Anticorticotropin-releasing hormone antibodies and polycystic ovary syndrome

Abstract

Methods of using antibodies and antigen-binding fragments thereof that specifically bind to corticotropin-releasing hormone (CRH) in the treatment of polycystic ovary syndrome (PCOS) and related disorders are disclosed. Also disclosed are kits containing antibodies and antigen-binding fragments thereof that specifically bind to CRH and instructions for using them in the aforementioned methods.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of 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, which provisional applications are incorporated herein by reference in their entireties.

[0002] Reference to an electronically submitted sequence listing The contents of the electronically submitted sequence listing (Name: 4857_002PC02_SequenceListing_ST26, Size: 686,199 bytes, Created: July 25, 2023) are incorporated herein by reference in their entirety.

[0003] Field The present disclosure relates to methods of using antibodies and antigen-binding fragments thereof that specifically bind to corticotropin-releasing hormone (CRH) in the treatment of polycystic ovary syndrome (PCOS) and related disorders. The present disclosure also relates to kits comprising such antibodies and antigen-binding fragments thereof and instructions for using them in the aforementioned methods. [Background technology]

[0004] Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among women of reproductive age, affecting 4–50% of women worldwide. (Deswal et al., J. Hum. Reprod. Sci., 13(4):261–271 (2020). Women with PCOS may suffer from (i) infrequent, irregular, or prolonged menstrual cycles; (ii) excessive androgen levels, which can lead to excessive hair growth (hirsutism), acne, alopecia, or skin abnormalities; and (iii) polycystic ovaries, which can lead to infertility. (Azziz, Obstet. Gynecol., 132(2):321–336 (2018).) Key features of PCOS include hyperandrogenism, anovulation, insulin resistance, and neuroendocrine disruption. Crespo et al., Arch. Endocrin. Metab., 62(3):352-361 (2018).

[0005] Due to this complexity, managing PCOS remains challenging. Treatment is often based on presenting symptoms, which may include androgen-related symptoms, infertility related to ovulatory dysfunction, and menstrual disorders. Badawy et al., Int. J. Women's Health, 3:25-35 (2011). Evidence suggests that both clinicians and consumers are dissatisfied with current treatment options. Hoeger et al., J. Clin. Endocrinol. Metab., 106(3):e1071-el083 (2021). Therefore, improved treatments for PCOS and related disorders are needed. Summary of the Invention

[0006] Provided herein are methods for treating polycystic ovary syndrome (PCOS) in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds corticotropin-releasing hormone (CRH).

[0007] Also provided herein is a method for treating PCOS in a subject in need thereof, the method comprising the step of administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to CRH, wherein the antibody or antigen-binding fragment comprises: (a) a heavy chain variable domain (VH) CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 22 to 35, a VH CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 53 to 78, and a VH CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 113 to 122 or the amino acid sequence of a PDV or GID; and (b) a light chain variable domain (VL) CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 152 to 174, a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 193 to 198, and a VL CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 223 to 232.

[0008] In one embodiment, the antibody or antigen-binding fragment thereof comprises (a) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to any one of SEQ ID NOs: 240-295, 459, and 461, and / or (b) a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to any one of SEQ ID NOs: 296-347 and 462.

[0009] In another embodiment, the antibody or antigen-binding fragment thereof 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 to any one of SEQ ID NOs: 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 to any one of SEQ ID NOs: 404-455 and 464.

[0010] Also provided herein is 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.

[0011] Also provided herein is a method for reducing androgen levels in a subject with PCOS, comprising the step of administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to CRH, wherein the antibody or antigen-binding fragment comprises (a) a VH CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 22 to 35, a VH CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 53 to 78, and a VH CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 113 to 122 or the amino acid sequence of a PDV or GID; and (b) a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 152 to 174, a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 193 to 198, and a VL CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 223 to 232.

[0012] In one embodiment, the antibody or antigen-binding fragment thereof comprises (a) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to any one of SEQ ID NOs: 240-295, 459, and 461, and / or (b) a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to any one of SEQ ID NOs: 296-347 and 462.

[0013] In another embodiment, the antibody or antigen-binding fragment thereof 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 to any one of SEQ ID NOs: 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 to any one of SEQ ID NOs: 404-455 and 464.

[0014] In another embodiment, the androgen is testosterone, free testosterone, androstenedione, an 11-oxygenated androgen, or a combination thereof. In another embodiment, the 11-oxygenated androgen is 11-hydroxyandrostenedione (11OHA4), 11-hydroxytestosterone (11OHT), 11-ketoandrostenedione (11KA4), 11-ketotestosterone (11KT), or a combination thereof.

[0015] Also provided herein is a method for reducing the severity of one or more symptoms or signs of PCOS in a subject, the method comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to CRH.

[0016] Also provided herein is a method for reducing the severity of one or more symptoms or signs of PCOS in a subject, the method comprising the step of administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to CRH, wherein the antibody or antigen-binding fragment comprises (a) a VH CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 22 to 35, a VH CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 53 to 78, and a VH CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 113 to 122 or the amino acid sequence of a PDV or GID, and (b) a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 152 to 174, a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 193 to 198, and a VL CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 223 to 232.

[0017] In one embodiment, the antibody or antigen-binding fragment thereof comprises (a) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to any one of SEQ ID NOs: 240-295, 459, and 461, and / or (b) a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to any one of SEQ ID NOs: 296-347 and 462.

[0018] In one embodiment, the antibody or antigen-binding fragment thereof 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 to any one of SEQ ID NOs: 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 to any one of SEQ ID NOs: 404-455 and 464.

[0019] In another embodiment, the one or more symptoms or signs are anovulation, irregular or amenorrheal periods, hyperandrogenism, elevated testosterone, free testosterone, androstenedione, 11-oxygenated androgens, or a combination thereof, abnormal uterine bleeding, polycystic ovarian enlargement, infertility, obesity, insulin resistance, hyperinsulinemia, hypertension, hyperlipidemia, type 2 diabetes, excessive facial hair growth, hirsutism, alopecia, or acne. In another embodiment, the 11-oxygenated androgen is 11-hydroxyandrostenedione (11OHA4), 11-hydroxytestosterone (11OHT), 11-ketoandrostenedione (11KA4), 11-ketotestosterone (11KT), or a combination thereof.

[0020] In another embodiment, the antibody is polyclonal, monoclonal, human, humanized, or chimeric.

[0021] In another embodiment, the antigen-binding fragment is a Fab, scFab, Fab', F(ab')2, Fv, scFv, diabody, or triabody.

[0022] In another embodiment, the antibody or antigen-binding fragment thereof is administered in a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

[0023] In another embodiment, the antibody or antigen-binding fragment thereof is administered in combination with one or more PCOS therapies. In another embodiment, the one or more PCOS therapies are selected from the group consisting of combined contraceptives, progestin therapy, clomiphene, tamoxifen, aromatase inhibitors, gonadotropins, ovarian drilling, spironolactone, eflornithine, electrolysis, and antidiabetic drugs. In another embodiment, the combined contraceptive is an oral contraceptive containing estrogen and progestin. In another embodiment, the antidiabetic drug is metformin. In another embodiment, the aromatase inhibitor is letrozole.

[0024] In another embodiment, the one or more PCOS therapies are selected from the group consisting of spironolactone, metformin, and a combined contraceptive, hi another embodiment, the combined contraceptive is an oral contraceptive containing estrogen and progestin.

[0025] In another embodiment, the subject is a human.

[0026] Also provided herein are kits comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to CRH, and (ii) instructions for use in treating PCOS in a subject in need thereof, reducing androgen levels in a subject with PCOS, or reducing the severity of one or more symptoms or signs of PCOS in a subject with PCOS.

[0027] Also provided herein are kits comprising: (i) a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that specifically binds to CRH and a pharmaceutically acceptable carrier; and (ii) instructions for use in treating PCOS in a subject in need thereof, reducing androgen levels in a subject with PCOS, or reducing the severity of one or more symptoms or signs of PCOS in a subject with PCOS. [Brief explanation of the drawings]

[0028] Some aspects of the present invention are herein described, by way of example only, with reference to the accompanying drawings. Referring now specifically to the drawings in detail, it is emphasized that the particulars shown are by way of example only and are for the purpose of illustrative discussion of aspects of the invention.

[0029] [Figure 1] FIG. 1 shows the coding sequence open reading frame of mouse CRH (GenBank NM 205769) along with the CRISPR targeting gRNA and PCR primers used in Example 1. [Figure 2A]Figure 2A shows the identification of CRH knockout founder mice by PCR. The expected PCR DNA fragment sizes from wild-type mice are 146 base pairs (bp) and 103 bp. PCR fragments smaller than these expected sizes indicated deletions in founder mice. PCR primers CRHbp70F and CRHbp215R flanking gRNA1 were used to amplify the expected 146 bp size from wild-type H2L2 Harbour Mice® (Figure 2A, left panel). PCR primers CRHbp208F and CRHbp310R were used to amplify the expected 103 bp size from wild-type H2L2 Harbour Mice® (Figure 2A, center panel). PCR primers CRHbp70F and CRHbp310R yielded a fragment size of 241 bp (Figure 2A, right panel). PCR products smaller than these expected sizes indicated mutations. Founder mice 1, 2, 4, and 5 were identified as carrying the mutation. Founder mouse 5 from litter 12198 in FIG. 2B had an 11 bp deletion (5′-CAGCCGGTTCT-3′) (SEQ ID NO: 465) and was used for subsequent breeding. [Figure 2B]Figure 2A shows the identification of CRH knockout founder mice by PCR. The expected PCR DNA fragment sizes from wild-type mice are 146 base pairs (bp) and 103 bp. PCR fragments smaller than these expected sizes indicated deletions in founder mice. PCR primers CRHbp70F and CRHbp215R flanking gRNA1 were used to amplify the expected 146 bp size from wild-type H2L2 Harbour Mice® (Figure 2A, left panel). PCR primers CRHbp208F and CRHbp310R were used to amplify the expected 103 bp size from wild-type H2L2 Harbour Mice® (Figure 2A, center panel). PCR primers CRHbp70F and CRHbp310R yielded a fragment size of 241 bp (Figure 2A, right panel). PCR products smaller than these expected sizes indicated mutations. Founder mice 1, 2, 4, and 5 were identified as carrying the mutation. Founder mouse 5 from litter 12198 in FIG. 2B had an 11 bp deletion (5′-CAGCCGGTTCT-3′) (SEQ ID NO: 465) and was used for subsequent breeding. [Figure 3] Figure 4 shows the location of an 11-bp deletion (5'-CAGCCGGTTCT-3') (SEQ ID NO: 465) in the CRH gene coding region 157-167, which results in a downstream reading frameshift and a premature stop codon. This defective CRH open reading frame is unable to synthesize the CRH peptide. Founders with this deletion were selected for further breeding to homozygosity, rescue, and immunization. [Figure 4A] Figure 1 shows the CRH ELISA binding activity of the indicated anti-CRH monoclonal antibodies (mAbs) in PR numbers. An irrelevant human IgG1 was used as a negative control. [Figure 4B] Figure 1 shows the CRH ELISA binding activity of the indicated anti-CRH monoclonal antibodies (mAbs) in PR numbers. An irrelevant human IgG1 was used as a negative control. [Figure 4C]Figure 1 shows the human CRHR1 (hCRHR1) luciferase reporter blocking activity of anti-CRH monoclonal antibodies (mAbs). An irrelevant human IgG1 was used as a negative control. [Figure 4D] Figure 1 shows the CRH ELISA binding activity of the indicated anti-CRH monoclonal antibodies (mAbs) in PR numbers. An irrelevant human IgG1 was used as a negative control. [Figure 4E] Figure 1 shows the CRH ELISA binding activity of the indicated anti-CRH monoclonal antibodies (mAbs) in PR numbers. An irrelevant human IgG1 was used as a negative control. [Figure 4F] Figure 1 shows the human CRHR1 (hCRHR1) luciferase reporter blocking activity of anti-CRH monoclonal antibodies (mAbs). An irrelevant human IgG1 was used as a negative control. [Figure 4G] Figure 1 shows the CRH ELISA binding activity of the indicated anti-CRH monoclonal antibodies (mAbs) in PR numbers. An irrelevant human IgG1 was used as a negative control. [Figure 4H] Figure 1 shows the CRH ELISA binding activity of the indicated anti-CRH monoclonal antibodies (mAbs) in PR numbers. An irrelevant human IgG1 was used as a negative control. [Figure 4I] Figure 1 shows the human CRHR1 (hCRHR1) luciferase reporter blocking activity of anti-CRH monoclonal antibodies (mAbs). An irrelevant human IgG1 was used as a negative control. [Figure 4J] Figure 1 shows the CRH ELISA binding activity of the indicated anti-CRH monoclonal antibodies (mAbs) in PR numbers. An irrelevant human IgG1 was used as a negative control. [Figure 4K] Figure 1 shows the CRH ELISA binding activity of the indicated anti-CRH monoclonal antibodies (mAbs) in PR numbers. An irrelevant human IgG1 was used as a negative control. [Figure 4L]Figure 1 shows the CRH ELISA binding activity of the indicated anti-CRH monoclonal antibodies (mAbs) in PR numbers. An irrelevant human IgG1 was used as a negative control. [Figure 4M] Figure 1 shows the CRH ELISA binding activity of the indicated anti-CRH monoclonal antibodies (mAbs) in PR numbers. An irrelevant human IgG1 was used as a negative control. [Figure 4N] Figure 1 shows the human CRHR1 (hCRHR1) luciferase reporter blocking activity of anti-CRH monoclonal antibodies (mAbs). An irrelevant human IgG1 was used as a negative control. [Figure 5A] FIG. 1 shows the CRH ELISA binding activity of series 1 mAb PR004290 and its ΔPTM variants. [Figure 5B] FIG. 1 shows the CRH ELISA binding activity of series 1 mAb PR004290 and its ΔPTM variants. [Figure 5C] FIG. 1 shows the CRH ELISA binding activity of series 1 mAb PR004290 and its ΔPTM variants. [Figure 5D] FIG. 1 shows the CRH ELISA binding activity of series 1 mAb PR004290 and its ΔPTM variants. [Figure 5E] FIG. 1 shows the luciferase reporter blocking activity of series 1 mAb PR004290 and its ΔPTM variants for human CRHR1 (hCRHR1), human CRHR2 (hCRHR2), mouse CRHR1 (mCRHR1), and mouse CRHR2 (mCRHR2). [Figure 5F] FIG. 1 shows the luciferase reporter blocking activity of series 1 mAb PR004290 and its ΔPTM variants for human CRHR1 (hCRHR1), human CRHR2 (hCRHR2), mouse CRHR1 (mCRHR1), and mouse CRHR2 (mCRHR2). [Figure 5G]FIG. 1 shows the luciferase reporter blocking activity of series 1 mAb PR004290 and its ΔPTM variants for human CRHR1 (hCRHR1), human CRHR2 (hCRHR2), mouse CRHR1 (mCRHR1), and mouse CRHR2 (mCRHR2). [Figure 5H] FIG. 1 shows the luciferase reporter blocking activity of series 1 mAb PR004290 and its ΔPTM variants for human CRHR1 (hCRHR1), human CRHR2 (hCRHR2), mouse CRHR1 (mCRHR1), and mouse CRHR2 (mCRHR2). [Figure 5I] FIG. 1 shows the luciferase reporter blocking activity of series 1 mAb PR004290 and its ΔPTM variants for human CRHR1 (hCRHR1), human CRHR2 (hCRHR2), mouse CRHR1 (mCRHR1), and mouse CRHR2 (mCRHR2). [Figure 5J] FIG. 1 shows the luciferase reporter blocking activity of series 1 mAb PR004290 and its ΔPTM variants for human CRHR1 (hCRHR1), human CRHR2 (hCRHR2), mouse CRHR1 (mCRHR1), and mouse CRHR2 (mCRHR2). [Figure 5K] FIG. 1 shows the luciferase reporter blocking activity of series 1 mAb PR004290 and its ΔPTM variants for human CRHR1 (hCRHR1), human CRHR2 (hCRHR2), mouse CRHR1 (mCRHR1), and mouse CRHR2 (mCRHR2). [Figure 5L] FIG. 1 shows the luciferase reporter blocking activity of series 1 mAb PR004290 and its ΔPTM variants for human CRHR1 (hCRHR1), human CRHR2 (hCRHR2), mouse CRHR1 (mCRHR1), and mouse CRHR2 (mCRHR2). [Figure 6A]FIG. 1 shows the luciferase reporter blocking activity of series 2 mAb PR006669 and its ΔPTM variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 6B] FIG. 1 shows the luciferase reporter blocking activity of series 2 mAb PR006669 and its ΔPTM variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 6C] FIG. 1 shows the luciferase reporter blocking activity of series 2 mAb PR006669 and its ΔPTM variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 6D] FIG. 1 shows the luciferase reporter blocking activity of series 2 mAb PR006669 and its ΔPTM variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 7A] FIG. 1 shows the CRH ELISA binding activity of series 5 mAb PR301777 and its humanized variants. [Figure 7B] FIG. 1 shows the CRH ELISA binding activity of series 5 mAb PR301777 and its humanized variants. [Figure 7C] FIG. 1 shows the CRH ELISA binding activity of series 5 mAb PR301777 and its humanized variants. [Figure 7D] FIG. 1 shows the CRH ELISA binding activity of series 5 mAb PR301777 and its humanized variants. [Figure 7E] FIG. 1 shows the luciferase reporter blocking activity of series 5 mAb PR301777 and its humanized variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 7F]FIG. 1 shows the luciferase reporter blocking activity of series 5 mAb PR301777 and its humanized variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 7G] FIG. 1 shows the luciferase reporter blocking activity of series 5 mAb PR301777 and its humanized variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 7H] FIG. 1 shows the luciferase reporter blocking activity of series 5 mAb PR301777 and its humanized variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 7I] FIG. 1 shows the luciferase reporter blocking activity of series 5 mAb PR301777 and its humanized variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 7J] FIG. 1 shows the luciferase reporter blocking activity of series 5 mAb PR301777 and its humanized variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 7K] FIG. 1 shows the luciferase reporter blocking activity of series 5 mAb PR301777 and its humanized variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 7L] FIG. 1 shows the luciferase reporter blocking activity of series 5 mAb PR301777 and its humanized variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 8A] FIG. 1 shows the human CRHR1 luciferase reporter blocking activity of series 7 mAb PR302309 and its humanized variants. [Figure 8B]FIG. 1 shows the human CRHR1 luciferase reporter blocking activity of series 7 mAb PR302309 and its humanized variants. [Figure 8C] FIG. 1 shows the human CRHR1 luciferase reporter blocking activity of series 7 mAb PR302309 and its humanized variants. [Figure 8D] FIG. 1 shows the human CRHR1 luciferase reporter blocking activity of series 7 mAb PR302309 and its humanized variants. [Figure 9A] FIG. 1 shows the luciferase reporter blocking activity of series 9 mAb PR302334 and its humanized variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 9B] FIG. 1 shows the luciferase reporter blocking activity of series 9 mAb PR302334 and its humanized variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 9C] FIG. 1 shows the luciferase reporter blocking activity of series 9 mAb PR302334 and its humanized variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 9D] FIG. 1 shows the luciferase reporter blocking activity of series 9 mAb PR302334 and its humanized variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 9E] FIG. 1 shows the luciferase reporter blocking activity of series 9 mAb PR302334 and its humanized variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 9F] FIG. 1 shows the luciferase reporter blocking activity of series 9 mAb PR302334 and its humanized variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 10A] FIG. 1 shows the luciferase reporter blocking activity of series 10 mAb PR302341 and its humanized variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 10B] FIG. 1 shows the luciferase reporter blocking activity of series 10 mAb PR302341 and its humanized variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 10C] FIG. 1 shows the luciferase reporter blocking activity of series 10 mAb PR302341 and its humanized variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 10D] FIG. 1 shows the luciferase reporter blocking activity of series 10 mAb PR302341 and its humanized variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 10E] FIG. 1 shows the luciferase reporter blocking activity of series 10 mAb PR302341 and its humanized variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 10F] FIG. 1 shows the luciferase reporter blocking activity of series 10 mAb PR302341 and its humanized variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 10G] FIG. 1 shows the luciferase reporter blocking activity of series 10 mAb PR302341 and its humanized variants for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 11A]FIG. 1 shows the luciferase reporter blocking activity of series 10 humanized mAbs PR302341-10, PR302341-20, and PR302341-23, and their ΔPTM variants, for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 11B] FIG. 1 shows the luciferase reporter blocking activity of series 10 humanized mAbs PR302341-10, PR302341-20, and PR302341-23, and their ΔPTM variants, for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 11C] FIG. 1 shows the luciferase reporter blocking activity of series 10 humanized mAbs PR302341-10, PR302341-20, and PR302341-23, and their ΔPTM variants, for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 11D] FIG. 1 shows the luciferase reporter blocking activity of series 10 humanized mAbs PR302341-10, PR302341-20, and PR302341-23, and their ΔPTM variants, for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 11E] FIG. 1 shows the luciferase reporter blocking activity of series 10 humanized mAbs PR302341-10, PR302341-20, and PR302341-23, and their ΔPTM variants, for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 11F] FIG. 1 shows the luciferase reporter blocking activity of series 10 humanized mAbs PR302341-10, PR302341-20, and PR302341-23, and their ΔPTM variants, for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 11G]FIG. 1 shows the luciferase reporter blocking activity of series 10 humanized mAbs PR302341-10, PR302341-20, and PR302341-23, and their ΔPTM variants, for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 11H] FIG. 1 shows the luciferase reporter blocking activity of series 10 humanized mAbs PR302341-10, PR302341-20, and PR302341-23, and their ΔPTM variants, for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 11I] FIG. 1 shows the luciferase reporter blocking activity of series 10 humanized mAbs PR302341-10, PR302341-20, and PR302341-23, and their ΔPTM variants, for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 11J] FIG. 1 shows the luciferase reporter blocking activity of series 10 humanized mAbs PR302341-10, PR302341-20, and PR302341-23, and their ΔPTM variants, for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 11K] FIG. 1 shows the luciferase reporter blocking activity of series 10 humanized mAbs PR302341-10, PR302341-20, and PR302341-23, and their ΔPTM variants, for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 11L] FIG. 1 shows the luciferase reporter blocking activity of series 10 humanized mAbs PR302341-10, PR302341-20, and PR302341-23, and their ΔPTM variants, for human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2. [Figure 12A]FIG. 1 shows by ELISA that some anti-CRH mAbs bind to human UCN1 but not to human UCN2 or human UCN3. [Figure 12B] FIG. 1 shows by ELISA that some anti-CRH mAbs bind to human UCN1 but not to human UCN2 or human UCN3. [Figure 13A] FIG. 1 shows ELISA binding of selected mAbs to CRH and human UCN1. [Figure 13B] FIG. 1 shows ELISA binding of selected mAbs to CRH and human UCN1. [Figure 13C] FIG. 1 shows ELISA binding of selected mAbs to CRH and human UCN1. [Figure 13D] FIG. 1 shows ELISA binding of selected mAbs to CRH and human UCN1. [Figure 14A] FIG. 1 shows human UCN1-mediated inhibition of human CRHR1 and CRHR2 reporter activity. [Figure 14B] FIG. 1 shows human UCN1-mediated inhibition of human CRHR1 and CRHR2 reporter activity. [Figure 14C] FIG. 1 shows human UCN1-mediated inhibition of human CRHR1 and CRHR2 reporter activity. [Figure 14D] FIG. 1 shows human UCN1-mediated inhibition of human CRHR1 and CRHR2 reporter activity. [Figure 14E] FIG. 1 shows human UCN1-mediated inhibition of human CRHR1 and CRHR2 reporter activity. [Figure 14F] FIG. 1 shows human UCN1-mediated inhibition of human CRHR1 and CRHR2 reporter activity. [Figure 14G] FIG. 1 shows human UCN1-mediated inhibition of human CRHR1 and CRHR2 reporter activity. [Figure 14H]FIG. 1 shows human UCN1-mediated inhibition of human CRHR1 and CRHR2 reporter activity. [Figure 15A] FIG. 1 shows that anti-CRH mAb binds to the N-terminal region of the CRH peptide. [Figure 15B] FIG. 1 shows that anti-CRH mAb binds to the N-terminal region of the CRH peptide. [Figure 15C] FIG. 1 shows that anti-CRH mAb binds to the N-terminal region of the CRH peptide. [Figure 15D] FIG. 1 shows that anti-CRH mAb binds to the N-terminal region of the CRH peptide. [Figure 16] FIG. 1 is a diagram of epitope binding experiments using the Octet® platform. [Figure 17] FIG. 1 shows that anti-CRH mAb PR302050 reduces restraint stress-induced ACTH and corticosterone levels in wild-type mice. [Figure 18] FIG. 1 shows that anti-CRH mAb PR302050 reduces constitutively high ACTH in Mrap1 knockout mice. [Figure 19A] FIG. 1 shows that anti-CRH mAb reduces constitutively high ACTH in Mrap1 knockout mice. [Figure 19B] FIG. 1 shows that anti-CRH mAb reduces constitutively high ACTH in Mrap1 knockout mice. [Figure 19C] FIG. 1 shows that anti-CRH mAb reduces constitutively high ACTH in Mrap1 knockout mice. [Figure 20A] FIG. 1 shows additional anti-CRH mAbs that reduce constitutively high ACTH in Mrap1 knockout mice. [Figure 20B] FIG. 1 shows additional anti-CRH mAbs that reduce constitutively high ACTH in Mrap1 knockout mice. [Figure 21A]Figure 21A shows that anti-CRH mAbs PR302038, PR005660, and PR302050 dose-dependently reduced plasma ACTH concentrations in Mrap1 KO mice at doses of 20 mg / kg and 5 mg / kg of antibody. Figure 21A shows the results two days after mAb administration. [Figure 21B] Figure 21B shows that anti-CRH mAbs PR302038, PR005660, and PR302050 dose-dependently reduced plasma ACTH concentrations in Mrap1 KO mice at doses of 20 mg / kg and 5 mg / kg of antibody. Figure 21B shows the results 16 days after mAb administration. [Figure 22A] This figure shows that 20 mg / kg of anti-CRH mAbs PR302334-24 and PR005660 significantly reduced plasma ACTH concentrations in Mrap1 KO mice, whereas PR301429, an anti-CRH mAb with similar binding affinity but a nonblocking agent in vitro, did not inhibit ACTH production. PR303394, an analog of CTRND05, was used as a comparison. Figure 22A shows the results two days after mAb administration. [Figure 22B] Figure 22B shows that 20 mg / kg of anti-CRH mAbs PR302334-24 and PR005660 significantly reduced plasma ACTH concentrations in Mrap1 KO mice, whereas PR301429, an anti-CRH mAb with similar binding affinity but a nonblocking agent in vitro, did not inhibit ACTH production. PR303394, an analog of CTRND05, was used as a comparison. Figure 22B shows the results 16 days after mAb administration. [Figure 23A] FIG. 1 shows that anti-CRH mAb PR302064 is more effective than SSR125543A in inhibiting constraint-induced ACTH release. [Figure 23B] FIG. 1 shows that anti-CRH mAb PR302064 is more effective than SSR125543A in inhibiting restraint-induced corticosterone release. [Figure 24A]

[0023] Figure 1 shows that the anti-CRH mAbs PR302064 and PR302334-24 were more effective than SSR125543A in a model of constitutively high ACTH in Mrap1 KO mice. ACTH levels are shown on day 2 after mAb administration. [Figure 24B]

[0023] Figure 1 shows that anti-CRH mAbs PR302064 and PR302334-24 were more effective than SSR125543A in a model of constitutively high ACTH in Mrap1 KO mice. ACTH levels 14 days after mAb administration are shown. [Figure 25A]

[0023] Figure 1 shows that anti-CRH mAb PR302334-24 was more effective than SSR125543A in the restraint stress-induced high ACTH and corticosterone model in wild-type mice. Plasma levels of inducible ACTH after mAb administration are shown. [Figure 25B] 1 shows that anti-CRH mAb PR302334-24 was more effective than SSR125543A in the restraint stress-induced high ACTH and corticosterone model in wild-type mice. Plasma levels of corticosterone are shown. [Figure 26A] 1 shows a method for testing the pharmacokinetics of anti-CRH mAb. Specifically, anti-CRH mAb in human IgG1-WT-Fc present in mouse serum was captured with a goat anti-human Fc polyclonal antibody and then detected with an HRP-conjugated goat anti-human (H+L) secondary antibody. [Figure 26B] FIG. 1 shows the mean serum concentration time profile of anti-CRH mAb following a single intravenous dose of 5 mg / kg in female C57BL / 6 wild-type mice. [Figure 26C] FIG. 1 shows the mean serum concentration time profile of anti-CRH mAb following a single intravenous dose of 5 mg / kg in female C57BL / 6 wild-type mice. [Figure 26D] FIG. 1 shows the mean serum concentration time profile of anti-CRH mAb following a single intravenous dose of 5 mg / kg in female C57BL / 6 wild-type mice. DETAILED DESCRIPTION OF THE INVENTION

[0030] Various terms relating to aspects of the present disclosure are used throughout the specification and claims. Such terms will be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms will be interpreted in a manner consistent with the definitions provided herein.

[0031] 1.Definition The term "antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin that specifically binds to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) interspersed with highly conserved regions called framework regions (FRs). VH and VL each comprise three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate 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 specified 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.

[0032] As used herein, the term "monoclonal antibody" refers to an antibody produced by a single clone of B cells and binding to the same epitope. In contrast, the term "polyclonal antibody" refers to a population of antibodies produced by different B cells and binding to different epitopes of the same antigen. The term "antibody" includes, by way of example, monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human or non-human antibodies, fully synthetic antibodies, and single-chain antibodies. Non-human antibodies may be humanized by recombinant methods to reduce immunogenicity in humans.

[0033] The antibody may be a modified antibody (e.g., by mutation, deletion, substitution, conjugation to a non-antibody moiety). For example, the antibody may contain one or more variant amino acids (compared to naturally occurring antibodies) that alter the properties (e.g., functional properties) of the antibody. Many such modifications are known in the art that affect, for example, half-life, effector functions, and / or the immune response to the antibody in patients. The term antibody also includes artificial polypeptide constructs that contain at least one antibody-derived antigen-binding site.

[0034] An "antigen-binding fragment" of an antibody refers to one or more fragments or portions of an antibody that retain the ability to specifically bind to the antigen to which the whole antibody binds. It has been shown that the antigen-binding function of an antibody can be performed by fragments or portions of a full-length antibody. As an example of a binding fragment encompassed by the term "antigen-binding portion" or "antigen-binding fragment" of an antibody disclosed herein, the term "antibody fragment" refers to a portion of an intact antibody. An "antigen-binding fragment," "antigen-binding domain," or "antigen-binding region" refers to a portion of an intact antibody that binds to an antigen. An antigen-binding fragment can contain an antigen-determining region (e.g., a complementarity-determining region (CDR)) of an intact 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 produced.

[0035] Furthermore, the two domains of an Fv fragment, VL and VH, are encoded by separate genes but can be recombinantly linked by a synthetic linker that allows the VL and VH regions to pair together to form a single protein chain, known as a monovalent molecule (known as 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 by the term "antigen-binding portion" or "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. In some embodiments, an antibody is an antigen-binding fragment.

[0036] 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 either the light or heavy chain, typically the amino-terminal approximately 110-120 or 110-125 amino acids in the mature heavy chain and approximately 90-115 amino acids in the mature light chain, which vary extensively in sequence among antibodies and are used to determine the binding and specificity of a particular antibody for its specific antigen. Sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while the more highly conserved regions in the variable domain are called framework regions (FRs). While not wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In some embodiments, the variable region is a mammalian variable region, e.g., a human or rabbit variable region. In some embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In some embodiments, the variable region is a primate (e.g., non-human primate) variable region. In some embodiments, the variable region comprises rodent or mouse CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0037] As used herein, the term "complementarity determining region" or "CDR" refers to each region of an antibody variable domain that is a hypervariable sequence and / or forms a structurally defined loop (hypervariable loop) and / or contains antigen-contacting residues. An antibody can contain six CDRs, e.g., three CDRs in the VH and three CDRs in the VL.

[0038] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0039] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0040] The term "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding fragment thereof. In some embodiments, 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, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 (1991). Using the Kabat numbering system, the CDRs within an antibody heavy chain molecule are typically located at amino acids 31-35, which can optionally include one or two additional amino acids, followed by 35 (designated 35A and 35B in the Kabat numbering scheme) (CDR1), amino acids 50-65 (CDR2), and amino acids 95-102 (CDR3). Using the Kabat numbering system, the CDRs within an antibody light chain molecule are typically located at amino acids 24-34 (CDR1), amino acids 50-56 (CDR2), and amino acids 89-97 (CDR3).

[0041] Chothia instead refers to the location of the structural loops. Chothia et al., J. Mol. Biol., 196:901-917 (1987). The end of the Chothia CDR-H1 loop, when numbered using the Kabat numbering convention, varies between H32 and H34 depending on the length of the loop (because the Kabat numbering scheme places the insertion at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software.

[0042] As used herein, the terms "constant region" or "constant domain" are interchangeable and have their common meaning in the art. The constant region is the portion of an antibody, e.g., the carboxyl-terminal portion of the light and / or heavy chain, that is not directly involved in binding the antibody to an antigen, but can exhibit various effector functions, such as interacting with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence than the immunoglobulin variable domain. In some embodiments, the antibody or antigen-binding fragment comprises a constant region or portion thereof sufficient for antibody-dependent cell-mediated cytotoxicity (ADCC).

[0043] As used herein, the term "heavy chain," when used in reference to an antibody, can refer to any of the distinct types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain, which give rise to antibodies of the IgA, IgD, IgE, IgG, and IgM classes, respectively, and include the subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4. Heavy chain amino acid sequences are well known in the art. In some embodiments, the heavy chain is a human heavy chain.

[0044] As used herein, the term "light chain," when used in reference to an antibody, can refer to either distinct type, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In some aspects, the light chain is a human light chain.

[0045] The term "chimeric" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof whose amino acid sequences are derived 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 antigen-binding fragment thereof derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capacity, while the constant regions are homologous to sequences in an antibody or antigen-binding fragment thereof derived from another species (usually human) to avoid eliciting an immune response in that species.

[0046] The term "humanized" antibody or antigen-binding fragment thereof refers to a form of a specific immunoglobulin chain, a non-human (e.g., murine) antibody or antigen-binding fragment, a chimeric immunoglobulin, or a fragment thereof that contains minimal non-human (e.g., murine) sequence. Typically, a humanized antibody or antigen-binding fragment thereof is a human immunoglobulin in which residues from the complementarity-determining regions (CDRs) are replaced ("CDR-grafted") by residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, hamster) having the desired specificity, affinity, and capacity (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 instances, Fv framework region (FR) residues of a human immunoglobulin are replaced with corresponding residues in an antibody or fragment from a non-human species having the desired specificity, affinity, and capacity. Humanized antibodies or antigen-binding fragments thereof can be further modified by substituting any additional residues within the Fv framework regions and / or within the substituted non-human residues to improve and optimize the specificity, affinity, and / or capacity of the antibody or antigen-binding fragment. Generally, a humanized antibody or antigen-binding fragment thereof will comprise substantially all of at least one, typically two or three, variable domains containing all or substantially all of the CDR regions corresponding to the non-human immunoglobulin, while all or substantially all of the FR regions are human immunoglobulin consensus sequence regions. A humanized antibody or antigen-binding fragment thereof may also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate 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 embodiments, a "humanized antibody" is a resurfaced antibody.

[0047] The term "human" antibody or antigen-binding fragment thereof means an antibody or antigen-binding fragment thereof having an amino acid sequence derived from the human immunoglobulin locus, and such an antibody or antigen-binding fragment thereof is made using any technique known in the art. The definition of a human antibody or antigen-binding fragment thereof includes intact or full-length antibodies and fragments thereof.

[0048] "Binding affinity" generally refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or antigen-binding fragment thereof) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody or antigen-binding fragment thereof and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D Affinity can be expressed as the equilibrium dissociation constant (K D ) and the equilibrium association constant (K A It can be measured and / or expressed in several ways known in the art, including but not limited to, K D is K off / k on is calculated from the quotient of A is k on / k off It is calculated from the quotient of k on refers to the association rate constant of, for example, an antibody or antigen-binding fragment thereof to an antigen, and k off k refers to the dissociation rate constant of, for example, an antibody or antigen-binding fragment thereof from an antigen. on and k off can be determined by techniques known to those skilled in the art, such as Octet® BLI, BIAcore®, or KinExA.

[0049] As used herein, "epitope" is a term used in the art and refers to a localized region of an antigen to which an antibody or antigen-binding fragment thereof can specifically bind. An epitope can be, for example, consecutive amino acids of a polypeptide (a linear or continuous epitope), or an epitope can be derived from, for example, two or more non-contiguous regions of one or more polypeptides (a conformational, non-linear, punctuated, or discontinuous epitope). In some embodiments, the epitope to which an antibody or antigen-binding fragment thereof binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA assay, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assay, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). In the case of X-ray crystallography, crystallization can be achieved using any method known in the art. See, for example, Giege et al., Acta Crystallogr D Biol. 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 / antigen-binding fragment thereof:antigen crystals can be examined using well-known X-ray diffraction techniques, such as X-PLOR (Yale University, 1992, published by Molecular Simulations, Inc.; see, e.g., Meth. Enzymol. (1985) volumes 114 & 115, Wyckoff et al., eds., U.S. Patent Application Publication No. 2004 / 0014194), and BUSTER (Bricogne, Acta Crystallogr. D Biol. Crystallogr., 49(Pt 1):37-60 (1993); Bricogne, Meth. Enzymol., 276A:361-423, Carter CW, ed. (1997), Roversi et al., Acta Crystallogr. D Biol. Crystallogr., 56(Pt 10):1316-1323 (2000)). Mutagenesis mapping studies can be accomplished using any method known to those skilled in the art. For a description of mutagenesis techniques, including alanine scanning mutagenesis, see, for example, Champe et al., J. Biol. Chem., 270:1388-1394 and Cunningham et al., Science, 244:1081-1085 (1989).

[0050] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in a form found in nature. In some embodiments, an isolated antibody, polynucleotide, vector, cell, or composition is substantially pure. As used herein, "substantially pure" refers to 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.

[0051] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers can be linear or branched, can comprise modified amino acids, and can be interrupted by non-amino acids. The term also encompasses amino acid polymers modified, naturally or by intervention, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within this definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art. Because the polypeptides of the present invention are based on antibodies, it is understood that in some aspects the polypeptides can occur as single chains or associated chains.

[0052] As used herein, the term "host cell" can be any type of cell, for example, a primary cell, a cell in culture, or a cell from a cell line. In some aspects, the term "host cell" refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due, for example, to mutations or environmental influences that may occur in subsequent generations or upon integration of the nucleic acid molecule into the host cell genome.

[0053] The term "pharmaceutically acceptable carrier" includes any and all solvents, cosolvents, complexing agents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are biologically or otherwise undesirable. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in therapeutic formulations is contemplated. Supplementary active ingredients can also be incorporated into the formulation. In addition, various excipients, such as those commonly used in the art, can be included. These and other such compounds are described in literature, for example, in The Merck Index, Merck & Company, Rahway, NJ. Considerations for including various ingredients in pharmaceutical compositions are described, for example, in Gilman et al. (eds.) (2010); Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 12th Edition, The McGraw-Hill Companies.

[0054] As used herein, the term "subject" refers to a human or non-human mammal, such as a dog, cat, mouse, rat, cow, sheep, pig, goat, non-human primate, or bird, such as a chicken, as well as any other vertebrate or invertebrate. In some embodiments, the subject is a human. In some embodiments, the subject is a female. In some embodiments, the female is of reproductive age. In some embodiments, the subject suffers from and / or shows at least one symptom of PCOS. In some embodiments, the subject has been identified or diagnosed as having PCOS. In some embodiments, the subject is suspected of having PCOS.

[0055] 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 important roles in stress response, anxiety and depression, wakefulness, feeding behavior, energy metabolism, and the control of digestive and cardiovascular functions. CRH is a 41-amino acid peptide derived by enzymatic cleavage from a 196-amino acid preprohormone and is 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).

[0056] CRH functions through two distinct G protein-coupled receptors, CRHR1 and CRHR2. CRHR1 expression is prevalent in brain regions responsible for sensory and motor control, including the palpebral cortex, olfactory bulb, hippocampus, amygdala, basal ganglia, medial and lateral hypothalamic nuclei, and cerebellum. In contrast, CRHR2 expression is prevalent in subcortical regions, including the lateral septum, bed nucleus of the stria terminalis, ventromedial hypothalamic nucleus, and medial and cortical nuclei of the amygdala. In the anterior pituitary, CRHR1 mediates the release of ACTH in response to CRH.

[0057] In response to stress, the hypothalamus releases CRH, which triggers the release of ACTH from the anterior pituitary gland into the circulation. ACTH then binds to its receptors on the adrenal cortex, triggering the release of stress hormones such as cortisol. This entire system, known as the hypothalamic-pituitary-adrenal (HPA) axis, plays an important role in the fight-or-flight response to stress.

[0058] As used herein, the term "polycystic ovary syndrome" is used interchangeably with the terms "polycystic ovarian syndrome," "PCOS," "polycystic ovarian disease," "polycystic ovarian disease," and "Stein-Leventhal syndrome." Women diagnosed with PCOS exhibit one or more of the following symptoms: anovulation (e.g., irregular or amenorrheal periods), hyperandrogenism (e.g., elevated testosterone, free testosterone, androstenedione, 11-oxygenated androgens, or a combination thereof), abnormal uterine bleeding, polycystic ovarian enlargement, infertility, obesity, insulin resistance, hyperinsulinemia, hypertension, hyperlipidemia, type 2 diabetes, hirsutism, excessive facial hair growth, hair loss, and acne. In some embodiments, a diagnosis of PCOS is based on biochemically documented hyperandrogenism (e.g., testosterone, free testosterone, androstenedione, 11-oxygenated androgens, or a combination thereof, more than two standard deviations above the control mean), chronic anovulation (e.g., fewer than six menses per year or dysfunctional uterine bleeding), and / or polycystic ovaries present on a vaginal ovarian ultrasound.

[0059] As used herein, the term "polycystic ovary syndrome therapy" or "PCOS therapy" refers to any therapy for PCOS. PCOS therapy includes, but is not limited to, combined contraceptives (e.g., oral contraceptives containing estrogen and progestin) or progestin therapy, for example, to regulate menstrual cycle. PCOS therapy also includes, but is not limited to, clomiphene, tamoxifen, aromatase inhibitors (e.g., letrozole), metformin, gonadotropin, or ovarian drilling, for example, to promote ovulation. PCOS therapy also includes, but is not limited to, combined contraceptives (e.g., oral contraceptives containing estrogen and progestin), spironolactone, eflornithine, or electrolysis, for example, to reduce excessive hair growth. PCOS therapy also includes, but is not limited to, antidiabetic drugs (e.g., metformin) for example, to reduce insulin and / or blood sugar levels.

[0060] 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-oxygenated androgens, or combinations thereof), for example, in serum.

[0061] As used herein, the term "11-oxygenated androgens" refers to 19-carbon steroids that originate primarily from the adrenal gland and share an oxygen atom on carbon 11. Examples of 11-oxygenated androgens include, but are not limited to, 11-hydroxyandrostenedione (11OHA4), 11-hydroxytestosterone (11OHT), 11-ketoandrostenedione (11KA4), and 11-ketotestosterone (11KT).

[0062] As used herein, the term "hirsutism" refers to a condition in which a subject exhibits abnormal hair formation.

[0063] As used herein, the term "hyperinsulinemia" refers to a condition in which a subject exhibits elevated plasma insulin levels.

[0064] As used herein, the term "hyperlipidemia" refers to a condition in which a subject exhibits elevated levels of any or all lipids in the plasma.

[0065] As used herein, the term "hypertension" refers to a condition in which a subject suffers from persistently high blood pressure (ie, systolic blood pressure equal to or greater than 140 mmHg and diastolic blood pressure equal to or greater than 90 mmHg).

[0066] As used herein, the term "type 2 diabetes" refers to a disease also known as non-insulin dependent diabetes mellitus (NIDDM) or adult-onset diabetes mellitus (AODM), in which a subject has high levels of blood glucose.

[0067] As used herein, the term "insulin resistance" refers to a subnormal biological response to a given concentration of insulin (eg, reduced glucose transport across cell membranes in response to insulin).

[0068] As used herein, the term "treat" or "treatment" refers to curative or palliative measures. Beneficial or desired clinical results include, but are not limited to, the total or partial alleviation of symptoms associated with a disease or disorder or condition, whether detectable or undetectable, a decrease in the extent of the disease, stabilization of the disease state (i.e., not worsening), a delay or slowing of disease progression, relief or alleviation of the disease state (e.g., one or more symptoms of the disease), and remission (whether partial or total). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0069] As used herein, a "therapeutically effective amount" is the amount of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein required to reduce or alleviate the severity of PCOS or any of its symptoms (e.g., insulin resistance, hyperinsulinemia, type 2 diabetes, obesity, hypertension, hyperlipidemia, ovulation or irregular ovulation, infertility, hyperandrogenism, hirsutism, alopecia, acne, polycystic ovarian enlargement, and abnormal uterine bleeding over a period of time). A therapeutically effective amount also refers to the amount required to improve clinical symptoms in a subject in need thereof.

[0070] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to refer to "one or more" of all listed or enumerated elements.

[0071] The term "and / or," as used herein, should be construed as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or" used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: 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).

[0072] Whenever an embodiment is described herein with the word "comprising," it is to be understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0073] The term "about" refers to a value or composition that falls within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one or more standard deviations, as practiced in the art. Alternatively, "about" can mean a range of up to 10% or 20% (i.e., ±10% or ±20%). For example, about 3 mg can include all numbers between 2.7 mg and 3.3 mg (in the 10% case) or between 2.4 mg and 3.6 mg (in the 20% case). Furthermore, particularly with respect to biological systems or processes, the term can mean within an order of magnitude or up to five times the value. When a particular value or composition is provided in this application and claims, unless otherwise specified, the meaning of "about" should be assumed to be within an acceptable error range for that particular value or composition.

[0074] As disclosed herein, any concentration range, percentage range, ratio range, or integer range should be understood to include all integer values ​​within the recited range, and fractions thereof (such as integer tenths and hundredths), where appropriate, unless otherwise indicated.

[0075] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 5th ed., 2013, Academic Press, and Oxford Dictionary of Biochemistry And Molecular Biology, 2006, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0076] Units, prefixes, and symbols are denoted in the form accepted by the Systeme International de Unites (SI). Numerical ranges are inclusive of the numbers defining the range. The headings provided herein are not intended to limit the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are fully defined by reference to the specification as a whole.

[0077] Various aspects are described in further detail in the following sections.

[0078] 2. Anti-CRH antibody or antigen-binding fragment thereof The methods and kits of the disclosure contain an antibody or antigen-binding fragment thereof that specifically binds to corticotropin-releasing hormone (CRH) (ie, an anti-CRH antibody or antigen-binding fragment thereof).

[0079] Examples of anti-CRH antibodies and antigen-binding fragments thereof are known, and are described, for example, in International Publication No. 2019 / 241127, International Publication No. 2020 / 115555, Chinese Patent Application Publication No. 107043752, 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), which are incorporated by reference in their entireties.

[0080] Other examples of anti-CRH antibodies and antigen-binding fragments thereof are described, for example, in US Provisional Application No. 63 / 234,130, which is incorporated by reference herein in its entirety.

[0081] In some embodiments, the anti-CRH antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) CDR1 comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of SEQ ID NOs: 22-35; a VH CDR2 comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of SEQ ID NOs: 53-78; and a VH CDR3 comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of SEQ ID NOs: 113-122 or the amino acid sequence of a PDV or GID. and / or a light chain variable domain (VL) CDR1 comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of SEQ ID NOs: 152-174, a VL CDR2 comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of SEQ ID NOs: 193-198, and a VL CDR3 comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of SEQ ID NOs: 223-232.

[0082] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 53, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 113, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 152, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 193, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 223.

[0083] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 54, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 113, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 152, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 193, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 223.

[0084] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 55, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 113, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 152, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 193, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 223.

[0085] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 53, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 113, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 153, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 193, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 223.

[0086] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 54, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 113, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 153, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 193, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 223.

[0087] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 55, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 113, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 153, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 193, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 223.

[0088] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 54, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 113, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 154, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 193, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 223.

[0089] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 54, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 113, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 155, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 193, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 223.

[0090] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 54, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 113, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 156, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 193, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 223.

[0091] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 54, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 113, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 157, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 193, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 223.

[0092] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 55, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 113, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 154, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 193, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 223.

[0093] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 55, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 113, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 155, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 193, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 223.

[0094] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 55, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 113, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 156, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 193, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 223.

[0095] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 55, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 113, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 157, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 193, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 223.

[0096] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 56, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 113, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 154, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 193, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 223.

[0097] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 56, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 113, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 155, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 193, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 223.

[0098] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 56, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 113, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 156, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 193, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 223.

[0099] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 56, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 113, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 157, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 193, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 223.

[0100] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 23, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 57, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 114, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 158, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 193, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 224.

[0101] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 23, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 58, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 114, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 158, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 193, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 224.

[0102] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 23, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 59, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 114, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 158, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 193, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 224.

[0103] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 23, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 60, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 114, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 158, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 193, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 224.

[0104] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 24, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 61, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 115, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 159, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 194, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 225.

[0105] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 25, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 62, the VH CDR3 comprises the amino acid sequence of PDV, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 160, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 195, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 226.

[0106] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 24, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 63, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 115, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 161, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 196, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 225.

[0107] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 26, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 64, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 116, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 162, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 227.

[0108] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 24, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 63, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 115, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 163, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 196, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 225.

[0109] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 24, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 65, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 115, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 161, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 196, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 225.

[0110] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 27, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 66, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 117, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 164, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 228.

[0111] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 28, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 67, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 118, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 165, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 227.

[0112] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 29, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 68, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 119, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 166, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 227.

[0113] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 30, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 69, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 120, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 167, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 229.

[0114] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 29, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 70, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 121, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 166, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 230.

[0115] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 31, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 71, the VH CDR3 comprises the amino acid sequence of GID, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 168, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 198, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 231.

[0116] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 32, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 72, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 118, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 166, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 227.

[0117] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 33, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 73, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 118, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 169, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 227.

[0118] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 34, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 71, the VH CDR3 comprises the amino acid sequence of GID, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 168, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 198, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 231.

[0119] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 30, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 74, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 122, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 170, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 232.

[0120] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 35, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 71, the VH CDR3 comprises the amino acid sequence of GID, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 168, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 198, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 231.

[0121] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 29, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 75, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 121, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 171, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 230.

[0122] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 29, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 76, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 121, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 171, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 230.

[0123] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 29, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 77, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 121, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 171, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 230.

[0124] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 29, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 75, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 121, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 172, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 230.

[0125] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 29, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 75, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 121, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 173, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 230.

[0126] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 29, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 76, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 121, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 172, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 230.

[0127] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 29, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 77, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 121, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 173, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 230.

[0128] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 29, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 76, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 121, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 173, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 230.

[0129] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 29, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 77, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 121, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 172, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 230.

[0130] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 29, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 78, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 121, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 172, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 230.

[0131] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 29, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 78, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 121, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 173, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 230.

[0132] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 29, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 76, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 121, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 174, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 230.

[0133] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 29, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 77, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 121, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 174, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 230.

[0134] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 29, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 78, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 121, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 174, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 230.

[0135] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 29, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 78, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 121, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 171, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 230.

[0136] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO:29, the VH CDR2 comprises the amino acid sequence of SEQ ID NO:75, the VH CDR3 comprises the amino acid sequence of SEQ ID NO:121, the VL CDR1 comprises the amino acid sequence of SEQ ID NO:174, the VL CDR2 comprises the amino acid sequence of SEQ ID NO:197, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:230; or the VH CDR1 comprises the amino acid sequence of SEQ ID NO:22, the VH CDR2 comprises the amino acid sequence of SEQ ID NO:53, the VH CDR3 comprises the amino acid sequence of SEQ ID NO:113, the VL CDR1 comprises the amino acid sequence of SEQ ID NO:152, the VL CDR2 comprises the amino acid sequence of SEQ ID NO:193, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:223.

[0137] In some embodiments, the anti-CRH antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of SEQ ID NOs: 240-295, 459, and 461, and / or a VL comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 296-347 and 462. In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising the amino acid sequence of any one of SEQ ID NOs: 240-295, 459, and 461, and / or a VL comprising the amino acid sequence of any one of SEQ ID NOs: 296-347 and 462.

[0138] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:240 and / or the VL comprises the amino acid sequence of SEQ ID NO:296.

[0139] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:241 and / or the VL comprises the amino acid sequence of SEQ ID NO:296.

[0140] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:242 and / or the VL comprises the amino acid sequence of SEQ ID NO:296.

[0141] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:240 and / or the VL comprises the amino acid sequence of SEQ ID NO:297.

[0142] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:241 and / or the VL comprises the amino acid sequence of SEQ ID NO:297.

[0143] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:242 and / or the VL comprises the amino acid sequence of SEQ ID NO:297.

[0144] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:241 and / or the VL comprises the amino acid sequence of SEQ ID NO:298.

[0145] In some aspects, the VH comprises the amino acid sequence of SEQ ID NO:241 and the VL comprises the amino acid sequence of SEQ ID NO:299.

[0146] In some aspects, the VH comprises the amino acid sequence of SEQ ID NO:241 and the VL comprises the amino acid sequence of SEQ ID NO:300.

[0147] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:241 and / or the VL comprises the amino acid sequence of SEQ ID NO:301.

[0148] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:242 and / or the VL comprises the amino acid sequence of SEQ ID NO:298.

[0149] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:242 and / or the VL comprises the amino acid sequence of SEQ ID NO:299.

[0150] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:242 and / or the VL comprises the amino acid sequence of SEQ ID NO:300.

[0151] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:242 and / or the VL comprises the amino acid sequence of SEQ ID NO:301.

[0152] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:243 and / or the VL comprises the amino acid sequence of SEQ ID NO:298.

[0153] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:243 and / or the VL comprises the amino acid sequence of SEQ ID NO:299.

[0154] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:243 and / or the VL comprises the amino acid sequence of SEQ ID NO:300.

[0155] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:243 and / or the VL comprises the amino acid sequence of SEQ ID NO:301.

[0156] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:244 and / or the VL comprises the amino acid sequence of SEQ ID NO:302.

[0157] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:245 and / or the VL comprises the amino acid sequence of SEQ ID NO:302.

[0158] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:246 and / or the VL comprises the amino acid sequence of SEQ ID NO:302.

[0159] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:247 and / or the VL comprises the amino acid sequence of SEQ ID NO:302.

[0160] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:248 and / or the VL comprises the amino acid sequence of SEQ ID NO:303.

[0161] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:249 and / or the VL comprises the amino acid sequence of SEQ ID NO:304.

[0162] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:250 and / or the VL comprises the amino acid sequence of SEQ ID NO:305.

[0163] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:251 and / or the VL comprises the amino acid sequence of SEQ ID NO:306.

[0164] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 252 and / or the VL comprises the amino acid sequence of SEQ ID NO: 305.

[0165] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 252 and / or the VL comprises the amino acid sequence of SEQ ID NO: 307.

[0166] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:253 and / or the VL comprises the amino acid sequence of SEQ ID NO:308.

[0167] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:254 and / or the VL comprises the amino acid sequence of SEQ ID NO:309.

[0168] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 255 and / or the VL comprises the amino acid sequence of SEQ ID NO: 310.

[0169] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:255 and / or the VL comprises the amino acid sequence of SEQ ID NO:311.

[0170] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 255 and / or the VL comprises the amino acid sequence of SEQ ID NO: 312.

[0171] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:256 and / or the VL comprises the amino acid sequence of SEQ ID NO:310.

[0172] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:256 and / or the VL comprises the amino acid sequence of SEQ ID NO:311.

[0173] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:256 and / or the VL comprises the amino acid sequence of SEQ ID NO:312.

[0174] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:257 and / or the VL comprises the amino acid sequence of SEQ ID NO:310.

[0175] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:257 and / or the VL comprises the amino acid sequence of SEQ ID NO:311.

[0176] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:257 and / or the VL comprises the amino acid sequence of SEQ ID NO:312.

[0177] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:258 and / or the VL comprises the amino acid sequence of SEQ ID NO:310.

[0178] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:258 and / or the VL comprises the amino acid sequence of SEQ ID NO:311.

[0179] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:258 and / or the VL comprises the amino acid sequence of SEQ ID NO:312.

[0180] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:259 and / or the VL comprises the amino acid sequence of SEQ ID NO:310.

[0181] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:259 and / or the VL comprises the amino acid sequence of SEQ ID NO:311.

[0182] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:259 and / or the VL comprises the amino acid sequence of SEQ ID NO:312.

[0183] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:260 and / or the VL comprises the amino acid sequence of SEQ ID NO:317.

[0184] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:260 and / or the VL comprises the amino acid sequence of SEQ ID NO:313.

[0185] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:261 and / or the VL comprises the amino acid sequence of SEQ ID NO:314.

[0186] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:261 and / or the VL comprises the amino acid sequence of SEQ ID NO:315.

[0187] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:261 and / or the VL comprises the amino acid sequence of SEQ ID NO:316.

[0188] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:262 and / or the VL comprises the amino acid sequence of SEQ ID NO:317.

[0189] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:261 and / or the VL comprises the amino acid sequence of SEQ ID NO:317.

[0190] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:263 and / or the VL comprises the amino acid sequence of SEQ ID NO:317.

[0191] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:264 and / or the VL comprises the amino acid sequence of SEQ ID NO:317.

[0192] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:262 and / or the VL comprises the amino acid sequence of SEQ ID NO:318.

[0193] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:263 and / or the VL comprises the amino acid sequence of SEQ ID NO:318.

[0194] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:264 and / or the VL comprises the amino acid sequence of SEQ ID NO:318.

[0195] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:260 and / or the VL comprises the amino acid sequence of SEQ ID NO:318.

[0196] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:264 and / or the VL comprises the amino acid sequence of SEQ ID NO:314.

[0197] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:264 and / or the VL comprises the amino acid sequence of SEQ ID NO:316.

[0198] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:260 and / or the VL comprises the amino acid sequence of SEQ ID NO:319.

[0199] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:260 and / or the VL comprises the amino acid sequence of SEQ ID NO:314.

[0200] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:260 and / or the VL comprises the amino acid sequence of SEQ ID NO:315.

[0201] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:260 and / or the VL comprises the amino acid sequence of SEQ ID NO:316.

[0202] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:261 and / or the VL comprises the amino acid sequence of SEQ ID NO:313.

[0203] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:261 and / or the VL comprises the amino acid sequence of SEQ ID NO:318.

[0204] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:261 and / or the VL comprises the amino acid sequence of SEQ ID NO:319.

[0205] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:265 and / or the VL comprises the amino acid sequence of SEQ ID NO:320.

[0206] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:266 and / or the VL comprises the amino acid sequence of SEQ ID NO:321.

[0207] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:267 and / or the VL comprises the amino acid sequence of SEQ ID NO:322.

[0208] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:268 and / or the VL comprises the amino acid sequence of SEQ ID NO:323.

[0209] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:269 and / or the VL comprises the amino acid sequence of SEQ ID NO:324.

[0210] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:270 and / or the VL comprises the amino acid sequence of SEQ ID NO:325.

[0211] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:271 and / or the VL comprises the amino acid sequence of SEQ ID NO:326.

[0212] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:272 and / or the VL comprises the amino acid sequence of SEQ ID NO:331.

[0213] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:272 and / or the VL comprises the amino acid sequence of SEQ ID NO:327.

[0214] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:273 and / or the VL comprises the amino acid sequence of SEQ ID NO:328.

[0215] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:273 and / or the VL comprises the amino acid sequence of SEQ ID NO:329.

[0216] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:273 and / or the VL comprises the amino acid sequence of SEQ ID NO:330.

[0217] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:273 and / or the VL comprises the amino acid sequence of SEQ ID NO:331.

[0218] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:274 and / or the VL comprises the amino acid sequence of SEQ ID NO:331.

[0219] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:275 and / or the VL comprises the amino acid sequence of SEQ ID NO:331.

[0220] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:276 and / or the VL comprises the amino acid sequence of SEQ ID NO:331.

[0221] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:277 and / or the VL comprises the amino acid sequence of SEQ ID NO:331.

[0222] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:272 and / or the VL comprises the amino acid sequence of SEQ ID NO:332.

[0223] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:276 and / or the VL comprises the amino acid sequence of SEQ ID NO:332.

[0224] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:275 and / or the VL comprises the amino acid sequence of SEQ ID NO:328.

[0225] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:275 and / or the VL comprises the amino acid sequence of SEQ ID NO:330.

[0226] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:277 and / or the VL comprises the amino acid sequence of SEQ ID NO:328.

[0227] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:277 and / or the VL comprises the amino acid sequence of SEQ ID NO:330.

[0228] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:272 and / or the VL comprises the amino acid sequence of SEQ ID NO:333.

[0229] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:272 and / or the VL comprises the amino acid sequence of SEQ ID NO:328.

[0230] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:272 and / or the VL comprises the amino acid sequence of SEQ ID NO:329.

[0231] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:272 and / or the VL comprises the amino acid sequence of SEQ ID NO:330.

[0232] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:273 and / or the VL comprises the amino acid sequence of SEQ ID NO:327.

[0233] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:273 and / or the VL comprises the amino acid sequence of SEQ ID NO:332.

[0234] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:273 and / or the VL comprises the amino acid sequence of SEQ ID NO:333.

[0235] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:278 and / or the VL comprises the amino acid sequence of SEQ ID NO:334.

[0236] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:279 and / or the VL comprises the amino acid sequence of SEQ ID NO:323.

[0237] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:280 and / or the VL comprises the amino acid sequence of SEQ ID NO:339.

[0238] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:280 and / or the VL comprises the amino acid sequence of SEQ ID NO:335.

[0239] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:281 and / or the VL comprises the amino acid sequence of SEQ ID NO:336.

[0240] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:281 and / or the VL comprises the amino acid sequence of SEQ ID NO:337.

[0241] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:281 and / or the VL comprises the amino acid sequence of SEQ ID NO:338.

[0242] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:282 and / or the VL comprises the amino acid sequence of SEQ ID NO:339.

[0243] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:281 and / or the VL comprises the amino acid sequence of SEQ ID NO:339.

[0244] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:283 and / or the VL comprises the amino acid sequence of SEQ ID NO:339.

[0245] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:284 and / or the VL comprises the amino acid sequence of SEQ ID NO:339.

[0246] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:285 and / or the VL comprises the amino acid sequence of SEQ ID NO:339.

[0247] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:286 and / or the VL comprises the amino acid sequence of SEQ ID NO:339.

[0248] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:282 and / or the VL comprises the amino acid sequence of SEQ ID NO:340.

[0249] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:280 and / or the VL comprises the amino acid sequence of SEQ ID NO:340.

[0250] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:283 and / or the VL comprises the amino acid sequence of SEQ ID NO:340.

[0251] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:284 and / or the VL comprises the amino acid sequence of SEQ ID NO:340.

[0252] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:285 and / or the VL comprises the amino acid sequence of SEQ ID NO:340.

[0253] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:286 and / or the VL comprises the amino acid sequence of SEQ ID NO:340.

[0254] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:284 and / or the VL comprises the amino acid sequence of SEQ ID NO:336.

[0255] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:284 and / or the VL comprises the amino acid sequence of SEQ ID NO:338.

[0256] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:286 and / or the VL comprises the amino acid sequence of SEQ ID NO:336.

[0257] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:286 and / or the VL comprises the amino acid sequence of SEQ ID NO:338.

[0258] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:287 and / or the VL comprises the amino acid sequence of SEQ ID NO:340.

[0259] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:288 and / or the VL comprises the amino acid sequence of SEQ ID NO:340.

[0260] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:280 and / or the VL comprises the amino acid sequence of SEQ ID NO:341.

[0261] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:286 and / or the VL comprises the amino acid sequence of SEQ ID NO:342.

[0262] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:286 and / or the VL comprises the amino acid sequence of SEQ ID NO:343.

[0263] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:287 and / or the VL comprises the amino acid sequence of SEQ ID NO:342.

[0264] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:288 and / or the VL comprises the amino acid sequence of SEQ ID NO:343.

[0265] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:287 and / or the VL comprises the amino acid sequence of SEQ ID NO:343.

[0266] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:288 and / or the VL comprises the amino acid sequence of SEQ ID NO:342.

[0267] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:289 and / or the VL comprises the amino acid sequence of SEQ ID NO:342.

[0268] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:289 and / or the VL comprises the amino acid sequence of SEQ ID NO:343.

[0269] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:287 and / or the VL comprises the amino acid sequence of SEQ ID NO:344.

[0270] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:288 and / or the VL comprises the amino acid sequence of SEQ ID NO:344.

[0271] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:280 and / or the VL comprises the amino acid sequence of SEQ ID NO:336.

[0272] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:289 and / or the VL comprises the amino acid sequence of SEQ ID NO:344.

[0273] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:289 and / or the VL comprises the amino acid sequence of SEQ ID NO:340.

[0274] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:286 and / or the VL comprises the amino acid sequence of SEQ ID NO:344.

[0275] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:290 and / or the VL comprises the amino acid sequence of SEQ ID NO:336.

[0276] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:291 and / or the VL comprises the amino acid sequence of SEQ ID NO:336.

[0277] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:292 and / or the VL comprises the amino acid sequence of SEQ ID NO:336.

[0278] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:290 and / or the VL comprises the amino acid sequence of SEQ ID NO:345.

[0279] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:291 and / or the VL comprises the amino acid sequence of SEQ ID NO:345.

[0280] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:292 and / or the VL comprises the amino acid sequence of SEQ ID NO:345.

[0281] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:290 and / or the VL comprises the amino acid sequence of SEQ ID NO:346.

[0282] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:280 and / or the VL comprises the amino acid sequence of SEQ ID NO:337.

[0283] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:291 and / or the VL comprises the amino acid sequence of SEQ ID NO:346.

[0284] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:292 and / or the VL comprises the amino acid sequence of SEQ ID NO:346.

[0285] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:290 and / or the VL comprises the amino acid sequence of SEQ ID NO:347.

[0286] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:291 and / or the VL comprises the amino acid sequence of SEQ ID NO:347.

[0287] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:292 and / or the VL comprises the amino acid sequence of SEQ ID NO:347.

[0288] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:293 and / or the VL comprises the amino acid sequence of SEQ ID NO:340.

[0289] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:294 and / or the VL comprises the amino acid sequence of SEQ ID NO:340.

[0290] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:295 and / or the VL comprises the amino acid sequence of SEQ ID NO:340.

[0291] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:293 and / or the VL comprises the amino acid sequence of SEQ ID NO:343.

[0292] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:294 and / or the VL comprises the amino acid sequence of SEQ ID NO:343.

[0293] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:280 and / or the VL comprises the amino acid sequence of SEQ ID NO:338.

[0294] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:295 and / or the VL comprises the amino acid sequence of SEQ ID NO:343.

[0295] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:293 and / or the VL comprises the amino acid sequence of SEQ ID NO:342.

[0296] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:294 and / or the VL comprises the amino acid sequence of SEQ ID NO:342.

[0297] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:295 and / or the VL comprises the amino acid sequence of SEQ ID NO:342.

[0298] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:293 and / or the VL comprises the amino acid sequence of SEQ ID NO:344.

[0299] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:294 and / or the VL comprises the amino acid sequence of SEQ ID NO:344.

[0300] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:295 and / or the VL comprises the amino acid sequence of SEQ ID NO:344.

[0301] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:281 and / or the VL comprises the amino acid sequence of SEQ ID NO:345.

[0302] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:281 and / or the VL comprises the amino acid sequence of SEQ ID NO:346.

[0303] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:281 and / or the VL comprises the amino acid sequence of SEQ ID NO:347.

[0304] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:281 and / or the VL comprises the amino acid sequence of SEQ ID NO:335.

[0305] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:283 and / or the VL comprises the amino acid sequence of SEQ ID NO:343.

[0306] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:283 and / or the VL comprises the amino acid sequence of SEQ ID NO:342.

[0307] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:283 and / or the VL comprises the amino acid sequence of SEQ ID NO:344.

[0308] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:281 and / or the VL comprises the amino acid sequence of SEQ ID NO:340.

[0309] In some aspects, the VH comprises the amino acid sequence of SEQ ID NO: 281, and / or the VL comprises the amino acid sequence of SEQ ID NO: 341, the VH comprises the amino acid sequence of SEQ ID NO: 461, and / or the VL comprises the amino acid sequence of SEQ ID NO: 462, or the VH comprises the amino acid sequence of SEQ ID NO: 459, and / or the VL comprises the amino acid sequence of SEQ ID NO: 296.

[0310] In some embodiments, the anti-CRH antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of SEQ ID NOs: 348-403, 460, and 463, and / or a light chain comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of SEQ ID NOs: 404-455 and 464. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 348-403, 460, and 463, and / or a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 404-455 and 464.

[0311] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:348 and / or the light chain comprises the amino acid sequence of SEQ ID NO:404.

[0312] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:349 and / or the light chain comprises the amino acid sequence of SEQ ID NO:404.

[0313] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:350 and / or the light chain comprises the amino acid sequence of SEQ ID NO:404.

[0314] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:348 and / or the light chain comprises the amino acid sequence of SEQ ID NO:405.

[0315] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:349 and / or the light chain comprises the amino acid sequence of SEQ ID NO:405.

[0316] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:350 and / or the light chain comprises the amino acid sequence of SEQ ID NO:405.

[0317] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:349 and / or the light chain comprises the amino acid sequence of SEQ ID NO:406.

[0318] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:349 and / or the light chain comprises the amino acid sequence of SEQ ID NO:407.

[0319] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:349 and / or the light chain comprises the amino acid sequence of SEQ ID NO:408.

[0320] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:349 and / or the light chain comprises the amino acid sequence of SEQ ID NO:409.

[0321] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:350 and / or the light chain comprises the amino acid sequence of SEQ ID NO:406.

[0322] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:350 and / or the light chain comprises the amino acid sequence of SEQ ID NO:407.

[0323] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:350 and / or the light chain comprises the amino acid sequence of SEQ ID NO:408.

[0324] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:350 and / or the light chain comprises the amino acid sequence of SEQ ID NO:409.

[0325] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:351 and / or the light chain comprises the amino acid sequence of SEQ ID NO:406.

[0326] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:351 and / or the light chain comprises the amino acid sequence of SEQ ID NO:407.

[0327] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:351 and / or the light chain comprises the amino acid sequence of SEQ ID NO:408.

[0328] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:351 and / or the light chain comprises the amino acid sequence of SEQ ID NO:409.

[0329] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:352 and / or the light chain comprises the amino acid sequence of SEQ ID NO:410.

[0330] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:353 and / or the light chain comprises the amino acid sequence of SEQ ID NO:410.

[0331] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:354 and / or the light chain comprises the amino acid sequence of SEQ ID NO:410.

[0332] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:355 and / or the light chain comprises the amino acid sequence of SEQ ID NO:410.

[0333] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:356 and / or the light chain comprises the amino acid sequence of SEQ ID NO:411.

[0334] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:357 and / or the light chain comprises the amino acid sequence of SEQ ID NO:412.

[0335] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:358 and / or the light chain comprises the amino acid sequence of SEQ ID NO:413.

[0336] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:359 and / or the light chain comprises the amino acid sequence of SEQ ID NO:414.

[0337] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:360 and / or the light chain comprises the amino acid sequence of SEQ ID NO:413.

[0338] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:360 and / or the light chain comprises the amino acid sequence of SEQ ID NO:415.

[0339] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:361 and / or the light chain comprises the amino acid sequence of SEQ ID NO:416.

[0340] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:362 and / or the light chain comprises the amino acid sequence of SEQ ID NO:417.

[0341] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:363 and / or the light chain comprises the amino acid sequence of SEQ ID NO:418.

[0342] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:363 and / or the light chain comprises the amino acid sequence of SEQ ID NO:419.

[0343] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:363 and / or the light chain comprises the amino acid sequence of SEQ ID NO:420.

[0344] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:364 and / or the light chain comprises the amino acid sequence of SEQ ID NO:418.

[0345] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:364 and / or the light chain comprises the amino acid sequence of SEQ ID NO:419.

[0346] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:364 and / or the light chain comprises the amino acid sequence of SEQ ID NO:420.

[0347] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:365 and / or the light chain comprises the amino acid sequence of SEQ ID NO:418.

[0348] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:365 and / or the light chain comprises the amino acid sequence of SEQ ID NO:419.

[0349] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:365 and / or the light chain comprises the amino acid sequence of SEQ ID NO:420.

[0350] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:366 and / or the light chain comprises the amino acid sequence of SEQ ID NO:418.

[0351] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:366 and / or the light chain comprises the amino acid sequence of SEQ ID NO:419.

[0352] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:366 and / or the light chain comprises the amino acid sequence of SEQ ID NO:420.

[0353] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:367 and / or the light chain comprises the amino acid sequence of SEQ ID NO:418.

[0354] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:367 and / or the light chain comprises the amino acid sequence of SEQ ID NO:419.

[0355] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:367 and / or the light chain comprises the amino acid sequence of SEQ ID NO:420.

[0356] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:368 and / or the light chain comprises the amino acid sequence of SEQ ID NO:425.

[0357] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:368 and / or the light chain comprises the amino acid sequence of SEQ ID NO:421.

[0358] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:369 and / or the light chain comprises the amino acid sequence of SEQ ID NO:422.

[0359] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:369 and / or the light chain comprises the amino acid sequence of SEQ ID NO:423.

[0360] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:369 and / or the light chain comprises the amino acid sequence of SEQ ID NO:424.

[0361] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:370 and / or the light chain comprises the amino acid sequence of SEQ ID NO:425.

[0362] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:369 and / or the light chain comprises the amino acid sequence of SEQ ID NO:425.

[0363] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:371 and / or the light chain comprises the amino acid sequence of SEQ ID NO:425.

[0364] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:372 and / or the light chain comprises the amino acid sequence of SEQ ID NO:425.

[0365] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:370 and / or the light chain comprises the amino acid sequence of SEQ ID NO:426.

[0366] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:371 and / or the light chain comprises the amino acid sequence of SEQ ID NO:426.

[0367] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:372 and / or the light chain comprises the amino acid sequence of SEQ ID NO:426.

[0368] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:368 and / or the light chain comprises the amino acid sequence of SEQ ID NO:426.

[0369] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:372 and / or the light chain comprises the amino acid sequence of SEQ ID NO:422.

[0370] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:372 and / or the light chain comprises the amino acid sequence of SEQ ID NO:424.

[0371] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:368 and / or the light chain comprises the amino acid sequence of SEQ ID NO:427.

[0372] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:368 and / or the light chain comprises the amino acid sequence of SEQ ID NO:422.

[0373] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:368 and / or the light chain comprises the amino acid sequence of SEQ ID NO:423.

[0374] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:368 and / or the light chain comprises the amino acid sequence of SEQ ID NO:424.

[0375] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:369 and / or the light chain comprises the amino acid sequence of SEQ ID NO:421.

[0376] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:369 and / or the light chain comprises the amino acid sequence of SEQ ID NO:426.

[0377] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:369 and / or the light chain comprises the amino acid sequence of SEQ ID NO:427.

[0378] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:373 and / or the light chain comprises the amino acid sequence of SEQ ID NO:428.

[0379] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:374 and / or the light chain comprises the amino acid sequence of SEQ ID NO:429.

[0380] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:375 and / or the light chain comprises the amino acid sequence of SEQ ID NO:430.

[0381] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:376 and / or the light chain comprises the amino acid sequence of SEQ ID NO:431.

[0382] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:377 and / or the light chain comprises the amino acid sequence of SEQ ID NO:432.

[0383] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:378 and / or the light chain comprises the amino acid sequence of SEQ ID NO:433.

[0384] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:379 and / or the light chain comprises the amino acid sequence of SEQ ID NO:434.

[0385] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:380 and / or the light chain comprises the amino acid sequence of SEQ ID NO:439.

[0386] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:380 and / or the light chain comprises the amino acid sequence of SEQ ID NO:435.

[0387] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:381 and / or the light chain comprises the amino acid sequence of SEQ ID NO:436.

[0388] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:381 and / or the light chain comprises the amino acid sequence of SEQ ID NO:437.

[0389] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:381 and / or the light chain comprises the amino acid sequence of SEQ ID NO:438.

[0390] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:381 and / or the light chain comprises the amino acid sequence of SEQ ID NO:439.

[0391] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:382 and / or the light chain comprises the amino acid sequence of SEQ ID NO:439.

[0392] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:383 and / or the light chain comprises the amino acid sequence of SEQ ID NO:439.

[0393] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:384 and / or the light chain comprises the amino acid sequence of SEQ ID NO:439.

[0394] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:385 and / or the light chain comprises the amino acid sequence of SEQ ID NO:439.

[0395] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:380 and / or the light chain comprises the amino acid sequence of SEQ ID NO:440.

[0396] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:384 and / or the light chain comprises the amino acid sequence of SEQ ID NO:440.

[0397] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:383 and / or the light chain comprises the amino acid sequence of SEQ ID NO:436.

[0398] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:383 and / or the light chain comprises the amino acid sequence of SEQ ID NO:438.

[0399] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:385 and / or the light chain comprises the amino acid sequence of SEQ ID NO:436.

[0400] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:385 and / or the light chain comprises the amino acid sequence of SEQ ID NO:438.

[0401] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:380 and / or the light chain comprises the amino acid sequence of SEQ ID NO:441.

[0402] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:380 and / or the light chain comprises the amino acid sequence of SEQ ID NO:436.

[0403] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:380 and / or the light chain comprises the amino acid sequence of SEQ ID NO:437.

[0404] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:380 and / or the light chain comprises the amino acid sequence of SEQ ID NO:438.

[0405] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:381 and / or the light chain comprises the amino acid sequence of SEQ ID NO:435.

[0406] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:381 and / or the light chain comprises the amino acid sequence of SEQ ID NO:440.

[0407] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:381 and / or the light chain comprises the amino acid sequence of SEQ ID NO:441.

[0408] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:386 and / or the light chain comprises the amino acid sequence of SEQ ID NO:442.

[0409] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:387 and / or the light chain comprises the amino acid sequence of SEQ ID NO:431.

[0410] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:388 and / or the light chain comprises the amino acid sequence of SEQ ID NO:447.

[0411] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:388 and / or the light chain comprises the amino acid sequence of SEQ ID NO:443.

[0412] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:389 and / or the light chain comprises the amino acid sequence of SEQ ID NO:444.

[0413] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:389 and / or the light chain comprises the amino acid sequence of SEQ ID NO:445.

[0414] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:389 and / or the light chain comprises the amino acid sequence of SEQ ID NO:446.

[0415] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:390 and / or the light chain comprises the amino acid sequence of SEQ ID NO:447.

[0416] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:389 and / or the light chain comprises the amino acid sequence of SEQ ID NO:447.

[0417] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:391 and / or the light chain comprises the amino acid sequence of SEQ ID NO:447.

[0418] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:392 and / or the light chain comprises the amino acid sequence of SEQ ID NO:447.

[0419] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:393 and / or the light chain comprises the amino acid sequence of SEQ ID NO:447.

[0420] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:394 and / or the light chain comprises the amino acid sequence of SEQ ID NO:447.

[0421] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:390 and / or the light chain comprises the amino acid sequence of SEQ ID NO:448.

[0422] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:388 and / or the light chain comprises the amino acid sequence of SEQ ID NO:448.

[0423] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:391 and / or the light chain comprises the amino acid sequence of SEQ ID NO:448.

[0424] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:392 and / or the light chain comprises the amino acid sequence of SEQ ID NO:448.

[0425] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:393 and / or the light chain comprises the amino acid sequence of SEQ ID NO:448.

[0426] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:394 and / or the light chain comprises the amino acid sequence of SEQ ID NO:448.

[0427] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:392 and / or the light chain comprises the amino acid sequence of SEQ ID NO:444.

[0428] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:392 and / or the light chain comprises the amino acid sequence of SEQ ID NO:446.

[0429] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:394 and / or the light chain comprises the amino acid sequence of SEQ ID NO:444.

[0430] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:394 and / or the light chain comprises the amino acid sequence of SEQ ID NO:446.

[0431] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:395 and / or the light chain comprises the amino acid sequence of SEQ ID NO:448.

[0432] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:396 and / or the light chain comprises the amino acid sequence of SEQ ID NO:448.

[0433] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:388 and / or the light chain comprises the amino acid sequence of SEQ ID NO:449.

[0434] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:394 and / or the light chain comprises the amino acid sequence of SEQ ID NO:450.

[0435] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:394 and / or the light chain comprises the amino acid sequence of SEQ ID NO:451.

[0436] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:395 and / or the light chain comprises the amino acid sequence of SEQ ID NO:450.

[0437] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:396 and / or the light chain comprises the amino acid sequence of SEQ ID NO:451.

[0438] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:395 and / or the light chain comprises the amino acid sequence of SEQ ID NO:451.

[0439] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:396 and / or the light chain comprises the amino acid sequence of SEQ ID NO:450.

[0440] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:397 and / or the light chain comprises the amino acid sequence of SEQ ID NO:450.

[0441] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:397 and / or the light chain comprises the amino acid sequence of SEQ ID NO:451.

[0442] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:395 and / or the light chain comprises the amino acid sequence of SEQ ID NO:452.

[0443] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:396 and / or the light chain comprises the amino acid sequence of SEQ ID NO:452.

[0444] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:388 and / or the light chain comprises the amino acid sequence of SEQ ID NO:444.

[0445] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:397 and / or the light chain comprises the amino acid sequence of SEQ ID NO:452.

[0446] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:397 and / or the light chain comprises the amino acid sequence of SEQ ID NO:448.

[0447] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:394 and / or the light chain comprises the amino acid sequence of SEQ ID NO:452.

[0448] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:398 and / or the light chain comprises the amino acid sequence of SEQ ID NO:444.

[0449] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:399 and / or the light chain comprises the amino acid sequence of SEQ ID NO:444.

[0450] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:400 and / or the light chain comprises the amino acid sequence of SEQ ID NO:444.

[0451] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:398 and / or the light chain comprises the amino acid sequence of SEQ ID NO:453.

[0452] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:399 and / or the light chain comprises the amino acid sequence of SEQ ID NO:453.

[0453] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:400 and / or the light chain comprises the amino acid sequence of SEQ ID NO:453.

[0454] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:398 and / or the light chain comprises the amino acid sequence of SEQ ID NO:454.

[0455] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:388 and / or the light chain comprises the amino acid sequence of SEQ ID NO:445.

[0456] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:399 and / or the light chain comprises the amino acid sequence of SEQ ID NO:454.

[0457] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:400 and / or the light chain comprises the amino acid sequence of SEQ ID NO:454.

[0458] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:398 and / or the light chain comprises the amino acid sequence of SEQ ID NO:455.

[0459] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:399 and / or the light chain comprises the amino acid sequence of SEQ ID NO:455.

[0460] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:400 and / or the light chain comprises the amino acid sequence of SEQ ID NO:455.

[0461] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:401 and / or the light chain comprises the amino acid sequence of SEQ ID NO:448.

[0462] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:402 and / or the light chain comprises the amino acid sequence of SEQ ID NO:448.

[0463] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:403 and / or the light chain comprises the amino acid sequence of SEQ ID NO:448.

[0464] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:401 and / or the light chain comprises the amino acid sequence of SEQ ID NO:451.

[0465] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:402 and / or the light chain comprises the amino acid sequence of SEQ ID NO:451.

[0466] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:388 and / or the light chain comprises the amino acid sequence of SEQ ID NO:446.

[0467] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:403 and / or the light chain comprises the amino acid sequence of SEQ ID NO:451.

[0468] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:401 and / or the light chain comprises the amino acid sequence of SEQ ID NO:450.

[0469] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:402 and / or the light chain comprises the amino acid sequence of SEQ ID NO:450.

[0470] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:403 and / or the light chain comprises the amino acid sequence of SEQ ID NO:450.

[0471] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:401 and / or the light chain comprises the amino acid sequence of SEQ ID NO:452.

[0472] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:402 and / or the light chain comprises the amino acid sequence of SEQ ID NO:452.

[0473] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:403 and / or the light chain comprises the amino acid sequence of SEQ ID NO:452.

[0474] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:389 and / or the light chain comprises the amino acid sequence of SEQ ID NO:453.

[0475] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:389 and / or the light chain comprises the amino acid sequence of SEQ ID NO:454.

[0476] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:389 and / or the light chain comprises the amino acid sequence of SEQ ID NO:455.

[0477] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:389 and / or the light chain comprises the amino acid sequence of SEQ ID NO:443.

[0478] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:391 and / or the light chain comprises the amino acid sequence of SEQ ID NO:451.

[0479] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:391 and / or the light chain comprises the amino acid sequence of SEQ ID NO:450.

[0480] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:391 and / or the light chain comprises the amino acid sequence of SEQ ID NO:452.

[0481] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:389 and / or the light chain comprises the amino acid sequence of SEQ ID NO:448.

[0482] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 389 and / or the light chain comprises the amino acid sequence of SEQ ID NO: 449, the heavy chain comprises the amino acid sequence of SEQ ID NO: 463 and / or the light chain comprises the amino acid sequence of SEQ ID NO: 464, or the heavy chain comprises the amino acid sequence of SEQ ID NO: 460 and / or the light chain comprises the amino acid sequence of SEQ ID NO: 404.

[0483] In some embodiments, the anti-CRH antibody or antigen-binding fragment is an antibody or antigen-binding fragment thereof in Table 1.

[0484] [Table 1-1]

[0485] [Table 1-2]

[0486] [Table 1-3]

[0487] [Table 1-4]

[0488] [Table 1-5]

[0489] [Table 1-6]

[0490] [Table 1-7]

[0491] [Table 1-8]

[0492] [Table 1-9]

[0493] [Table 1-10]

[0494] [Table 1-11]

[0495] In some aspects, the methods and kits of the present disclosure include a pharmaceutical composition comprising an anti-CRH antibody or antigen-binding fragment thereof disclosed herein and a pharmaceutically acceptable carrier.

[0496] In some embodiments, the anti-CRH antibodies used in the methods and kits disclosed herein are polyclonal, monoclonal, human, humanized, or chimeric.

[0497] In other embodiments, the anti-CRH antigen-binding fragment used in the methods and kits disclosed herein is a Fab, scFab, Fab', F(ab')2, Fv, scFv, diabody, or triabody.

[0498] 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, and / or light chain sequences disclosed herein, including, for example, using in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination to construct expression vectors containing sequences encoding such antibodies or antigen-binding fragments thereof or domains thereof (e.g., CDR, VH, VL, heavy chain, and / or light chain) and appropriate transcriptional and translational control signals.

[0499] The expression vector can be transferred into a cell (e.g., a host cell) by conventional techniques, and the resulting cells can then be cultured by conventional techniques to produce an antibody or antigen-binding fragment thereof (e.g., an antibody or antigen-binding fragment thereof comprising the six CDRs of an antibody disclosed herein, VH, VL, VH and VL, a heavy chain, a light chain, or a heavy chain and a light chain) or domain (e.g., the VH, VL, VH and VL, a heavy chain, or a light chain of an antibody disclosed herein) disclosed herein.

[0500] In some embodiments, when expressing a double-chain antibody or antigen-binding fragment thereof, vectors encoding both the heavy and light chains individually can be simultaneously expressed in a host cell to express the entire immunoglobulin. In some embodiments, the host cell contains a vector comprising polynucleotides encoding both the heavy and light chains of an antibody or domain thereof disclosed herein. In some embodiments, the host cell comprises two different vectors: a first vector comprising polynucleotides encoding the heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof disclosed herein, and a second vector comprising polynucleotides encoding the light chain or light chain variable region of an antibody or domain thereof disclosed herein. In some embodiments, the first host cell comprises a first vector comprising polynucleotides encoding the heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof disclosed herein, and the second host cell comprises a second vector comprising polynucleotides encoding the light chain or light chain variable region of an antibody or antigen-binding fragment thereof disclosed herein. In some embodiments, 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 an antibody or antigen-binding fragment thereof disclosed herein. In some embodiments herein, a host cell population comprising such a first host cell and such a second host cell is provided.

[0501] A variety of host-expression vector systems can be utilized to express the antibodies and antigen-binding fragments thereof disclosed herein. Such host-expression systems represent vehicles in which a coding sequence of interest may be produced and subsequently purified, but also represent cells that, when transformed or transfected with the appropriate nucleotide coding sequence, can express the proteins or antibodies or antigen-binding fragments thereof disclosed herein in situ. These include microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing antibody coding sequences; and plant cell systems (e.g., Chlamydomonas reinhardtii) infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus or CaMV, tobacco mosaic virus or TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences. reinhardtii); or mammalian cell lines (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK293, NS0, PER.C6, VERO, CRL7030, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells) harboring a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter, a vaccinia virus 7.5K promoter). In some embodiments, the cells for expressing the proteins or antibodies and antigen-binding fragments thereof disclosed herein are CHO cells, e.g., CHO cells from the CHO GS System™ (Lonza).In some embodiments, cells for expressing the antibodies and antigen-binding fragments thereof disclosed herein are human cells, e.g., human cell lines. In some embodiments, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In some embodiments, bacterial cells such as Escherichia coli, particularly for expression of whole recombinant antibody molecules, or eukaryotic cells (e.g., mammalian cells) are used for expression of recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary (CHO) cells in combination with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus are effective expression systems for antibodies (Foecking et al., Gene, 45:101-105 (1986) and Cockett et al., Biotechnology, 8:662-667 (1990)). In some embodiments, the proteins or antibodies or antigen-binding fragments thereof disclosed herein are produced by HEK-293T cells.

[0502] In addition, a host cell strain can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of the protein product may contribute to the function of the protein. To this end, eukaryotic host cells that possess the cellular machinery for proper processing, glycosylation, and phosphorylation of the primary transcript of the gene product can be used. Such 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.

[0503] Once produced by recombinant expression, the antibodies or antigen-binding fragments thereof disclosed herein can be purified by any method known in the art for the purification of proteins or immunoglobulin molecules, such as, for example, by chromatography (e.g., ion exchange, particularly protein A followed by affinity for the specific antigen, and size exclusion chromatography), centrifugation, differential solubility, or any other standard technique for the purification of proteins. Additionally, the antibodies or antigen-binding fragments thereof disclosed herein can be fused to heterologous polypeptide sequences disclosed herein or otherwise known in the art to facilitate purification.

[0504] In some embodiments, the methods and kits disclosed herein contain an isolated or purified antibody or antigen-binding fragment thereof. Generally, an isolated protein or antibody or antigen-binding fragment thereof is substantially free of other proteins or antibodies or antigen-binding fragments thereof that have antigen specificities different from those of the isolated antibody or antigen-binding fragment thereof. For example, in some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein are substantially free of cellular material and / or chemical precursors.

[0505] 3.How to use Patients with polycystic ovary syndrome (PCOS) synthesize and secrete excessive amounts of androgens from the adrenal glands and ovaries. Recent analyses of androgen sources in PCOS patients have revealed that the majority of these increased androgens originate from the adrenal glands (O'Reilly et al., J Clin Endocrinol Metab 102:840, 2017). In humans, the synthesis and secretion of adrenal androgens requires ACTH (Weber et al., Clin Endocrinol 46:431, 2003), and the synthesis and secretion of ACTH requires CRH (Muglia et al., Journal of Clinical Investigation, 105:1269, 2000). Therefore, blocking CRH action with an anti-CRH antibody in PCOS patients should reduce CRH, ACTH, and adrenal androgens in these patients.

[0506] In some embodiments, the present disclosure provides a method for treating PCOS and related disorders.The method comprises administering to a subject in need of treatment a therapeutically effective amount of the anti-CRH antibody or its antigen-binding fragment disclosed herein.In some embodiments, the method comprises administering to a subject a therapeutically effective amount of the pharmaceutical composition of the present disclosure, which comprises the anti-CRH antibody or its antigen-binding fragment disclosed herein.

[0507] In some embodiments, the present disclosure provides a method for reducing androgen levels in a subject with PCOS. The method comprises administering to the subject a therapeutically effective amount of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present disclosure containing an anti-CRH antibody or antigen-binding fragment thereof disclosed herein. In some embodiments, the androgen is testosterone, free testosterone, androstenedione, an 11-oxygenated androgen, or a combination thereof. In another embodiment, the 11-oxygenated androgen is 11-hydroxyandrostenedione (11OHA4), 11-hydroxytestosterone (11OHT), 11-ketoandrostenedione (11KA4), 11-ketotestosterone (11KT), or a combination thereof.

[0508] In some embodiments, the present disclosure provides a method for reducing the severity of one or more symptoms or signs in a subject with PCOS.The method comprises administering to a subject with PCOS a therapeutically effective amount of the anti-CRH antibody or its antigen-binding fragment disclosed herein.In some embodiments, the method comprises administering to a subject a therapeutically effective amount of the pharmaceutical composition of the present disclosure, which comprises the anti-CRH antibody or its antigen-binding fragment disclosed herein.

[0509] In some embodiments, one or more symptoms or signs of PCOS are anovulation (e.g., irregular menstrual periods or amenorrhea), hyperandrogenism (e.g., elevated testosterone, free testosterone, androstenedione, 11-oxygenated androgens, or a combination thereof), abnormal uterine bleeding, polycystic ovarian enlargement, infertility, obesity, insulin resistance, hyperinsulinemia, hypertension, hyperlipidemia, type 2 diabetes, excessive facial hair growth, hirsutism, alopecia, and acne. In another embodiment, the 11-oxygenated androgen is 11-hydroxyandrostenedione (11OHA4), 11-hydroxytestosterone (11OHT), 11-ketoandrostenedione (11KA4), 11-ketotestosterone (11KT), or a combination thereof.

[0510] In some embodiments, after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein, the subject exhibits a decrease in the severity of one or more symptoms of PCOS or an improvement in one or more symptoms of PCOS. In some embodiments, the one or more symptoms are anovulation (e.g., irregular menstrual periods or amenorrhea), hyperandrogenism (e.g., elevated testosterone, free testosterone, androstenedione, 11-oxygenated androgens, or a combination thereof), abnormal uterine bleeding, polycystic ovarian enlargement, infertility, obesity, insulin resistance, hyperinsulinemia, hypertension, hyperlipidemia, type 2 diabetes, excessive facial hair growth, hirsutism, alopecia, or acne. In another embodiment, the 11-oxygenated androgen is 11-hydroxyandrostenedione (11OHA4), 11-hydroxytestosterone (11OHT), 11-ketoandrostenedione (11KA4), 11-ketotestosterone (11KT), or a combination thereof.

[0511] In some embodiments, the subject exhibits reduced anovulation after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to before administration of the antibody or antigen-binding fragment thereof, hi some embodiments, the subject resumes ovulation after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein.

[0512] In some embodiments, the subject exhibits reduced menstrual irregularity after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to the menstrual irregularity before administration of the antibody or antigen-binding fragment thereof. In some embodiments, the subject resumes menstrual periods after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein. In some embodiments, the subject resumes regular menstrual periods after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein.

[0513] In some embodiments, a subject exhibits a decrease in hyperandrogenism after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to before administration of the antibody or antigen-binding fragment thereof. In some embodiments, a subject exhibits a decrease in testosterone, free testosterone, androstenedione, 11-oxygenated androgen, or a combination thereof after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to before administration of the antibody or antigen-binding fragment thereof. In another embodiment, the 11-oxygenated androgen is 11-hydroxyandrostenedione (11OHA4), 11-hydroxytestosterone (11OHT), 11-ketoandrostenedione (11KA4), 11-ketotestosterone (11KT), or a combination thereof.

[0514] In some embodiments, the subject exhibits a decrease in abnormal uterine bleeding after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to before administration of the antibody or antigen-binding fragment thereof.

[0515] In some embodiments, a subject exhibits fewer ovarian cysts and / or less polyfollicular ovaries after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to before administration of the antibody or antigen-binding fragment thereof.

[0516] In some aspects, the subject exhibits improved fertility after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to before administration of the antibody or antigen-binding fragment thereof.

[0517] In some aspects, the subject exhibits improved insulin resistance after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to before administration of the antibody or antigen-binding fragment thereof.

[0518] In some aspects, the subject exhibits improved hyperinsulinemia after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to before administration of the antibody or antigen-binding fragment thereof.

[0519] In some embodiments, the subject exhibits improvement in hypertension after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to before administration of the antibody or antigen-binding fragment thereof.

[0520] In some aspects, the subject exhibits an improvement in hyperlipidemia after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to before administration of the antibody or antigen-binding fragment thereof.

[0521] In some embodiments, a subject exhibits a decrease in excess hair growth after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to before administration of the antibody or antigen-binding fragment thereof.

[0522] In some aspects, the subject exhibits a decrease in hirsutism after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to before administration of the antibody or antigen-binding fragment thereof.

[0523] In some aspects, a subject exhibits a decrease in hair loss (e.g., male pattern baldness) after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to before administration of the antibody or antigen-binding fragment thereof.

[0524] In some embodiments, the subject exhibits a decrease in acne after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to before administration of the antibody or antigen-binding fragment thereof.

[0525] In some embodiments of any of the methods provided herein, the anti-CRH antibody or antigen-binding fragment thereof disclosed herein is administered in combination with one or more PCOS therapies. In some embodiments, the one or more PCOS therapies are selected from the group consisting of combined contraceptives (e.g., oral contraceptives containing estrogen and progestin), progestin therapy, clomiphene, tamoxifen, aromatase inhibitors (e.g., letrozole), gonadotropins, ovarian drilling, spironolactone, eflornithine, electrolysis, and antidiabetic agents (e.g., metformin). In some embodiments, the anti-CRH antibody or antigen-binding fragment thereof disclosed herein is administered in combination with one or more PCOS therapies selected from the group consisting of spironolactone, metformin, and combined contraceptives (e.g., oral contraceptives containing estrogen and progestin).

[0526] In some embodiments, the subject is a human. In some embodiments, the subject is a female. In some embodiments, the female is of reproductive age.

[0527] It may be desirable to detect the presence or measure the amount of CRH in a sample (e.g., a biological sample). In some aspects, the present disclosure provides a method for using an anti-CRH antibody or antigen-binding fragment thereof disclosed herein to measure the amount of CRH in a sample from a subject diagnosed with or suspected of having PCOS. To determine the measured amount of CRH, the subject's biological sample is contacted with an anti-CRH antibody or antigen-binding fragment thereof disclosed herein for a time sufficient to form an immune complex. The immune complex formed between the antibody and CRH in the sample is then detected. The amount of CRH in the biological sample is optionally quantified by measuring the amount of immune complex formed between the antibody and CRH. For example, the antibody can be quantitatively measured if it has a detectable label, or a secondary antibody can be used to quantify the immune complex.

[0528] In some aspects, the biological sample comprises a tissue sample, a cell sample, or a biological fluid sample such as blood, saliva, serum, or plasma.

[0529] Conditions for incubating the antibody with the test sample vary. Incubation conditions depend on the format used in the assay, the detection method employed, and the type and properties of the antibody used in the assay. Those skilled in the art will recognize that any one of the commonly available immunological assay formats can be easily adapted to utilize the antibodies (or fragments thereof) of the present disclosure. Examples of such assays can be found in An Introduction to Radioimmunoassay and Related Techniques by Chard, T., Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Techniques in Immunocytochemistry by Bullock et al., Academic Press, Orlando, FL, Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); and Practice and Theory of Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology by Tijssen, Elsevier Science Publishers, Amsterdam, The Netherlands (1985). The test sample used in the above-described methods will vary based on the assay format, nature of the detection method, and the tissues, cells, or fluids used as the sample to be assayed.

[0530] Such methods are useful, for example, in evaluating the effectiveness of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring treatment for individual patients.

[0531] In some embodiments, the CRH or anti-CRH antibody or antigen-binding fragment thereof is attached to a solid support, and binding is detected by detecting a complex between the CRH and the anti-CRH antibody or antigen-binding fragment thereof on the solid support. The anti-CRH antibody or antigen-binding fragment thereof optionally comprises a detectable label, and binding is detected by detecting the label in the CRH-antibody complex.

[0532] Detection of the presence or absence of CRH-antibody complexes can be achieved using any method known in the art. For example, transcripts obtained from reporter gene transcription assays of CRH peptides interacting with target molecules (e.g., antibodies) typically encode directly or indirectly detectable products (e.g., β-galactosidase activity and luciferase activity). In the case of cell-free binding assays, one of the components usually contains or is bound to a detectable label. A wide variety of labels can be used, including those that provide direct detection (radioactivity, luminescence, optical density, or electron density) or indirect detection (epitope tags such as FLAG epitopes, enzyme tags such as horseradish peroxidase, etc.). The label can be bound to the antibody or incorporated into the antibody structure.

[0533] According to the nature of label and other assay components, label can be detected by various methods.For example, label can be detected while being bound to solid substrate or after being separated from solid substrate.Label can be directly detected by optical density or electron density, radioactive emission, non-radioactive energy transfer, or can be indirectly detected by using antibody conjugate or streptavidin-biotin conjugate.The method for detecting label is well known in the art.

[0534] 4. Kit In some aspects, the present disclosure provides kits comprising an anti-CRH antibody or antigen-binding fragment thereof disclosed herein.

[0535] In some embodiments, the kit comprises (i) an anti-CRH antibody or antigen-binding fragment disclosed herein and (ii) instructions for using the anti-CRH antibody or antigen-binding fragment in the treatment of PCOS in a subject in need thereof.

[0536] In some embodiments, the kit includes (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 levels (e.g., testosterone, free testosterone, androstenedione, 11-oxygenated androgens, or a combination thereof) in a subject with PCOS. In another embodiment, the 11-oxygenated androgen is 11-hydroxyandrostenedione (11OHA4), 11-hydroxytestosterone (11OHT), 11-ketoandrostenedione (11KA4), 11-ketotestosterone (11KT), or a combination thereof.

[0537] In some embodiments, the kit comprises (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.

[0538] In some embodiments, the anti-CRH antibody or antigen-binding fragment in the kit is present in a pharmaceutical composition disclosed herein.

[0539] In some embodiments, the anti-CRH antibody, antigen-binding fragment, or pharmaceutical composition is stored in a container such as a sterile vial as a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated or lyophilized powder. Such embodiments can be provided in either a ready-to-use form or a form that is reconstituted before administration (e.g., lyophilized form). In some embodiments, the kit comprises a first container containing the anti-CRH antibody, antigen-binding fragment, or pharmaceutical composition in a dehydrated or lyophilized powder, and a second container containing an aqueous formulation. In some embodiments, the kit comprises a pre-filled syringe containing the anti-CRH antibody, antigen-binding fragment, or pharmaceutical composition. [Example]

[0540] The following examples are provided to further illustrate aspects of the present disclosure, but are not meant to limit the disclosure to any particular application or theory of operation.

[0541] Example 1 Generation of CRH knockout mice on the H2L2 Harbour Mice® background Corticotropin-releasing hormone (CRH) knockout (KO) mice were generated on the H2L2 Harbour Mice® background using standard transgenic methods described in the laboratory manual "Manipulating the Mouse Embryo" by Nagy et al., Cold Spring Harbor Laboratory Press. Generation of CRH KO H2L2 mice was performed under animal license ADV101002016512, animal protocol 16-512-11, approved by the Dutch Central Authority for Scientific Procedures on Animals (CCD) and the EDC animal facility.

[0542] Briefly, superovulated H2L2 Harbour Mice® females and males were used for mating. The collected fertilized eggs were used for microinjection of 20 ng / microliter of two guide RNAs (gRNAs): gRNA1 Mm.Cas9.CRH.1.AA and gRNA2 Mm.Cas9.CRH.1.AB, and Cas9 protein in a microinjection buffer of 5 mM tris(hydroxymethyl)aminomethane (TRIS), 0.1 mM ethylenediaminetetraacetic acid (EDTA) in ultrapure water, adjusted to pH 7.4. The following gRNA1 sequence was used:

[0543] gRNA1=Sequence-Mm.Cas9CHR.1.AA:

[0544] 5'-AltR1 / rArArC rUrCrC rArCrG rCrCrC rCrUrC rArCrC rGrCrG rUrUrU rUrArG rArGrC rUrArU rGrCrU / altR2 / -3'

[0545] gRNA2=Mm.Cas9.CRH.1.AB:

[0546] 5'-AltR1 / rUrCrA rCrCrC rArUrG rCrGrG rArUrC rArGrArArCrG rUrUrU rUrArG rArGrC rUrArU rGrCrU / altR2 / -3'

[0547] Guide RNA and CAS9 were obtained from Integrated DNA Technologies (IDT).

[0548] Injected eggs were transferred to foster mothers (B6CBAF1). Knockout pups were identified from toe DNA by amplification of DNA spanning the gRNA region. Polymerase chain reaction (PCR) fragments smaller than these predicted sizes indicated deletions in founder mice. PCR primers CRHbp70F and CRHbp215R flanking gRNA1 were used to amplify a fragment of 146 bp expected from wild-type H2L2 Harbour Mice® (Figure 2A, left panel). PCR primers CRHbp208F and CRHbp310R were used to amplify a fragment of 103 bp expected from wild-type H2L2 Harbour Mice® (Figure 2A, center panel). PCR primers CRHbp70F and CRHbp310R yielded a fragment size of 241 bp (Figure 2A, right panel). PCR products smaller than these predicted sizes indicated mutations. Founder mice 1, 2, 4, and 5 were identified as carrying the mutation. PCR products deviating from the size expected for the wild-type were further analyzed by excising the PCR product from an agarose gel, isolating the DNA, and sequencing it using the same primers used for amplification. Founder mouse 5 from litter 12198 in Figure 2B carried an 11-bp deletion (5'-CAGCCGGTTCT-3') (SEQ ID NO: 465) and was used for subsequent breeding.

[0549] Rescue of CRH Knockout H2L2 Harbour Mice® Rescue and immunization of CRH knockout mice were performed under Animal License ADV101002016512, Animal Work Protocol 16-512-19. Briefly, homozygous CRH knockout H2L2 Harbour Mice® males and females were housed in a single cage. Upon observation of a copulatory plug (0.5 days after conception), females were removed from their cages and isolated. After 10 days, cortisol (30 μg / ml) was added to the drinking water (dark bottle) and allowed to be freely available to pregnant females. This resulted in the birth of live knockout offspring used for immunization.

[0550] Immunization of H2L2 Harbour Mice® with CRH Knockout Ten homozygous CRH knockout H2L2 Harbour Mice® of both sexes, approximately 8 weeks of age, were immunized on days 0, 14, 28, 42, and 50 with subcutaneous (SC) and intraperitoneal (IP) injections of the CRH-KLH conjugate in a maximum volume of 100 μl.

[0551] The following conjugates were used:

[0552] CRH-KLH: SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII-KLH (C-terminus) (GenScript) (SEQ ID NO: 467)

[0553] The CRH-KLH conjugate was mixed with the adjuvant Stimmune for the first injection and with the adjuvant Ribi for the subsequent booster immunizations. The final booster immunization was IP. 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 sacrificed according to standard protocols, and the spleens and lymph nodes were used for fusion experiments with myeloma fusion partners for hybridoma production. Sera were screened for anti-CRH antibody titers and subsequently for hybridomas by enzyme-linked immunosorbent assay (ELISA) using biotinylated CRH captured on streptavidin (SA)-coated plates. The detailed protocol is as follows:

[0554] CRH ELISA ELISA plates were coated with 5 μg / ml streptavidin in phosphate-buffered saline (PBS) (Sigma 189730-1mg) overnight at 4°C or for 2 hours at room temperature (RT). 50 μl / well was used for 96-well plates, or less for 384-well plates. After incubation, the streptavidin solution was removed and biotinylated peptide was added (ThermoFisher; biotin is at the N-terminus of the peptide). After a 30-minute incubation at room temperature, the plates were washed with 1X phosphate-buffered saline (PBS) / 0.1% Tween-20 and blocked with 1% milk / 1% bovine serum albumin (BSA) in the wash solution. After washing with 3X PBS / 0.1% Tween-20, 50 μl of antibody was added in blocking buffer and incubated for 2 hours at room temperature. The plates were then washed in 3X PBS / 0.1% Tween-20, and 50 μl of anti-rat IgG-HRP (horseradish peroxidase) at a dilution of 1:2,000 was added and incubated for 2 hours at room temperature. After washing in 3X PBS / 0.1% Tween-20, 50 μl of peroxidase substrate (POD or equivalent) was added, and the absorbance was measured at 450 nm.

[0555] Immunization of Balb / c wild-type mice Anti-CRH antibodies were generated by immunizing Balb / c wild-type mice with CRH-KLH protein. The CRH and KLH conjugates were either N-terminally conjugated CRH (peptide 1:KLH-CRH) or C-terminally conjugated CRH (peptide 2:CRH-KLH), via an extra cysteine ​​(C) at either the N- or C-terminus of CRH, respectively. Each mouse received 50 μg of protein for the initial IP priming with complete Freund's adjuvant (CFA, Sigma, catalog no. F5881), followed by 25 μg for the subsequent IP boost with Ribi (Sigma S6322). Boosts were administered four times every other week. At the indicated time points, mouse sera were sampled and titrated by indirect ELISA to test for binding to human CRH.

[0556] Mice with high serum titers and specific immune responses to human CRH were selected and boosted with 25 μg of protein in PBS. Three days later, the mice were sacrificed and their spleen and lymph node cells were harvested.

[0557] Preparation of CRH-KLH conjugate KLH carrier protein (Sigma, H7017-50MG) was weighed and dialyzed against 0.1 M sodium phosphate buffer, pH 7.5. Sulfo-MBS (BBI, C100314-0050) solution was added to the KLH carrier protein solution at a 1:10 ratio. After 1 h of incubation at room temperature, the reaction mixture was applied to a G-50 desalting column (Sigma, G50150-10G) pre-equilibrated with conjugation buffer (0.1 M sodium phosphate buffer, pH 6.5, containing 1 mM EDTA). The fraction corresponding to the first protein peak at A280 nm was collected. KLH carrier protein-MBS and CRH peptides with an extra cysteine ​​at either the N- or C-terminus were mixed and incubated at room temperature for 2–4 h. The reaction was terminated by adding 2-mercaptoethanol (Sigma, 3148-25ML) to a final concentration of 10 mM and incubated at room temperature for at least 30 min. The CRH-KLH conjugate was purified by passing it through a Sephadex G-25 desalting column to separate the unconjugated peptide from the CRH-KLH conjugate. The fractions corresponding to the first protein peak were collected. The protein concentration of the CRH-KLH conjugate was determined by NanoPhotometer.

[0558] The following peptides were used for immunization of Balb / c wild-type mice:

[0559] Peptide 1 (KLH-CRH) (SEQ ID NO: 468):

[0560] KLH-C-SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII-CONH2

[0561] Peptide 2 (CRH-KLH) (SEQ ID NO: 469):

[0562] SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII-C-KLH

[0563] The following peptides were used in the ELISA:

[0564] Peptide 3 (Biotin-CRH) (SEQ ID NO: 470):

[0565] Biotin-K-SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII-CONH2

[0566] Peptide 4 (CRH-biotin) (SEQ ID NO: 471):

[0567] SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII-K-Biotin

[0568] Biotin-CRH (SEQ ID NO: 472):

[0569] Biotin-SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII-COOH (ThermoFisher)

[0570] Single B-cell screening using the Beacon® optofluidic system We used a nanofluidic optoelectronic B lymphocyte antibody screening technology (NanOBlast) built around a commercially available integrated culture and imaging platform, Beacon™ (Berkeley Lights).

[0571] The NanOBlast workflow begins with the generation of antigen-specific antibody-secreting cells (ASCs) through in vivo immunization. Selectively enriched antigen-experienced murine ASCs are harvested from the spleen and lymph nodes. ASCs are microfluidically transferred into the chip and then isolated in individual nanopens for screening by optoelectronic positioning (OEP). ASCs secreting antigen-specific IgG are detected using a bead-based colorimetric binding assay, which produces a characteristic fluorescent bloom. Individual cells of interest are then unpenned by OEP and ejected directly from the chip into a 96-well plate containing cell lysis buffer. Single-cell aspirate amplification of cDNA ends (RACE) is used to recover antibody heavy and light variable domain (VH and VL) sequences, which are then cloned and recombinantly expressed as standard antibodies using standard methods. The recombinant antibodies are used for binding confirmation and final validation in relevant downstream assays.

[0572] Antibody production and purification Recombinant plasmids encoding target antibodies were transiently co-transfected into HEK293-6E or ExpiCHO cell cultures using polyethyleneimine (PEI) (Polyscience, 24885). One day before transfection, cells were plated in Corning Erlenmeyer flasks. On the day of transfection, recombinant plasmids encoding the target protein and transfection reagent were mixed at an optimal ratio (heavy chain:light chain = 2:3) and then added to the seeded flasks for transfection. On day 6, cell culture supernatants were harvested and used for purification. The cell culture broth 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 pooled and buffer exchanged with storage buffer. The purified proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size-exclusion chromatography-high-performance liquid chromatography (SEC-HPLC) analysis to determine molecular weight and purity. The concentration was determined by light absorbance at 280 nm.

[0573] antibody manipulation The heavy chain variable domain (VH) and light chain variable domain (VL) sequences of the anti-CRH chimeric antibody were further optimized by humanization and post-translational modification (PTM) removal procedures. In the humanization procedure, the VH and VL sequences were first aligned to the closest human germline sequences using an algorithm, such as NCBI / Ig-BLAST, and then the positions within the framework regions were reverted to the human germline sequences. All important reversion positions were scanned using Chothia numbering. If mismatches existed, they were replaced with the mouse counterpart residues. Antibodies composed of the humanized sequence variants were then recombinantly produced using standard molecular biology techniques.

[0574] For PTM removal, the VH and VL sequences were scanned for the presence of PTM motifs, such as isomerization motifs (e.g., DG). "Hot spot" residues (e.g., D or G in the DG motif) were mutated to either the corresponding residue in the germline sequence or another residue with similar biophysical properties. Antibodies containing the sequence variants after PTM removal were then recombinantly produced by standard molecular biology techniques.

[0575] Binding of anti-CRH antibodies to CRH or human UCN1 by ELISA Streptavidin (Thermo, catalog no. 21125) was diluted to a concentration of 2 μg / ml in PBS. 100 μl of diluted streptavidin was added per well to an ELISA microplate, and the plate was incubated overnight at 4°C. The plate was blocked with ELISA blocking solution (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) for 1 hour at 37°C. The plate was then washed and incubated with 15 μg / ml diluted anti-CRH antibody (100 nM, 10 dilutions, 8 points) at 37°C for 1 hour. The plate was 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 3,3',5,5'-tetramethylbenzidine (TMB) base (Biopanda, catalog number TMB-S-003) was added, and the plate was incubated at room temperature for 15 minutes. The reaction was stopped by adding 100 μL of ELISA stop solution (Solarbio, catalog number C1058), and the optical density at 450 nm (OD450nm) was determined using an ELISA plate reader (Molecular Devices, Spectra max 384 plus).

[0576] Plasmid and cell line preparation The CHO-K1 (Cat. No. CCL-61) cell line was obtained from the American Type Culture Collection (ATCC), and pGL4.29 / luc2P / CRE / Hygro (Cat. No. E8471) and Bio-Gio Luciferase Assay System (Cat. No. G7940) were obtained from Promega. Plasmids 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) carrying G418 resistance were synthesized in GenScript®. Human CRH peptide was obtained from Peptide International (Cat. No. 4136-s). Forskolin (catalog no. 1099) and human CRHR1 antagonist small molecule (Antalarmin, catalog no. 2778) were obtained from Tocris. Anti-human CRHR1 antibody (catalog no. MAB3930) was obtained from R&D System. Electroporation equipment (Electro Cell Manipulator, catalog no. ECM630) and electroporation cuvettes plus (2 mm Gap cuvette, catalog no. 45-0125) were used from BTX. Electroporation buffer (Ingenio Solution, catalog no. MIR50111) was obtained from Mirus, and sheep anti-human CRH polyclonal antibody was obtained from Salk Institute.

[0577] Generation of CHO-CRE-luciferase-human CRHR1, -human CRHR2, -mouse CRHR1, and -mouse CRHR2 reporter cell lines To generate CHO-CRE-luciferase reporter stable cell lines, a mixture of 2 million CHO-K1 cells and 10 μg of pGL4.29 / luc2P / CRE / Hygro plasmid in 100 μl of Ingenio solution was transferred to an electroporation cuvette and subjected to one or two pulses using an electroporator (950 μF and 120 V). The treated cells were transferred to a 6-well plate containing 2 mL of Dulbecco's Modified Eagle Medium (DMEM) / F12 containing 10% fetal bovine serum (FBS). After 24 hours, 500 μg / mL of the selection antibiotic hygromycin was added to the cells. This selection process took approximately 10–14 days. Using matrix dilution, pools of CHO-CRE cells were subcloned into 96-well plates. When selected colonies were evident, individual stable clones were harvested. Positive clones were verified using a series of forskolin dilutions. Validated positive clones were frozen for future use.

[0578] To generate CHO-CRE-hCRHR1, -hCRHR2, -mCRHR1, and -mCRHR2 reporter cell lines, pcDNA3.1-hCRHR1, -hCRHR2, -mCRHR1, and -mCRHR2 were introduced into the established CHO-CRE cells by electroporation as described above. The selection antibiotics hygromycin (500 μg / ml) and G418 (1,000 μg / ml) were used. Each established stable cell line was characterized by stimulation with both CRH and forskolin.

[0579] Development of luciferase reporter assays Forskolin stimulates adenylate cyclase, increasing intracellular cyclic adenosine monophosphate (cAMP) levels in cells. To examine whether cells respond to elevated cAMP, CHO-CRE cells were stimulated with a range of concentrations of forskolin, and cAMP-dependent luciferase expression was determined using the Bio-Glo Luciferase Assay System. CHO-CRE cells increased luciferase expression in a dose-dependent manner in response to forskolin stimulation (data not shown). CRH binds to the Gas-coupled G protein-coupled receptors (GPCRs), CRHR1 and CRHR2.

[0580] Activated GPCR proteins increase intracellular cAMP production. CHO-CRE-human CRHR1 stable clone 11 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 verify that the cell line was functional, CHO-CRE-hCRHR1 clone 11 cells were treated with forskolin and CRH peptide. CHO-CRE-hCRHR1 clone 11 cells showed a dramatic increase in luciferase expression upon CRH treatment compared with the parent CHO-CRE, while both cell lines showed comparable levels of intracellular cAMP in response to forskolin treatment. Subsequently, CHO-CRE-hCRHR2 clone 32, -mCRHR1 clone 22, and -mCRHR2 clone 8 cell lines were established using a similar methodology and validated by CRH treatment (data not shown).

[0581] Blocking CRHR1 with small molecules or neutralizing CRH peptides can reduce luciferase expression when the CRH-CRHR1 system is functional in CHO-CRE-hCRHR1 cells. The small molecule CRHR1 antagonist, antalarmin, 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).

[0582] Affinity determination by Octet® Octet® Red 96e was used for affinity measurements. Octet® running buffer was prepared by diluting kinetics buffer 10X (Fortebio, catalog no. 18-1105) in PBS according to the manufacturer's instructions. Two columns of SA sensors (Fortebio, catalog no. 18-5019) were hydrated in running buffer for 10 minutes, one for ligand capture and the other for reference. Antibodies were diluted 2-fold and added to a 96-well plate. 30 nM biotinylated CRH (GL Biochem, catalog no. 784751) was captured by the first column of the SA sensors for 20 seconds, while the reference sensors were immersed in the buffer wells. After a 60-second baseline step, the sensors were sequentially immersed in the analyte and buffer wells with association and dissociation times of 180 and 800 seconds, respectively. The sensors were regenerated in glycine pH 1.5 (Cytiva, Cat. No. BR-1003-54) before the next cycle. Equilibrium dissociation constant (KD) values ​​were estimated using Data Analysis Software 11.0 and a fitting model of 1:1 global fitting. The signals of the reference well and reference sensor were subtracted by selecting "Double Reference" before data fitting.

[0583] Blocking activity of CRH-induced signaling in reporter cell lines (CHO-CRE-hCRHR1, CHO-CRE-hCRHR2, CHO-CRE-mCRHR1, and CHO-CRE-mCRHR2) Cells were digested with trypsin-EDTA (Life Technologies, Catalog No. 12604-013), centrifuged at 1,000 rpm for 5 minutes, resuspended in growth medium, counted using a cell counter, and plated at 5,000 cells / 100 μl in a 96-well flat plate. Fifty μl of 4X serially diluted antibody was added to the plate containing CHO-CRE-hCRHR1 cells (eight-point, 1 / 4 dilution with growth medium), and 50 μl of 4X CRH (4X is 2 nM, final 0.5 nM) was added. Similarly, 4X CRH (4X is 0.4 nM, final 0.1 nM) was added to CHO-CRE-hCRHR2 and CHO-CRE-mCRHR1 cells, and 4X CRH (4X is 20 nM, final 5 nM) was added to CHO-CRE-mCRHR2 cells. The plates were then incubated in a CO2 incubator at 37°C for 4-6 hours. 60 μl of culture medium was removed and 60 μl of Bio-Gio luciferase was added, followed by incubation at room temperature for 10 minutes. The plates were read in a luciferase plate reader (EnVision, PerkinElmer).

[0584] ELISA for human UCN1, human UCN2, and human UCN3 Streptavidin (Thermo, catalog no. 21125) was diluted in PBS to a concentration of 2 μg / mL. 100 μl of diluted streptavidin was added per well to an ELISA microplate, and the plate was incubated overnight at 4°C. The plate was blocked with ELISA blocking solution (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 of hUCN1(NT biotin), hUCN1(CT biotin), hUCN2(NT biotin), hUCN2(CT biotin), hUCN3(NT biotin), or hUCN3(CT biotin) for 1 hour at 37°C. The plate was then washed and incubated with 100 nM and 10 nM anti-CRH antibody at 37°C for 1 hour. The plate was then washed and incubated with HRP-conjugated goat anti-human IgG (H+L) antibody (Jackson, Cat. No. 109-035-088) for 1 hour at 37°C. 100 μl of TMB base (Biopanda, Cat. No. TMB-S-003) was added, and the plate was incubated for 15 minutes at room temperature. 100 μL of ELISA stop solution (Solarbio, Cat. No. C1058) was then added to stop the reaction, and the OD450nm was determined using an ELISA plate reader (Molecular Devices, Spectra max 384 plus).

[0585] Human UCN1-inducible luciferase reporter assay CHO-CRE-hCRHR1 and CHO-CRE-hCRHR2 cells were harvested with 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 plated at 5,000 cells / 100 μl into a 96-well flat plate and incubated overnight. 50 μl of 4X serially diluted antibody (8-point, 1 / 3 dilution with growth medium) or CP376395 hydrochloride (TOCRIS, catalog number: 3212) (8-point, 1 / 10 dilution with growth medium) was added to the plate, followed by the addition of 50 μl of 4X hUCN1 (4X at 4 nM, final 1 nM) to the CHO-CRE-hCRHR1 or CHO-CRE-hCRHR2 cells. The plates were then incubated for 4-6 hours at 37°C in a CO2 incubator. After this time, 60 μl of culture medium was removed and 60 μl of Bio-Gio luciferase was added, followed by a 10-minute incubation at room temperature. The plates were then read in a luciferase plate reader (EnVision, PerkinElmer).

[0586] In Vivo Efficacy Model - Wild-Type Female C57BL / 6 Mice and Mrap1 KO Mice Eight- to ten-week-old wild-type female C57BL / 6 mice were obtained from Charles River Laboratories. Mrap1 KO mice were obtained from Queen Mary University of London, UK. The small molecule CRHR1 antagonist, SSR125543A, 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 Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Charles River Laboratories Institutional Animal Care and Use Committee (IACUC).

[0587] Breeding, ear tagging, and genotyping of Mrap1 KO mice To generate Mrap1 KO mice, Mrap1- / - male mice were mated with Mrap1+ / - female mice. Corticosterone hormone replacement was administered to the female mice at 50 μg / ml in their drinking water once they became pregnant and the pregnancy became palpable (approximately embryonic day 10). The drinking water was replaced weekly, and treatment was discontinued at weaning. Mice were individually identified using ear tags at the time of tail clipping to ensure future genotype matching. DNA was extracted from tail tissue and used to identify the genotype of the mice.

[0588] Genotyping of Mrap1− / − mice was performed using PCR and the following PCR primers:

[0589] For the wild-type allele of exon 1 of mrap1:

[0590] Forward 5'-GCGTCTCTTAGTAGCCTTTGG (SEQ ID NO: 473)

[0591] Reverse 5'-CTTGTTGGCTTTCAGCTTCT (SEQ ID NO: 474)

[0592] For mutant alleles of Mrap1:

[0593] Forward 5'-GAACTTGCCTGGTCAACTGTTAAAAGGAC (SEQ ID NO: 475)

[0594] Reverse 5'-TCTTTAGGCTCACTTCCTCCACTGACC (SEQ ID NO: 476)

[0595] PCR band sizes: 345 bp for the wild-type allele and 459 bp for the mutant allele.

[0596] Physical limitations for wild-type mice To determine the effect of nonpainful physical restraint on stress responses, mice were restrained in 50 ml polypropylene ventilated tubes, the tip of which was cut with a razor blade, allowing the mice to breathe easily for 15–20 minutes. The mice were then tucked into the open tube. A paper towel was placed behind the mouse to prevent escape, and the cap was screwed on. The mouse was then returned to its cage, ventral side down on the bedding. This stressor was not considered painful because the mouse was confined to a limited space (within a ventilated restraining tube) but not in an unnatural physical position. The investigator was present during the entire procedure. At the end of the procedure, the mouse's tail was decapitated.

[0597] Effect of CRH Ab on wild-type mice in a restriction stress model On day 1, each mouse was intraperitoneally injected with 20 mg / kg of anti-CRH mAb, hIgG1, or PBS (five mice per group). On days 2, 3, and 14, each mouse was subjected to the above-described physical restraint stress for 15 or 20 minutes. Immediately after restraint stress, 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 × g for 10 minutes) and flash-frozen in dry ice for subsequent ACTH and corticosterone ELISA measurements.

[0598] Effect of anti-CRH Ab on Mrap1 KO mouse model On day 1, each mouse was injected (IP) with 20 mg / kg of anti-CRH Ab or hIgG1 (5 or 6 mice 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 × g for 10 min) and flash-frozen in dry ice for subsequent ACTH ELISA measurements.

[0599] Side-by-side comparison of anti-CRH mAb and SSR125543A in wild-type mouse restraint stress model On day 1, half of the mice were intraperitoneally injected with 20 mg / kg anti-CRH Ab or hIgG1 (five mice per group). On day 3, the other half of the mice were intraperitoneally injected with 30 mg / kg SSR125543A or vehicle (five mice per group). Two hours later, mice in four groups were subjected to the restraint stress described above for 20 minutes. Immediately after restraint stress, 200 μl of blood was collected in EDTA tubes from the submandibular vein of each mouse in all four groups. Plasma was separated as described above and snap-frozen in dry ice for subsequent ACTH and corticosterone ELISA measurements. On day 4, mice treated with SSR125543A or vehicle were subjected to a second restraint stress for 20 minutes, and plasma was collected. Two weeks later, mice treated with anti-CRH mAb or hIgG1 were subjected to physical stress for 20 minutes, after which plasma was collected.

[0600] In Vivo PK Study Dosing and Blood Collection For each antibody tested, six female C57BL / 6 mice weighing 18-22 grams were selected and intravenously administered a single dose of 5 mg / kg of antibody. Whole blood was collected from three mice before administration and 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 5 hours, 2 days, 7 days, and 14 days after administration. The whole blood was allowed to clot for 30 minutes and then centrifuged. The separated serum was then frozen at -80°C until analysis.

[0601] Analysis method ELISA was used to quantitatively determine drug concentrations in mouse serum. Test antibodies (including human Fc) in mouse serum were captured by a goat anti-human Fc polyclonal antibody (Rockland, #609-101-017) pre-coated on a 96-well plate. HRP-labeled goat anti-human (H+L) secondary antibody (Abcam, #ab97175) was then added to the wells. The plate was then sealed and incubated at 37°C for 30 minutes, after which it was washed and TMB base and ELISA stop solution were added. The 96-well plate was read at 450 / 630 nm wavelengths using a SpectraMax M2. Data were processed using Soft Max Pro.

[0602] The pharmacokinetic (PK) parameters were analyzed by Phoenix WinNonlin version 8.2 using a non-compartmental model (NCA).

[0603] Determination of the binding region of CRH for anti-CRH antibodies by ELISA Streptavidin (Thermo, catalog no. 21125) was diluted to a concentration of 2 μg / ml in PBS. 100 μl of diluted streptavidin was added per well to an ELISA microplate, and the plate was incubated overnight at 4°C. The plate was blocked with ELISA blocking solution (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 of CRH N-terminal region + central region (CT biotin), central region only (NT biotin or CT biotin), or central region + C-terminal region (NT biotin) for 1 hour at 37°C. The plate was then washed and incubated with 15 μg / ml of diluted anti-CRH antibody (Ab) (100 nM, 10 dilutions, 8 points) at 37°C for 1 hour. The plate was then washed and incubated with HRP-conjugated goat anti-human IgG (H+L) antibody (Jackson, catalog number 109-035-088) for 1 hour at 37°C. 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) base (Biopanda, catalog number TMB-S-003) was added, and the plate was incubated for 15 minutes at room temperature. The reaction was stopped by adding 100 μL of ELISA stop solution (Solarbio, catalog number C1058), and the optical density at 450 nm (OD450nm) was determined using an ELISA plate reader (Molecular Devices, Spectra max 384 plus).

[0604] Epitope binding by competitive ELISA Competitive ELISA for epitope binding was performed. Specifically, 1 μg / ml of capture antibody was coated onto a 96-well plate at 4°C overnight (100 μl / well). The plate was blocked with ELISA blocking solution (containing 2% w / v BSA, 0.05% (v / v) Tween-20, pH 7.4 PBS buffer) at 37°C for 1 hour. Next, 50 μl / well of a mixture of competing antibody (40 μg / ml) and biotin-labeled CRH (50 μl / well) at appropriate concentrations was added to the 96-well plate, followed by incubation at 37°C for 1 hour. The plate was then washed and incubated with streptavidin-peroxidase (SIGMA, catalog number S2438-250UG) at 37°C for 1 hour. 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) base (Biopanda, catalog no. TMB-S-003) was added, and the plate was incubated for 15 minutes at room temperature. The reaction was stopped by adding 100 μL of ELISA stop solution (Solarbio, catalog no. C1058), and the optical density at 450 nm (OD450nm) was determined using an ELISA plate reader (Molecular Devices, Spectra max 384 plus).

[0605] Epitope Binning with Octet® Epitope binding was performed in Octet® Red 96e. Running buffer was prepared by diluting kinetics buffer 10x (Fortebio, catalogue number 18-1105) in PBS according to the manufacturer's instructions.

[0606] SA sensors (Fortebio, catalog no. 18-5019) were hydrated in running buffer for 10 minutes. The in-tandem epitope binding assay required a four-step binding cycle: 1) a 30-second regeneration step, 2) 30 nM capture of CRH with a loading threshold of 0.12 nM, 3) 60 nM antibody (primary Ab) loading to saturate the immobilized peptide for 200 seconds, and 4) a mixture of the primary Ab and competing antibody from step 3 was allowed to bind for 200 seconds.

[0607] Octet® data were processed with Fortebio's Data Analysis Software 11.0. After exporting the signals, the inhibition ratio was calculated as follows: Inhibition ratio (%) = (ab) / a*100% Taking antibodies (Abn) as an example, Variable a: signal of Abn binding alone to the antigen (also called 100% signal of Abn) If the variable b:Abn is a second Ab, its signal binds to the antigen

[0608] Example 2 Generation of CRH knockout mice on the H2L2 Harbour Mice® background CRH knockout mice were generated on the H2L2 Harbour Mice® background using the materials and methods described above. Figure 1 shows the coding sequence open reading frame of mouse CRH (GenBank NM_205769), along with the CRISPR targeting gRNA and PCR primers used. Specifically, as shown in Figure 1, the following primers were used for amplification on genotype conductors, i.e., genomic DNA:

[0609] CRHbp70F: 5'-GCC CTG CTC AGC AGG GGA TCC GT-3' (SEQ ID NO: 477)

[0610] CRHbp215R: 5'-CTG TTG AGA TTC CCC AGG CGG AG-3' (SEQ ID NO: 478)

[0611] CRHbp208F:5'-CTC AAC AGA AGT CCC GCT CGG-3' (SEQ ID NO: 479)

[0612] CRHbp310R: 5'-GGA AAA AGT TAG CCG CAG CCT GG-3' (SEQ ID NO: 480)

[0613] The expected PCR DNA fragment sizes from wild-type mice are 146 bp and 103 bp, respectively. PCR fragments smaller than these expected sizes indicated deletions in the founder mice. Figures 2A-2B show the identification of CRH knockout founder mice using this method.

[0614] Figure 3 shows the location of an 11-bp deletion (5'-CAGCCGGTTCT-3') (SEQ ID NO: 465) in the CRH gene coding region 157-167, which results in a downstream reading frameshift and a premature stop codon. This defective CRH open reading frame is unable to synthesize the CRH peptide. Founders with this deletion were selected for further breeding to homozygosity, rescue, and immunization.

[0615] Example 3 Identification of anti-CRH mAbs from CRH knockout mice on the H2L2 Harbour Mice® background Multiple hybridoma clone condensations performed on CRH knockout mice in the H2L2 Harbour Mice® background, immunized with CRH peptide, identified many positive hybridoma clones. Monoclonal antibodies (mAbs) purified from hybridoma supernatants bound to CRH with high affinity in ELISA. However, three functional monoclonal antibody clones, hybridoma clones 16g2, 28b3, and 52g7, were identified with blocking activity in the human CRHR1 luciferase reporter assay. These three clones were recombinantly expressed with human IgG1 wild-type Fc as PR004289, PR004290, and PR006669, respectively. PR004289 (derived from hybridoma clone 16g2), PR004290 (derived from hybridoma clone 28b3), and PR006669 (derived from hybridoma clone 52g7) share the same germline sequences of IGHV3-7 and IGKV2-28. PR004289 and PR004290 have one residue difference at position 28 of IGHV, which is I28 in PR004289 and T28 in PR004290, respectively. Both PR004289 and PR004290 have complementarity-determining regions (CDRs) that differ from those of PR006669. These three mAbs were designated series 1 (PR004289 and PR004290) and series 2 (PR006669) mAbs. See Table 2.

[0616] [Table 2]

[0617] Example 4 Identification of anti-CRH mAb from wild-type mice Using the materials and methods described above, more than 200,000 single B cells from wild-type mice immunized with a CRH-KLH conjugate were screened using the Beacon system. More than 500 monoclonal antibodies (mAbs) with binding activity in bead-binding assays were identified. More than 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. Eight series of functional mAbs with blocking activity in a human CRHR1 luciferase reporter assay were identified using the materials and methods described above. Different series of mAbs have different germline sequences and different CDRs. mAbs within each series share the same germline but have different CDRs.

[0618] These mAbs were designated as Series 3 to Series 10 as follows:

[0619] Series 3 includes PR301306.

[0620] Series 4 includes PR301612.

[0621] Series 5 includes PR301777, PR301788, PR301790, and PR301803.

[0622] Series 6 includes PR301806.

[0623] Series 7 includes PR302309, PR302332, and PR302333.

[0624] Series 8 includes PR302315 and PR302335.

[0625] Series 9 includes PR302328, PR302331, PR302334, and PR302339.

[0626] Series 10 includes PR302312, PR302324, and PR302341.

[0627] See also Table 2 and Figures 4A-4N.

[0628] Example 5 antibody manipulation PTM removal of series 1 mAb PR004290 PR004290 contains two possible post-translational modifications (PTMs): DG in the heavy chain CDR2 (HCDR2) and NS in the light chain CDR1 (LCDR1). PR006669 has one possible PTM, DG, in HCDR2. These possible PTMs were individually mutated in either HCDR2 alone, LCDR1 alone, or various combinations of mutations, as detailed in Table 3. Their binding activity in CRH ELISA and functional blocking activity in human CRHR1 (hCRHR1), human CRHR2 (hCRHR2), mouse CRHR1 (mCRHR1), and mouse CRHR2 (mCRHR2) luciferase reporter assays are shown in Table 3 and Figures 5A–5L. Most ΔPTM variants retained functional activity. PR005660 had a median inhibitory concentration (IC50) of approximately 10 nM in the hCRHR1 reporter assay, compared to the parent PR004290 at approximately 5 nM.

[0629] [Table 3]

[0630] PTM removal of series 2 mAb PR006669 PR006745 and PR006746 are two ΔPTM variants of PR006669 that have somewhat weaker ELISA binding and functional blocking activities (see Table 4 and Figures 6A-6D).

[0631] [Table 4]

[0632] Humanization of series 5 mAb PR301777 Using the materials and methods described above, 15 humanized variants of PR301777 were recombinantly expressed and tested. Many humanized variants maintained highly potent in vitro ELISA binding activity and functional blocking activity. See Table 5 and Figures 7A-7L.

[0633] [Table 5-1]

[0634] [Table 5-2]

[0635] Humanization of series 7 mAb PR302309 Using the materials and methods described above, 21 humanized variants of PR302309 were recombinantly expressed and tested. Many humanized variants maintained highly potent in vitro ELISA binding activity and functional blocking activity. See Table 6 and Figures 8A-8D.

[0636] [Table 6-1]

[0637] [Table 6-2]

[0638] Humanization of series 9 mAb PR302334 Using the materials and methods described above, 22 humanized variants of PR302334 were recombinantly expressed and tested. Many humanized variants maintained highly potent in vitro ELISA binding activity and functional blocking activity. See Table 7 and Figures 9A-9F.

[0639] [Table 7-1]

[0640] [Table 7-2]

[0641] Humanization of series 10 mAb PR302341 Using the materials and methods described above, 27 humanized variants of PR302341 were recombinantly expressed and tested. Many humanized variants maintained highly potent in vitro ELISA binding activity and functional blocking activity. See Table 8 and Figures 10A-10G.

[0642] [Table 8-1]

[0643] [Table 8-2]

[0644] PTM removal of humanized series 10 mAbs PR302341-10, PR302341-20, and PR302341-23 Using the materials and methods described above, humanized series 10 mAbs PR302341-10, PR302341-20, and PR302341-23 were subjected to 4X4 double PTM removal mutagenesis to generate a total of 48 mAbs (3X4X4), including the three parent humanized mAbs described above. Many humanized variants maintained highly potent in vitro ELISA binding activity and functional blocking activity. See Table 9 and Figures 11A-11L. PR302341-34, PR302341-48, PR302341-60, PR302341-28, and PR302341-55 were scaled up for in vivo testing.

[0645] [Table 9-1]

[0646] [Table 9-2]

[0647] [Table 9-3]

[0648] Example 6 Selectivity for human UCNL, human UCN2, and human UCN3 peptides All tested mAbs, except PR302334-26, bound to the human UCN1 peptide in ELISA assays. None of the tested mAbs bound to the human UCN2 or UCN3 peptides in ELISA assays. The binding affinity of these mAbs for human UCN1 was comparable to that of CRH by ELISA. However, only mAbs from the PR302341 series inhibited hUCN1-induced hCRHR1 luciferase reporter activity. Furthermore, these mAbs inhibited hUCN1-induced hCRHR1 reporter activity with potencies 30- to 267-fold lower than those of CRH-induced hCRHR1 reporter activity. None of the mAbs blocked hUCN1-induced hCRHR2 luciferase reporter activity. See Table 10, Figures 12A-B, 13A-D, and 14A-H.

[0649] [Table 10]

[0650] Example 7 Anti-CRH mAb binds to the N-terminal region of the CRH peptide To determine the binding region of the CRH peptide, the N-terminal region plus central region, central region only, and central region plus C-terminal region of the CRH peptide were used for ELISA capture of CRH using the materials and methods described above. All mAbs bound to the N-terminal region plus central region of the CRH peptide. Therefore, these mAbs likely bind to the N-terminal region of CRH. See Table 11 and Figures 15A-15D.

[0651] [Table 11]

[0652] Example 8 Affinity determination of mAbs by Octet® Using the materials and methods described above, the binding affinity of anti-CRH antibodies was determined by the Octet® platform. The affinities of the identified mAbs were very high, with equilibrium dissociation constant (KD) values ​​of approximately one to two digit pM. See Table 12.

[0653] [Table 12]

[0654] Example 9 Binding of antibody-binding epitopes by ELISA Using the materials and methods described above, the selected mAbs were tested for their ability to block each other's binding to biotin-CRH in a pairwise competitive ELISA. Table 13 shows that these mAbs blocked each other's binding to CRH and therefore likely bind to a similar epitope, presumably at the N-terminus of CRH.

[0655] [Table 13]

[0656] Example 10 Antibody-binding epitope binding by Octet® Using the materials and methods described above, the selected mAbs were tested for their ability to block each other for binding to biotin-CRH in a pairwise competition Octet® assay. Figure 16 shows a diagram of the competition assay using the Octet® platform. Table 14 shows that these mAbs blocked each other's binding to CRH and therefore likely bind to a similar epitope, presumably at the N-terminus of CRH.

[0657] [Table 14]

[0658] Example 11 In Vivo Efficacy Studies Transient increase in ACTH and corticosterone induced by restraint in wild-type mice Treatment with anti-CRH mAbs PR004290 (series 1 mAb), PR006152 (series 1 mAb), and PR302050 (series 5 mAb) (20 mg / kg IP) significantly inhibited adrenocorticotropic hormone (ACTH) and corticosterone levels in wild-type mice subjected to the restraint-induced stress model discussed above. One day after mAb administration, plasma ACTH and corticosterone levels were inhibited to basal, non-stressed levels. 14 days after a single mAb administration, ACTH and corticosterone levels remained partially inhibited. See Figure 17.

[0659] A model of constitutively high ACTH in Mrap1 KO mice Mrap1 KO mice have constitutively high plasma ACTH concentrations of approximately 5,000 pg / ml, which is approximately 50-fold higher than the basal ACTH concentration in wild-type mice and approximately 10-fold higher than that observed in wild-type mice following restraint-induced stress. A single administration of 20 mg / kg of anti-CRH mAb PR302050 (series 5 mAb) significantly reduced the constitutively high ACTH concentration in Mrap1 KO mice 2 and 14 days after administration. See Figure 18.

[0660] A single dose of the anti-CRH mAb PR302064 (series 5 mAb) significantly reduced constitutively high levels of ACTH in Mrap1 KO mice at doses of 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-dose. See Figures 19A-C.

[0661] Additional anti-CRH mAbs were tested in the Mrap1 KO model. A single dose of 20 mg / kg of PR302334-26 (series 9 mAb), PR302341-28, PR302341-34, PR302341-55, and PR302341-60 (all series 10 mAbs) significantly reduced plasma ACTH concentrations in Mrap1 KO mice (see Figures 20A-B).

[0662] PR302038, PR005660, and PR302050 dose-dependently reduced plasma ACTH concentrations in Mrap1 KO mice at doses of 20 mg / kg and 5 mg / kg of antibody (see Figures 21A-B).

[0663] Anti-CRH mAbs PR302334-24 and PR005660 at 20 mg / kg significantly reduced plasma ACTH concentrations in Mrap1 KO mice, whereas PR301429, an anti-CRH mAb with similar binding affinity but a nonblocking agent in vitro, did not inhibit ACTH production. PR303394, an analog of CTRND05, was used as a comparison. See Figures 22A-B.

[0664] Anti-CRH mAb PR302064 was more effective than SSR125543A in the restraint-induced ACTH model in wild-type mice The anti-CRH mAb PR302064 (series 5 mAb) at 20 mg / kg was more effective than the CRHR1 small molecule antagonist SSR125543A (Crinecerfont) at 30 mg / kg in reducing restraint-induced ACTH in wild-type mice. PR302064 more completely inhibited both ACTH and corticosterone and had a longer duration of action. The effects of this mAb persisted for at least 16 days. See Figures 23A-B.

[0665] Anti-CRH mAbs PR302064 and PR302334-24 were more effective than SSR125543A in a model of constitutively high ACTH in Mrap1 KO mice The anti-CRH mAh PR302064 at 20 mg / kg and PR302334-24 at 20 mg / kg, 10 mg / kg, and 5 mg / kg, respectively, were all more effective than the CRHR1 small molecule antagonist SSR125543A (Crinecerfont) at 30 mg / kg in reducing plasma ACTH levels in a model of constitutively elevated ACTH in Mrap1 KO mice. PR302064 and PR302334-24 provided more complete inhibition and, more importantly, a longer duration of action. The effects of these two mAbs persisted for at least 14 days. See Figures 24A-B.

[0666] Anti-CRH mAb PR302334-24 was more effective than SSR125543A in preventing restraint stress-induced hyperACTH and hypercorticosterone in wild-type mice The anti-CRH mAb PR302334-24 reduced plasma ACTH and corticosterone levels at both 20 mg / kg and 5 mg / kg more effectively than the CRHR1 small molecule antagonist SSR125543A (Crinecerfont) at 30 mg / kg in the restraint stress-induced hyperACTH and hypercorticosterone model in wild-type mice. PR302334-24 provided more complete inhibition and, more importantly, a longer duration of action. The effects of PR302334-24 persisted for at least 17 days. See Figures 25A-B.

[0667] In Vivo PK Study The pharmacokinetics of mAbs PR004290, PR302050, and PR302064 were tested in C57BL / 6 wild-type mice after a single IV dose of 5 mg / kg. Specifically, anti-CRH mAbs in human IgG1-WT-Fc present in mouse serum were captured with a goat anti-human Fc polyclonal antibody and detected with an HRP-conjugated goat anti-human (H+L) secondary antibody. Figure 26A shows a diagram of this method.

[0668] As shown in Table 15 and Figures 26B-26D, the serum half-lives (T 1 / 2 ) were 14, 21, and 18 days, respectively. These data demonstrate that these mAbs have a typical human IgG1PK profile and T 1 / 2 This indicates that it is desirable for clinical application.

[0669] [Table 15]

[0670] All publications, patents, and patent applications mentioned in this application are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference herein. Furthermore, citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. These, to the extent that section headings are used, should not be construed as necessarily limiting.

Claims

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

2. 1. A method of treating PCOS in a subject in need thereof, comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds CRH, wherein the antibody or antigen-binding fragment thereof: (a) a heavy chain variable domain (VH) CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 22 to 35, a VH CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 53 to 78, and a VH CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 113 to 122 or the amino acid sequence of a PDV or GID; (b) a light chain variable domain (VL) CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 152-174, a VL CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 193-198, and a VL CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 223-232; A method comprising:

3. the antibody or antigen-binding fragment thereof (a) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to any one of SEQ ID NOs: 240-295, 459, and 461; and / or (b) a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to any one of SEQ ID NOs: 296-347 and 462; 3. The method of claim 1 or 2, comprising:

4. the antibody or antigen-binding fragment thereof (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 to any one of SEQ ID NOs: 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 to any one of SEQ ID NOs: 404-455 and 464. The method according to any one of claims 1 to 3, comprising:

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

6. 1. A method of reducing androgen levels in a subject with PCOS, comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds CRH, wherein the antibody or antigen-binding fragment thereof: (a) a VH CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 22 to 35, a VH CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 53 to 78, and a VH CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 113 to 122 or the amino acid sequence of a PDV or GID; (b) a VL CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 152 to 174, a VL CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 193 to 198, and a VL CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 223 to 232; A method comprising:

7. the antibody or antigen-binding fragment thereof (a) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to any one of SEQ ID NOs: 240-295, 459, and 461; and / or (b) a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to any one of SEQ ID NOs: 296-347 and 462; 7. The method of claim 5 or 6, comprising:

8. the antibody or antigen-binding fragment thereof (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 to any one of SEQ ID NOs: 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 to any one of SEQ ID NOs: 404-455 and 464. The method according to any one of claims 5 to 7, comprising:

9. 9. The method of claim 5, wherein the androgen is testosterone, free testosterone, androstenedione, an 11-oxygenated androgen, or a combination thereof.

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

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

12. 1. A method for reducing the severity of one or more symptoms or signs in a subject having PCOS, comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds CRH, wherein the antibody or antigen-binding fragment thereof: (a) a VH CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 22 to 35, a VH CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 53 to 78, and a VH CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 113 to 122 or the amino acid sequence of a PDV or GID; (b) a VL CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 152 to 174, a VL CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 193 to 198, and a VL CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 223 to 232; A method comprising:

13. the antibody or antigen-binding fragment thereof (a) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to any one of SEQ ID NOs: 240-295, 459, and 461; and / or (b) a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to any one of SEQ ID NOs: 296-347 and 462; 13. The method of claim 11 or 12, comprising:

14. the antibody or antigen-binding fragment thereof (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 to any one of SEQ ID NOs: 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 to any one of SEQ ID NOs: 404-455 and 464. The method according to any one of claims 11 to 13, comprising:

15. 15. The method of any one of claims 11 to 14, wherein the one or more symptoms or signs are anovulation, irregular or amenorrheal periods, hyperandrogenism, elevated testosterone, free testosterone, androstenedione, 11-oxygenated androgens, or a combination thereof, abnormal uterine bleeding, polycystic ovarian enlargement, infertility, obesity, insulin resistance, hyperinsulinemia, hypertension, hyperlipidemia, type 2 diabetes, excessive facial hair growth, hirsutism, alopecia, or acne.

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

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

18. The antigen-binding fragment may be Fab, scFab, Fab', F(ab') ２ , Fv, scFv, diabody, or triabody.

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

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

21. 21. The method of claim 20, wherein the one or more PCOS therapies are selected from the group consisting of combined contraceptives, progestin therapy, clomiphene, tamoxifen, aromatase inhibitors, gonadotropins, ovarian drilling, spironolactone, eflornithine, electrolysis, and antidiabetic agents.

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

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

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

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

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

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

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

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