ANTI-CORTICOTROPIN-RELEASING HORMONE ANTIBODIES AND POLYCYSTIC OVARY SYNDROME

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of 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 are incorporated herein by reference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The content of the electronically submitted sequence listing (Name: 4857_002PC02_SequenceListing_ST26; Size: 686,199 bytes; and Date of Creation: July 25, 2023) is herein incorporated by reference in its entirety.

FIELD

[0003] 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 containing such antibodies and antigen-binding fragments, and instructions for their use in such methods.

BACKGROUND

[0004] Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in 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 experience (i) infrequent, irregular or prolonged menstrual cycles, (ii) excess androgen levels that can result in excess hair growth (hirsutism), acne, alopecia, or skin abnormalities, and (iii) polycystic ovaries that can result in infertility.

Azziz, Obstet. Gynecol., 132(2):321-336 (2018). The primary characteristics of PCOS include hyperandrogenism, anovulation, insulin resistance, and neuroendocrine disruption. Crespo et al., Arch. Endocrin. Metab., 62(3):352-361 (2018).

SUBSTITUTE SHEET ( RULE 26) [0005] Due to this complexity, there remain many challenges to managing PCOS. Treatment is often based on the symptoms being experienced, which can include androgen-related symptoms, ovulatory dysfunction-related infertility, and menstrual disorders. Badawy et al., Int.

J. Womens Health, 3:25-35 (2011). Evidence suggests that both clinicians and consumers are not satisfied with current treatment options. Hoeger et al., J. Clin. Endocrinol. Metab., 106(3): e 1071 - el083 (2021). As such, there is a need for improved therapies for PCOS and related disorders.

BRIEF SUMMARY

[0006] Provided herein is 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).

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

[0008] In one aspect, 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 aspect, 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.

SUBSTITUTE SHEET ( RULE 26) [0010] Also provided herein is a method of reducing androgen concentration in a subject having PCOS, comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to CRH.

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

[0012] In one aspect, 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 aspect, 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 aspect, the androgen is testosterone, free testosterone, androstenedione, an 11 -oxygenated androgen, or a combination thereof. In another aspect, the 11 -oxygenated androgen is 11 -hydroxyandrostenedione (110HA4), 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 in a subject having PCOS, 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 in a subject having PCOS, comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to CRH, wherein the antibody or

SUBSTITUTE SHEET ( RULE 26) antigen-binding fragment thereof comprises (a) a VH CDR1 comprising an amino acid sequence of any one of SEQ ID NOs: 22-35, a VH CDR2 comprising an amino acid sequence of any one of SEQ ID NOs: 53-78, and a VH CDR3 comprising an amino acid sequence of any one of SEQ ID NOs: 113-122 or the amino acid sequence of PDV or GID; and (b) a VL CDR1 comprising an amino acid sequence of any one of SEQ ID NOs: 152-174, a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 193-198, and a VL CDR3 comprising an amino acid sequence of any one of SEQ ID NOs: 223-232.

[0017] In one aspect, 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 aspect, 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 aspect, the one or more symptoms or signs is anovulation; irregular or absent menstrual periods; hyperandrogenism; elevated testosterone, free testosterone, androstenedione, 11 -oxygenated androgen, or a combination thereof; abnormal uterine bleeding; enlarged multifollicular ovaries; infertility; obesity; insulin resistance; hyperinsulinemia; hypertension; hyperlipidemia; type-2 diabetes mellitus; excess facial hair growth; hirsutism; hair loss; or acne. In another aspect, the 11 -oxygenated androgen is 11-hydroxyandrostenedione (11OHA4), 11 -hydroxytestosterone (11OHT), 11 -ketoandrostenedione (11KA4), 11- ketotestosterone (11KT), or a combination thereof.

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

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

[0022] In another aspect, 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.

SUBSTITUTE SHEET ( RULE 26) [0023] In another aspect, the antibody or antigen-binding fragment thereof is administered in combination with one or more PCOS therapy. In another aspect, the one or more PCOS therapy is selected from the group consisting of a combination birth control, progestin therapy, clomiphene, tamoxifen, an aromatase inhibitor, a gonadotropin, ovarian drilling, spironolactone, eflornithine, electrolysis, and an antidiabetic agent. In another aspect, the combination birth control is an oral contraceptive containing estrogen and progestin. In another aspect, the antidiabetic agent is metformin. In another aspect, the aromatase inhibitor is letrozole.

[0024] In another aspect, the one or more PCOS therapy is selected from the group consisting of spironolactone, metformin, and a combination birth control. In another aspect, the combination birth control is an oral contraceptive containing estrogen and progestin.

[0025] In another aspect, the subject is human.

[0026] Also provided herein is 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, for reducing androgen concentration in a subject having PCOS, or for reducing the severity of one or more symptoms or signs in a subject having PCOS.

[0027] Also provided herein is 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, for reducing androgen concentration in a subject having PCOS, or for reducing the severity of one or more symptoms or signs in a subject having PCOS.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Some aspects of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of aspects of the invention.

[0029] FIG. 1 shows the coding sequence open reading frame of mouse CRH (GenBank NM 205769), along with the CRISPR targeting gRNAs and PCR primers used in Example 1. [0030] FIGs. 2A-2B show 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 deletion in founder mice. PCR Primers CRHbp70F and CRHbp215R flanking the gRNAl were used to amplify an expected size of 146

SUBSTITUTE SHEET ( RULE 26) bp from wildtype H2L2 Harbour Mice® (FIG. 2A, left panel). PCR primers CRHbp208F and CRHbp310R were used to amplify an expected size of 103 bp from wildtype H2L2 Harbour Mice® (FIG. 2A, middle panel). PCR Primers CRHbp70F and CRHbp310R resulted in PCR fragment size of 241 bp (FIG. 2A, right panel). PCR product smaller than these expected sizes indicated mutations. Founder Mice 1, 2, 4 and 5 were identified as carrying mutations. Founder Mouse 5 from litter 12198 in FIG. 2B carried the 11 bp deletion (5'-CAGCCGGTTCT-3') (SEQ ID NO: 465), which was used for subsequent breedings.

[0031] FIG. 3 shows the location of an 11 bp deletion (5'-CAGCCGGTTCT-3') (SEQ ID NO: 465) in CRH gene coding region 157-167, that leads to a downstream reading frame shift and premature stop codon. This defective CRH open reading frame is not able to synthesize CRH peptide. A founder having this deletion was chosen for further breeding to homozygosity, rescue and immunization.

[0032] FIGs. 4A-4N show CRH ELISA binding activities of the indicated antibodies in PR numbers (FIGs. 4A, 4B, 4D, 4E, 4G, 4H, 4J, 4K, 4L and 4M) and human CRHR1 (hCRHRl) luciferase reporter blocking activities (FIGs. 4C, 4F, 41 and 4N) of anti-CRH monoclonal antibodies (mAbs). An unrelated human IgGl was used as a negative control.

[0033] FIGs. 5A-5L show CRH ELISA binding activities (FIGs. 5A-5D) and human CRHR1 (hCRHRl), human CRHR2 (hCRHR2), mouse CRHR1 (mCRHRl), and mouse CRHR2 (mCRHR2) luciferase reporter blocking activities (FIGs. 5E-5L) of Series 1 mAb PR004290 and its APTM variants.

[0034] FIGs. 6A-6D show human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2 luciferase reporter blocking activities of Series 2 mAb PR006669 and its APTM variants.

[0035] FIGs. 7A-7L show CRH ELISA binding activities (FIGs. 7A-7D) and human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2 luciferase reporter blocking activities (FIGs. 7E-7L) of Series 5 mAb PR301777 and its humanized variants.

[0036] FIGs. 8A-8D show human CRHR1 luciferase reporter blocking activities of Series 7 mAb PR302309 and its humanized variants.

[0037] FIGs. 9A-9F show human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2 luciferase reporter blocking activities of Series 9 mAb PR302334 and its humanized variants.

SUBSTITUTE SHEET ( RULE 26) [0038] FIGs. 10A-10G show human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2 luciferase reporter blocking activities of Series 10 mAh PR302341 and its humanized variants.

[0039] FIG. 11 A-l IL show human CRHR1, human CRHR2, mouse CRHR1, and mouse CRHR2 luciferase reporter blocking activities of Series 10 humanized mAbs PR302341-10, PR302341-20, and PR302341-23 and their APTM variants.

[0040] FIGs. 12A-12B show that some anti-CRH mAbs bind to human UCN1, but not human UCN2 or human UCN3 by ELISA.

[0041] FIGs. 13A-13D show ELISA binding of selected mAbs to CRH and human UCN1.

[0042] FIGs. 14A-14H show inhibition of human UCNl-mediated human CRHR1 reporter activity and human CRHR2 reporter activity.

[0043] FIGs. 15A-15D show that anti-CRH mAbs bind to the N-terminal region of CRH peptide.

[0044] FIG. 16 is a diagram of an epitope binding experiment using the Octet® platform.

[0045] FIG. 17 shows anti-CRH mAb PR302050 reduces restraint stress-induced ACTH and corticosterone concentrations in wildtype mice.

[0046] FIG. 18 shows anti-CRH mAb PR302050 reduces constitutively high ACTH in Mrapl knockout mice.

[0047] FIGs. 19A-19C show anti-CRH mAbs reduce constitutively high ACTH in Mrapl knockout mice.

[0048] FIGs. 20A-20B show additional anti-CRH mAbs that reduce constitutively high ACTH in Mrapl knockout mice.

[0049] FIGs. 21 A-21B show anti-CRH mAbs PR302038, PR005660, and PR302050 reduced plasma ACTH concentrations in Mrapl KO mice, in a dose dependent manner, at 20 mg/kg and 5 mg/kg doses of antibody. FIG. 21 A shows results at day 2 after mAb dosing. FIG. 21B shows results at day 16 after mAb dosing.

[0050] FIGs. 22A-22B show anti-CRH mAbs PR302334-24 and PR005660 at 20 mg/kg significantly reduced plasma ACTH concentrations in Mrapl KO mice, whereas PR301429, an anti-CRH mAb with comparable binding affinity, but a non-blocker in vitro, did not inhibit ACTH production. PR303394, an analog of CTRND05, was used as a comparator. FIG. 22A shows results at day 2 after mAb dosing. FIG. 22B shows results at day 16 after mAb dosing.

SUBSTITUTE SHEET ( RULE 26) [0051] FIGs. 23A-23B show anti-CRH mAb PR302064 is more efficacious than SSR125543A at inhibiting restraint induced ACTH (FIG. 23 A) and restraint induced corticosterone (FIG. 23B) release.

[0052] FIGs. 24A-24B show anti-CRH mAbs PR302064 and PR302334-24 had superior efficacy to SSR125543A in the Mrapl KO mice constitutively high ACTH model. ACTH levels at day 2 (FIG. 24 A) and day 14 (FIG. 24B) after mAb dosing are shown.

[0053] FIGs. 25A-25B show anti-CRH mAb PR302334-24 had superior efficacy to SSR125543A in the wildtype mice restraint stress induced high ACTH and corticosterone model. Induced ACTH (FIG. 25A) and corticosterone (FIG. 25B) plasma levels after mAb dosing are shown.

[0054] FIG. 26A is a diagram of a method to test the pharmacokinetics of anti-CRH mAbs. Specifically, anti-CRH mAbs in human IgGl-WT-Fc present in mouse serum were captured by goat anti -human Fc polyclonal antibody and then detected with goat anti-human (H+L) secondary antibody conjugated with HRP.

[0055] FIGs. 26B-26D show mean serum concentration time profiles of anti-CRH mAbs administered in a single intravenous dose of 5 mg/kg in female C57BL/6 wildtype mice.

DETAILED DESCRIPTION

[0056] Various terms relating to aspects of disclosure are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein.

/. Definitions

[0057] The term "antibody" (Ab) includes, without limitation, a glycoprotein immunoglobulin which binds specifically 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, CHI, 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, termed

SUBSTITUTE SHEET ( RULE 26) complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. A heavy chain may have the C-terminal lysine or not. Unless specified otherwise herein, the amino acids in the variable regions are numbered using the Kabat numbering system and those in the constant regions are numbered using the EU system.

[0058] The term "monoclonal antibodies," as used herein, refers to antibodies that are produced by a single clone of B-cells and bind to the same epitope. In contrast, the term "polyclonal antibodies" refers to a population of antibodies that are produced by different B-cells and bind 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 nonhuman antibodies; wholly synthetic antibodies; and single chain antibodies. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man. [0059] The antibody may be an antibody that has been altered (e.g., by mutation, deletion, substitution, conjugation to a non-antibody moiety). For example, an antibody may include one or more variant amino acids (compared to a naturally occurring antibody) which change a property (e.g., a functional property) of the antibody. For example, numerous such alterations are known in the art which affect, e.g., half-life, effector function, and/or immune responses to the antibody in a patient. The term antibody also includes artificial polypeptide constructs which comprise at least one antibody-derived antigen binding site.

[0060] An "antigen-binding fragment" of an antibody refers to one or more fragments or portions of an antibody that retain the ability to bind specifically to the antigen bound by the whole antibody. It has been shown that the antigen-binding function of an antibody can be performed by fragments or portions of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" or "antigen-binding fragment" of an antibody disclosed herein, include: 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 the antigenic determining regions of an intact antibody (e.g., the

SUBSTITUTE SHEET ( RULE 26) complementarity determining regions (CDR)). 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. An antigen-binding fragment of an antibody can be derived from any animal species, such as rodents (e.g., mouse, rat, or hamster) and humans or can be artificially produced.

[0061] Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv). See, e.g., Bird et al., Science, 242:423- 426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" or "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins. In some aspects, an antibody is an antigen-binding fragment.

[0062] As used herein, the terms "variable region" or "variable domain" are used interchangeably and are common in the art. The variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids or 110 to 125 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen. The variability in sequence is concentrated in those regions called complementarity determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR). Without 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 antigen. In some aspects, the variable region is a mammalian variable region, e.g., a human or rabbit variable region. In some aspects, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In some aspects, the variable region is a primate (e.g., non-human primate) variable region. In some aspects, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).

SUBSTITUTE SHEET ( RULE 26) [0063] The term "complementarity determining region" or "CDR" as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops (hypervariable loops) and/or contain the antigen-contacting residues. Antibodies can comprise six CDRs, e.g., three in the VH and three in the VL.

[0064] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0065] The terms "VH" and " VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0066] The term "Kabat numbering" and like terms are recognized in the art and refer to a system of numbering amino acid residues in the heavy and light chain variable regions of an antibody or an antigen-binding fragment thereof. In some aspects, CDRs can be determined according to the Kabat numbering system. See, e.g., Kabat et al., Ann. NY Acad. Sci., 190:382- 391 (1971) and Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991). Using the Kabat numbering system, CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35, which optionally can include one or two additional amino acids, following 35 (referred to in the Kabat numbering scheme as 35 A and 35B) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3).

[0067] Chothia refers instead 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 (this is because the Kabat numbering scheme places the insertions 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; if both 35 A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software.

[0068] As used herein, the term "constant region" or "constant domain" are interchangeable and have its meaning common in the art. The constant region is an antibody portion, e.g., a carboxyl terminal portion of a light and/or heavy chain which is not directly involved in binding of an antibody to antigen but which can exhibit various effector functions, such as interaction

SUBSTITUTE SHEET ( RULE 26) with the Fc receptor. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to an immunoglobulin variable domain. In some aspects, an antibody or antigen-binding fragment comprises a constant region or portion thereof that is sufficient for antibody-dependent cell-mediated cytotoxicity (ADCC).

[0069] As used herein, the term "heavy chain" when used in reference to an antibody can refer to any distinct type, e.g., alpha (a), delta (5), epsilon (a), gamma (y), and mu (p), based on the amino acid sequence of the constant domain, which give rise to IgA, IgD, IgE, IgG and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgGl, IgG2, IgG3 and IgG4. Heavy chain amino acid sequences are well known in the art. In some aspects, the heavy chain is a human heavy chain.

[0070] As used herein, the term "light chain" when used in reference to an antibody can refer to any distinct type, e.g., kappa (K) or lambda (X) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. In some aspects, the light chain is a human light chain.

[0071] The term "chimeric" antibodies or antigen-binding fragments thereof refers to antibodies or antigen-binding fragments thereof wherein the amino acid sequence is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies or antigen-binding fragments thereof derived from one species of mammals (e.g. mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability while the constant regions are homologous to the sequences in antibodies or antigen-binding fragments thereof derived from another (usually human) to avoid eliciting an immune response in that species.

[0072] The term "humanized" antibody or antigen-binding fragment thereof refers to forms of non-human (e.g. murine) antibodies or antigen-binding fragments that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies or antigen-binding fragments thereof are human immunoglobulins in which residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g. mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capability ("CDR grafted") (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, the Fv framework region (FR) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody or fragment from a non-human species that has the desired specificity, affinity, and

SUBSTITUTE SHEET ( RULE 26) capability. The humanized antibody or antigen-binding fragment thereof can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody or antigen-binding fragment thereof specificity, affinity, and/or capability. In general, the humanized antibody or antigenbinding fragment thereof will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody or antigen-binding fragment thereof can 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. Pat. 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 aspects, a "humanized antibody" is a resurfaced antibody.

[0073] The term "human" antibody or antigen-binding fragment thereof means an antibody or antigen-binding fragment thereof having an amino acid sequence derived from a human immunoglobulin gene locus, where such antibody or antigen-binding fragment is made using any technique known in the art. This definition of a human antibody or antigen-binding fragment thereof includes intact or full-length antibodies and fragments thereof.

[0074] "Binding affinity" generally refers to the strength of the sum total 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 indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody or antigen-binding fragment thereof and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured and/or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD), and equilibrium association constant (KA). The KD is calculated from the quotient of k _O ff/k _O n, whereas KA is calculated from the quotient of kon/koff. k _on refers to the association rate constant of, e.g., an antibody or antigen-binding fragment thereof to an antigen, and k _O ff refers to the dissociation of, e.g., an antibody or antigen-binding fragment thereof from an antigen. The kon and k _O ff can be determined by techniques known to one of ordinary skill in the art, such as Octet® BLI, BIAcore® or KinExA.

SUBSTITUTE SHEET ( RULE 26) [0075] As used herein, an "epitope" is a term 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, contiguous amino acids of a polypeptide (linear or contiguous epitope) or an epitope can, for example, come together from two or more non-contiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous, or non-contiguous epitope). In some aspects, the epitope to which an antibody or antigen-binding fragment thereof binds can be determined by, e.g., NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays, and/or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization may be accomplished using any of the known methods in the art. See, e.g., 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 studied using well known X-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth. Enzymol. (1985) volumes 114 & 115, eds Wyckoff et al.,; U.S. Pub. No. 2004/0014194), and BUSTER (Bricogne, Acta Crystallogr. D Biol.

Crystallogr., 49(Pt l):37-60 (1993); Bricogne, Meth. Enzymol., 276A:361-423, ed. Carter CW (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 one of skill in the art. See, e.g., Champe et al., J. Biol. Chem., 270: 1388-1394 (1995) and Cunningham et al., Science, 244:1081-1085 (1989) for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques.

[0076] A polypeptide, antibody, polynucleotide, vector, cell, or composition which is "isolated" is a polypeptide, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cell or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some aspects, an antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure. As used herein, "substantially pure" refers to material which 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.

SUBSTITUTE SHEET ( RULE 26) [0077] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been 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 the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that, because the polypeptides of this invention are based upon antibodies, in some aspects, the polypeptides can occur as single chains or associated chains.

[0078] As used herein, the term "host cell" can be any type of cell, e.g., 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. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule, e.g., due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

[0079] 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, which are not 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 the therapeutic formulations is contemplated. Supplementary active ingredients can also be incorporated into the formulations. In addition, various excipients, such as are commonly used in the art, can be included. These and other such compounds are described in the literature, e.g., in the Merck Index, Merck & Company, Rahway, NJ. Considerations for the inclusion of various components in pharmaceutical compositions are described, e.g., in Gilman et al. (Eds.) (2010); Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 12th Ed., The McGraw-Hill Companies.

[0080] As used herein, the term "subject" refers to a human or a non-human mammal, e.g., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-human primate or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate. In some aspects, the subject is a human. In some aspects, the subject is a woman. In some aspects, the woman is of reproductive age. In

SUBSTITUTE SHEET ( RULE 26) some aspects, a subject has experienced and/or exhibited at least one symptom of PCOS. In some aspects, the subject has been identified or diagnosed as having PCOS. In some aspects, the subject is suspected of having PCOS.

[0081] 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 crucial roles in controlling stress response, anxiety and depression, arousal, feeding behavior, energy metabolism, and digestive and cardiovascular function. 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 No. EAW86897.1) is: SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII (SEQ ID NO: 466).

[0082] CRH acts via two distinct G protein-coupled receptors, CRHR1 and CRHR2. CRHR1 expression is prevalent in brain areas responsible for sensory and motor control, such as the cortical mantle, olfactory bulb, hippocampus, amygdala, basal ganglia, medial and lateral hypothalamic nuclei, and cerebellum. In contrast, CRHR2 is predominant 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.

[0083] In response to stress, the hypothalamus releases CRH and triggers the release of ACTH from the anterior pituitary into the circulation. Subsequently, ACTH binds to its receptor on the adrenal cortex and triggers the release of stress hormones such as cortisol. This entire system is known as the hypothalamic-pituitary-adrenal (HP A) axis, which plays a crucial role in modulating fight-or-flight responses to stress.

[0084] As used herein, the term "polycystic ovary syndrome" is used interchangeably with the terms "polycystic ovarian syndrome", "PCOS", "polycystic ovary 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 absent menstrual periods), hyperandrogenism (e.g., elevated testosterone, free testosterone, androstenedione, 11 -oxygenated androgen, or a combination thereof), abnormal uterine bleeding, enlarged multifollicular ovaries, infertility, obesity, insulin resistance, hyperinsulinemia, hypertension, hyperlipidemia, type-2

SUBSTITUTE SHEET ( RULE 26) diabetes mellitus, hirsutism, excess facial hair growth, hair loss, and acne. In some aspects, a diagnosis of PCOS is based on biochemically documented hyperandrogenism (e.g., testosterone, free testosterone, androstenedione, 11 -oxygenated androgen, or a combination thereof two standard deviations or more above the control mean), chronic anovulation (e.g., <6 menses/year or dysfunctional uterine bleeding), and/or polycystic ovaries present on vaginal ovarian ultrasound.

[0085] As used herein, the terms "polycystic ovary syndrome therapy" or "PCOS therapy" refer to any treatment for PCOS. A PCOS therapy includes, but is not limited to, a combination birth control (e.g., an oral contraceptive containing estrogen and progestin) or progestin therapy, e.g., for regulating the menstrual cycle. A PCOS therapy also includes, but it not limited to, clomiphene, tamoxifen, an aromatase inhibitor (e.g., letrozole), metformin, a gonadotropin, or ovarian drilling, e.g., to promote ovulation. A PCOS therapy also includes, but is not limited to, a combination birth control (e.g., an oral contraceptive containing estrogen and progestin), spironolactone, eflornithine or electrolysis, e.g., to reduce excessive hair growth. A PCOS therapy also includes, but is not limited to, an antidiabetic agent (e.g., metformin), e.g., to reduce insulin and/or blood sugar levels.

[0086] 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 androgen, or a combination thereof), for example, in serum.

[0087] As used herein, the term "11 -oxygenated androgen" refers to 19-carbon steroids that originate primarily in the adrenal gland and share an oxygen atom on carbon 11. Examples of an 11 -oxygenated androgen include, but are not limited to, 11 -hydroxy androstenedione (110HA4), 11 -hydroxytestosterone (11OHT), 11 -ketoandrostenedione (11KA4), and 11 -ketotestosterone (11KT).

[0088] As used herein, the term "hirsutism" refers to a condition in which a subject exhibits abnormal hairiness.

[0089] As used herein, the term "hyperinsulinemia" refers to a condition in which a subject exhibits elevated plasma insulin levels.

[0090] As used herein, the term "hyperlipidemia" refers to a condition in which a subject exhibits elevated concentrations of any or all lipids in plasma.

[0091] As used herein, the term "hypertension" refers to a condition in which a subject experiences persistently high blood pressure (i.e., a systolic pressure equal or greater than 140 mm Hg and a diastolic pressure equal to or greater than 90 mm Hg).

SUBSTITUTE SHEET ( RULE 26) [0092] As used herein, the term "type-2 diabetes mellitus" refers to a disease, also known as non-insulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes mellitus (AODM), in which a subject has elevated concentrations of blood sugar levels.

[0093] As used herein, the term "insulin resistance" describes a subnormal biological response to a given concentration of insulin (e.g., decreased glucose transport across the cell membrane in response to insulin).

[0094] As used herein, the terms "treat" or "treatment" refer to therapeutic or palliative measures. Beneficial or desired clinical results include, but are not limited to, alleviation, in whole or in part, of symptoms associated with a disease or disorder or condition, diminishment of the extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state (e.g., one or more symptoms of the disease), and remission (whether partial or total), whether detectable or undetectable.

"Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0095] As used herein, "therapeutically effective amount" is an amount of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein required to reduce or lessen the severity of PCOS or any of its symptoms (e.g., of insulin resistance, hyperinsulinemia, type-2 diabetes mellitus, obesity, hypertension, hyperlipidemia, anovulation or irregular ovulation, infertility, hyperandrogenism, hirsutism, alopecia, acne, enlarged multifollicular ovaries and abnormal uterine bleeding, for some period of time). A therapeutically effective amount also means the amount required to improve the clinical symptoms of a subject in need thereof.

[0096] 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 articles "a" or "an" should be understood to refer to "one or more" of any recited or enumerated component.

[0097] The term "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: 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).

[0098] It is understood that wherever aspects are disclosed herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of and/or "consisting essentially of are also provided.

SUBSTITUTE SHEET ( RULE 26) [0099] The term "about" refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation per the practice 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 any number between 2.7 mg and 3.3 mg (for 10%) or between 2.4 mg and 3.6 mg (for 20%). Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the application and claims, unless otherwise stated, the meaning of "about" should be assumed to be within an acceptable error range for that particular value or composition.

[0100] As disclosed herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated.

[0101] Unless defined otherwise, 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 is related. 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 the Oxford Dictionary Of Biochemistry And Molecular Biology, 2006, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0102] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of 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 more fully defined by reference to the specification in its entirety.

[0103] Various aspects are described in further detail in the following sections.

2. Anti-CRH Antibodies or Antigen-Binding Fragments Thereof

[0104] The methods and kits of the present disclosure contain an antibody or antigen-binding fragment thereof that specifically binds to corticotropin-releasing hormone (CRH) (i.e., an antiCRH antibody or antigen-binding fragment thereof).

SUBSTITUTE SHEET ( RULE 26) [0105] Examples of anti-CRH antibodies and antigen-binding fragments thereof are known and described, for example, in Int'l Pub. No. WO 2019/241127, Int'l Pub. No. WO 2020/115555, CN107043752, Futch et al., J. Exp. Med., 216(11):2479-2491 (2019), Kawahito et al., Gut, 37(4):544-551 (1995), Kageyama et al., Neuroimmunomod., 2(3): 137-140 (1995), Carnes et al., Life Sci., 45(12): 1049-1056 (1989), Okamoto et al., Brain Res., 855(1): 192-197 (2000), Kawahito et al., Gastroenterol., 106(4):859-865 (1994), Miyazaki et al., Mod. Rheumatol., 12(3):206-212 (2002), Perkins et al., Placenta, 16(3):233-243 (1995), Yamada et al., Intern. Med., 41(7)549-554 (2002), and Sasaki et al., Neuroimmunomod., 2(3): 134-136 (1995), which are incorporated by reference in their entireties.

[0106] Other examples of anti-CRH antibodies and antigen-binding fragments thereof are described, for example, in U.S. Provisional Appl. No. 63/234,130, which is incorporated by reference herein in its entirety.

[0107] In some aspects, 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 of 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 of any one of SEQ ID NOs: 113-122 or the amino acid sequence of 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 of 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 of any one of SEQ ID NOs: 223-232.

[0108] In some aspects, 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.

[0109] In some aspects, 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

SUBSTITUTE SHEET ( RULE 26) 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.

[0110] In some aspects, 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.

[OHl] In some aspects, 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.

[0112] In some aspects, 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.

[0113] In some aspects, 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.

[0114] In some aspects, 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.

[0115] In some aspects, 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.

SUBSTITUTE SHEET ( RULE 26) [0116] In some aspects, 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.

[0117] In some aspects, 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.

[0118] In some aspects, 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.

[0119] In some aspects, 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.

[0120] In some aspects, 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.

[0121] In some aspects, 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.

[0122] In some aspects, 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

SUBSTITUTE SHEET ( RULE 26) 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.

[0123] In some aspects, 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.

[0124] In some aspects, 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.

[0125] In some aspects, 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.

[0126] In some aspects, 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.

[0127] In some aspects, 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.

[0128] In some aspects, 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.

SUBSTITUTE SHEET ( RULE 26) [0129] In some aspects, 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.

[0130] In some aspects, 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.

[0131] In some aspects, 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.

[0132] In some aspects, 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.

[0133] In some aspects, 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.

[0134] In some aspects, 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.

[0135] In some aspects, 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

SUBSTITUTE SHEET ( RULE 26) 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.

[0136] In some aspects, 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.

[0137] In some aspects, 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.

[0138] In some aspects, 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.

[0139] In some aspects, 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.

[0140] In some aspects, 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.

[0141] In some aspects, 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.

SUBSTITUTE SHEET ( RULE 26) [0142] In some aspects, 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.

[0143] In some aspects, 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.

[0144] In some aspects, 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.

[0145] In some aspects, 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.

[0146] In some aspects, 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.

[0147] In some aspects, 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.

[0148] In some aspects, 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

SUBSTITUTE SHEET ( RULE 26) 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.

[0149] In some aspects, 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.

[0150] In some aspects, 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.

[0151] In some aspects, 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.

[0152] In some aspects, 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.

[0153] In some aspects, 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.

[0154] In some aspects, 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.

SUBSTITUTE SHEET ( RULE 26) [0155] In some aspects, 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.

[0156] In some aspects, 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.

[0157] In some aspects, 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.

[0158] In some aspects, 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.

[0159] In some aspects, 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.

[0160] In some aspects, 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.

[0161] In some aspects, 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

SUBSTITUTE SHEET ( RULE 26) 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.

[0162] In some aspects, 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.

[0163] In some aspects, 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%, at least 99% identical to any one of SEQ ID NOs: 296-347 and 462. In some aspects, the antibody or antigen-binding fragment comprises a VH comprising an amino acid sequence of any one of SEQ ID NOs: 240-295, 459 and 461; and/or a VL comprising an amino acid sequence of any one of SEQ ID NOs: 296-347 and 462.

[0164] In some aspects, 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.

[0165] In some aspects, 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.

[0166] In some aspects, 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.

[0167] In some aspects, 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.

[0168] In some aspects, 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.

[0169] In some aspects, 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.

SUBSTITUTE SHEET ( RULE 26) [0170] In some aspects, 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.

[0171] 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.

[0172] 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.

[0173] In some aspects, 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.

[0174] In some aspects, 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.

[0175] In some aspects, 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.

[0176] In some aspects, 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.

[0177] In some aspects, 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.

[0178] In some aspects, 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.

[0179] In some aspects, 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.

[0180] In some aspects, 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.

[0181] In some aspects, 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.

[0182] In some aspects, 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.

[0183] In some aspects, 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.

[0184] In some aspects, 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.

[0185] In some aspects, 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.

SUBSTITUTE SHEET ( RULE 26) [0186] In some aspects, 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.

[0187] In some aspects, 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.

[0188] In some aspects, 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.

[0189] In some aspects, 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.

[0190] In some aspects, 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.

[0191] In some aspects, 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.

[0192] In some aspects, 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.

[0193] In some aspects, 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.

[0194] In some aspects, 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.

[0195] In some aspects, 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.

[0196] In some aspects, 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.

[0197] In some aspects, 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.

[0198] In some aspects, 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.

[0199] In some aspects, 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.

[0200] In some aspects, 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.

[0201] In some aspects, 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.

SUBSTITUTE SHEET ( RULE 26) [0202] In some aspects, 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.

[0203] In some aspects, 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.

[0204] In some aspects, 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.

[0205] In some aspects, 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.

[0206] In some aspects, 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.

[0207] In some aspects, 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.

[0208] In some aspects, 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.

[0209] In some aspects, 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.

[0210] In some aspects, 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.

[0211] In some aspects, 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.

[0212] In some aspects, 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.

[0213] In some aspects, 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.

[0214] In some aspects, 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.

[0215] In some aspects, 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.

[0216] In some aspects, 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.

[0217] In some aspects, 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.

SUBSTITUTE SHEET ( RULE 26) [0218] In some aspects, 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.

[0219] In some aspects, 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.

[0220] In some aspects, 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.

[0221] In some aspects, 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.

[0222] In some aspects, 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.

[0223] In some aspects, 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.

[0224] In some aspects, 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.

[0225] In some aspects, 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.

[0226] In some aspects, 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.

[0227] In some aspects, 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.

[0228] In some aspects, 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.

[0229] In some aspects, 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.

[0230] In some aspects, 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.

[0231] In some aspects, 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.

[0232] In some aspects, 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.

[0233] In some aspects, 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.

SUBSTITUTE SHEET ( RULE 26) [0234] In some aspects, 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.

[0235] In some aspects, 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.

[0236] In some aspects, 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.

[0237] In some aspects, 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.

[0238] In some aspects, 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.

[0239] In some aspects, 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.

[0240] In some aspects, 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.

[0241] In some aspects, 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.

[0242] In some aspects, 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.

[0243] In some aspects, 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.

[0244] In some aspects, 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.

[0245] In some aspects, 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.

[0246] In some aspects, 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.

[0247] In some aspects, 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.

[0248] In some aspects, 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.

[0249] In some aspects, 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.

SUBSTITUTE SHEET ( RULE 26) [0250] In some aspects, 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.

[0251] In some aspects, 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.

[0252] In some aspects, 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.

[0253] In some aspects, 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.

[0254] In some aspects, 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.

[0255] In some aspects, 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.

[0256] In some aspects, 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.

[0257] In some aspects, 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.

[0258] In some aspects, 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.

[0259] In some aspects, 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.

[0260] In some aspects, 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.

[0261] In some aspects, 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.

[0262] In some aspects, 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.

[0263] In some aspects, 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.

[0264] In some aspects, 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.

[0265] 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: 336.

SUBSTITUTE SHEET ( RULE 26) [0266] 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: 337.

[0267] 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: 338.

[0268] In some aspects, 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.

[0269] 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: 339.

[0270] In some aspects, 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.

[0271] In some aspects, 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.

[0272] In some aspects, 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.

[0273] In some aspects, 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.

[0274] In some aspects, 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.

[0275] In some aspects, 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.

[0276] In some aspects, 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.

[0277] In some aspects, 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.

[0278] In some aspects, 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.

[0279] In some aspects, 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.

[0280] In some aspects, 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.

[0281] In some aspects, 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.

SUBSTITUTE SHEET ( RULE 26) [0282] In some aspects, 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.

[0283] In some aspects, 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.

[0284] In some aspects, 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.

[0285] In some aspects, 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.

[0286] In some aspects, 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.

[0287] In some aspects, 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.

[0288] In some aspects, 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.

[0289] In some aspects, 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.

[0290] In some aspects, 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.

[0291] In some aspects, 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.

[0292] In some aspects, 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.

[0293] In some aspects, 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.

[0294] In some aspects, 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.

[0295] In some aspects, 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.

[0296] In some aspects, 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.

[0297] In some aspects, 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.

SUBSTITUTE SHEET ( RULE 26) [0298] In some aspects, 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.

[0299] In some aspects, 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.

[0300] In some aspects, 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.

[0301] In some aspects, 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.

[0302] In some aspects, 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.

[0303] In some aspects, 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.

[0304] In some aspects, 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.

[0305] In some aspects, 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.

[0306] In some aspects, 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.

[0307] In some aspects, 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.

[0308] In some aspects, 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.

[0309] In some aspects, 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.

[0310] In some aspects, 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.

[0311] In some aspects, 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.

[0312] In some aspects, 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.

[0313] In some aspects, 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.

SUBSTITUTE SHEET ( RULE 26) [0314] In some aspects, 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.

[0315] In some aspects, 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.

[0316] In some aspects, 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.

[0317] In some aspects, 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.

[0318] In some aspects, 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.

[0319] In some aspects, 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.

[0320] In some aspects, 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.

[0321] In some aspects, 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.

[0322] In some aspects, 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.

[0323] In some aspects, 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.

[0324] In some aspects, 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.

[0325] In some aspects, 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.

[0326] In some aspects, 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.

[0327] 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: 345.

[0328] 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: 346.

[0329] 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: 347.

SUBSTITUTE SHEET ( RULE 26) [0330] 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: 335.

[0331] In some aspects, 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.

[0332] In some aspects, 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.

[0333] In some aspects, 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.

[0334] 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: 340.

[0335] 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.

[0336] In some aspects, 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 aspects, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of any one of SEQ ID NOs: 348-403, 460 and 463; and/or a light chain comprising an amino acid sequence of any one of SEQ ID NOs: 404-455 and 464.

[0337] In some aspects, 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.

[0338] In some aspects, 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.

[0339] In some aspects, 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.

[0340] In some aspects, 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.

[0341] In some aspects, 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.

SUBSTITUTE SHEET ( RULE 26) [0342] In some aspects, 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.

[0343] In some aspects, 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.

[0344] In some aspects, 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.

[0345] In some aspects, 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.

[0346] In some aspects, 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.

[0347] In some aspects, 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.

[0348] In some aspects, 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.

[0349] In some aspects, 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.

[0350] In some aspects, 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.

[0351] In some aspects, 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.

[0352] In some aspects, 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.

[0353] In some aspects, 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.

[0354] In some aspects, 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.

[0355] In some aspects, 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.

[0356] In some aspects, 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.

[0357] In some aspects, 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.

SUBSTITUTE SHEET ( RULE 26) [0358] In some aspects, 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.

[0359] In some aspects, 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.

[0360] In some aspects, 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.

[0361] In some aspects, 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.

[0362] In some aspects, 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.

[0363] In some aspects, 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.

[0364] In some aspects, 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.

[0365] In some aspects, 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.

[0366] In some aspects, 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.

[0367] In some aspects, 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.

[0368] In some aspects, 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.

[0369] In some aspects, 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.

[0370] In some aspects, 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.

[0371] In some aspects, 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.

[0372] In some aspects, 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.

[0373] In some aspects, 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.

SUBSTITUTE SHEET ( RULE 26) [0374] In some aspects, 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.

[0375] In some aspects, 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.

[0376] In some aspects, 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.

[0377] In some aspects, 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.

[0378] In some aspects, 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.

[0379] In some aspects, 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.

[0380] In some aspects, 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.

[0381] In some aspects, 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.

[0382] In some aspects, 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.

[0383] In some aspects, 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.

[0384] In some aspects, 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.

[0385] In some aspects, 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.

[0386] In some aspects, 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.

[0387] In some aspects, 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.

[0388] In some aspects, 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.

[0389] In some aspects, 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.

SUBSTITUTE SHEET ( RULE 26) [0390] In some aspects, 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.

[0391] In some aspects, 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.

[0392] In some aspects, 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.

[0393] In some aspects, 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.

[0394] In some aspects, 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.

[0395] In some aspects, 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.

[0396] In some aspects, 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.

[0397] In some aspects, 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.

[0398] In some aspects, 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.

[0399] In some aspects, 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.

[0400] In some aspects, 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.

[0401] In some aspects, 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.

[0402] In some aspects, 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.

[0403] In some aspects, 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.

[0404] In some aspects, 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.

[0405] In some aspects, 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.

SUBSTITUTE SHEET ( RULE 26) [0406] In some aspects, 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.

[0407] In some aspects, 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.

[0408] In some aspects, 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.

[0409] In some aspects, 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.

[0410] In some aspects, 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.

[0411] In some aspects, 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.

[0412] In some aspects, 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.

[0413] In some aspects, 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.

[0414] In some aspects, 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.

[0415] In some aspects, 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.

[0416] In some aspects, 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.

[0417] In some aspects, 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.

[0418] In some aspects, 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.

[0419] In some aspects, 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.

[0420] In some aspects, 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.

[0421] In some aspects, 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.

SUBSTITUTE SHEET ( RULE 26) [0422] In some aspects, 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.

[0423] In some aspects, 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.

[0424] In some aspects, 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.

[0425] In some aspects, 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.

[0426] In some aspects, 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.

[0427] In some aspects, 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.

[0428] In some aspects, 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.

[0429] In some aspects, 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.

[0430] In some aspects, 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.

[0431] In some aspects, 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.

[0432] In some aspects, 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.

[0433] In some aspects, 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.

[0434] In some aspects, 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.

[0435] In some aspects, 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.

[0436] In some aspects, 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.

[0437] In some aspects, 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.

SUBSTITUTE SHEET ( RULE 26) [0438] In some aspects, 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.

[0439] In some aspects, 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.

[0440] In some aspects, 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.

[0441] In some aspects, 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.

[0442] In some aspects, 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.

[0443] In some aspects, 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.

[0444] In some aspects, 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.

[0445] In some aspects, 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.

[0446] In some aspects, 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.

[0447] In some aspects, 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.

[0448] In some aspects, 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.

[0449] In some aspects, wherein 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.

[0450] In some aspects, 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.

[0451] In some aspects, 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.

[0452] In some aspects, 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.

[0453] In some aspects, 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.

SUBSTITUTE SHEET ( RULE 26) [0454] In some aspects, 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.

[0455] In some aspects, 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.

[0456] In some aspects, 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.

[0457] In some aspects, 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.

[0458] In some aspects, 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.

[0459] In some aspects, 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.

[0460] In some aspects, 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.

[0461] In some aspects, 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.

[0462] In some aspects, 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.

[0463] In some aspects, 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.

[0464] In some aspects, 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.

[0465] In some aspects, 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.

[0466] In some aspects, 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.

[0467] In some aspects, 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.

[0468] In some aspects, 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.

[0469] In some aspects, 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.

SUBSTITUTE SHEET ( RULE 26) [0470] In some aspects, 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.

[0471] In some aspects, 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.

[0472] In some aspects, 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.

[0473] In some aspects, 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.

[0474] In some aspects, 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.

[0475] In some aspects, 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.

[0476] In some aspects, 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.

[0477] In some aspects, 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.

[0478] In some aspects, 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.

[0479] In some aspects, 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.

[0480] In some aspects, 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.

[0481] In some aspects, 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.

[0482] In some aspects, 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.

[0483] In some aspects, 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.

[0484] In some aspects, 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.

[0485] In some aspects, 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.

SUBSTITUTE SHEET ( RULE 26) [0486] In some aspects, 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.

[0487] In some aspects, 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.

[0488] In some aspects, 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.

[0489] In some aspects, 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.

[0490] In some aspects, 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.

[0491] In some aspects, 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.

[0492] In some aspects, 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.

[0493] In some aspects, 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.

[0494] In some aspects, 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.

[0495] In some aspects, 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.

[0496] In some aspects, 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.

[0497] In some aspects, 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.

[0498] In some aspects, 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.

[0499] In some aspects, 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.

[0500] In some aspects, 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.

[0501] In some aspects, 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.

SUBSTITUTE SHEET ( RULE 26) [0502] In some aspects, 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.

[0503] In some aspects, 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.

[0504] In some aspects, 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.

[0505] In some aspects, 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.

[0506] In some aspects, 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.

[0507] In some aspects, 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.

[0508] In some aspect, 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.

[0509] In some aspects, the anti-CRH antibody or antigen-binding fragment is an antibody or antigen-binding fragment thereof of Table 1.

SUBSTITUTE SHEET ( RULE 26) Table 1

[0510] In some aspects, the methods and kits of the present disclosure comprise a pharmaceutical composition comprising an anti-CRH antibody or antigen-binding fragment thereof disclosed herein and a pharmaceutically acceptable carrier.

[0511] In some aspects, an anti-CRH antibody used in the methods and kits disclosed herein is polyclonal, monoclonal, human, humanized, or chimeric.

[0512] In other aspects, an anti-CRH antigen-binding fragment used in the methods and kits disclose herein is a Fab, scFab, Fab', F(ab')2, Fv, scFv, diabody, or triabody.

[0513] Methods which are 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. These methods include, for example, constructing expression vectors containing such antibodies or antigen-binding fragments thereof or domain thereof (e.g., CDR, VH, VL, heavy chain and/or light chain) coding sequences and appropriate transcriptional and translational control signals, using in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

[0514] An expression vector can be transferred to a cell (e.g., 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 disclosed herein (e.g., an antibody or antigenbinding fragment thereof comprising the six CDRs, the VH, the VL, the VH and the VL, the heavy chain, the light chain, or the heavy and the light chain of an antibody disclosed herein) or a domain thereof (e.g., the VH, the VL, the VH and the VL, the heavy chain, or the light chain of an antibody disclosed herein).

[0515] In some aspects, for the expression of double-chained antibodies or antigen-binding fragments thereof, vectors encoding both the heavy and light chains, individually, can be coexpressed in the host cell for expression of the entire immunoglobulin. In some aspects, a host cell contains a vector comprising a polynucleotide encoding both the heavy chain and light chain of an antibody disclosed herein, or a domain thereof. In some aspects, a host cell contains two different vectors, a first vector comprising a polynucleotide encoding a heavy chain or a heavy chain variable region of an antibody or antigen-binding fragment thereof disclosed herein, and a second vector comprising a polynucleotide encoding a light chain or a light chain variable region of an antibody disclosed herein, or a domain thereof. In some aspects, a first host cell comprises a first vector comprising a polynucleotide encoding a heavy chain or a heavy chain variable region of an antibody or antigen-binding fragment thereof disclosed herein, and a second host cell comprises a second vector comprising a polynucleotide encoding a light chain or a light chain

SUBSTITUTE SHEET ( RULE 26) variable region of an antibody or antigen-binding fragment thereof disclosed herein. In some aspects, a heavy chain/heavy chain variable region expressed by a first cell associated with a light chain/light chain variable region of a second cell to form an antibody or antigen-binding fragment thereof disclosed herein. In some aspects, provided herein is a population of host cells comprising such first host cell and such second host cell.

[0516] 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 by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, express a protein or an antibody or antigen-binding fragment thereof disclosed herein in situ. These include but are not limited to 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 virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems (e.g., green algae such as Chlamydomonas reinhardtii) infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NSO, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210, Rl. l, B-W, L-M, BSC1, BSC40, YB/20, and BMT10 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). In some aspects, cells for expressing proteins or antibodies and antigen-binding fragments thereof disclosed herein are CHO cells, for example CHO cells from the CHO GS System™ (Lonza). In some aspects, cells for expressing the antibodies and antigenbinding fragments thereof disclosed herein are human cells, e.g., human cell lines. In some aspects, a mammalian expression vector is pOptiVEC™ or pcDNA3.3. In some aspects, bacterial cells such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells), especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary (CHO) cells in conjunction with a vector such as the major intermediate early gene promoter

SUBSTITUTE SHEET ( RULE 26) element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene, 45: 101-105 (1986), and Cockett et al., Biotechnology 8:662-667 (1990)). In some aspects, proteins or antibodies or antigen-binding fragments thereof disclosed herein are produced by HEK-293T cells.

[0517] In addition, a host cell strain can be chosen which 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 protein products can contribute to the function of the protein. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO (a murine 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, Rl.l, B-W, L-M, BSC1, BSC40, YB/20, BMT10 and HsS78Bst cells.

[0518] Once an antibody or antigen-binding fragment thereof disclosed herein has been produced by recombinant expression, it can be purified by any method known in the art for purification of a protein or an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and size exclusion chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, 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.

[0519] In some aspects, the methods and kits disclosed herein contain an antibody or antigenbinding fragment thereof that is isolated or purified. Generally, an isolated protein or antibody or antigen-binding fragment thereof is one that is substantially free of other proteins or antibodies or antigen-binding fragments thereof with different antigenic specificities than the isolated antibody or antigen-binding fragment thereof. For example, in some aspects, an antibody or antigenbinding fragment thereof disclosed herein is substantially free of cellular material and/or chemical precursors.

SUBSTITUTE SHEET ( RULE 26) 3. Methods of Use

[0520] Patients with polycystic ovary syndrome (PCOS) synthesize and secrete excessive amounts of androgens from their adrenal glands and ovaries. Recent analysis of the source of androgens in PCOS patients has found that the majority of these increased androgens come from the adrenal gland (O’Reilly et al. J Clin Endocrinol Metab 102: 840, 2017). The synthesis and secretion of adrenal androgens in humans 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, anti-CRH antibody blockade of CRH action in patients with PCOS should reduce CRH, ACTH, and adrenal androgens in these patients.

[0521] In some aspects, the present disclosure provides methods of treating PCOS and related disorders. The methods include administering to a subject in need thereof a therapeutically effective amount of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein. In some aspects, the method includes administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present disclosure that contains an anti-CRH antibody or antigen-binding fragment thereof disclosed herein.

[0522] In some aspects, the present disclosure provides methods of reducing androgen concentration in a subject having PCOS. The methods include administering to the subject a therapeutically effective amount of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein. In some aspects, the method includes administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present disclosure that contains an anti-CRH antibody or antigen-binding fragment thereof disclosed herein. In some aspects, the androgen is testosterone, free testosterone, androstenedione, an 11 -oxygenated androgen, or a combination thereof. In another aspect, the 11 -oxygenated androgen is 11 - hydroxyandrostenedione (110HA4), 11 -hydroxytestosterone (11OHT), 11 -ketoandrostenedione (11KA4), 11 -ketotestosterone (11KT), or a combination thereof..

[0523] In some aspects, the present disclosure provides methods for reducing the severity of one or more symptoms or signs in a subject having PCOS. The methods include administering to the subject having PCOS a therapeutically effective amount of an anti-CRH antibody or antigenbinding fragment thereof disclosed herein. In some aspects, the method includes administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present disclosure that contains an anti-CRH antibody or antigen-binding fragment thereof disclosed herein.

SUBSTITUTE SHEET ( RULE 26) [0524] In some aspects, the one or more symptoms or signs of PCOS is anovulation (e.g., irregular or absent menstrual periods), hyperandrogenism (e.g., elevated testosterone, free testosterone, androstenedione, 11 -oxygenated androgen, or a combination thereof), abnormal uterine bleeding, enlarged multifollicular ovaries, infertility, obesity, insulin resistance, hyperinsulinemia, hypertension, hyperlipidemia, type-2 diabetes mellitus, excess facial hair growth, hirsutism, hair loss, and acne. In another aspect, the 11 -oxygenated androgen is 11 - hydroxyandrostenedione (110HA4), 11 -hydroxytestosterone (11OHT), 11 -ketoandrostenedione (11KA4), 11 -ketotestosterone (11KT), or a combination thereof.

[0525] In some aspects, the subject exhibits a reduction in the severity of one or more symptoms of PCOS or an improvement of one or more symptoms of PCOS after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein. In some aspects, the one or more symptoms is anovulation (e.g., irregular or absent menstrual periods), hyperandrogenism (e.g., elevated testosterone, free testosterone, androstenedione, 11 -oxygenated androgen, or a combination thereof), abnormal uterine bleeding, enlarged multifollicular ovaries, infertility, obesity, insulin resistance, hyperinsulinemia, hypertension, hyperlipidemia, type-2 diabetes mellitus, excess facial hair growth, hirsutism, hair loss, or acne. In another aspect, the 11-oxygenated androgen is 11-hydroxyandrostenedione (110HA4), 11 -hydroxytestosterone (11OHT), 11 -ketoandrostenedione (11KA4), 11 -ketotestosterone (11KT), or a combination thereof.

[0526] In some aspects, the subject exhibits reduced anovulation after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to prior to administration of the antibody or antigen-binding fragment thereof. In some aspects, the subject resumes ovulation after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein.

[0527] In some aspects, the subject exhibits reduced irregularity of menstrual periods after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to the irregularity of menstrual periods prior to administration of the antibody or antigen-binding fragment thereof. In some aspects, the subject resumes menstrual periods after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein. In some aspects, the subject resumes regular menstrual periods after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein.

[0528] In some aspects, the subject exhibits reduced hyperandrogenism after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to prior

SUBSTITUTE SHEET ( RULE 26) to administration of the antibody or antigen-binding fragment thereof. In some aspects, the subject exhibits reduced testosterone, free testosterone, androstenedione, 11 -oxygenated androgen, or a combination thereof after administration of an anti-CRH antibody or antigenbinding fragment thereof disclosed herein compared to prior to administration of the antibody or antigen-binding fragment thereof. In another aspect, the 11 -oxygenated androgen is 11 - hydroxyandrostenedione (110HA4), 11 -hydroxytestosterone (110HT), 11 -ketoandrostenedione (11KA4), 11 -ketotestosterone (11KT), or a combination thereof.

[0529] In some aspects, the subject exhibits reduced abnormal uterine bleeding after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to prior to administration of the antibody or antigen-binding fragment thereof.

[0530] In some aspects, the subject exhibits reduced ovarian cysts and/or reduced multifollicular ovaries after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to prior to administration of the antibody or antigen-binding fragment thereof.

[0531] In some aspects, the subject exhibits improved fertility after administration of an anti- CRH antibody or antigen-binding fragment thereof disclosed herein compared to prior to administration of the antibody or antigen-binding fragment thereof.

[0532] 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 prior to administration of the antibody or antigen-binding fragment thereof.

[0533] In some aspects, the subject exhibits improved hyperinsulinemia after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to prior to administration of the antibody or antigen-binding fragment thereof.

[0534] In some aspects, the subject exhibits improved hypertension after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to prior to administration of the antibody or antigen-binding fragment thereof.

[0535] In some aspects, the subject exhibits improved hyperlipidemia after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to prior to administration of the antibody or antigen-binding fragment thereof.

[0536] In some aspects, the subject exhibits reduced excess hair growth after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to prior to administration of the antibody or antigen-binding fragment thereof.

SUBSTITUTE SHEET ( RULE 26) [0537] In some aspects, the subject exhibits reduced hirsutism after administration of an antiCRH antibody or antigen-binding fragment thereof disclosed herein compared to prior to administration of the antibody or antigen-binding fragment thereof.

[0538] In some aspects, the subject exhibits reduced hair loss (e.g., male pattern baldness) after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to prior to administration of the antibody or antigen-binding fragment thereof. [0539] In some aspects, the subject exhibits reduced acne after administration of an anti-CRH antibody or antigen-binding fragment thereof disclosed herein compared to prior to administration of the antibody or antigen-binding fragment thereof.

[0540] In some aspects of any of the methods provided herein, an anti-CRH antibody or antigen-binding fragment thereof disclosed herein is administered in combination with one or more PCOS therapy. In some aspects, the one or more PCOS therapy is selected from the group consisting of a combination birth control (e.g., an oral contraceptive containing estrogen and progestin); progestin therapy; clomiphene; tamoxifen; an aromatase inhibitor (e.g., letrozole); a gonadotropin; ovarian drilling; spironolactone; eflornithine; electrolysis; and an antidiabetic agent (e.g., metformin). In some aspects, an anti-CRH antibody or antigen-binding fragment thereof disclosed herein is administered in combination with one or more PCOS therapy selected from the group consisting of spironolactone, metformin, and a combination birth control (e.g., an oral contraceptive containing estrogen and progestin).

[0541] In some aspects, the subject is a human. In some aspects, the subject is a woman. In some aspects, the woman is of reproductive age.

[0542] It is sometimes 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 of 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 a measurement of CRH, a biological sample from a subject is contacted with an anti- CRH antibody or antigen-binding fragment thereof disclosed herein for a time sufficient to allow immunocomplexes to form. Immunocomplexes formed between the antibody and CRH in the sample are then detected. The amount of CRH in the biological sample is optionally quantitated by measuring the amount of the immunocomplex formed between the antibody and the 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 immunocomplex.

SUBSTITUTE SHEET ( RULE 26) [0543] 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.

[0544] Conditions for incubating an antibody with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the antibody used in the assay. One skilled in the art will recognize that any one of the commonly available immunological assay formats can readily be adapted to employ the antibodies (or fragments thereof) of the present disclosure. Examples of such assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock et al., Techniques in Immunocytochemistry, Academic Press, Orlando, FL Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, Practice and Theory of immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985). The test sample used in the above-described method 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.

[0545] Such methods are useful in, e.g., evaluating the efficacy of a particular therapeutic treatment regime in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.

[0546] In some aspects, CRH or the 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.

[0547] Detection of the presence or absence of a CRH-antibody complex be achieved using any method known in the art. For example, the transcript resulting from a reporter gene transcription assay of a CRH peptide interacting with a target molecule (e.g., antibody) typically encodes a directly or indirectly detectable product (e.g., P-galactosidase activity and luciferase activity). For cell free binding assays, one of the components usually includes, or is coupled to, a detectable label. A wide variety of labels can be used, such as those that provide direct detection (such as radioactivity, luminescence, optical or electron density) or indirect detection (such as epitope tag such as the FLAG epitope, enzyme tag such as horseradish peroxidase). The label can be bound to the antibody, or incorporated into the structure of the antibody.

[0548] A variety of methods can be used to detect the label, depending on the nature of the label and other assay components. For example, the label can be detected while bound to the

SUBSTITUTE SHEET ( RULE 26) solid substrate or subsequent to separation from the solid substrate. Labels can be directly detected through optical or electron density, radioactive emissions, nonradiative energy transfers or indirectly detected with antibody conjugates, or streptavidin-biotin conjugates. Methods for detecting the labels are well known in the art.

4. Kits

[0549] In some aspects, the present disclosure provides a kit comprising an anti-CRH antibody or antigen-binding fragment thereof disclosed herein.

[0550] In some aspects, the kit comprises (i) an anti-CRH antibody or antigen-binding fragment disclosed herein, and (ii) instructions for use of the anti-CRH antibody or antigenbinding fragment in treating PCOS in a subject in need thereof.

[0551] In some aspects, the kit comprises (i) an anti-CRH antibody or antigen-binding fragment disclosed herein, and (ii) instructions for use of the anti-CRH antibody or antigenbinding for reducing androgen concentration (e.g., testosterone, free testosterone, androstenedione, 11-oxygenated androgen, or a combination thereof) in a subject having PCOS. In another aspect, the 11-oxygenated androgen is 11-hydroxyandrostenedione (110HA4), 11- hydroxytestosterone (11OHT), 11 -ketoandrostenedione (11KA4), 11 -ketotestosterone (11KT), or a combination thereof.

[0552] In some aspects, the kit comprises (i) an anti-CRH antibody or antigen-binding fragment disclosed herein, and (ii) instructions for use of the anti-CRH antibody or antigenbinding for reducing the severity of one or more symptoms or signs in a subject having PCOS. [0553] In some aspects, the anti-CRH antibody or antigen-binding fragment in the kit is present in a pharmaceutical composition disclosed herein.

[0554] In some aspects, 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 aspects can be provided either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration. In some aspects, the kit comprises a first container having the anti-CRH antibody, antigen-binding fragment, or pharmaceutical composition in a dehydrated or lyophilized powder and a second container having an aqueous formulation. In some aspects, the kit comprises a prefilled syringe containing the anti-CRH antibody, antigen-binding fragment, or pharmaceutical composition.

SUBSTITUTE SHEET ( RULE 26) EXAMPLES

[0555] The following Examples are provided to further illustrate aspects of the disclosure, and are not meant to constrain the disclosure to any particular application or theory of operation.

EXAMPLE 1

Generation of CRH Knockout Mouse on H2L2 Harbour Mice® Background

[0556] Corticotropin-releasing hormone (CRH) knockout (KO) mice were generated on a H2L2 Harbour Mice® background, using the standard transgenesis method described in "Manipulating the Mouse Embryo," a laboratory manual by Nagy et al. Cold Spring Harbor Laboratory Press. Generation of CRH KO H2L2 mice was done under animal license ADV101002016512, animal protocol 16-512-11, approved by the Dutch Central Authority for Scientific Procedures on Animals of (CCD) and EDC animal facility.

[0557] In short, super-ovulated H2L2 Harbour Mice® females and males were used for mating. Collected fertilized eggs were used for microinjection of two guide RNAs (gRNAs) at 20 ng/microliter in a microinjection buffer of 5 mM tris(hydroxymethyl)aminomethane (TRIS), 0.1 mM ethylenediaminetetraacetic acid (EDTA) in ultrapure water, adjusted to pH7.4: gRNAl Mm.Cas9.CRH.l.AA and gRNA2 Mm.Cas9.CRH. l.AB and the CAS9 protein. The following gRNAl sequences were used:

[0558] gRNAl = Sequence-Mm.Cas9CHR.1. AA:

[0559] 5'-AltRl/rArArC rUrCrC rArCrG rCrCrC rCrUrC rArCrC rGrCrG rUrUrU rUrArG rArGrC rUrArU rGrCrU/altR2/-3'

[0560] gRNA2 = Mm.Cas9.CRH.l.AB:

[0561] 5'-AltRl/rUrCrA rCrCrC rArUrG rCrGrG rArUrC rArGrArArCrG rUrUrU rUrArG rArGrC rUrArU rGrCrU /altR2/-3'

[0562] Guide RNAs and CAS9 were obtained from Integrated DNA Technologies (IDT).

[0563] Injected eggs were transferred into foster mothers (B6CBAF1). Knockout pups were identified from toe DNA by amplification of DNA spanning the region over the gRNAs. Polymerase chain reaction (PCR) fragments smaller than these expected sizes indicated deletion in founder mice. PCR Primers CRHbp70F and CRHbp215R flanking the gRNAl were used to amplify an expected size of 146 bp from wildtype H2L2 Harbour Mice® (FIG. 2A, left panel). PCR primers CRHbp208F and CRHbp310R were used to amplify an expected size of 103 bp

SUBSTITUTE SHEET ( RULE 26) from wildtype H2L2 Harbour Mice® (FIG. 2A, middle panel). PCR Primers CRHbp70F and CRHbp310R resulted in PCR fragment size of 241 bp (FIG. 2 A, right panel). PCR product smaller than these expected sizes indicated mutations. Founder Mice 1, 2, 4 and 5 were identified as carrying mutations. PCR products deviating from the expected size for the wild type were further analyzed by cutting the PCR amplification products from the agarose gel, isolation of DNA and sequencing, using the same primers that were used for amplification. Founder Mouse 5 from litter 12198 in FIG. 2B carried the 11 bp deletion (5'-CAGCCGGTTCT-3') (SEQ ID NO: 465), which was used for subsequent breedings.

Rescue of CRH Knockout H2L2 Harbour Mice®

[0564] Rescue and immunization of CRH knockout mice were done under animal license ADV101002016512, animal working protocol 16-512-19. In short, homozygous CRH knockout H2L2 Harbour Mice® males and females were set in cages one to one. By observation of the copulation plug (0.5 d.p.c), females were taken out of the cage and separated. Ten days later, cortisol (30 pg/ml) was given in the drinking water (dark bottles) ad libitum to the pregnant females. This resulted in the birth of live knock out progenies that were used for immunization.

Immunization of H2L2 Harbour Mice® with CRH Knockout

[0565] Ten homozygous CRH knockout H2L2 Harbour Mice® of both genders at the age of about 8 weeks were immunized with subcutaneous (SC) and intraperitoneal (IP) injections of CRH-KLH conjugates on days 0, 14, 28, 42 and 50 in a maximum volume of 100 pl.

[0566] The following conjugates were used:

[0567] CRH-KLH: SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEILKLH

(C-Terminus) (GenScript) (SEQ ID NO: 467)

[0568] CRH-KLH conjugates were mixed with adjuvant Stimmune for the first injection and adjuvant Ribi for the following boosts. The last boost was IP. Blood samples were collected after the 4th injection and at the end of successful immunization experiment. Three to five days after the last IP injection, mice were euthanized, and spleens and lymph nodes were used in the fusion experiment with myeloma fusion partner for hybridoma production, according to standard protocol. Sera were screened for anti-CRH antibody titers and hybridomas later by the enzyme- linked immunosorbent assay (ELISA) with biotinylated CRH captured on streptavidin (SA)- coated plates. A detailed protocol follows.

SUBSTITUTE SHEET ( RULE 26) CRH ELISA

[0569] ELISA plates were coated with 5 pg/ml streptavidin in phosphate buffered saline (PBS) (Sigma 189730-lmg) at 4°C overnight or at room temperature (RT) for 2 hours. 50 pl/well was used for 96-well plates, or less for 384-well plates. After incubation, the strepavidin solution was removed and biotinylated peptide was added (ThermoFisher, biotin is at the N terminus of the peptide). Following incubation for 30 min at RT, plates were washed with IX phosphate buffered saline (PBS)/0.1% Tween-20 and blocked with 1% milk/1% bovine serum albumin (BSA) in washing solution. Following a wash in 3X PBS/0.1% Tween-20, 50 pl of antibody was added in blocking buffer and incubated at RT for 2 hours. Plates were then washed in 3X PBS/0.1% Tween-20, and 50 pl of anti-rat IgG-HRP (horseradish peroxidase) at a 1 :2,000 dilution was added and incubated at RT for 2 hours. Following a wash in 3X PBS/0.1% Tween- 20, 50 pl peroxidase substrate (POD or equivalent) was added and absorbance was measured at 450 nm.

Immunization of Balb/c Wildtype Mice

[0570] Balb/c wildtype mice were immunized with CRH-KLH protein to generate anti-CRH antibodies. The CRH and KLH conjugates are 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 N-terminus or C-terminus of CRH, respectively. 50 pg of protein was administered to each mouse for the first prime via IP with complete Freund's adjuvant (CFA, Sigma, Cat # F5881) and 25 pg for the following boosts via IP with Ribi (Sigma S6322). Boosts were conducted bi-weekly for 4 times. At designated time points, mouse serum was sampled and titrated by indirect ELISA to test binding against human CRH.

[0571] Mice with high serum titers and specific immune responses against human CRH were selected and injected with 25 pg of protein in PBS to boost immunity. Three days later, the mice were sacrificed, and their spleen cells and lymph node cells were collected.

Preparation of CRH-KLH Conjugate

[0572] The KLH carrier protein (Sigma, H7017-50MG) was weighed and dialyzed against 0.1M sodium phosphate buffer, pH7.5. Sulfo-MBS (BBI, C100314-0050) solution was added to the KLH carrier protein solution at a ratio of 1 : 10. One hour after incubation at room temperature, the reaction mixture was applied to a G-50 desalting column (Sigma, G50150-10G) pre-equilibrated with conjugation buffer (0.1M sodium phosphate buffer, pH6.5, with 1 mM

SUBSTITUTE SHEET ( RULE 26) EDTA). Fractions corresponding to the first protein peak based on A280nm were collected. KLH carrier protein-MBS and CRH peptide with an extra cysteine either at N-terminus or at C- terminus were mixed and incubated at room temperature for 2-4 hours. The reaction was terminated by adding 2-mercaptoethanol (Sigma, M3148-25ML) to a final concentration of 10 mM and incubated at room temperature for more than 30 minutes. The CRH-KLH conjugate was purified through a Sephadex G-25 desalting column to separate the unconjugated peptide from the CRH-KLH conjugate. Fractions corresponding to the first protein peak were collected. The protein concentration of CRH-KLH conjugate was determined by NanoPhotometer.

[0573] The following peptides were used for immunizing Balb/c wildtype mice:

[0574] Peptide 1 (KLH-CRH) (SEQ ID NO: 468):

[0575] KLH-C-SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII-CONH2

[0576] Peptide 2 (CRH-KLH) (SEQ ID NO: 469):

[0577] SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII-C-KLH

[0578] And, the following peptides were used for ELISA:

[0579] Peptide 3 (Biotin-CRH) (SEQ ID NO: 470):

[0580] Biotin-K-SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII-CONH2

[0581] Peptide 4 (CRH-Biotin) (SEQ ID NO: 471):

[0582] SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII-K-Biotin

[0583] Biotin-CRH (SEQ ID NO: 472):

[0584] Biotin-SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII-COOH (ThermoFisher)

Single B Cell Screening Based on the Beacon® Optofluidic System

[0585] A nanofluidic optoelectronic B lymphocyte antibody screening technique (NanOBlast) was used that was built around Beacon™, a commercially available (Berkeley Lights), integrated culture and imaging platform.

[0586] The NanOBlast workflow begins with generating antigen-specific antibody secreting cells (ASCs) via in vivo immunization. Selectively enriched, antigen-experienced murine ASCs were harvested from spleen and lymph nodes. ASCs are microfluidically imported into the chip and sequestered into individual nanopens for screening via OptoElectro Positioning (OEP). ASCs that secrete antigen-specific IgG are detected using a bead based, color fluorescent binding assay that produces a characteristic fluorescent bloom. Individual cells of interest are then un-penned using OEP and exported from the chip directly into 96-well plates containing cell lysis buffer.

SUBSTITUTE SHEET ( RULE 26) Antibody heavy chain variable domain (VH) and light chain variable domain (VL) sequences are recovered using single cell rapid amplification of cDNA ends (RACE), cloned and recombinantly expressed as canonical antibodies using standard methods. The recombinant antibodies are used for binding confirmation and eventual validation in relevant downstream assays.

Antibody Production and Purification

[0587] Recombinant plasmids encoding target antibodies were transiently co-transfected into HEK293-6E or ExpiCHO cell cultures using polyethylenimine (PEI) (Polyscience, 24885). One day before transfection, cells were seeded in Corning Erlenmeyer Flasks. On the day of transfection, recombinant plasmids encoding target protein and transfection reagent were mixed at an optimal ratio (Heavy ChaimLight Chain = 2:3) and then added to the seeded flasks for transfection. Cell culture supernatants were collected on day 6 and used for purification. Cell culture broth was centrifuged followed by filtration. Filtered cell culture supernatant was loaded onto an affinity purification column. Following washing and elution, the eluted fractions were pooled and the buffer was exchanged to storage buffer. The purified protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) analysis to determine molecular weight and purity. The concentration was determined by absorbance of light at 280 nm.

Antibody Engineering

[0588] Heavy chain variable domain (VH) and light chain variable domain (VL) sequences of anti-CRH chimeric antibodies were further optimized by humanization and posttranslational modification (PTM) removal procedures. In the humanization procedure, VH and VL sequences were first aligned to the closest human germline sequence by algorithms, e.g., NCBEIg-BLAST, and then positions in the framework regions were reverted to human germline sequence. All key backmutation positions were scanned by Chothia numbering. If there was a mismatch, it was replaced with the mouse counterpart residue. Antibodies composed of sequence variants after humanization were then recombinantly produced by standard molecular biology techniques. [0589] For PTM removal, VH and VL sequences were scanned for the presence of PTM motifs, e.g., isomerization motifs (e.g., DG). "Hotspot" residues (e.g., D or G in a DG motif) were mutated to either the counterpart residue in the germline sequence or another residue with

SUBSTITUTE SHEET ( RULE 26) similar biophysical properties. Antibodies containing sequence variants after PTM removal were then recombinantly produced by standard molecular biology techniques.

Binding of Anti-CRH Antibodies to CRH or Human UCN1 by ELISA

[0590] Streptavidin (Thermo, Cat: 21125) was diluted in PBS to a concentration of 2 pg/ml. 100 pl of diluted streptavidin was added per well to ELISA microplates, and the plates were incubated overnight at 4°C. Plates were 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 pg/mL CRH (NT biotin), 0.5 pg/mL CRH (CT biotin), 1 pg/mL human UCN1 (NT biotin), or 1 pg/mL human UCN1 (CT biotin) for 1 hour at 37°C. Plates were then washed and incubated with diluted anti-CRH antibody (Ab) at 15 pg/ml (100 nM, 10 diluted, 8 points) for 1 hour at 37°C. Subsequently, plates were washed and incubated with HRP-conjugated goat anti-human IgG (H+L) antibody (Jackson, Cat: 109-035-088) at 37°C for 1 hour. 100 pL of 3, 3', 5,5'- tetramethylbenzidine (TMB) substrate (Biopanda, Cat: TMB-S-003) was added, and the plates were incubated at room temperature for 15 minutes. 100 pL of ELISA stopping solution (Solarbio, Cat#: C1058) was added to terminate the reaction, and the optical density at 450 nm (OD450nm) was determined by an ELISA plate reader (Molecular Devices, Spectra max 384 plus).

Preparation of Plasmids and Cell Lines

[0591] The CHO-K1 (Cat# CCL-61) cell line was obtained from the American Type Culture Collection (ATCC), and pGL4.29/luc2P/CRE/Hygro (Cat# E8471) and Bio-Gio Luciferase Assay System (Cat# G7940) were obtained from Promega. Plasmids pcDNA3.1 -human CRHR1 (Clone ID: OHu27188C), pcDNA3.1 -human CRHR2 (Clone ID: OHul0803), pcDNA3.1 -mouse CRHR1 (Clone ID: OMu00753), and pcDNA3.1 -mouse CRHR2 (Clone ID: OMu23246) were synthesized in GenScript®, which are G418-resistant. Human CRH peptide was obtained from Peptide International (Cat# 4136-s). Forskolin (Cat# 1099) and human CRHR1 antagonist small molecule (Antalarmin, Cat# 2778) were obtained from Tocris. Anti-human CRHR1 antibody (Cat# MAB3930) was obtained from R&D System. An electroporation instrument (Electro Cell Manipulator Cat# ECM630) and electroporation Cuvettes plus (2 mm Gap Cuvette, Cat# 45- 0125) were used from BTX. Electroporation buffer (Ingenio Solution, Cat# MIR50111) was obtained from Minis, and sheep anti-human CRH polyclonal antibody was obtained from the Salk Institute.

SUBSTITUTE SHEET ( RULE 26) Generation of CHO-CRE-Luciferase-human CRHRL -human CRHR2. -mouse CRHRL and - mouse CRHR2 Reporter Cell Lines

[0592] To generate a CHO-CRE-Luciferase Reporter stable cell line, a mixture of 2 million CHO-K1 cells with 10 pg of pGL4.29/luc2P/CRE/Hygro plasmid in 100 pl of Ingenio solution was transferred into electroporation cuvettes and subjected to 1 or 2 pulses using an Electro Cell Manipulator (950uF and 120V). The processed cells were transferred into a 6-well plate with 2 mL of Dulbecco's Modified Eagle's Medium (DMEM)/F12 with 10% fetal bovine serum (FBS). After 24 hours, selection antibiotic hygromycin at 500 pg/ml was added to the cells. This selection took about 10-14 days. Using matrix dilution, the pool of CHO-CRE cells was subcloned into 96-well plates. Individual stable clones were harvested when selected colonies became obvious. Positive clones were validated using a series of diluted forskolin. Validated positive clones were frozen for the future use.

[0593] To create CHO-CRE-hCRHRl, -hCRHR2, -mCRHRl, and -mCRHR2 reporter cell lines, pcDNA3.1-hCRHRl, -hCRHR2, -mCRHRl, and -mCRHR2 were introduced into CHO- CRE cells established above by electroporation as described above. Selection antibiotics hygromycin at 500 pg/ml and G418 at 1,000 pg/ml were employed. Individual established stable cell lines were characterized by both CRH and forskolin stimulation.

Luciferase Reporter Assay Development

[0594] Forskolin stimulates adenylate cyclase and increases intracellular cyclic adenosine monophosphate (cAMP) levels in cells. To validate if cells respond to cAMP elevation, CHO- CRE cells were stimulated with a series of concentrations of forskolin, and cAMP-dependent luciferase expression was determined using the Bio-Gio Luciferase Assay System. CHO-CRE cells increased luciferase expression in response to forskolin stimulation in a dose-dependent manner (data not shown). CRH binds to CRHR1 and CRHR2, which are Gas coupled G-protein coupled receptors (GPCRs).

[0595] Activated GPCR proteins increase production of the intracellular cAMP. CHO-CRE- human CRHR1 stable clone 11 was generated by introduction of pcDNA3.1 -human CRHR1 under the selection of G418 and hygromycin. Human CRHR1 expression in CHO-CRE-hCRHRl Clone 11 was confirmed by flow cytometry (data not shown). To further validate that the cell line was functional, CHO-CRE-hCRHRl Clone 11 cells were treated with forskolin and CRH peptide. CHO-CRE-hCRHRl Clone 11 cells showed a dramatic increase of luciferase expression upon CRH treatment compared with the parental CHO-CRE, while both cell lines showed a

SUBSTITUTE SHEET ( RULE 26) comparable level of intracellular cAMP in response to forskolin treatment. Subsequently CHO- CRE-hCRHR2 Clone 32, -mCRHRl Clone 22, and -mCRHR2 Clone 8 cell lines were established using similar methodology and validated with CRH treatment (data not shown).

[0596] Blocking CRHR1 by small molecules or neutralization of CRH peptide can reduce luciferase expression if the CRH-CRHR1 system works in CHO-CRE-hCRHRl cells. Antalarmin, a small molecule CRHR1 antagonist, blocked CRH-mediated luciferase expression in CHO-CRE-hCRHRl Clone 11 (data not shown). Furthermore, sheep anti-CRH polyclonal antibody reduced CRH-mediated luciferase expression in these cells (data not shown). Likewise, sheep anti-CRH polyclonal antibody blocked CRH-mediated luciferase expression in CHO-CRE- hCRHR2, CHO-CRE-mCRHRl, and CHO-CRE-mCRHR2 cells (data not shown).

Affinity Determination with Octet®

[0597] Octet® Red 96e was used for affinity measurement. Octet® running buffer was prepared by diluting kinetics buffer 10X (Fortebio, Cat# 18-1105) in PBS according to the manufacturer's instructions. Two columns of SA sensors (Fortebio, Cat#18-5019) were hydrated in running buffer for 10 mins, one for ligand capturing and the other for reference. Antibodies were 2-fold diluted and added into a 96-well plate. 30 nM biotinylated CRH (GL Biochem, Cat#784751) was captured by the first column of SA sensors for 20 seconds, while reference sensors were dipped into buffer wells. After a 60-second baseline step, sensors were dipped into analyte and buffer wells successively, with the association and dissociation time of 180 seconds and 800 seconds, respectively. Sensors were regenerated in glycine pH1.5 (Cytiva, Cat#BR- 1003-54) before the next cycle. The equilibrium dissociation constant (KD) value was evaluated using Data Analysis Software 11.0 and the fitting model of 1 : 1 global fitting. Signals of reference wells and reference sensors were subtracted by selecting "Double reference" before data fitting.

Blocking Activity of CRH Induced Signaling in Reporter Cell Lines (CHO-CRE-hCRHRL CHO-CRE-hCRHR2. CHO-CRE-mCRHRl and CHO-CRE-mCRHR2)

[0598] Cells were digested by trypsin-EDTA (Life technologies, Cat#: 12604-013), centrifuged at 1,000 rpm for 5 min, resuspended in growth medium, counted using a cell counter, seeded at a concentration of 5,000 cells per 100 pl in 96-well flat plates, and incubated overnight. 50 pl of 4X serially diluted antibodies was added (8 points, 1/4 diluted by growth medium) to plates containing CHO-CRE-hCRHRl cells, and 50 pl of 4X CRH (4X is 2 nM, final is 0.5 nM) was added. Similarly, 4X CRH (4X is 0.4 nM, final is 0.1 nM) was added to CHO-CRE-

SUBSTITUTE SHEET ( RULE 26) hCRHR2 cells and CHO-CRE-mCRHRl cells, and 4X CRH (4X is 20 nM, final is 5 nM) was added to CHO-CRE-mCRHR2 cells. Next, plates were incubated 4-6 hours at 37°C in a CO2 incubator. 60 pl of culture medium was removed, to which was added 60 pl of Bio-Gio luciferase, followed by incubation for 10 min at RT. Plates were read in a luciferase plate reader (EnVision, PerkinElmer).

Human UCNL Human UCN2 and Human UCN3 ELISA

[0599] Streptavidin (Thermo, Cat: 21125) was diluted in PBS with a concentration of 2 pg/mL. 100 pL of diluted streptavidin was added per well to ELISA microplates, and the plates were incubated overnight at 4°C. Plates were 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 either 1 pg/mL hUCNl (NT Biotin), hUCNl (CT Biotin), hUCN2 (NT Biotin), hUCN2 (CT Biotin), hUCN3 (NT Biotin), or hUCN3 (CT Biotin) for 1 hour at 37°C. Plates were then washed and incubated with 100 nM and 10 nM of anti-CRH antibody for 1 hour at 37 °C. Plates were subsequently washed and incubated with HRP-conjugated goat anti-human IgG (H+L) antibody (Jackson, Cat: 109-035-088) at 37°C for 1 hour. 100 pl of TMB Substrate (Biopanda, Cat: TMB- S-003) was added, and the plates were incubated at room temperature for 15 minutes. 100 pL of ELISA stopping solution (Solarbio, Cat#: C1058) was then added to terminate the reaction, and the OD450nm was determined by an ELISA plate reader (Molecular Devices, Spectra max 384 plus).

Human UCN1 -Induced Luciferase Reporter Assay

[0600] CHO-CRE-hCRHRl and CHO-CRE-hCRHR2 cells were harvested with trypsin- EDTA (Life Technologies, Cat#: 12604-013). Cells were centrifuged at 1,000 rpm for 5 min and resuspended in growth medium. Cells were counted using a cell counter, seeded at 5,000 cells per 100 pl to 96-well flat plates, and incubated overnight. 50 pl of 4X serially diluted antibody (8 points, 1/3 diluted by growth medium) or CP 376395 hydrochloride (TOCRIS, Cat#:3212) (8 points, 1/10 diluted by growth medium) was added to plates, followed by 50 pl 4X hUCNl (4X is 4 nM, final is 1 nM) to CHO-CRE-hCRHRl cells or CHO-CRE-hCRHR2 cells. Plates were then incubated for 4-6 hours at 37°C in a CO2 incubator. Following this, 60 pl of culture medium was removed, to which was added 60 pl of Bio-Gio luciferase, followed by incubation for 10 min at RT. Plates were then read in a luciferase plate reader (EnVision, PerkinElmer).

SUBSTITUTE SHEET ( RULE 26) In Vivo Efficacy Models - Wildtype Female C57BL/6 Mice and Mrapl KO Mice

[0601] 8 -10 week old wildtype female C57BL/6 mice were obtained from Charles River

Laboratories. Mrapl KO mice were obtained from Queen Mary University of London, UK. SSR125543A, a small molecule CRHR1 antagonist, was obtained from Axon Medchem (Cat# Axon 1799). Mouse Corticosterone (55-Corms-E01) and adrenocorticotropic hormone (ACTH) ELISA kits (21-ACTHU-E01) were obtained from ALPCO, USA. All procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Protocols were approved by Charles River Laboratories Institutional Animal Care and Use Committee (IACUC).

Mrapl KO Mice Breeding, Ear Tagging and Genotyping

[0602] To generate Mrapl KO mice, Mrapl-/- male mice were bred with Mrapl+/- female mice. Corticosterone hormone replacement was performed by administering 50 pg/ml in drinking water to breeder cages when female mice were pregnant and their pregnancies palpable (approximately embryonic day 10). Drinking water was changed weekly, and treatment was stopped at weaning. Mice were uniquely identified with ear tags at the time of tail clipping to match future genotype with each mouse. DNA was extracted from tail tissue and used to genotype mice.

[0603] Genotyping of Mrapl-/- mice was conducted using PCR and the following PCR primers:

[0604] For the wild type allele of exon 1 of Mrapl :

[0605] Forward 5'-GCGTCTCTTAGTAGCCTTTGG (SEQ ID NO: 473)

[0606] Reverse 5'-CTTGTTGGCTTTCAGCTTCT (SEQ ID NO: 474)

[0607] For the mutant allele of Mrapl :

[0608] Forward 5'-GAACTTGCCTGGTCAACTGTTAAAAGGAC (SEQ ID NO: 475) [0609] Reverse 5'-TCTTTAGGCTCACTTCCTCCACTGACC (SEQ ID NO: 476) [0610] PCR band sizes: wild type allele is 345 bp and mutant allele is 459 bp.

Physical Restraint in Wildtype Mice

[0611] To determine the effect of nonpainful physical restraint upon stress responses, mice were restrained in 50 ml polypropylene ventilated tubes in which the tip was cut off with a razor blade to allow the mouse to breathe easily for 15-20 minutes. A mouse was directed to the open tube and crawled in. A piece of paper towel was put behind the mouse so that it could not back

SUBSTITUTE SHEET ( RULE 26) out, and the cap was screwed on. The mouse was placed back in its cage, ventral size down on the bedding. This stressor is not considered painful, as mice are confined to a limited space (in a ventilated restraint tube) but not in an unnatural physical position. The investigator was present in the room during the entire procedure. At the end of the procedure, the mouse was removed by the tail.

Effect of CRH Abs in Wildtype Mice in Restraint-Stress Model

[0612] On day 1, each mouse was injected intraperitoneally with 20 mg/kg of anti-CRH mAbs, hlgGl or PBS (5 mice per group). On day 2 or 3, and 14 each mouse was subjected to physical restraint-stress described above for 15 or 20 min. Immediately after restraint-stress, 200 pl of blood was collected in EDTA tubes from the submandibular vein of each mouse. Plasma was separated using a refrigerated centrifuge (4°C at 500 x g for 10 min) and snap-frozen in dry ice for subsequent ACTH and corticosterone ELISA measurement.

Effect of Anti-CRH Abs in Mrapl KO Mouse Model

[0613] On day 1, each mouse was injected (IP) with 20 mg/kg of anti-CRH Ab or hlgGl (5 or 6 mice each group). On day 2 or 3, 14 and 28, 200 pl of blood was collected in EDTA tubes from the submandibular vein of each mouse. Plasma was separated using a refrigerated centrifuge (4°C at 500 x g for 10 min) and snap-frozen in dry ice for subsequent ACTH ELISA measurement.

Parallel Comparison of Anti-CRH mAb with SSR125543A in Wildtype Mice Restraint- Stress Model

[0614] On day 1, one half of mice were injected IP with 20 mg/kg of anti-CRH Ab or hlgGl (5 mice per group). On day 3, the other half of mice were injected IP with 30 mg/kg of SSR125543A or solvent (5 mice each group). Two hours later, four groups of mice were subjected to restraint-stress as described above for 20 min. Immediately after restraint-stress, 200 pl 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 measurement. On day 4, SSR125543A or solvent-treated mice were subjected to a second round of restraint stress for 20 min and plasma was collected. Two weeks later, anti-CRH mAb- or hlgGl -treated mice were subjected to physical stress for 20 min followed by plasma collection.

SUBSTITUTE SHEET ( RULE 26) In Vivo PK Study

Administration and Blood Collection

[0615] For each antibody tested, 6 female C57BL/6 mice with a weight of 18-22 grams were selected, and antibody was administered intravenously at a single dose of 5 mg/kg. Whole blood was collected from 3 mice before and 15 minutes, 24 hours (1 day), 4, and 10 days after administration. Whole blood from the remaining 3 mice was collected before and 5 hours, 2, 7, and 14 days after administration. Whole blood was allowed to stand for 30 minutes to coagulate and then centrifuged. Separated serum was then frozen at -80°C until analysis.

Analysis Method

[0616] ELISA was used to quantitatively determine the drug concentration in mouse serum. Tested antibodies (with human Fc) in mice serum were captured by 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 (Abeam, #ab97175) was then added into the wells. Plates were then sealed and incubated at 37°C for 30 minutes, followed by washing and adding TMB substrate and ELISA stopping solution. 96-well plates were read at 450/630 nm wavelength using SpectraMax M2. Data was processed by Soft Max Pro.

[0617] Pharmacokinetic (PK) parameters were analyzed by Phoenix WinNonlin version 8.2 with non-compartmental model (NCA).

Determining the Binding Region of CRH for Anti-CRH Antibodies by ELISA

[0618] Streptavidin (Thermo, Cat: 21125) was diluted in PBS to a concentration of 2 pg/ml. 100 pl of diluted streptavidin was added per well to ELISA microplates, and the plates were incubated overnight at 4°C. Plates were 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 pg/mL N-terminal region plus the central region (CT biotin), the central region only (NT biotin or CT biotin), or the central region plus C-terminal region (NT biotin) of CRH for 1 hour at 37°C. Plates were then washed and incubated with diluted anti-CRH antibody (Ab) at 15 pg/ml (100 nM, 10 diluted, 8 points) for 1 hour at 37°C. Subsequently, plates were washed and incubated with HRP-conjugated goat anti-human IgG (H+L) antibody (Jackson, Cat: 109-035-088) at 37°C for 1 hour. 100 pL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Biopanda, Cat: TMB-S- 003) was added, and the plates were incubated at room temperature for 15 minutes. 100 pL of

SUBSTITUTE SHEET ( RULE 26) ELISA stopping solution (Solarbio, Cat#: C1058) was added to terminate the reaction, and the optical density at 450 nm (OD450nm) was determined by an ELISA plate reader (Molecular Devices, Spectra max 384 plus).

Epitope Binding by Competitive ELISA

[0619] For epitope binding, a competitive ELISA was performed. Specifically, 1 pg/ml of capture antibody, 100 pl/well, was coated on a 96 well-plate at 4°C overnight. Plates were 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. Then, a mixture of 50 pl/well of competitive antibody (40 pg/ml) and 50 pl/well biotin-labeled CRH with proper concentration was added to 96 well plates followed by incubation for 1 hour at 37°C. Subsequently, plates were washed and incubated with Streptavidin-Peroxidase (SIGMA, Cat: S2438-250UG) at 37°C for 1 hour. 100 pL of 3, 3', 5,5'- tetramethylbenzidine (TMB) substrate (Biopanda, Cat: TMB-S-003) was added, and the plates were incubated at room temperature for 15 minutes. 100 pL of ELISA stopping solution (Solarbio, Cat#: C1058) was added to terminate the reaction, and the optical density at 450 nm (OD450nm) was determined by an ELISA plate reader (Molecular Devices, Spectra max 384 plus).

Epitope Binning Using Octet®

[0620] Epitope binding was performed in Octet® Red 96e. Running buffer was prepared by diluting kinetics buffer lOx (Fortebio, Cat# 18-1105) in PBS according to the manufacturer's instructions.

[0621] SA sensors (Fortebio, Cat#18-5019) were hydrated in running buffer for 10 mins. Performing an in-tandem epitope binding assays involved a four-step binding cycle: 1) a regeneration step was established for 30 s, 2) CRH was captured at 30 nM with the loading threshold at 0.12 nm, 3) 60 nM antibodies (1st Ab) were loaded to saturate the immobilized peptide for 200 s, 4) the mixture of 1st Ab in step 3 and competing antibodies were bound for 200 s.

[0622] Octet® data were processed in Fortebio's Data Analysis Software 11.0. After exporting the signal, the inhibition ratio was calculated as below:

Inhibition ratio (%) = (a-b)/a*100% Take antibody Abn, for example

SUBSTITUTE SHEET ( RULE 26) Variable a: The signal of the binding of Abn alone to the antigen (also refers to 100% signal of Abn)

Variable b: When Abn is the 2nd Ab, the signal of it binding to antigen

EXAMPLE 2

Generation of CRH Knockout Mice on H2L2 Harbour Mice® Background

[0623] CRH knockout mice 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 gRNAs and PCR primers used. Specifically, the following primers were used for genotyping, i.e., amplification on genomic DNA, as depicted in Figure 1 :

[0624] CRHbp70F: 5'-GCC CTG CTC AGC AGG GGA TCC GT-3' (SEQ ID NO: 477) [0625] CRHbp215R: 5'-CTG TTG AGA TTC CCC AGG CGG AG-3' (SEQ ID NO: 478)

[0626] CRHbp208F: 5'-CTC AAC AGA AGT CCC GCT CGG-3' (SEQ ID NO: 479)

[0627] CRHbp310R: 5'-GGA AAA AGT TAG CCG CAG CCT GG-3' (SEQ ID NO: 480)

[0628] The expected PCR DNA fragment sizes from wildtype mice are 146 bp and 103 bp, respectively. PCR fragments smaller than these expected sizes indicated deletion in founder mice. Figures 2A-2B show the identification of CRH knockout founder mice using this method. [0629] Figure 3 shows location of an 11 bp deletion (5'-CAGCCGGTTCT-3') (SEQ ID NO: 465) in CRH gene coding region 157-167, that leads to a downstream reading frame shift and premature stop codon. This defective CRH open reading frame is not able to synthesize CRH peptide. A founder having this deletion was chosen for further breeding to homozygosity, rescue and immunization.

EXAMPLE 3

Identification of Anti-CRH mAbs from CRH Knockout Mice on H2L2 Harbour Mice® Background

[0630] Many positive hybridoma clones were identified from multiple rounds of hybridoma fusions from immunization with CRH peptide in CRH knockout mice on a H2L2 Harbour Mice® background. Purified monoclonal antibodies (mAbs) from hybridoma supernatants bound to CRH with high affinity in ELISA. However, three functional monoclonal antibody clones,

SUBSTITUTE SHEET ( RULE 26) hybridoma clones 16g2, 28b3 and 52g7, were identified with blocking activity in the human CRHR1 luciferase reporter assay. These three clones were expressed recombinantly with human IgGl wildtype Fc as PR004289, PR004290 and PR006669, respectively. PR004289 (from Hybridoma Clone 16g2), PR004290 (from Hybridoma Clone 28b3), and PR006669 (from Hybridoma Clone 52g7) share the same germline sequences of IGHV3-7 and IGKV2-28. PR004289 and PR004290 have one residue difference at IGHV position 28, being 128 in PR004289 and T28 in PR004290, respectively. Both PR004289 and PR004290 have complementarity determining regions (CDRs) different from that of PR006669. These three mAbs were designated as Series 1 (PR004289 and PR004290) and Series 2 (PR006669) mAbs. See Table 2.

SUBSTITUTE SHEET ( RULE 26) Table 2. Identification of Anti-CRH mAbs

EXAMPLE 4

Identification of Anti-CRH mAbs from Wildtype Mice

[0631] Over 200,000 single B cells from wildtype mice immunized with CRH-KLH conjugates were screened with the Beacon system, using the materials and methods described above. More than 500 monoclonal antibodies (mAbs) with binding activities in bead binding assay were identified. Over 300 of these mAbs were expressed recombinantly with human IgGl wildtype Fc. Many of these recombinantly-expressed mAbs bound to CRH with high affinity in ELISA. Eight series of functional mAbs with blocking activities in human CRHR1 luciferase reporter assay were identified, using the materials and methods described above. mAbs from different series have different germline sequences and different CDRs. mAbs within each series share same germline sequences, but have different CDRs.

[0632] These mAbs were designated as Series 3 to Series 10 as follows.

[0633] Series 3 includes PR301306.

[0634] Series 4 includes PR301612.

[0635] Series 5 includes PR301777, PR301788, PR301790 and PR301803.

[0636] Series 6 includes PR301806.

[0637] Series 7 includes PR302309, PR302332 and PR302333.

[0638] Series 8 includes PR302315 and PR302335.

[0639] Series 9 includes PR302328, PR302331, PR302334 and PR302339.

[0640] Series 10 includes PR302312, PR302324 and PR302341.

[0641] See also Table 2 and Figures 4A-4N.

EXAMPLE 5

Antibody Engineering

PTM Removal of Series 1 mAb PR004290

[0642] PR004290 contains two potential posttranslational modifications (PTMs), DG in heavy chain CDR2 (HCDR2) and NS in light chain CDR1 (LCDR1). PR006669 has one potential PTM of DG in HCDR2. These potential PTMs were mutated individually either in HCDR2 alone, in LCDR1 alone, or in various combinations of mutations, as detailed in Table 3. Their binding activities in CRH ELISA and functional blocking activities in human CRHR1 (hCRHRl), human CRHR2 (hCRHR2), mouse CRHR1 (mCRHRl), and mouse CRHR2

SUBSTITUTE SHEET ( RULE 26) (mCRHR2) luciferase reporter assays are shown in Table 3 and Figures 5A-5L. Most APTM variants retained functional activities. PR005660 had a 50% inhibitory concentration (IC50) of about 10 nM in the hCRHRl reporter assay, vis-a-vis about 5 nM of parental PR004290.

SUBSTITUTE SHEET ( RULE 26) Table 3. CRH ELISA Binding Activities and Functional Blocking Activities of Series 1 mAb PR004290 and Its APTM Variants

PTM Removal of Series 2 mAb PR006669

[0643] PR006745 and PR006746 are two APTM variants of PR006669 and with somewhat weaker ELISA binding activities and functional blocking activities. See Table 4 and Figures 6A- 6D.

SUBSTITUTE SHEET ( RULE 26) Table 4. Functional Blocking Activities of Series 2 mAb PR006669 and Its APTM Variants

Humanization of Series 5 mAb PR301777

[0644] Fifteen humanized variants of PR301777 were recombinantly expressed and tested, using the materials and methods described above. Many humanization variants maintained high potency in vitro ELISA binding activities and functional blocking activities. See Table 5 and Figures 7A-7L.

Table 5. CRH ELISA Binding Activities and Functional Blocking Activities of Series 5 mAb PR301777 and Its Humanized Variants

SUBSTITUTE SHEET ( RULE 26)

Humanization of Series 7 mAb PR302309

[0645] Twenty-one humanized variants of PR302309 were recombinantly expressed and tested, using the materials and methods described above. Many humanization variants maintained high potency in vitro ELISA binding activities and functional blocking activities. See Table 6 and Figures 8A-8D.

SUBSTITUTE SHEET ( RULE 26) Table 6. Functional Blocking Activities of Series 7 mAb PR302309 and Its Humanized Variants

Humanization of Series 9 mAb PR302334

[0646] Twenty-two humanized variants of PR302334 were recombinantly expressed and tested, using the materials and methods described above. Many humanization variants maintained high potency in vitro ELISA binding activities and functional blocking activities. See Table 7 and Figures 9A-9F.

SUBSTITUTE SHEET ( RULE 26) Table 7. Functional Blocking Activities of Series 9 mAb PR302334 and Its Humanized Variants

Humanization of Series 10 mAb PR302341

[0647] Twenty-seven humanized variants of PR302341 were recombinantly expressed and tested, using the materials and methods described above. Many humanization variants maintained high potency in vitro ELISA binding activities and functional blocking activities. See Table 8 and Figures 10A-10G.

SUBSTITUTE SHEET ( RULE 26) Table 8. Functional Blocking Activities of Series 10 mAb PR302341 and Its Humanized Variants

PTM Removal of Humanized Series 10 mAbs PR302341-10, PR302341-20, and PR302341-23

[0648] 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 above three parental humanized mAbs, using the materials and methods described above. Many humanization variants maintained high potency in vitro ELISA binding activities and functional blocking activities. See Table 9 and Figures 11 A-l IL. PR302341-34, PR302341- 48, PR302341-60, PR302341-28 and PR302341-55 were scaled up for in vivo studies.

SUBSTITUTE SHEET ( RULE 26) Table 9. Functional Blocking Activities of Series 10 Humanized mAbs PR302341-10, PR302341-20 and PR302341-23 and their PTM Removed Variants

SUBSTITUTE SHEET (RULE 26) EXAMPLE 6

Selectivity Against Human UCNL Human UCN2, and Human UCN3 peptides

[0649] All tested mAbs, except for PR302334-26, bound to human UCN1 peptides in ELISA assays. None of the tested mAbs bound either human UCN2 or human UCN3 peptides in ELISA assays. The binding affinities of these mAbs to human UCN1 are comparable to that of CRH by ELISA. However, only mAbs from PR302341 series inhibited hUCNl-induced hCRHRl luciferase reporter activity. In addition, these mAbs inhibited hUCNl -induced hCRHRl reporter activity with a potency 30X to 267X lower than that of CRH-induced hCRHRl reporter activity. None of the mAbs blocked hIJCNl -induced hCRHR2 luciferase reporter activity. See Table 10 and Figures 12A-12B, 13A-13D and 14A-14H.

SUBSTITUTE SHEET ( RULE 26) Table 10. Anti-CRH mAbs Block CRH-Induced Signaling with Higher Potency than that of hUCNl-Induced Signaling

EXAMPLE 7

Anti-CRH mAbs Bind to N-Terminal Region of CRH Peptide

[0650] To determine the binding regions of the CRH peptide, the N-terminal region plus the central region, the central region only, and the central region plus C-terminal region of CRH peptides were used for ELISA capture of CRH, using the materials and methods described above. All mAbs bound to the N-terminal 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.

Table 11. Anti-CRH mAbs bind to N-terminal region of CRH peptide

CRH Region : N-Terminal -Central Region- C-Terminal

Full Length CRH : SEEPPISLDL TFHLLREVLE MARAEQLAQQ AHSNRKLMEI I -NH2

(SEQ ID NO : 481) N- Terminal CRH : SEEPPISLDL TFHLLREVLE MARAEQLAQQ A-K-Biotin

(SEQ ID NO : 482 ) C-Terminal CRH : Biotin-K-VLE MARAEQLAQQ AHSNRKLMEI I -NH2

(SEQ ID NO : 483 ) Central CRH : Biotin-K-LREVLE MARAEQLAQQ AHSN-NH2

(SEQ ID NO : 484 ) Central CRH : LREVLE MARAEQLAQQ AHSN-K-Biotin

(SEQ ID NO : 485 )

EXAMPLE 8

Affinity Determination of mAbs with Octet®

[0651] The binding affinities of anti-CRH antibodies were determined using the Octet® platform, using the materials and methods described above. The identified mAbs have very high affinities, with equilibrium dissociation constant (KD) values around single digit to double digit pM. See Table 12.

SUBSTITUTE SHEET ( RULE 26) Table 12. Affinity Determination of mAbs with Octet®

EXAMPLE 9

Antibody Binding Epitope Binding with ELISA

[0652] Selected mAbs were tested for their ability to block each other for binding to biotin- CRH in a pairwise competition ELISA, using materials and methods described above. Table 13 shows that these mAbs blocked binding of each other to CRH, and therefore, likely bind to a similar epitope, possibly at the N-terminus of CRH.

SUBSTITUTE SHEET ( RULE 26) Table 13. Antibody Binding Epitope Binding with ELISA

Note: PR303394 is an analog of CTRND05, from Patent WO2019241127A1, reformatted with human IgGl wildtype Fc.

EXAMPLE 10

Antibody Binding Epitope Binding with Octet®

[0653] Selected mAbs were tested for their ability to block each other for binding to biotin- CRH in a pairwise competition Octet® assay, using the materials and methods described above. Figure 16 shows a diagram of the competition assay using the Octet® platform. Table 14 shows that these mAbs blocked binding of each other to CRH, and therefore, likely bind to a similar epitope, possibly at the N-terminus of CRH.

Table 14. Antibody Binding Epitope Binding with Octet®

EXAMPLE 11

In Vivo Efficacy Studies

Wildtype Mice Restraint-Induced Transient Increase of ACTH and Corticosterone

[0654] Treatment with anti-CRH mAbs PR004290 (Series 1 mAb), PR006152 (Series 1 mAb), and PR302050 (Series 5 mAb) (20 mg/kg IP) markedly inhibited adrenocorticotropic hormone (ACTH) and corticosterone concentrations in wildtype mice subjected to the restraint- induced stress model discussed above. Day 1 post-mAb dosing inhibited plasma ACTH and corticosterone concentrations down to basal unstressed levels. Fourteen days after a single dose of mAb, ACTH and corticosterone concentrations remained partially inhibited. See Figure 17.

Mrapl KO Mice Constitutively High ACTH Model

[0655] Mrapl KO mice have constitutively high plasma ACTH concentrations of -5,000 pg/ml, approximately 50-fold higher than the basal ACTH in wildtype mice, and -10-fold higher than those observed in restraint-induced stress in wildtype mice. A single dose of anti-CRH mAb

SUBSTITUTE SHEET ( RULE 26) PR302050 (Series 5 mAb) at 20 mg/kg markedly reduced the constitutively high concentrations of ACTH in Mrapl KO mice both 2 and 14 days after administration. See Figure 18.

[0656] A single dose of anti-CRH mAb PR302064 (Series 5 mAb) markedly reduced constitutively high levels of ACTH in Mrapl KO mice in a dose-dependent manner at 20 mg/kg, 5 mg/kg, and 1 mg/kg, on post-dosing Days 2, 16, and 28, respectively. PR302064 at 20 mg/kg had 73%, 82%, and 55% reduction of ACTH on Days 2, 16, and 28, respectively. PR302064 at 5 mg/kg had 74%, 45%, and 34% reduction of ACTH on Days 2, 16, and 28, respectively.

PR302064 at 1 mg/kg had 47%, 31%, and 23% reduction of ACTH 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 post-dosing days 2 and 16. See Figures 19A-19C.

[0657] Additional anti-CRH mAbs were tested in a Mrapl KO model. Single doses of PR302334-26 (Series 9 mAb), PR302341-28, PR302341-34, PR302341-55, and PR302341-60 (all Series 10 mAbs) at 20 mg/kg also markedly reduced plasma ACTH concentrations in Mrapl KO mice. See Figures 20A-20B.

[0658] PR302038, PR005660, and PR302050 reduced plasma ACTH concentrations in

Mrapl KO mice, in a dose dependent manner, at 20 mg/kg and 5 mg/kg doses of antibody. See Figures 21A-21B.

[0659] PR302334-24 and PR005660 at 20 mg/kg significantly reduced plasma ACTH concentrations in Mrapl KO mice, whereas PR301429, an anti-CRH mAb with comparable binding affinity, but a non-blocker in vitro, did not inhibit ACTH production. PR303394, an analog of CTRND05, was used as a comparator. See Figures 22A-22B.

Anti-CRH mAb PR302064 had Superior Efficacy to SSR125543 A in a Wildtype Mice Restraint- Induced ACTH Model

[0660] Anti-CRH mAb PR302064 (Series 5 mAb) at 20 mg/kg was more efficacious than CRHR1 small molecule antagonist SSR125543A (Crinecerfont) at 30 mg/kg in reducing restraint-induced ACTH in wildtype mice. PR302064 had a more complete inhibition of both ACTH and corticosterone, and a longer duration of action. The effects of this mAb lasted at least 16 days. See Figures 23A-23B.

SUBSTITUTE SHEET ( RULE 26) Anti-CRH mAbs PR302064 and PR302334-24 Had Superior Efficacy to SSR125543 A in the Mrapl KO Mice Constitutively High ACTH Model

[0661] 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 efficacious than CRHR1 small molecule antagonist SSR125543A (Crinecerfont) at 30 mg/kg in reducing plasma ACTH levels in the Mrapl KO Mice constitutively high ACTH model. PR302064 and PR302334-24 had more complete inhibition, and more importantly had longer duration of action. The effects of these two mAbs lasted for at least 14 days. See Figures 24A-24B.

Anti-CRH mAb PR302334-24 Had Superior Efficacy to SSR125543 A in the Wildtype Mice Restraint Stress Induced High ACTH and Corticosterone Model

[0662] Anti-CRH mAb PR302334-24 at 20 mg/kg and 5 mg/kg were both more efficacious than CRHR1 small molecule antagonist SSR125543A (Crinecerfont) at 30 mg/kg in reducing plasma ACTH and corticosterone levels in a wildtype mice restraint stress induced high ACTH and corticosterone model. PR302334-24 had more complete inhibition, and more importantly had longer duration of action. The effects of PR302334-24 lasted for at least 17 days. See Figures 25A-25B.

In Vivo PK Study

[0663] The pharmacokinetics of mAbs PR004290, PR302050, and PR302064 were tested in C57BL/6 wildtype mice in a single IV dose of 5 mg/kg. Specifically, anti-CRH mAbs in human IgGl-WT-Fc present in mouse serum were captured by goat anti-human Fc polyclonal antibody and detected with goat anti-human (H+L) secondary antibody conjugated with HRP. Figure 26A shows a diagram of this method.

[0664] As shown in Table 15 and Figures 26B-26D, serum half-life (T1/2) of PR004290, PR302050 and PR302064 were 14 days, 21 days and 18 days, respectively. These data indicate that these mAbs have typical human IgGl PK profiles, with desirable T1/2 for clinical application.

Table 15. PK Analysis of Antibodies in Single IV Dose of 5 mg/kg in Female C57BL/6 Wildtype Mice (N=6)

SUBSTITUTE SHEET ( RULE 26)

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

SUBSTITUTE SHEET ( RULE 26)