISA binding and functional blocking activity. See Table 4 and... Figures 6A to 6D .

[0716] Table 4. Functional blocking activity of series 2 mAb PR006669 and its ΔPTM variants

[0717]

[0718] Humanization of Series 5 mAb PR301777

[0719] Fifteen humanized variants of PR301777 were recombinantly expressed and tested using the materials and methods described above. Many of these humanized variants maintained high levels of in vitro ELISA binding activity and functional blocking activity. See Table 5 and... Figures 7A to 7L .

[0720] Table 5. CRH ELISA binding activity and functional blocking activity of series 5 mAb PR301777 and its humanized variants

[0721]

[0722]

[0723] Humanization of Series 7 mAb PR302309

[0724] Twenty-one humanized variants of PR302309 were recombinantly expressed and tested using the materials and methods described above. Many of these humanized variants maintained high levels of in vitro ELISA binding activity and functional blocking activity. See Table 6 and... Figures 8A to 8D .

[0725] Table 6. Functional blocking activities of series 7 mAb PR302309 and its humanized variants

[0726]

[0727]

[0728]

[0729] Humanization of Series 9 mAb PR302334

[0730] Twenty-two humanized variants of PR302334 were recombinantly expressed and tested using the materials and methods described above. Many of these variants maintained high levels of in vitro ELISA binding activity and functional blocking activity. See Table 7 and... Figures 9A to 9F .

[0731] Table 7. Functional blocking activities of series 9 mAb PR302334 and its humanized variants

[0732]

[0733]

[0734]

[0735] Humanization of Series 10 mAb PR302341

[0736] Twenty-seven humanized variants of PR302341 were recombinantly expressed and tested using the materials and methods described above. Many of these humanized variants maintained high levels of in vitro ELISA binding activity and functional blocking activity. See Table 8 and... Figures 10A to 10G .

[0737] Table 8. Functional blocking activities of the series 10 mAb PR302341 and its humanized variants

[0738]

[0739]

[0740]

[0741] PTM removal in humanized series 10 mAb PR302341-10, PR302341-20 and PR302341-23

[0742] Using the materials and methods described above, the humanized series 10 mAb PR302341-10, PR302341-20, and PR302341-23 were subjected to 4×4 double PTM removal mutagenesis, yielding a total of 48 mAbs (3×4×4), including the three parental humanized mAbs mentioned above. Many of the humanized variants maintained highly efficient in vitro ELISA binding and functional blocking activities. See Table 9 and... Figures 11A to 11L PR302341-34, PR302341-48, PR302341-60, PR302341-28 and PR302341-55 are scaled up for in vivo studies.

[0743] Table 9. Functional blocking activity and PTM-removed variants of series 10 humanized mAbs PR302341-10, PR302341-20 and PR302341-23

[0744]

[0745]

[0746]

[0747]

[0748] Example 6

[0749] Selectivity for human UCN1, human UCN2 and human UCN3 peptides

[0750] In ELISA assays, all mAbs tested, except PR302334-26, bound to the human UCN1 peptide. None of the mAbs tested bound to the human UCN2 or human UCN3 peptides in ELISA assays. By ELISA, the binding affinity of these mAbs to human UCN1 was comparable to that to CRH. However, only mAbs from the PR302341 series inhibited hUCN1-induced hCRHR1 luciferase reporter gene activity. Furthermore, these mAbs inhibited hUCN1-induced hCRHR1 reporter gene activity at a potency 30X to 267X lower than that induced by CRH. None of these mAbs blocked hUCN1-induced hCRHR2 luciferase reporter gene activity. See Table 10 and... Figures 12A to 12B , Figures 13A to 13D as well as Figures 14A to 14H .

[0751] Table 10. The efficacy of anti-CRH mAb in blocking CRH-induced signal transduction is higher than that in blocking hUCN1-induced signal transduction.

[0752]

[0753]

[0754] Example 7

[0755] Anti-CRH mAb binds to the N-terminal region of CRH peptides

[0756] To determine the binding region of the CRH peptide, using the materials and methods described above, the N-terminal region plus a central region, the central region only, and the central region plus a C-terminal region of the CRH peptide were used for ELISA capture of CRH. All mAbs bound to the N-terminal plus central region of the CRH peptide. Therefore, these mAbs may bind to the N-terminal region of CRH. See Table 11 and... Figures 15A to 15D .

[0757] Table 11. N-terminal region of anti-CRH mAb binding to CRH peptide

[0758] CRH zone: N-terminal-central zone-C-terminal

[0759] Full length CRH: SEEPPISLDL TFHLLREVLE MARAEQLAQQ AHSNRKLMEI I-NH2

[0760] (SEQ ID NO:481)

[0761] N-terminal CRH: SEEPPISLDL TFHLLREVLE MARAEQLAQQ AK-Biotin

[0762] (SEQ ID NO:482)

[0763] C-terminal CRH: Biotin-K-VLE MARAEQLAQQ AHSNRKLMEI I-NH2

[0764] (SEQ ID NO:483)

[0765] Central CRH: Biotin-K-LREVLE MARAEQLAQQ AHSN-NH2

[0766] (SEQ ID NO:484)

[0767] CRH Center: LREVLE MARAEQLAQQ AHSN-K-Biotin

[0768] (SEQ ID NO:485)

[0769]

[0770] Example 8

[0771] use Determining the affinity of mAbs

[0772] use The platform was used to determine the binding affinity of the anti-CRH antibodies using the materials and methods described above. The identified mAbs exhibited very high affinity, with equilibrium dissociation constants (KD) of approximately single to double digits pM. See Table 12.

[0773] Table 12. Using Determining the affinity of mAbs

[0774]

[0775]

[0776] Example 9

[0777] ELISA binding of antibody-binding epitopes

[0778] Using the materials and methods described above, the ability of selected mAbs to block each other's binding to biotin-CRH was tested in a pairwise competitive ELISA. Table 13 shows that these mAbs block each other's binding to CRH and therefore may bind similar epitopes, possibly at the N-terminus of CRH.

[0779] Table 13. ELISA binding of antibody-binding epitopes

[0780]

[0781]

[0782] Note: PR303394 is an analogue of CTRND05, derived from patent WO2019241127A1, reformatted with human IgG1 wild-type Fc.

[0783] Example 10

[0784] Antibody-binding epitopes Combination

[0785] Using the above materials and methods, in pairwise competition The assay tested the ability of the selected mAbs to block the binding of biotin-CRH to each other. Figure 16 It shows the use of A schematic diagram of the competition assays on the platform. Table 14 shows that these mAbs block each other's binding to CRH, and therefore may bind to similar epitopes, possibly at the N-terminus of CRH.

[0786] Table 14. Antibody-binding epitopes Combination

[0787]

[0788] Example 11

[0789] In vivo efficacy study

[0790] Transient increase in ACTH and corticosterone induced by constraint in wild-type mice

[0791] Treatment with anti-CRH mAbs PR004290 (series 1 mAb), PR006152 (series 1 mAb), and PR302050 (series 5 mAb) (20 mg / kg IP) significantly suppressed adrenocorticotropic hormone (ACTH) and corticosterone concentrations in wild-type mice subjected to the aforementioned constraint-induced stress model. Plasma ACTH and corticosterone concentrations were suppressed to baseline stress-free levels on day 1 after mAb administration. Partial suppression of mAb, ACTH, and corticosterone concentrations was maintained for fourteen days following a single dose. See also Figure 17 .

[0792] Mrap1KO mouse constitutive high ACTH model

[0793] Mrap1 KO mice exhibited constitutively high plasma ACTH concentrations of approximately 5,000 pg / ml, about 50 times higher than the basal ACTH levels observed in wild-type mice and about 10 times higher than those observed in wild-type mice under constraint-induced stress. A single dose of 20 mg / kg of the anti-CRH mAb PR302050 (serialized 5 mAb) significantly reduced the constitutively high ACTH concentrations in Mrap1 KO mice at both 2 and 14 days post-administration. See also Figure 18 .

[0794] A single dose of the anti-CRH mAb PR302064 (a series of 5 mAbs) significantly reduced constitutively high ACTH levels in Mrap1KO mice in a dose-dependent manner, at 20 mg / kg, 5 mg / kg, and 1 mg / kg on days 2, 16, and 28, respectively. At 20 mg / kg, PR302064 reduced ACTH by 73%, 82%, and 55% on days 2, 16, and 28, respectively. At 5 mg / kg, PR302064 reduced ACTH by 74%, 45%, and 34% on days 2, 16, and 28, respectively. At 1 mg / kg, PR302064 reduced ACTH by 47%, 31%, and 23% on days 2, 16, and 28, respectively. Other mAbs at 5 mg / kg, including PR302050 (series 5 mAb), PR005660 (series 1 mAb), and PR006669 (series 2 mAb), also significantly reduced ACTH on days 2 and 16 post-administration. See also Figures 19A to 19C .

[0795] Other anti-CRH mAbs were tested in the Mrap1 KO model. Single doses of PR302334-26 (serial 9 mAbs), PR302341-28, PR302341-34, PR302341-55, and PR302341-60 (all 10 mAbs) at 20 mg / kg also significantly reduced plasma ACTH concentrations in Mrap1 KO mice. See also Figures 20A to 20B .

[0796] PR302038, PR005660, and PR302050 reduced plasma ACTH concentrations in Mrap1 KO mice in a dose-dependent manner at antibody doses of 20 mg / kg and 5 mg / kg. See also Figures 21A to 21B .

[0797] PR302334-24 and PR005660 significantly reduced plasma ACTH concentrations in Mrap1 KO mice at 20 mg / kg, while PR301429 (an anti-CRH mAb with considerable binding affinity, but a non-blocker in vitro) did not inhibit ACTH production. PR303394, an analogue of CTRND05, was used as a comparative. See also Figures 22A to 22B .

[0798] In a wild-type mouse constraint-induced ACTH model, the anti-CRH mAb PR302064 was superior to SSR125543A in efficacy. effect

[0799] The 20 mg / kg anti-CRH mAb PR302064 (a series of 5 mAbs) was more effective than the 30 mg / kg CRHR1 small molecule antagonist SSR125543A (Crinecerfont) in reducing constraint-induced ACTH in wild-type mice. PR302064 showed more complete inhibition of both ACTH and corticosterone, with a longer duration of action. The effect of this mAb lasted for at least 16 days. See also Figures 23A to 23B .

[0800] In the Mrap1 KO mouse constitutive high ACTH model, anti-CRH mAb PR302064 and PR302334-24 were compared... SSR125543A has superior efficacy.

[0801] In a constitutive high ACTH model in Mrap1 KO mice, the anti-CRH mAb PR302064 at 20 mg / kg and PR302334-24 at 20 mg / kg, 10 mg / kg, and 5 mg / kg were more effective than the CRHR1 small molecule antagonist SSR125543A (Crinecerfont) at 30 mg / kg in reducing plasma ACTH levels. PR302064 and PR302334-24 exhibited more complete inhibition and, more importantly, a longer duration of action. The effects of both mAbs lasted for at least 14 days. See also Figures 24A to 24B .

[0802] In a wild-type mouse model of constipation stress-induced high ACTH and corticosterone levels, anti-CRH mAb PR302334-24... It has superior efficacy compared to SSR125543A.

[0803] In a wild-type mouse model of constipation stress-induced high ACTH and corticosterone levels, 20 mg / kg and 5 mg / kg of the anti-CRHmAb PR302334-24 were more effective than 30 mg / kg of the CRHR1 small molecule antagonist SSR125543A (Crinecerfont) in reducing plasma ACTH and corticosterone levels. PR302334-24 exhibited more complete inhibition and, more importantly, a longer duration of action. The effect of PR302334-24 lasted for at least 17 days. See also Figures 25A to 25B .

[0804] In vivo PK research

[0805] The pharmacokinetics of mAbs PR004290, PR302050, and PR302064 were tested in C57BL / 6 wild-type mice at a single IV dose of 5 mg / kg. Specifically, anti-CRH mAbs in human IgG1-WT-Fc present in mouse serum were captured by goat anti-human Fc polyclonal antibody and detected with HRP-conjugated goat anti-human (H+L) secondary antibody. Figure 26A A schematic diagram of the method is shown.

[0806] As shown in Table 15 and Figures 26B to 26D As shown, the serum half-lives (T004290, PR302050, and PR302064 are... 1 / 2 The timeframes were 14 days, 21 days, and 18 days, respectively. These data indicate that these mAbs have typical human IgG1 PK curves and the Tp values ​​required for clinical application. 1 / 2 .

[0807] Table 15. PK analysis of antibodies in female C57BL / 6 wild-type mice (N=6) at a single IV dose of 5 mg / kg.

[0808]

[0809] All publications, patents, and patent applications mentioned in this application are incorporated herein by reference in their entirety, to the extent that each individual publication, patent, or patent application is specifically and individually identified and incorporated herein by reference. Furthermore, any reference or identification of any reference in this application should not be construed as an admission that such references are prior art to the invention. The use of partial headings should not be construed as necessarily limiting.

Claims

1. A method for treating polycystic ovary syndrome (PCOS) in a subject in need, the method comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to corticotropin-releasing hormone (CRH).

2. A method for treating PCOS in a subject of need, the method comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to CRH, wherein the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable domain (VH) CDR1 comprising an amino acid sequence of any one of SEQ ID NO: 22-35, a VH CDR2 comprising an amino acid sequence of any one of SEQ ID NO: 53-78, and a VH CDR3 comprising an amino acid sequence of any one of SEQ ID NO: 113-122 or an amino acid sequence of PDV or GID; and (b) A light chain variable domain (VL) CDR1 comprising an amino acid sequence of any one of SEQ ID NO:152-174, a VL CDR2 comprising an amino acid sequence of any one of SEQ ID NO:193-198, and a VL CDR3 comprising an amino acid sequence of any one of SEQ ID NO:223-232.

3. The method according to claim 1 or 2, wherein the antibody or its antigen-binding fragment comprises: (a) VH, wherein the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NO:240-295, 459, and 461; and / or (b) VL, wherein the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NO:296-347 and 462.

4. The method according to any one of claims 1 to 3, wherein the antibody or its antigen-binding fragment comprises: (a) a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NO:348-403, 460, and 463; and / or (b) A light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NO:404-455 and 464.

5. A method for reducing androgen levels in a subject suffering from PCOS, the method comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to CRH.

6. A method for reducing androgen levels in a subject suffering from PCOS, the method comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to CRH, wherein the antibody or antigen-binding fragment thereof comprises: (a) VH CDR1 comprising the amino acid sequence of any one of SEQ ID NO:22-35, VH CDR2 comprising the amino acid sequence of any one of SEQ ID NO:53-78, and VH CDR3 comprising the amino acid sequence of any one of SEQ ID NO:113-122 or the amino acid sequence of PDV or GID; and (b) VL CDR1 containing the amino acid sequence of any one of SEQ ID NO:152-174, VL CDR2 containing the amino acid sequence of any one of SEQ ID NO:193-198, and VL CDR3 containing the amino acid sequence of any one of SEQ ID NO:223-232.

7. The method according to claim 5 or 6, wherein the antibody or its antigen-binding fragment comprises: (a) VH, wherein the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NO:240-295, 459, and 461; and / or (b) VL, wherein the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NO:296-347 and 462.

8. The method according to any one of claims 5 to 7, wherein the antibody or its antigen-binding fragment comprises: (a) a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NO:348-403, 460, and 463; and / or (b) A light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NO:404-455 and 464.

9. The method according to any one of claims 5 to 8, wherein the androgen is testosterone, free testosterone, androstenedione, 11-oxyandrogens, or combinations thereof.

10. The method according to claim 9, wherein the 11-oxyandrogen is 11-hydroxyandrostenedione (11OHA4), 11-hydroxytestosterone (11OHT), 11-ketoandrostenedione (11KA4), 11-ketotestosterone (11KT), or a combination thereof.

11. A method for reducing the severity of one or more symptoms or signs in a subject suffering from PCOS, the method comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to CRH.

12. A method for reducing the severity of one or more symptoms or signs in a subject suffering from PCOS, the method comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to CRH, wherein the antibody or antigen-binding fragment thereof comprises: (a) VH CDR1 comprising the amino acid sequence of any one of SEQ ID NO:22-35, VH CDR2 comprising the amino acid sequence of any one of SEQ ID NO:53-78, and VH CDR3 comprising the amino acid sequence of any one of SEQ ID NO:113-122 or the amino acid sequence of PDV or GID; and (b) VL CDR1 containing the amino acid sequence of any one of SEQ ID NO:152-174, VL CDR2 containing the amino acid sequence of any one of SEQ ID NO:193-198, and VL CDR3 containing the amino acid sequence of any one of SEQ ID NO:223-232.

13. The method of claim 11 or 12, wherein the antibody or its antigen-binding fragment comprises: (a) VH, wherein the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NO:240-295, 459, and 461; and / or (b) VL, wherein the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NO:296-347 and 462.

14. The method according to any one of claims 11 to 13, wherein the antibody or its antigen-binding fragment comprises: (a) a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NO:348-403, 460, and 463; and / or (b) A light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NO:404-455 and 464.

15. The method according to any one of claims 11 to 14, wherein the one or more symptoms or signs are: anovulation; irregular menstruation or amenorrhea; hyperandrogenism; elevated levels of testosterone, free testosterone, androstenedione, 11-oxyandrogens, or combinations thereof; abnormal uterine bleeding; enlarged ovaries with multiple follicles; infertility; obesity; insulin resistance; hyperinsulinemia; hypertension; hyperlipidemia; type 2 diabetes; excessive facial hair growth; hirsutism; alopecia; or acne.

16. The method of claim 15, wherein the 11-oxyandrogen is 11-hydroxyandrostenedione (11OHA4), 11-hydroxytestosterone (11OHT), 11-ketoandrostenedione (11KA4), 11-ketotestosterone (11KT), or a combination thereof.

17. The method according to any one of claims 1 to 16, wherein the antibody is polyclonal, monoclonal, human, humanized, or chimeric.

18. The method according to any one of claims 1 to 16, wherein the antigen-binding fragment is Fab, scFab, Fab', F(ab')2, Fv, scFv, a biantibody, or a triantibody.

19. The method according to any one of claims 1 to 18, wherein the antibody or its antigen-binding fragment is administered in a pharmaceutical composition comprising the antibody or its antigen-binding fragment and a pharmaceutically acceptable carrier.

20. The method according to any one of claims 1 to 19, wherein the antibody or its antigen-binding fragment is administered in combination with one or more PCOS therapies.

21. The method of claim 20, wherein the one or more PCOS therapies are selected from combined contraception, progesterone therapy, clomiphene, tamoxifen, aromatase inhibitors, gonadotropins, ovarian drilling, spironolactone, efornithine, electrolytes, and antidiabetic drugs.

22. The method of claim 21, wherein the combined contraception is an oral contraceptive containing estrogen and progestin.

23. The method of claim 21, wherein the aromatase inhibitor is letrozole.

24. The method of claim 21, wherein the antidiabetic drug is metformin.

25. The method of claim 20, wherein the one or more PCOS therapies are selected from spironolactone, metformin, and combined contraception.

26. The method of claim 25, wherein the combined contraception is an oral contraceptive containing estrogen and progestin.

27. The method according to any one of claims 1 to 26, wherein the subject is a human.

28. A kit comprising (i) an antibody that specifically binds to CRH or an antigen-binding fragment thereof; and (ii) instructions for treating PCOS in a subject in need, for reducing androgen levels in a subject with PCOS, or for reducing the severity of one or more symptoms or signs in a subject with PCOS.

29. A kit comprising (i) a pharmaceutical composition comprising an antibody that specifically binds to CRH or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier; and (ii) instructions for use in treating PCOS in a subject in need, for reducing androgen levels in a subject with PCOS, or for reducing the severity of one or more symptoms or signs in a subject with PCOS.

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