Anti-PAPP-A antibodies and methods of use thereof

Abstract

Provided herein are anti-PAPP-A antibodies, as well as methods of using such antibodies to treat PAPP-A-associated disorders, e.g., kidney disease.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 383,875, filed November 15, 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing, which is incorporated herein by reference in its entirety. The XML copy, created on November 6, 2023, is named CLS102WO_ST.26.xml and is 112,848 bytes in size. [Background technology]

[0003] Autosomal dominant polycystic kidney disease (ADPKD) is a rare disease with unmet medical needs. Approximately 166,000 patients in the United States alone suffer from ADPKD. ADPKD is a leading cause of morbidity and accounts for approximately 5–10% of deaths related to end-stage renal disease (ESRD). Currently, only one approved treatment, tolvaptan, is available. However, tolvaptan has significant side effects and is prescribed under an FDA-mandated risk evaluation and mitigation strategy (REMS) to reduce the risk of serious, potentially fatal, liver damage associated with its administration. Therefore, additional therapeutic agents are needed to treat kidney diseases, including ADPKD. Summary of the Invention

[0004] In some aspects, provided herein is an isolated human antibody that binds to human pregnancy-associated plasma protein A (PAPP-A) (SEQ ID NO: 79), wherein the antibody comprises a variable heavy chain (VH) sequence comprising three heavy chain CDR sequences, CDR-H1, CDR-H2, and CDR-H3, and a variable light chain (VL) sequence comprising three light chain CDR sequences, CDR-L1, CDR-L2, and CDR-L3, wherein: CDR-H1 comprises the sequence SYAMH (SEQ ID NO: 3); CDR-H2 contains the sequence VISYDGSIKYYADAVKG (SEQ ID NO: 4); CDR-H3 comprises the sequence HNRIYSWGWHTFDI (SEQ ID NO: 5); CDR-L1 comprises the sequence RASQDISIYLN (SEQ ID NO: 8); CDR-L2 comprises the sequence GASSLQS (SEQ ID NO:9); and CDR-L3 contains the sequence QQADAGPWK (SEQ ID NO: 10).

[0005] In some embodiments, the VH sequence comprises the VH sequence shown in SEQ ID NO:2.

[0006] In some embodiments, the VL sequence comprises the VL sequence set forth in SEQ ID NO:7.

[0007] In some embodiments, the VH sequence comprises the VH sequence set forth in SEQ ID NO:2 and the VL sequence comprises the VL sequence set forth in SEQ ID NO:7.

[0008] In some embodiments, the antibody comprises the heavy chain sequence shown in SEQ ID NO:1.

[0009] In some embodiments, the antibody comprises the light chain sequence shown in SEQ ID NO:6.

[0010] In some embodiments, the heavy chain comprises the sequence set forth in SEQ ID NO:1 and the light chain sequence set forth in SEQ ID NO:6.

[0011] In another aspect, provided herein is an isolated human antibody that binds to human pregnancy-associated plasma protein A (PAPP-A) (SEQ ID NO: 79), wherein the antibody comprises two variable heavy chain (VH) sequences and two variable light chain (VL) sequences, wherein the VH sequences comprise the VH sequence set forth in SEQ ID NO: 2 and the VL sequences comprise the VL sequence set forth in SEQ ID NO: 7.

[0012] In another aspect, provided herein is an isolated human antibody that binds to human pregnancy-associated plasma protein A (PAPP-A) (SEQ ID NO: 79), wherein the antibody comprises a human IgG1 Fc region, two heavy chains comprising the sequences set forth in SEQ ID NO: 1, and two light chains comprising the sequences set forth in SEQ ID NO: 6.

[0013] In some aspects, provided herein is an isolated antibody that binds to pregnancy-associated plasma protein A (PAPP-A) (SEQ ID NO: 79).

[0014] In some embodiments, the antibody comprises a variable heavy chain (VH) sequence comprising three heavy chain CDR sequences, CDR-H1, CDR-H2, and CDR-H3, and a variable light chain (VL) sequence comprising three light chain CDR sequences, CDR-L1, CDR-L2, and CDR-L3; where: CDR-H1 comprises the sequence X1YX2MX3 (SEQ ID NO: 73), where X1 is S or T, X2 is A or G, and X3 is H or S; CDR-H2 comprises the sequence X1IX2X3X4X5X6X7X8YYADX9VKG (SEQ ID NO: 74), where X1 is V or A; X2 is S, Y, or R; X3 is Y or M; X4 is D or T; X5 is G or V; X6 is S, R, G, or Q; X7 is I, R, N, or E; X8 is K or T; and X9 is A or S; CDR-H3 comprises the sequence HX1RIX2X3WGX4HTFDI (SEQ ID NO: 75), where X1 is N or E, X2 is Y or P, X3 is S or P, and X4 is W or F, or the sequence ADMHRFDV (SEQ ID NO: 45), the sequence VWGGVRFDV (SEQ ID NO: 55), or the sequence YKPMPFDV (SEQ ID NO: 25 or 35); CDR-L1 comprises the sequence RASQX1IX2X3YLN (SEQ ID NO: 76), wherein X1 is D or S, X2 is S or I, and X3 is I, S, T, or R; CDR-L2 comprises the sequence X1ASX2LQS (SEQ ID NO: 77), where X1 is G, V, E, or A and X2 is S or I; CDR-L3 comprises the sequence X1QX2X3X4X5PX6X7 (SEQ ID NO: 78), where X1 is Q or G, X2 is A or S, X3 is D, Y, S, or H, X4 is A, S, G, Y, or P, X5 is G, P, or T, X6 is W, Y, or F, and X7 is K, T, or P.

[0015] In some embodiments, the VH sequence comprises a sequence selected from the sequences set forth in SEQ ID NOs: 2, 12, 22, 32, 42, or 52.

[0016] In some embodiments, the VL sequence comprises the VL sequence set forth in SEQ ID NO: 7, 17, 27, 37, 47, or 57.

[0017] In some embodiments, the VH sequence comprises the VH sequence set forth in SEQ ID NO: 2, 12, 22, 32, 42, or 52, and the VL sequence comprises the VL sequence set forth in SEQ ID NO: 7, 17, 27, 37, 47, or 57.

[0018] In some embodiments, the antibody comprises a heavy chain sequence selected from the sequences set forth in SEQ ID NOs: 1, 11, 21, 31, 41, or 51.

[0019] In some embodiments, the antibody comprises a light chain sequence selected from the sequences set forth in SEQ ID NOs: 6, 16, 26, 36, 46, or 56.

[0020] In some embodiments, the antibody comprises a heavy chain sequence selected from the sequences set forth in SEQ ID NO: 1, 11, 21, 31, 41, or 51, and a light chain sequence selected from the sequences set forth in SEQ ID NO: 6, 16, 26, 36, 46, or 56.

[0021] In some embodiments, the antibody comprises two heavy chain sequences selected from the sequences set forth in SEQ ID NO: 1, 11, 21, 31, 41, or 51, and two light chain sequences selected from the sequences set forth in SEQ ID NO: 6, 16, 26, 36, 46, or 56.

[0022] In some embodiments, the CDR-H3 comprises HNRIYSWGWHTFDI (SEQ ID NO: 5) or HERIPPWGFHTFDI (SEQ ID NO: 15).

[0023] In some embodiments, CDR-H1 comprises the sequence shown in SEQ ID NO:3; CDR-H2 comprises the sequence shown in SEQ ID NO:4; CDR-H3 comprises the sequence shown in SEQ ID NO:5; CDR-L1 comprises the sequence shown in SEQ ID NO:8; CDR-L2 comprises the sequence set forth in SEQ ID NO:9; and CDR-L3 comprises the sequence shown in SEQ ID NO:10.

[0024] In some embodiments, the VH sequence comprises the VH sequence shown in SEQ ID NO:2.

[0025] In some embodiments, the VL sequence comprises the VL sequence set forth in SEQ ID NO:7.

[0026] In some embodiments, the VH sequence comprises the VH sequence set forth in SEQ ID NO:2 and the VL sequence comprises the VL sequence set forth in SEQ ID NO:7.

[0027] In some embodiments, the antibody comprises the heavy chain sequence shown in SEQ ID NO:1.

[0028] In some embodiments, the antibody comprises the light chain sequence shown in SEQ ID NO:6.

[0029] In some embodiments, the antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO:1 and a light chain comprising the sequence set forth in SEQ ID NO:6.

[0030] In some embodiments, CDR-H1 comprises the sequence shown in SEQ ID NO: 13; CDR-H2 comprises the sequence shown in SEQ ID NO: 14; CDR-H3 comprises the sequence shown in SEQ ID NO: 15; CDR-L1 comprises the sequence shown in SEQ ID NO: 18; CDR-L2 comprises the sequence set forth in SEQ ID NO: 19; and CDR-L3 comprises the sequence shown in SEQ ID NO:20.

[0031] In some embodiments, the VH sequence comprises the VH sequence set forth in SEQ ID NO:12.

[0032] In some embodiments, the VL sequence comprises the VL sequence set forth in SEQ ID NO:17.

[0033] In some embodiments, the VH sequence comprises the VH sequence set forth in SEQ ID NO:12 and the VL sequence comprises the VL sequence set forth in SEQ ID NO:17.

[0034] In some embodiments, the antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO:11.

[0035] In some embodiments, the antibody comprises a light chain comprising the sequence set forth in SEQ ID NO:16.

[0036] In some embodiments, the antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO:11 and a light chain comprising the sequence set forth in SEQ ID NO:16.

[0037] In some embodiments, CDR-H1 comprises the sequence shown in SEQ ID NO: 23; CDR-H2 comprises the sequence shown in SEQ ID NO: 24; CDR-H3 comprises the sequence set forth in SEQ ID NO: 25; CDR-L1 comprises the sequence shown in SEQ ID NO: 28; CDR-L2 comprises the sequence set forth in SEQ ID NO: 29; and CDR-L3 comprises the sequence shown in SEQ ID NO:30.

[0038] In some embodiments, the VH sequence comprises the VH sequence set forth in SEQ ID NO:22.

[0039] In some embodiments, the VL sequence comprises the VL sequence set forth in SEQ ID NO:27.

[0040] In some embodiments, the VH sequence comprises the VH sequence set forth in SEQ ID NO:22 and the VL sequence comprises the VL sequence set forth in SEQ ID NO:27.

[0041] In some embodiments, the antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO:21.

[0042] In some embodiments, the antibody comprises a light chain comprising the sequence set forth in SEQ ID NO:26.

[0043] In some embodiments, the antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO:21 and a light chain comprising the sequence set forth in SEQ ID NO:26.

[0044] In some embodiments, CDR-H1 comprises the sequence shown in SEQ ID NO: 33; CDR-H2 comprises the sequence set forth in SEQ ID NO: 34; CDR-H3 comprises the sequence set forth in SEQ ID NO: 35; CDR-L1 comprises the sequence shown in SEQ ID NO: 38; CDR-L2 comprises the sequence set forth in SEQ ID NO: 39; and CDR-L3 comprises the sequence shown in SEQ ID NO:40.

[0045] In some embodiments, the VH sequence comprises the VH sequence set forth in SEQ ID NO:32.

[0046] In some embodiments, the VL sequence comprises the VL sequence set forth in SEQ ID NO:37.

[0047] In some embodiments, the VH sequence comprises the VH sequence set forth in SEQ ID NO:32 and the VL sequence comprises the VL sequence set forth in SEQ ID NO:37.

[0048] In some embodiments, the antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO:31.

[0049] In some embodiments, the antibody comprises a light chain comprising the sequence set forth in SEQ ID NO:36.

[0050] In some embodiments, the antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO:31 and a light chain comprising the sequence set forth in SEQ ID NO:36.

[0051] In some embodiments, CDR-H1 comprises the sequence shown in SEQ ID NO: 43; CDR-H2 comprises the sequence set forth in SEQ ID NO: 44; CDR-H3 comprises the sequence set forth in SEQ ID NO: 45; CDR-L1 comprises the sequence shown in SEQ ID NO: 48; CDR-L2 comprises the sequence set forth in SEQ ID NO: 49; and CDR-L3 comprises the sequence shown in SEQ ID NO:50.

[0052] In some embodiments, the VH sequence comprises the VH sequence set forth in SEQ ID NO:42.

[0053] In some embodiments, the VL sequence comprises the VL sequence set forth in SEQ ID NO:47.

[0054] In some embodiments, the VH sequence comprises the VH sequence set forth in SEQ ID NO:42 and the VL sequence comprises the VL sequence set forth in SEQ ID NO:47.

[0055] In some embodiments, the antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO:41.

[0056] In some embodiments, the antibody comprises a light chain comprising the sequence set forth in SEQ ID NO:46.

[0057] In some embodiments, the antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO:41 and a light chain comprising the sequence set forth in SEQ ID NO:46.

[0058] In some embodiments, CDR-H1 comprises the sequence set forth in SEQ ID NO: 53; CDR-H2 comprises the sequence set forth in SEQ ID NO: 54; CDR-H3 comprises the sequence set forth in SEQ ID NO: 55; CDR-L1 comprises the sequence shown in SEQ ID NO: 58; CDR-L2 comprises the sequence set forth in SEQ ID NO: 59; and CDR-L3 comprises the sequence shown in SEQ ID NO:60.

[0059] In some embodiments, the VH sequence comprises the VH sequence set forth in SEQ ID NO:52.

[0060] In some embodiments, the VL sequence comprises the VL sequence set forth in SEQ ID NO:57.

[0061] In some embodiments, the VH sequence comprises the VH sequence set forth in SEQ ID NO:52 and the VL sequence comprises the VL sequence set forth in SEQ ID NO:57.

[0062] In some embodiments, the antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO:51.

[0063] In some embodiments, the antibody comprises a light chain comprising the sequence set forth in SEQ ID NO:56.

[0064] In some embodiments, the antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO:51 and a light chain comprising the sequence set forth in SEQ ID NO:56.

[0065] In some embodiments, the antibody comprises a variable heavy chain (VH) sequence comprising three heavy chain CDR sequences, CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1, CDR-H2, and CDR-H3 comprise the CDRs of one of the variable heavy (VH) chain sequences set forth in SEQ ID NO: 2, 12, 22, 32, 42, or 52, as defined by the Kabat, AbM, IMGT, or Chothia numbering schemes.

[0066] In some embodiments, the antibody comprises a variable light chain (VL) sequence comprising three light chain CDR sequences, CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1, CDR-L2, and CDR-L3 comprise the CDRs of one of the variable light (VL) chain sequences set forth in SEQ ID NO: 7, 17, 27, 37, 47, or 57 as defined by the Kabat, AbM, IMGT, or Chothia numbering schemes.

[0067] In some embodiments, the antibody is a chimeric antibody, a human antibody, or a humanized antibody.

[0068] In some embodiments, the antibody is a monoclonal antibody.

[0069] In some embodiments, the antibody is a humanized antibody.

[0070] In some embodiments, the antibody is a human antibody.

[0071] In some embodiments, the antibody comprises an Fc region.

[0072] In some embodiments, the antibody comprises a human Fc region.

[0073] In some embodiments, the human Fc region comprises a human IgG1 Fc region.

[0074] In some embodiments, the Fc region comprises the L234A / L235A mutations according to the EU numbering system.

[0075] In another aspect, provided herein is an isolated human antibody that binds to pregnancy-associated plasma protein A (PAPP-A) (SEQ ID NO: 79), wherein the antibody comprises a human IgG1 Fc region, two heavy chains comprising the sequences set forth in SEQ ID NO: 1, and two light chains comprising the sequences set forth in SEQ ID NO: 6.

[0076] In another aspect, provided herein is an isolated human antibody that binds to pregnancy-associated plasma protein A (PAPP-A) (SEQ ID NO: 79), wherein the antibody comprises a human IgG1 Fc region, two heavy chains comprising the sequences set forth in SEQ ID NO: 11, and two light chains comprising the sequences set forth in SEQ ID NO: 16.

[0077] In another aspect, provided herein is an isolated human antibody that binds to pregnancy-associated plasma protein A (PAPP-A) (SEQ ID NO: 79), wherein the antibody comprises a human IgG1 Fc region, two heavy chains comprising the sequences set forth in SEQ ID NO: 21, and two light chains comprising the sequences set forth in SEQ ID NO: 26.

[0078] In another aspect, provided herein is an isolated human antibody that binds to pregnancy-associated plasma protein A (PAPP-A) (SEQ ID NO: 79), wherein the antibody comprises a human IgG1 Fc region, two heavy chains comprising the sequences set forth in SEQ ID NO: 31, and two light chains comprising the sequences set forth in SEQ ID NO: 36.

[0079] In another aspect, provided herein is an isolated human antibody that binds to pregnancy-associated plasma protein A (PAPP-A) (SEQ ID NO: 79), wherein the antibody comprises a human IgG1 Fc region, two heavy chains comprising the sequences set forth in SEQ ID NO: 41, and two light chains comprising the sequences set forth in SEQ ID NO: 46.

[0080] In another aspect, provided herein is an isolated human antibody that binds to pregnancy associated plasma protein A (PAPP-A) (SEQ ID NO: 79), wherein the antibody comprises a human IgG1 Fc region, two heavy chains comprising the sequences set forth in SEQ ID NO: 51, and two light chains comprising the sequences set forth in SEQ ID NO: 56.

[0081] In some embodiments, the antibody has a cytoplasmic affinity of about 1, 1.5, 2, 2.5, 10, 25, 50, 75, or 100×10 as measured by a surface plasmon resonance (SPR) assay. -12 K below M D and binds to human PAPP-A.

[0082] In some embodiments, the antibody has enzyme inhibiting or neutralizing activity, and optionally, the antibody has metalloprotease inhibiting activity.

[0083] In some embodiments, the antibody blocks PAPP-A cleavage of the IGF binding protein.

[0084] An isolated antibody as disclosed herein for use as a pharmaceutical.

[0085] An isolated antibody as disclosed herein for use in treating a PAPP-A associated disorder.

[0086] In another aspect, provided herein is an isolated polynucleotide or set of polynucleotides encoding an antibody, its VH, its VL, its light chain, its heavy chain, or an antigen-binding portion thereof of any of the above claims; optionally, the isolated polynucleotide or set of polynucleotides is cDNA.

[0087] In another aspect, provided herein is a vector or set of vectors comprising a polynucleotide or set of polynucleotides disclosed herein.

[0088] In another aspect, provided herein is a host cell comprising a polynucleotide or set of polynucleotides disclosed herein, or a vector or set of vectors disclosed herein.

[0089] In another aspect, provided herein is a method of making an antibody comprising expressing an antibody in a host cell disclosed herein and isolating the expressed antibody.

[0090] In another aspect, provided herein is a pharmaceutical composition comprising an isolated antibody disclosed herein and a pharmaceutically acceptable excipient.

[0091] In another aspect, provided herein is a kit comprising an isolated antibody disclosed herein or a pharmaceutical composition disclosed herein and instructions for use.

[0092] In another aspect, provided herein is a method of treating a PAPP-A associated disorder in a subject, comprising administering to the subject a composition comprising an anti-PAPP-A antibody.

[0093] In some embodiments, the PAPP-A associated disorder is kidney disease, polycystic kidney disease, or autosomal dominant polycystic kidney disease (ADPKD). [Brief explanation of the drawings]

[0094] These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description and accompanying drawings. [Figure 1]

[0023] Figure 1 is a diagram of the method used to assess in vitro analysis of PAPP-A activity as measured by cleavage of IGF-binding protein (IGFBP4). The resulting Western blot showed uncleaved and cleaved IGFBP bands, which were quantified using Compass for SW software (Bio-techne®). [Figure 2]High temperature stability analysis of Ab1, Ab2, and Ab3. After 3 weeks of storage at 40°C, a decrease in the proportion of monomer was observed. [Figure 3] Total kidney volume (TKV) in pcy mice treated with Ab8 (isotype control) or Ab7 (anti-PAPP-A). TKV was estimated by MRI at baseline, 12 weeks, and 21 weeks of treatment. pcy mice were treated with 10 mg / kg of Ab8 (isotype control, black circles) or Ab7 (anti-PAPP-A, gray squares). Data are presented as box plots. [Figure 4] Glomerular filtration rate (GFR) in pcy mice treated with Ab8 (isotype control) or Ab7 (anti-PAPP-A). Renal function (GFR) was assessed at baseline, 12 weeks, and 18 weeks of treatment using the FITC-sinistrin method (MediBeacon®). pcy mice were treated with either 10 mg / kg Ab8 (isotype control, black circles) or Ab7 (anti-PAPP-A, gray squares). Data are presented as box plots. DETAILED DESCRIPTION OF THE INVENTION

[0095] Detailed Description definition Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0096] The term "amelioration" refers to any therapeutically beneficial result in the treatment of a disease state, eg, a renal disease state, including prevention, reduction in severity or progression, remission, or cure thereof.

[0097] The term "in situ" refers to processes that occur in living cells grown apart from the organism, for example, grown in tissue culture.

[0098] The term "in vivo" refers to a process that occurs within a living organism.

[0099] As used herein, the term "mammal" includes both humans and non-humans, including, but not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0100] The term percent "identity," with respect to two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotide or amino acid residues that are identical when compared and aligned for maximum correspondence, as determined using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those of skill in the art), or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequences being compared, e.g., a functional domain, or over the full length of the two sequences being compared.

[0101] For sequence comparison, typically, one sequence serves as a reference sequence to which test sequences are compared.When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the designated program parameters.

[0102] Optimal alignment of sequences for comparison can be performed, for example, by the homology alignment algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA, 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., supra).

[0103] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov / ).

[0104] The term "sufficient amount" means an amount sufficient to produce a desired effect, for example, an amount sufficient to modulate protein aggregation in a cell.

[0105] The term "therapeutically effective amount" is an amount effective to ameliorate symptoms of disease. Prevention can be considered treatment, and thus a therapeutically effective amount can be a "prophylactically effective amount."

[0106] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "compound" includes not only a single compound but also one or more of the same or different compounds; reference to a "pharmaceutically acceptable carrier" means not only a single pharmaceutically acceptable carrier but also one or more pharmaceutically acceptable carriers, and so forth.

[0107] The term "ADPKD" as used herein defines autosomal dominant polycystic kidney disease.

[0108] As used herein, the term "APC" defines allophycocyanin.

[0109] The term "scFv" as used herein defines a single-chain variable fragment that is a fusion protein of immunoglobulin heavy (VH) and light (VL) chains connected by a short linker peptide. These chimeric proteins are used in yeast display technology.

[0110] The term "Fc region" as used herein defines the portion of IgG (IgG Fc) that interacts with effector proteins, including Fcγ receptors.

[0111] As used herein, the term "GFR" defines glomerular filtration rate.

[0112] As used herein, the term "ICH" defines the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, particularly guidelines relating to photostability.

[0113] As used herein, the term "neutralization" defines the inhibition of an enzyme target protein by an antibody.

[0114] As used herein, the term "NHP" defines a non-human primate, specifically a cynomolgus monkey or macaque.

[0115] As used herein, the term "PAPP-A" defines pregnancy-associated plasma protein A, which is produced by the placenta and is necessary for the implantation process and for maintaining the placenta during pregnancy.

[0116] As used herein, the term "SPR" defines surface plasmon resonance, an optical technique utilized to detect interactions between two molecules.

[0117] As used herein, the term "t-GFR" defines transcutaneous glomerular filtration rate.

[0118] As used herein, the term "TKV" defines total kidney volume.

[0119] As used herein, the term "WB" defines Western Blot.

[0120] antibody structure The present application provides antibodies that bind to pregnancy-associated plasma protein A (PAPP-A) and compositions comprising the antibodies. Such antibodies include antibodies that block, inhibit, or reduce PAPP-A enzymatic activity.

[0121] The term "antibody" is used herein in its broadest sense and includes a specific type of immunoglobulin molecule that contains one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies.

[0122] Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous other immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. The "class" of an antibody or immunoglobulin refers to the type of constant domain or region possessed by its heavy chain. There are five major antibody classes: IgA, IgD, IgE, IgG, and IgM, several of which can be further subdivided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

[0123] An exemplary immunoglobulin (antibody) structural unit consists of two pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kD) and one "heavy" chain (approximately 50-70 kD), e.g., a homodimer of paired light and heavy chains. In other words, an exemplary antibody comprises two heavy chains and two light chains. The N-terminal domain of each chain defines a variable region of approximately 100-110 amino acids primarily responsible for antigen recognition. The terms "variable light chain" (VL) and "variable heavy chain" (VH) refer to these light and heavy chain domains, respectively. An IgG1 heavy chain comprises, from the N-terminus to the C-terminus, the VH, CH1, CH2, and CH3 domains, respectively. The light chain is composed of a VL and a CL domain from the N-terminus to the C-terminus. An IgG1 heavy chain comprises a hinge between the CH1 and CH2 domains. In certain embodiments, the immunoglobulin constructs comprise at least one immunoglobulin domain from IgG, IgM, IgA, IgD, or IgE linked to a therapeutic polypeptide. In some embodiments, the immunoglobulin domains found in the antibodies provided herein are derived from or derived from immunoglobulin-based constructs such as diabodies or nanobodies. In certain embodiments, the immunoglobulin constructs described herein comprise at least one immunoglobulin domain from a heavy chain antibody, such as a camelid antibody. In certain embodiments, the immunoglobulin constructs provided herein comprise at least one immunoglobulin domain from a mammalian antibody, such as a bovine antibody, a human antibody, a camelid antibody, a murine antibody, or any chimeric antibody.

[0124] In some embodiments, an antibody provided herein comprises a heavy chain. In one embodiment, the heavy chain is IgA. In one embodiment, the heavy chain is IgD. In one embodiment, the heavy chain is IgE. In one embodiment, the heavy chain is IgG. In one embodiment, the heavy chain is IgM. In one embodiment, the heavy chain is IgG1. In one embodiment, the heavy chain is IgG2. In one embodiment, the heavy chain is IgG3. In one embodiment, the heavy chain is IgG4. In one embodiment, the heavy chain is IgA1. In one embodiment, the heavy chain is IgA2.

[0125] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops ("hypervariable loops"). Generally, a naturally occurring four-chain antibody comprises six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and / or from "complementarity-determining regions" (CDRs), the latter of which have the highest sequence variability and / or are involved in antigen recognition. With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. Hypervariable regions (HVRs) are also referred to as "complementarity-determining regions" (CDRs), and these terms are used interchangeably herein with respect to the portions of the variable domain that form the antigen-binding region. This particular region is described by Kabat et al., US Department of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia et al., J. Mol. Biol. 196:901-917 (1987), and the definitions include overlapping or subsets of amino acid residues when compared with each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The exact residue numbers encompassing a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the amino acid sequence of the variable region of an antibody.

[0126] The amino acid sequence boundaries of the CDRs can be determined by one of skill in the art using any of a number of known numbering schemes, including those described in Kabat et al., supra (the "Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (the "Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (the "Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (the "IMGT" numbering scheme); and Honegger and Pluckthun, J. Mol. Biol., 2001, 309:657-70 (the "AHo" numbering scheme), each of which is incorporated by reference in its entirety.

[0127] Table 1 presents the positions of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 as identified by the Kabat and Chothia scheme. For CDR-H1, residue numbering is provided using both the Kabat and Chothia numbering schemes.

[0128] CDRs can be assigned using antibody numbering software such as, for example, Abnum (available at bioinf.org.uk / abs / abnum / and described in Abhinandan and Martin, Immunology, 2008, 45:3832-3839, which is incorporated by reference in its entirety).

[0129] [Table 1]

[0130] The "EU numbering scheme" is generally used when referring to residues in antibody heavy chain constant regions (e.g., as reported in Kabat et al., supra). Unless otherwise specified, the EU numbering scheme is used to refer to residues in antibody heavy chain constant regions described herein.

[0131] As used herein, the term "single chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In certain such embodiments, the C-terminus of the Fab light chain is linked to the N-terminus of the Fab heavy chain of the single-chain Fab molecule. As described in more detail herein, an scFv has a light chain variable domain (VL) connected from its C-terminus to the N-terminus of the heavy chain variable domain (VH) by a polypeptide chain. Alternatively, an scFv is composed of a polypeptide chain in which the C-terminus of the VH is linked to the N-terminus of the VL by a polypeptide chain.

[0132] A "Fab fragment" (also called fragment antigen binding) contains the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1), along with the variable domains VL and VH on the light and heavy chains, respectively. The variable domains contain the complementarity-determining loops (CDRs, also called hypervariable regions) involved in antigen binding. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region.

[0133] "The F(ab')2 fragment contains two Fab' fragments linked near the hinge region by a disulfide bond. F(ab')2 fragments can be produced, for example, by recombinant methods or by pepsin digestion of intact antibody. F(ab')2 fragments can be dissociated, for example, by treatment with β-mercaptoethanol.

[0134] A "single-chain Fv" or "scFv" comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In one embodiment, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see, e.g., Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., SPRinger-Verlag, New York, pp. 269-315 (1994). HER2 antibody scFv fragments are described in WO 93 / 16185; U.S. Patent Nos. 5,571,894; and 5,587,458.

[0135] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a naturally occurring antibody structure and having a heavy chain that includes an Fc region. For example, when used to refer to an IgG molecule, a "full-length antibody" is an antibody that includes two heavy chains and two light chains.

[0136] The term "epitope" refers to the portion of an antigen that specifically binds to an antibody. Epitopes often consist of surface-accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural and charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter can be lost in the presence of denaturing solvents. An epitope may include amino acid residues directly involved in binding and other amino acid residues not directly involved in binding. The epitope bound by an antibody can be determined using known techniques for determining epitopes, such as testing antibody binding to PAPP-A variants with different point mutations or chimeric PAPP-A variants.

[0137] The term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies contains antibodies that are substantially similar and bind to the same epitope(s), excluding variations that may normally arise during the production of monoclonal antibodies. Such variations are generally present only in minor amounts. Monoclonal antibodies are typically obtained by a process that includes selection of a single antibody from a plurality of antibodies. For example, the selection process can be selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody can be further modified, for example, to improve affinity for the target ("affinity maturation"), to humanize the antibody, to improve production in cell culture, and / or to reduce immunogenicity in a subject.

[0138] "Effector function" refers to a biological activity mediated by the Fc region of an antibody, and the activity may vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding to activate complement-dependent cytotoxicity (CDC), Fc receptor binding to activate antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP), receptor-ligand blockade, agonism, or antagonism. An active Fc region is one that is capable of Fc-based effector functions such as ADCC, CDC, and / or ADCP.

[0139] Anti-PAPP-A antibodies can include those described herein, such as the clones shown in the tables. In some embodiments, the antibodies comprise an alternative scaffold. In some embodiments, the antibodies consist of an alternative scaffold. In some embodiments, the antibodies consist essentially of an alternative scaffold. In some embodiments, the antibodies comprise an antibody fragment. In some embodiments, the antibodies consist of an antibody fragment. In some embodiments, the antibodies consist essentially of an antibody fragment. A "PAPP-A antibody," "anti-PAPP-A antibody," or "PAPP-A-specific antibody" is an antibody provided herein that specifically binds to the antigen PAPP-A. In some embodiments, the antibody binds to the extracellular domain of PAPP-A. In certain embodiments, the PAPP-A antibodies provided herein bind to an epitope of PAPP-A that is conserved among PAPP-A proteins from different species.

[0140] The term "chimeric antibody" or "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0141] In some embodiments, the antibody comprises a murine PAPP-A antibody. In some embodiments, the antibody comprises a chimeric PAPP-A antibody. In some embodiments, the antibody comprises a humanized PAPP-A antibody. In some embodiments, the antibody comprises a human PAPP-A antibody.

[0142] In one embodiment, constant domain(s) from a human antibody are fused to variable domain(s) of a non-human species. In another embodiment, one or more amino acid residues in one or more CDR sequences of the non-human antibody are altered to reduce the potential immunogenicity of the non-human antibody when it is administered to a human subject, either because none of the altered amino acid residues are important for immunospecific binding of the antibody to the antigen, or because the changes made to the amino acid sequence are conservative changes such that binding of the humanized antibody to the antigen is significantly lower than binding of the non-human antibody to the antigen.

[0143] A "human antibody" is one having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire or human antibody coding sequences (e.g., obtained from a human source or designed de novo). Human antibodies specifically exclude humanized antibodies. In one embodiment, all of the variable and constant domains are derived from human immunoglobulin sequences (fully human antibodies). These antibodies may be prepared in a variety of ways, including immunization with the antigen of interest of mice that have been genetically modified to express antibodies derived from genes encoding human heavy and / or light chains.

[0144] In some embodiments, the antibodies provided herein comprise an antibody fragment. In some embodiments, the antibodies provided herein consist of an antibody fragment. In some embodiments, the antibodies provided herein consist essentially of an antibody fragment. In some embodiments, the antibody fragment is an Fv fragment. In some embodiments, the antibody fragment is a Fab fragment. In some embodiments, the antibody fragment is a F(ab')2 fragment. In some embodiments, the antibody fragment is a Fab' fragment. In some embodiments, the antibody fragment is an scFv (sFv) fragment. In some embodiments, the antibody fragment is an scFv-Fc fragment. In some embodiments, the antibody fragment is a fragment of a single domain antibody.

[0145] In some aspects, provided herein is an isolated polynucleotide or set of polynucleotides encoding any of the above-mentioned claimed antibodies, their VH, VL, light chain, heavy chain, or antigen-binding portion thereof; optionally, the isolated polynucleotide or set of polynucleotides is cDNA. Nucleotide sequences of the VH, VL, heavy chain, and light chain sequences of PAPP-A antibodies are provided in SEQ ID NOs: 80-103. For example, heavy chain sequences of PAPP-A antibodies disclosed herein are provided as sequences set forth in SEQ ID NOs: 80, 84, 88, 92, 96, and 100. Variable heavy chain sequences of PAPP-A antibodies disclosed herein are provided as sequences set forth in SEQ ID NOs: 81, 85, 89, 93, 97, and 101. Light chain sequences of PAPP-A antibodies disclosed herein are provided as sequences set forth in SEQ ID NOs: 82, 86, 90, 94, 98, and 102. The variable heavy chain sequences of the PAPP-A antibodies disclosed herein are provided as the sequences shown as SEQ ID NOs: 83, 87, 91, 95, 99, and 103.

[0146] In some embodiments, the PAPP-A antibody comprises a variable heavy (VH) chain nucleotide sequence comprising the sequence set forth in any one of SEQ ID NOs: 81, 85, 89, 93, 97, and 101. In some embodiments, the PAPP-A antibody comprises a variable light (VL) chain nucleotide sequence comprising the sequence set forth in any one of SEQ ID NOs: 83, 87, 91, 95, 99, and 103. In some embodiments, the PAPP-A antibody comprises a heavy chain nucleotide sequence comprising the sequence set forth in any one of SEQ ID NOs: 80, 84, 88, 92, 96, and 100. In some embodiments, the PAPP-A antibody comprises a light (VL) chain nucleotide sequence comprising the sequence set forth in any one of SEQ ID NOs: 82, 86, 90, 94, 98, and 102.

[0147] CDR In some embodiments, an isolated antibody that binds to human PAPP-A (SEQ ID NO: 79) comprises a variable heavy chain (VH) sequence comprising three heavy chain CDR sequences, CDR-H1, CDR-H2, and CDR-H3, and a variable light chain (VL) sequence comprising three light chain CDR sequences, CDR-L1, CDR-L2, and CDR-L3, wherein CDR-H1 comprises the sequence X1YX2MX3 (SEQ ID NO: 73), where X1 is S or T; X2 is G or A; and X3 is S or H. X6 is R, G, S, or Q; X7 is R, I, N, or E; X8 is K or T; and X9 is S or A; CDR-H3 comprises the sequence HX1RIX2X3WGX4HTFDI (SEQ ID NO: 75), and the sequence is X2 is P or Y; X3 is P or S; and X4 is F or W; or the sequence ADMHRFDV (SEQ ID NO:45), VWGGVRFDV (SEQ ID NO:55), or YKPMPFDV (SEQ ID NO:25 or SEQ ID NO:35); CDR-L1 comprises the sequence RASQX1IX2X3YLN (SEQ ID NO:76), where X1 is D or S; X2 is I or S; and X3 is S, T, I, or R. X4 is S, G, A, Y, or P; X5 is P, T, or G; X6 is Y, W, or F; and X7 is K, T, or P.

[0148] In some embodiments, CDR-H3 comprises the sequence HNRIYSWGWHTFDI (SEQ ID NO: 5). In some embodiments, CDR-H3 comprises the sequence HERIPPWGFHTFDI (SEQ ID NO: 15). In some embodiments, CDR-H3 comprises the sequence YKPMPFDV (SEQ ID NO: 25 or 35). In some embodiments, CDR-H3 comprises the sequence ADMHRFDV (SEQ ID NO: 45). In some embodiments, CDR-H3 comprises the sequence VWGGVRFDV (SEQ ID NO: 55).

[0149] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 5, a CDR-H2 of SEQ ID NO: 4, a CDR-H1 of SEQ ID NO: 3, a CDR-L3 of SEQ ID NO: 10, a CDR-L2 of SEQ ID NO: 9, and a CDR-L1 of SEQ ID NO: 8. In some embodiments, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H3 of SEQ ID NO: 5, the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H2 of SEQ ID NO: 4, and the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H1 of SEQ ID NO: 3. wherein CDR-L3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L3 of SEQ ID NO: 10; CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L2 of SEQ ID NO: 9; and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L1 of SEQ ID NO: 8. In some embodiments, CDR-H3 is CDR-H3 of SEQ ID NO: 5 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H2 is CDR-H2 of SEQ ID NO: 4 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H1 is CDR-H1 of SEQ ID NO: 3 with up to 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L3 is CDR-L3 of SEQ ID NO: 10 with up to 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L2 is CDR-L2 of SEQ ID NO: 9 with up to 1, 2, 3, or 4 amino acid substitutions; and CDR-L1 is CDR-L1 of SEQ ID NO: 8 with up to 1, 2, 3, 4, 5, or 6 amino acid substitutions.

[0150] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 15, a CDR-H2 of SEQ ID NO: 14, a CDR-H1 of SEQ ID NO: 13, a CDR-L3 of SEQ ID NO: 20, a CDR-L2 of SEQ ID NO: 19, and a CDR-L1 of SEQ ID NO: 18. In some embodiments, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H3 of SEQ ID NO: 15, the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H2 of SEQ ID NO: 14, and the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H1 of SEQ ID NO: 13. and CDR-L3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L3 of SEQ ID NO: 20, CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L2 of SEQ ID NO: 19, and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L1 of SEQ ID NO: 18. In some embodiments, CDR-H3 is CDR-H3 of SEQ ID NO: 15 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H2 is CDR-H2 of SEQ ID NO: 14 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H1 is CDR-H1 of SEQ ID NO: 13 with up to 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L3 is CDR-L3 of SEQ ID NO: 20 with up to 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L2 is CDR-L2 of SEQ ID NO: 19 with up to 1, 2, 3, or 4 amino acid substitutions; and CDR-L1 is CDR-L1 of SEQ ID NO: 18 with up to 1, 2, 3, 4, 5, or 6 amino acid substitutions.

[0151] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 25, a CDR-H2 of SEQ ID NO: 24, a CDR-H1 of SEQ ID NO: 23, a CDR-L3 of SEQ ID NO: 30, a CDR-L2 of SEQ ID NO: 29, and a CDR-L1 of SEQ ID NO: 28. In some embodiments, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H3 of SEQ ID NO: 25, the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H2 of SEQ ID NO: 24, and the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H1 of SEQ ID NO: 23. and CDR-L3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L3 of SEQ ID NO: 30, CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L2 of SEQ ID NO: 29, and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L1 of SEQ ID NO: 28. In some embodiments, CDR-H3 is CDR-H3 of SEQ ID NO: 25 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H2 is CDR-H2 of SEQ ID NO: 24 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H1 is CDR-H1 of SEQ ID NO: 23 with up to 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L3 is CDR-L3 of SEQ ID NO: 30 with up to 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L2 is CDR-L2 of SEQ ID NO: 29 with up to 1, 2, 3, or 4 amino acid substitutions; and CDR-L1 is CDR-L1 of SEQ ID NO: 28 with up to 1, 2, 3, 4, 5, or 6 amino acid substitutions.

[0152] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 35, a CDR-H2 of SEQ ID NO: 34, a CDR-H1 of SEQ ID NO: 33, a CDR-L3 of SEQ ID NO: 40, a CDR-L2 of SEQ ID NO: 39, and a CDR-L1 of SEQ ID NO: 38. In some embodiments, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H3 of SEQ ID NO: 35, the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H2 of SEQ ID NO: 34, and the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H1 of SEQ ID NO: 33. and CDR-L3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L3 of SEQ ID NO: 40, CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L2 of SEQ ID NO: 39, and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L1 of SEQ ID NO: 38. In some embodiments, CDR-H3 is CDR-H3 of SEQ ID NO: 35 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H2 is CDR-H2 of SEQ ID NO: 34 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H1 is CDR-H1 of SEQ ID NO: 33 with up to 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L3 is CDR-L3 of SEQ ID NO: 40 with up to 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L2 is CDR-L2 of SEQ ID NO: 39 with up to 1, 2, 3, or 4 amino acid substitutions; and CDR-L1 is CDR-L1 of SEQ ID NO: 38 with up to 1, 2, 3, 4, 5, or 6 amino acid substitutions.

[0153] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 45, a CDR-H2 of SEQ ID NO: 44, a CDR-H1 of SEQ ID NO: 43, a CDR-L3 of SEQ ID NO: 50, a CDR-L2 of SEQ ID NO: 49, and a CDR-L1 of SEQ ID NO: 48. In some embodiments, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H3 of SEQ ID NO: 45, the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H2 of SEQ ID NO: 44, and the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H1 of SEQ ID NO: 43. and CDR-L3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L3 of SEQ ID NO: 50, CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L2 of SEQ ID NO: 49, and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L1 of SEQ ID NO: 48. In some embodiments, CDR-H3 is CDR-H3 of SEQ ID NO: 45 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H2 is CDR-H2 of SEQ ID NO: 44 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H1 is CDR-H1 of SEQ ID NO: 43 with up to 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L3 is CDR-L3 of SEQ ID NO: 50 with up to 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L2 is CDR-L2 of SEQ ID NO: 49 with up to 1, 2, 3, or 4 amino acid substitutions; and CDR-L1 is CDR-L1 of SEQ ID NO: 48 with up to 1, 2, 3, 4, 5, or 6 amino acid substitutions.

[0154] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 55, a CDR-H2 of SEQ ID NO: 54, a CDR-H1 of SEQ ID NO: 53, a CDR-L3 of SEQ ID NO: 60, a CDR-L2 of SEQ ID NO: 59, and a CDR-L1 of SEQ ID NO: 58. In some embodiments, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H3 of SEQ ID NO: 55, the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H2 of SEQ ID NO: 54, and the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H1 of SEQ ID NO: 53. and CDR-L3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L3 of SEQ ID NO: 60, CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L2 of SEQ ID NO: 59, and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L1 of SEQ ID NO: 58. In some embodiments, CDR-H3 is CDR-H3 of SEQ ID NO: 55 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H2 is CDR-H2 of SEQ ID NO: 54 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H1 is CDR-H1 of SEQ ID NO: 53 with up to 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L3 is CDR-L3 of SEQ ID NO: 60 with up to 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L2 is CDR-L2 of SEQ ID NO: 59 with up to 1, 2, 3, or 4 amino acid substitutions; and CDR-L1 is CDR-L1 of SEQ ID NO: 58 with up to 1, 2, 3, 4, 5, or 6 amino acid substitutions.

[0155] In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described herein are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, but may be, for example, de novo isolated according to the methods provided herein for obtaining antibodies.

[0156] In some embodiments, the antibodies provided herein comprise a CDR-H3 selected from SEQ ID NO: 5, 15, 25, 35, 45, or 55. In some aspects, the CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H3 of SEQ ID NO: 5, 15, 25, 35, 45, or 55. In some embodiments, the CDR-H3 is a CDR-H3 selected from SEQ ID NO: 5, 15, 25, 35, 45, or 55 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

[0157] In some embodiments, the antibodies provided herein comprise a CDR-H2 of SEQ ID NO: 4, 14, 24, 34, 44, or 54. In some aspects, the CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H2 of SEQ ID NO: 4, 14, 24, 34, 44, or 54. In some embodiments, the CDR-H2 is a CDR-H2 selected from SEQ ID NO: 4, 14, 24, 34, 44, or 54 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

[0158] In some embodiments, the antibodies provided herein comprise a CDR-H1 of SEQ ID NO: 3, 13, 23, 33, 43, or 53. In some aspects, the CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-H1 of SEQ ID NO: 3, 13, 23, 33, 43, or 53. In some embodiments, the CDR-H1 is a CDR-H1 of SEQ ID NO: 3, 13, 23, 33, 43, or 53 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

[0159] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 5, 15, 25, 35, 45, or 55, a CDR-H2 of SEQ ID NO: 4, 14, 24, 34, 44, or 54, and a CDR-H1 of SEQ ID NO: 3, 13, 23, 33, 43, or 53. In some embodiments, CDR-H3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-H3 of SEQ ID NO: 5, 15, 25, 35, 45, or 55; CDR-H2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-H2 of SEQ ID NO: 4, 14, 24, 34, 44, or 54; and CDR-H1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-H1 of SEQ ID NO: 3, 13, 23, 33, 43, or 53. In some embodiments, the CDR-H3 is CDR-H3 of SEQ ID NO: 5, 15, 25, 35, 45, or 55 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; the CDR-H2 is CDR-H2 of SEQ ID NO: 34, 14, 24, 34, 44, or 54 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; and the CDR-H1 is CDR-H1 of SEQ ID NO: 3, 13, 23, 33, 43, or 53 with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

[0160] In some embodiments, the antibodies provided herein comprise a CDR-L3 of SEQ ID NO: 10, 20, 30, 40, 50, or 60. In some aspects, the CDR-L3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-L3 of SEQ ID NO: 10, 20, 30, 40, 50, or 60. In some embodiments, the CDR-L3 is a CDR-L3 of SEQ ID NO: 10, 20, 30, 40, 50, or 60 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

[0161] In some embodiments, the antibodies provided herein comprise a CDR-L2 of SEQ ID NO: 9, 19, 29, 39, 49, or 59. In some aspects, the CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-L2 of SEQ ID NO: 9, 19, 29, 39, 49, or 59. In some embodiments, the CDR-L2 is a CDR-L2 of SEQ ID NO: 9, 19, 29, 39, 49, or 59 with up to 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

[0162] In some embodiments, the antibodies provided herein comprise a CDR-L1 of SEQ ID NO: 8, 18, 28, 38, 48, or 58. In some aspects, the CDR-L1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to the CDR-L1 of SEQ ID NO: 8, 18, 28, 38, 48, or 58. In some embodiments, the CDR-L1 is a CDR-L1 of SEQ ID NO: 8, 18, 28, 38, 48, or 58 with up to 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions.

[0163] In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies provided herein comprise a CDR-L3 of SEQ ID NO: 10, 20, 30, 40, 50, or 60, and a CDR-L2 of SEQ ID NO: 9, 19, 29, 39, 49, or 59. In some embodiments, the antibodies provided herein comprise a CDR-L3 of SEQ ID NO: 10, 20, 30, 40, 50, or 60, a CDR-L2 of SEQ ID NO: 9, 19, 29, 39, 49, or 59, and a CDR-L1 of SEQ ID NO: 8, 18, 28, 38, 48, or 58. In some embodiments, CDR-L3 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L3 of SEQ ID NO: 10, 20, 30, 40, 50, or 60; CDR-L2 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L2 of SEQ ID NO: 9, 19, 29, 39, 49, or 59; and CDR-L1 has at least about 50%, 75%, 80%, 85%, 90%, or 95% identity to CDR-L1 of SEQ ID NO: 8, 18, 28, 38, 48, or 58. In some embodiments, CDR-L3 is CDR-L3 of SEQ ID NO: 10, 20, 30, 40, 50, or 60 with up to 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L2 is CDR-L2 of SEQ ID NO: 9, 19, 29, 39, 49, or 59 with up to 1, 2, 3, or 4 amino acid substitutions; and CDR-L1 is CDR-L1 of SEQ ID NO: 8, 18, 28, 38, 48, or 58 with up to 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

[0164] In some embodiments, the antibodies provided herein comprise one to three CDRs of a VH domain selected from SEQ ID NO: 2, 12, 22, 32, 42, or 52. In some embodiments, the antibodies provided herein comprise two to three CDRs of a VH domain selected from SEQ ID NO: 2, 12, 22, 32, 42, or 52. In some embodiments, the antibodies provided herein comprise three CDRs of a VH domain selected from SEQ ID NO: 2, 12, 22, 32, 42, or 52. In some aspects, the CDRs are Kabat CDRs. In some aspects, the CDRs are Chothia CDRs. In some aspects, the CDRs are AbM CDRs. In some aspects, the CDRs are Contact CDRs. In some aspects, the CDRs are IMGT CDRs.

[0165] In some embodiments, the CDR-H1 is the CDR-H1 of a VH domain selected from SEQ ID NO: 2, 12, 22, 32, 42, or 52 with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some embodiments, the CDR-H2 is the CDR-H2 of a VH domain selected from SEQ ID NO: 2, 12, 22, 32, 42, or 52 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some embodiments, the CDR-H3 is the CDR-H3 of a VH domain selected from SEQ ID NO: 2, 12, 22, 32, 42, or 52 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein, hi some embodiments, such variants are not derived from the sequences provided herein, but may be de novo isolated, e.g., according to the methods provided herein for obtaining antibodies.

[0166] In some embodiments, the antibodies provided herein comprise one to three CDRs of a VL domain selected from SEQ ID NO: 7, 17, 27, 37, 47, or 57. In some embodiments, the antibodies provided herein comprise two to three CDRs of a VL domain selected from SEQ ID NO: 7, 17, 27, 37, 47, or 57. In some embodiments, the antibodies provided herein comprise three CDRs of a VL domain selected from SEQ ID NO: 7, 17, 27, 37, 47, or 57. In some aspects, the CDRs are Kabat CDRs. In some aspects, the CDRs are Chothia CDRs. In some aspects, the CDRs are AbM CDRs. In some aspects, the CDRs are Contact CDRs. In some aspects, the CDRs are IMGT CDRs.

[0167] In some embodiments, CDR-L1 is the CDR-L1 of a VL domain selected from SEQ ID NO: 7, 17, 27, 37, 47, or 57 with up to 1, 2, 3, 4, or 5 amino acid substitutions. In some embodiments, CDR-L2 is the CDR-L2 of a VL domain selected from SEQ ID NO: 7, 17, 27, 37, 47, or 57 with up to 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions. In some embodiments, CDR-L3 is the CDR-L3 of a VL domain selected from SEQ ID NO: 7, 17, 27, 37, 47, or 57 with up to 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein, hi some embodiments, such variants are not derived from the sequences provided herein, but may be de novo isolated, e.g., according to the methods provided herein for obtaining antibodies.

[0168] In some embodiments, the antibodies provided herein comprise one to three CDRs of a VH domain selected from SEQ ID NO: 2, 12, 22, 32, 42, or 52, and one to three CDRs of a VL domain selected from SEQ ID NO: 7, 17, 27, 37, 47, or 57. In some embodiments, the antibodies provided herein comprise two to three CDRs of a VH domain selected from SEQ ID NO: 2, 12, 22, 32, 42, or 52, and two to three CDRs of a VL domain selected from SEQ ID NO: 7, 17, 27, 37, 47, or 57. In some embodiments, the antibodies provided herein comprise three CDRs of a VH domain selected from SEQ ID NO: 2, 12, 22, 32, 42, or 52, and three CDRs of a VL domain selected from SEQ ID NO: 7, 17, 27, 37, 47, or 57. In some aspects, the CDRs are Kabat CDRs. In some embodiments, the CDR is a Chothia CDR. In some embodiments, the CDR is an AbM CDR. In some embodiments, the CDR is a Contact CDR. In some embodiments, the CDR is an IMGT CDR.

[0169] V H domain In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs: 2, 12, 22, 32, 42, or 52. H In some embodiments, the antibodies provided herein comprise the V sequence of SEQ ID NO:2. H In some embodiments, the antibodies provided herein comprise the V sequence of SEQ ID NO: 12. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 22. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 32. H In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 42. H In some embodiments, the antibodies provided herein comprise the V sequence of SEQ ID NO: 52. H Contains arrays.

[0170] In some embodiments, V H The sequence has at least 70%, 80%, or 90% identity to SEQ ID NO:2, and any variation from SEQ ID NO:2 does not occur within CDR-H1, CDR-H2, or CDR-H3. H The sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 12, and any variation from SEQ ID NO: 12 does not occur within CDR-H1, CDR-H2, or CDR-H3. H The sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 22, and any variation from SEQ ID NO: 22 does not occur within CDR-H1, CDR-H2, or CDR-H3. H The sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 32, and any variation from SEQ ID NO: 32 does not occur within CDR-H1, CDR-H2, or CDR-H3. H The sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 42, and any variation from SEQ ID NO: 42 does not occur within CDR-H1, CDR-H2, or CDR-H3. H The sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 52, and any variation from SEQ ID NO: 52 does not occur within CDR-H1, CDR-H2, or CDR-H3.

[0171] In some embodiments, the antibodies provided herein comprise a V having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to SEQ ID NO:2. H In some embodiments, the antibodies provided herein comprise a V sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to SEQ ID NO: 12. HIn some embodiments, the antibodies provided herein comprise a V having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to SEQ ID NO:22. H In some embodiments, the antibodies provided herein comprise a V sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to SEQ ID NO: 32. H In some embodiments, the antibodies provided herein comprise a V having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to SEQ ID NO:42. H In some embodiments, the antibodies provided herein comprise a V sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to SEQ ID NO: 52. H Contains arrays.

[0172] In some embodiments, the antibodies provided herein comprise an exemplary VVL sequence provided in SEQ ID NO: 2, 12, 22, 32, 42, or 52. H V having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the sequence H In some embodiments, the antibodies provided herein comprise a V sequence as provided in SEQ ID NO: 2, 12, 22, 32, 42, or 52 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions. HThe antibodies described in this paragraph include sequences. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, but may be, for example, newly isolated according to the methods provided herein for obtaining antibodies.

[0173] V L domain In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs: 7, 17, 27, 37, 47, or 57. L In some embodiments, the antibodies provided herein comprise the V sequence of SEQ ID NO:7. L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 17. L In some embodiments, the antibodies provided herein comprise the V sequence of SEQ ID NO: 27. L In some embodiments, the antibodies provided herein comprise the V sequence of SEQ ID NO: 37. L In some embodiments, the antibodies provided herein comprise the V sequence of SEQ ID NO: 47. L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 57. L Contains arrays.

[0174] In some embodiments, V L The sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 7, and any variation from SEQ ID NO: 7 does not occur within CDR-L1, CDR-L2, or CDR-L3. LThe sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 17, and any variation from SEQ ID NO: 17 does not occur within CDR-L1, CDR-L2, or CDR-L3. L The sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 27, and any variation from SEQ ID NO: 27 does not occur within CDR-L1, CDR-L2, or CDR-L3. L The sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 37, and any variation from SEQ ID NO: 37 does not occur within CDR-L1, CDR-L2, or CDR-L3. L The sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 47, and any variation from SEQ ID NO: 47 does not occur within CDR-L1, CDR-L2, or CDR-L3. L The sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 57, and any variation from SEQ ID NO: 57 does not occur within CDR-L1, CDR-L2, or CDR-L3.

[0175] In some embodiments, the antibodies provided herein comprise a V having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to SEQ ID NO:7. L In some embodiments, the antibodies provided herein comprise a V having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to SEQ ID NO: 17. L In some embodiments, the antibodies provided herein comprise a V having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to SEQ ID NO:27. L In some embodiments, the antibodies provided herein comprise a V having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to SEQ ID NO: 37. LIn some embodiments, the antibodies provided herein comprise a V sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to SEQ ID NO:47. L In some embodiments, the antibodies provided herein comprise a V having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to SEQ ID NO:57. L Contains arrays.

[0176] In some embodiments, the antibodies provided herein comprise an exemplary VVL ... L V having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the sequence L In some embodiments, the antibodies provided herein comprise a V sequence as provided in SEQ ID NO: 7, 17, 27, 37, 47, or 57 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions. L The antibodies described in this paragraph include sequences. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein by, for example, affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein. In some embodiments, such variants are not derived from the sequences provided herein, but may be, for example, newly isolated according to the methods provided herein for obtaining antibodies.

[0177] V H -V L Combination of In some embodiments, the antibodies provided herein comprise a V selected from SEQ ID NOs: 2, 12, 22, 32, 42, or 52. Hand V selected from SEQ ID NOs: 7, 17, 27, 37, 47, or 57 L and an array.

[0178] In some embodiments, the antibodies provided herein comprise V of SEQ ID NO:2. H Sequence number 7, and V L In some embodiments, the antibodies provided herein comprise the V sequence of SEQ ID NO: 12. H Sequence, and V of SEQ ID NO: 17 L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 22. H Sequence number 27, and V L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 32. H Sequence number 37, and V L In some embodiments, the antibodies provided herein comprise the V of SEQ ID NO: 42. H Sequence, and V of SEQ ID NO: 47 L In some embodiments, the antibodies provided herein comprise the V sequence of SEQ ID NO: 52. H Sequence, and V of SEQ ID NO: 57 L Contains arrays.

[0179] In some embodiments, V H The sequence has at least 70%, 80%, or 90% identity to SEQ ID NO:2, and the variable region of the light chain has at least 70%, 80%, or 90% identity to SEQ ID NO:7, wherein no variants from SEQ ID NO:2 occur within CDR-H1, CDR-H2, or CDR-H3, and no variants from SEQ ID NO:7 occur within CDR-L1, CDR-L2, or CDR-L3. HThe sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 12, and the variable region of the light chain has at least 70%, 80%, or 90% identity to SEQ ID NO: 17, wherein no variants from SEQ ID NO: 12 occur within CDR-H1, CDR-H2, or CDR-H3, and no variants from SEQ ID NO: 17 occur within CDR-L1, CDR-L2, or CDR-L3. H The sequence has at least 70%, 80%, or 90% identity to SEQ ID NO:22, and the variable region of the light chain has at least 70%, 80%, or 90% identity to SEQ ID NO:27, wherein no variants from SEQ ID NO:22 occur within CDR-H1, CDR-H2, or CDR-H3, and no variants from SEQ ID NO:27 occur within CDR-L1, CDR-L2, or CDR-L3. H The sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 32, and the variable region of the light chain has at least 70%, 80%, or 90% identity to SEQ ID NO: 37, wherein no variants from SEQ ID NO: 32 occur within CDR-H1, CDR-H2, or CDR-H3, and no variants from SEQ ID NO: 37 occur within CDR-L1, CDR-L2, or CDR-L3. H The sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 42, and the variable region of the light chain has at least 70%, 80%, or 90% identity to SEQ ID NO: 47, wherein no variants from SEQ ID NO: 42 occur within CDR-H1, CDR-H2, or CDR-H3, and no variants from SEQ ID NO: 47 occur within CDR-L1, CDR-L2, or CDR-L3. H The sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 52, and the variable region of the light chain has at least 70%, 80%, or 90% identity to SEQ ID NO: 57, wherein no variants from SEQ ID NO: 52 occur within CDR-H1, CDR-H2, or CDR-H3, and no variants from SEQ ID NO: 57 occur within CDR-L1, CDR-L2, or CDR-L3.

[0180] In certain embodiments, any of SEQ ID NOs: 2, 12, 22, 32, 42, or 52 can be combined with any of SEQ ID NOs: 7, 17, 27, 37, 47, or 57. For example, SEQ ID NO: 2 can be combined with any of SEQ ID NOs: 7, 17, 27, 37, 47, or 57. As another example, SEQ ID NO: 17 can be combined with any of SEQ ID NOs: 2, 12, 22, 32, 42, or 52.

[0181] In some embodiments, the antibodies provided herein comprise an exemplary VVL ... H V having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the sequence H and VL sequences having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the exemplary VL sequences provided in SEQ ID NOs: 7, 17, 27, 37, 47, or 57. Land a VL sequence as provided in SEQ ID NO: 7, 17, 27, 37, 47, or 57, with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the antibodies described in this paragraph are referred to herein as "variants." In some embodiments, such variants are derived from the sequences provided herein, e.g., by affinity maturation, site-directed mutagenesis, random mutagenesis, or any other method known in the art or described herein, hi some embodiments, such variants are not derived from the sequences provided herein, but may be de novo isolated, e.g., according to the methods provided herein for obtaining antibodies.

[0182] In some embodiments, the percent homology of the heavy or light chain variable is calculated outside the CDRs, for example, the percent homology can be calculated in the framework regions.

[0183] In some embodiments, the antibody comprises a heavy chain provided in SEQ ID NO: 1, 11, 21, 31, 41, or 51.

[0184] In some embodiments, the antibody comprises a light chain provided in SEQ ID NO: 6, 16, 26, 36, 46, or 56.

[0185] In certain embodiments, any of SEQ ID NOs: 1, 11, 21, 31, 41, or 51 can be combined with any of SEQ ID NOs: 6, 16, 26, 36, 46, or 56.

[0186] In some embodiments, the antibodies provided herein comprise a heavy chain sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the exemplary heavy chain sequences provided in SEQ ID NOs: 1, 11, 21, 31, 41, or 51; and a light chain sequence having at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity to the exemplary light chain sequences provided in SEQ ID NOs: 6, 16, 26, 36, 46, or 56. In some embodiments, the antibodies provided herein comprise a heavy chain sequence provided in SEQ ID NO: 1, 11, 21, 31, 41, or 51 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions, and a light chain sequence provided in SEQ ID NO: 6, 16, 26, 36, 46, or 56 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid substitutions.

[0187] Fc area The term "Fc domain" or "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. The term includes native-sequence Fc regions and variant Fc regions. Unless otherwise specified herein, the numbering of amino acid residues in an Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, the "Fc polypeptide" of a dimeric Fc refers to one of the two polypeptides that form the dimeric Fc domain, i.e., the polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain capable of stable self-association. For example, the Fc polypeptide of a dimeric IgGFc comprises IgG CH2 and IgG CH3 constant domain sequences. The Fc can be of the classes IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0188] The terms "Fc receptor" and "FcR" are used to describe receptors that bind to the Fc region of an antibody. For example, an FcR can be a native-sequence human FcR. Generally, FcRs are those that bind IgG antibodies (gamma receptors) and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Immunoglobulins of other isotypes can also bind to specific FcRs (see, e.g., Janeway et al., ImmunoBiology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th ed., 1999)). Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (reviewed in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor FcRn, which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976); and Kim et al., J. Immunol. 24:249 (1994)).

[0189] In some embodiments, the antibody is an IgG1 antibody.

[0190] Modifications of the CH2 domain can affect FcR binding to Fc. Numerous amino acid modifications in the Fc region are known in the art to selectively alter the affinity of Fc for different Fc gamma (Fcγ) receptors. In one embodiment, the Fc contains one or more modifications to promote selective binding to Fc-gamma receptors.

[0191] In some embodiments, the antibodies described herein comprise an Fc region comprising the L234A / L235A mutations according to the EU numbering system.

[0192] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is produced by a hybridoma. In other embodiments, the antibody is produced by a recombinant cell engineered to express desired variable and constant domains. In some embodiments, the antibody is specific for a surface antigen such as the PAPP-A protein. In certain embodiments, the therapeutic antibody may have the Fc portion of a human or non-human primate IgG1.

[0193] join With respect to antibody binding to a target molecule, the terms "binds with," "specifically binding to," "specifically binds to," "specific for," "selectively binds to," and "selective for" a particular antigen (e.g., polypeptide target) or epitope on a particular antigen refer to binding that is measurably different from nonspecific or nonselective interactions (e.g., with non-target molecules). Specific binding can be measured, for example, by measuring binding to the target molecule and comparing it to binding to non-target molecules. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. In this case, specific binding is indicated if binding of the antibody to the target molecule is competitively inhibited by the control molecule. Cross-linking of an antigen target is a type of binding. In some embodiments, an anti-PAPP-A antibody cross-links PAPP-A to PAPP-A on PAPP-A+ cells.

[0194] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or epitope). Unless otherwise specified, as used herein, "affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen or epitope). The affinity of a molecule X for its partner Y is determined by the dissociation equilibrium constant (K D ) The kinetic factors that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described herein, such as surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).

[0195] As used herein, "k" d ”(seconds -1 The term k ) refers to the dissociation rate constant of a particular antibody-antigen interaction. This value is k off Also called value.

[0196] As used herein, "k" a " (M -1 × seconds -1 The term k ) refers to the association rate constant for a particular antibody-antigen interaction. This value is k on Also called value.

[0197] As used herein, "K" D The term "(M)" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. D =k d / k a In some embodiments, the affinity of an antibody is determined by the K D For clarity, as known in the art, the smaller K D values ​​indicate a high affinity interaction, but a large K D Values ​​indicate low affinity interactions.

[0198] As used herein, "K" A " (M -1 The term K ) refers to the association equilibrium constant of a particular antibody-antigen interaction. A =k a / k d

[0199] In some embodiments, the antibodies provided herein bind to human PAPP-A. In some embodiments, the antibodies provided herein bind to mouse PAPP-A. In some embodiments, the antibodies provided herein bind to macaque PAPP-A. In some embodiments, the antibodies provided herein bind to cynomolgus monkey PAPP-A. In some embodiments, the antibodies provided herein bind to human, rhesus monkey, and / or cynomolgus monkey PAPP-A.

[0200] In some embodiments, the antibodies provided herein have a cytoplasmic affinity of about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 1.95, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, or 100 x 10 as measured by a surface plasmon resonance assay. -12 K below M D In some embodiments, the K of the antibodies provided herein binds to human PAPP-A. D is approximately 0.5-1, 0.25-0.75, 0.25-0.5, 0.5-0.75, 0.75-1, 0.75-2, 1.1-1.2, 1.2-1.3, 1.3-1.4, 1.4-1.5, 1.5-1.6, 1.6-1.7, 1.7-1.8, 1.8-1.9, 1.9-2, 1-2, 1-5, 2-7, 3-8, 3-5, 4-6, 5-7, 6-8, 7-9, 7-10, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 × 10, as measured by surface plasmon resonance assay. -12 I am M.

[0201] In some embodiments, the antibodies provided herein have a cytoplasmic affinity of about 3, 2.5, 2.3, 2, 1.98, 1.95, 1.9, 1.85, 1.8, 1.75, 1.7, 1.65, 1.6, 1.55, 1.50, 1.45, or 1.4 x 10 as measured by a surface plasmon resonance assay. -12 K below M D In some embodiments, the antibodies provided herein bind to human PAPP-A at a ribozyme activity of 2.5 to 2.3, 2.5 to 2.0, 2.0 to 1.9, 1.9 to 1.8, 1.8 to 1.7, 1.7 to 1.6, 1.6 to 1.5, or 1.9 to 1.5 x 10 as measured by surface plasmon resonance. -12 K between M D and binds to human PAPP-A.

[0202] In some embodiments, the antibodies provided herein inhibit PAPP-A proteolytic activity with an IC50 of 3.5, 3, 2.5, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, or 0.04 nM or less as measured by flow cytometry or Western blotting. In some embodiments, the antibodies provided herein inhibit PAPP-A proteolytic activity towards IGFB-2, IGFBP-4, or IGFBP-5.

[0203] To screen for antibodies that bind to the epitope on the target antigen to which the antibody of interest (e.g., PAPP-A) binds, routine cross-blocking assays can be performed, such as those described in "Antibodies, A Laboratory Manual," Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Alternatively, or in addition, epitope mapping can be performed by methods known in the art.

[0204] function In some embodiments, the antibody is an antagonist antibody. An antagonist antibody can inhibit (e.g., reduce) one or more activities or functions of PAPP-A after the antibody binds to the PAPP-A protein. For example, an antagonist antibody may bind to the PAPP-A enzyme, inhibit the binding of the PAPP-A enzyme to its substrate, and prevent cleavage of the substrate.

[0205] method Methods for Treating PAPP-A-Associated Disorders In another aspect, provided herein is a method for treating a PAPP-A-associated disorder, comprising administering to a subject a composition comprising an anti-PAPP-A antibody. In some embodiments, the PAPP-A-associated disorder is a kidney disease, such as polycystic kidney disease or autosomal dominant polycystic kidney disease (ADPKD).

[0206] In some embodiments, the kidney disease is polycystic kidney disease, or autosomal dominant polycystic kidney disease (ADPKD).

[0207] In one embodiment, the subject is a human.

[0208] Pharmaceutical Composition The present disclosure also encompasses methods for treating PAPP-A-related disorders, comprising administering a therapeutically effective amount of an anti-PAPP-A antibody or antigen-binding fragment thereof. The PAPP-A antibody or antigen-binding fragment may be formulated into a pharmaceutical composition or medicament.

[0209] Kits and Articles of Manufacture The present application provides kits comprising any one or more of the antibody compositions described herein. In some embodiments, the kit further contains a component selected from any of a secondary antibody, an immunohistochemistry reagent, a pharmaceutically acceptable excipient, and instructions, and any combination thereof. In a specific embodiment, the kit comprises a pharmaceutical composition comprising any one or more of the antibody compositions described herein together with one or more pharmaceutically acceptable excipients.

[0210] The present application also provides an article of manufacture comprising any one of the antibody compositions or kits described herein. An example of an article of manufacture is a vial. [Example]

[0211] Below are examples of specific embodiments for carrying out the present disclosure. These examples are presented for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0212] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, within the skill of one in the art. Such techniques are fully explained in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); and Carey and Sundberg Advanced Organic Chemistry, 3rd Ed. (Plenum Press), Vols. A and B (1992).

[0213] Example 1: Identification of anti-PAPP-A monoclonal antibodies Yeast display technology was used to identify antibodies that specifically bind to PAPP-A. The goal of this study was to generate fully human antibodies that bind and neutralize human and cynomolgus monkey (NHP) PAPP-A with high affinity and potency. Briefly, six human synthetic scFv (single-chain fragment variable) antibody libraries were selected for binding to PAPP-A. Each library consisted of a single human VH germline, highly diverse HCDR3 fragments ranging in size from 7 to 18 amino acids, and a mixture of diverse VK germlines. Using biotinylated PAPP-A protein as a target, scFv fragments were selected using both magnetic and fluorescent activated cell sorting techniques. Selection results were analyzed by sequencing and sorted using GeneData Biologics® (GeneData, Lexington, MA, USA). Nonredundant clones were converted to IgG, expressed in EXPI293 (HEK293) cells (Gibco / Fisher), and screened for binding to PAPP-A. In total, 111 clones expressed as IgG showed specific binding to human PAPP-A.

[0214] Example 2. Inhibition of PAPP-A proteolytic activity by monoclonal antibodies Inhibition of PAPP-A proteolytic activity can modulate IGF bioavailability and downstream signaling. A graphical representation of the assay for inhibition of PAPP-A proteolytic activity and neutralization of PAPP-A activity is shown in Figure 1.

[0215] Protein expression Full-length PAPP-A protein was expressed in stably transfected HEK293 cell lines for humans, NHPs, and mice and purified by heparin column chromatography. Human IGFBP-4 protein with an N-terminal 6His tag and a C-terminal Flag tag was recombinantly produced by transient expression in HEK293 cells and purified by Ni-Sepharose column chromatography. Similarly, human IGFBP-2 and human IGFBP-5 proteins were expressed and purified using a 6His tag and a C-terminal Flag tag. Prior to initiating the study, the activity of the full-length PAPP-A protein was confirmed in a preliminary assay.

[0216] Enzyme cleavage experiments For the enzymatic cleavage reaction, IGFBP-2 and IGFBP-4 proteins were preincubated with human IGF-1 (Bio-Techne® / R&D Systems, Minneapolis, MN, USA) in an enzyme assay buffer consisting of Dulbecco's modified Eagle's medium (DMEM; ThermoFisher / Gibco, Waltham, MA, USA) containing 1% bovine serum albumin (ThermoFisher / Invitrogen) for 30 min at 37°C. The IGFBP / IGF-1 proteins were then mixed with PAPP-A in the enzyme assay buffer and incubated at 37°C for 2–4 h.

[0217] For the enzymatic cleavage reaction with IGFBP-5, the proteins were mixed directly with PAPP-A without preincubation with IGF-1, because the addition of IGF-1 inhibits the proteolytic activity of PAPP-A against IGFBP-5. The final concentrations for the IGFBP-4 cleavage reaction were 90 nM IGFBP-4, 566 nM IGF-1, and 0.5 nM PAPP-A. The final concentrations for the IGFBP-5 cleavage reaction were 80 nM IGFBP-5 and 0.05 nM PAPP-A. The final concentrations for the IGFBP-2 cleavage reaction were 80 nM IGFBP-2, 566 nM IGF-1, and 5 nM PAPP-A.

[0218] Dilutions of monoclonal antibodies were prepared in enzyme assay buffer from stock solutions, starting with a 1x dilution at an initial concentration of 30 μg / mL (200 nM) and followed by eight 3x serial dilutions. Each antibody concentration was tested in triplicate. PAPP-A protein was preincubated with a dilution series of anti-PAPP-A antibodies before addition to the IGFBP mix.

[0219] Analysis of PAPP-A activity Proteins were separated by capillary electrophoresis on a Wes™ instrument (Bio-Techne® / ProteinSimple, Minneapolis, MN, USA) using a capillary cartridge kit (Bio-Techne® / ProteinSimple, Minneapolis, MN, USA), probed with a polyclonal mouse anti-His tag antibody (GeneScript, Piscataway, NJ, USA), and visualized with an anti-mouse detection module (Bio-Techne® / ProteinSimple).

[0220] The relative proportions of uncleaved and cleaved IGFBP bands were quantified using Compass for Simple Western software (Bio-techne® / ProteinSimple). The inhibitory activity of the monoclonal antibodies for each tested concentration (S = IGFBP + PAPP-A + antibody) was evaluated by normalizing to the control lanes (A = IGFBP only, B = IGFBP + PAPP-A) using the following formula:

number

[0221] One hundred and eleven antibody clones expressed as IgG that showed binding to PAPP-A were screened for their ability to inhibit the proteolytic activity of PAPP-A. One hundred and one clones completely or partially inhibited the cleavage of IGFBP4, and 57 clones completely or partially inhibited the cleavage of both IGFBP4 and IGFBP2. However, only a subset of 16 clones was able to completely or partially block the cleavage of all three IGFBPs (IGFBP4, IGFBP2, and IGFBP5). Of these 16 clones, four clones with the best inhibitory (neutralizing) potency were selected. The two most active clones are designated Ab5 and Ab6 in the table below. Two other antibody clones with slightly lower neutralizing activity were subjected to affinity maturation by CDR mutagenesis. Affinity maturation provided a means to improve substrate binding and select antibodies with even better neutralizing activity. Two antibodies were obtained from each affinity-matured clone: ​​Ab1 and Ab2 from one clone, and Ab3 and Ab4 from the second clone, as screened below.

[0222] The IC of different inhibition assays was determined because different amounts of PAPP-A protein were required for complete cleavage of different IGFBPs. 50 Although the values ​​cannot be directly compared, they provide a relative measure of inhibitory activity. However, most of the Abs tested in each cleavage assay had IC 50 Values ​​were in the sub-nM range (Table U). Only Ab5 showed reduced activity against the proteolysis of IGFBP-4 and IGFBP-5 compared to the other five antibodies.

[0223] [Table U]

[0224] Example 3: Determination of substrate affinity of anti-PAPP-A antibodies to PAPP-A Surface plasmon resonance (SPR) binding analysis was used to determine the substrate affinity of the investigated anti-PAPP-A antibodies for PAPP-A. The substrate affinity (K ) of monoclonal antibodies for human, NHP, and mouse PAPP-A proteins was measured. D ) was evaluated.

[0225] Preparation of biosensor surface Monoclonal antibodies were captured using a goat antibody specific for the Fc region of human IgG (Thermo Fisher Scientific, Waltham, MA, USA). The Fc-specific antibody was covalently immobilized via amino groups onto the carboxymethyl dextran matrix of a Biacore™ CM5 biosensor chip (Cytiva Life Sciences, Marlborough, MA, USA) using an amine coupling kit (Cytiva) and the immobilization wizard option in the Biacore™ (Cytiva) instrument control software. The carboxyl groups of the dextran matrix on the chip were activated with 100 mM N-hydroxysuccinimide and 400 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. Goat anti-human IgG Fc (25 μg / mL, Thermo Fisher Scientific) diluted in 10 mM sodium acetate, pH 4.5, was injected across the activated surface. When the level of binding response reached a target value of 10,000 resonance units (RU), unreacted groups were inactivated by injection of 1 M ethanolamine. Approximately 10,000 RU of goat anti-human IgG Fc was immobilized on the chip surface of the flow cell, while a modified control matrix surface bearing a similarly conjugated goat anti-human IgG Fc antibody was used as a reference surface.

[0226] Binding of recombinant PAPP-A to immobilized anti-PAPP-A antibodies Anti-PAPP-A monoclonal antibody was diluted to a concentration of 1 μg / mL in running buffer (HEPES-buffered saline, HBS-P+, Cytiva) containing 0.1 mg / mL bovine serum albumin and injected over a goat anti-human IgG Fc surface at a flow rate of 50 μL / min for 25 seconds to achieve a capture level of approximately 100–110 RU. The net difference between the baseline signal and the signal after the end of antibody injection was used to determine the amount of bound monoclonal antibody.

[0227] Each antigen binding experiment consisted of an antigen association phase and an antigen dissociation phase. Aliquots of recombinant PAPP-A protein at different concentrations were injected over the captured monoclonal antibody and reference surfaces at a flow rate of 50 μL / min for 5 min to measure the association rate. Each PAPP-A protein was tested at the following concentrations: 0, 0.04, 0.12, 0.37, 1.11, 3.33, 10, and 30 nM. The PAPP-A dissociation phase was performed by continuously flowing a buffer solution (HEPES-buffered saline; HBS-EP+ plus 0.1 mg / mL bovine serum albumin) at 50 μL / min, with varying dissociation times (due to the slow off-rate). The dissociation time was 1 h for the higher concentrations of 3.33, 10, and 30 nM PAPP-A, and 5 min for the lower concentrations of 0.04, 0.12, 0.37, and 1.11 nM PAPP-A). The response of the instrument is measured in RU and is proportional to the mass of bound PAPP-A antigen.

[0228] Before injecting the next sample, the immobilized surface was regenerated with 10 mM glycine, pH 1.5 (two sequential injections of 25 μL at a flow rate of 50 μL / min). Each interaction between the monoclonal antibody and each PAPP-A antigen was performed in triplicate. Finally, the response of the reference surface was subtracted from the data of the reactive surface to remove refractive index changes and injection noise.

[0229] Determination of association and dissociation rates Association rate constant (k a , M -1 s -1 The dissociation rate constant (k) was derived from kinetic binding measurements at several antigen concentrations. d ,s -1) was determined by measuring the time course of the amount of antigen bound to the monoclonal antibody after the association step was completed. The association and dissociation rate constants were calculated by the instrument evaluation software using a global fit analysis based on values ​​extracted from the data. This analysis included a locally set R due to the variability in antibody capture levels. max The same values ​​could be set for each parameter in the data set, except for the overall apparent dissociation constant (K D ) to calculate the apparent dissociation rate constant (k d ) and the apparent association rate constant (k a ) into the following formula: K D =k d / k a Used in.

[0230] Using these parameters, the substrate affinity was calculated between each antibody and recombinant PAPP-A derived from human, NHP, and mouse. Data designated as less than a specified amount was below the resolution of the instrument. Of the six anti-PAPP-A monoclonal antibodies tested, Ab1 and Ab6 showed the most similar and higher affinity for human, NHP, and mouse PAPP-A compared with the other four antibodies (Table T).

[0231] [Table T]

[0232] Example 4. Antibody-mediated inhibition of AKT phosphorylation To evaluate the effect of monoclonal antibody-mediated inhibition of PAPP-A cleavage by IGFBP-4, we examined phosphorylated AKT in HEK293 cells using an assay with human full-length PAPP-A protein.

[0233] Preparation of cells and anti-PAPP-A antibodies HEK293 cells were seeded overnight in serum-free Eagle's minimum essential medium. The enzymatic reaction was set up the following day. Anti-PAPP-A monoclonal antibody dilutions were prepared from a stock solution at a working 1x initial concentration of 0.375 μg / mL (2.5 nM) in 0.015% bovine serum albumin / Dulbecco's phosphate-buffered saline medium, followed by nine 2.5-fold serial dilutions. Antibody titration started at 1 nM for human PAPP-A. All antibody concentrations were tested in triplicate.

[0234] Examination and analysis of phosphorylated AKT The monoclonal antibody was added to human PAPP-A and incubated at room temperature for 30 minutes. IGFBP-4 protein was mixed with IGF-1 and incubated at room temperature for 30 minutes. The antibody / PAPP-A mix was added to the IGFBP-4 / IGF-1 mix and incubated at 37°C for 5 hours. The final concentrations in the reaction were 1.05 nM IGFBP-4, 4.2 nM IGF-1, and 0.55 nM human PAPP-A.

[0235] The IGF-1 / IGFBP-4 / PAPP-A / antibody mixture was added to serum-deprived HEK293 cells and incubated for 20 min at 37°C. The medium was removed, and the cells were lysed with MSD Tris lysis buffer (Meso Scale Diagnostics, Rockville, MD, USA) and analyzed using the Phospho(Ser473) / Total Akt Whole Cell Lysate kit (Meso Scale Diagnostics) according to the manufacturer's protocol.

[0236] The data in Table V below show similar IC values ​​for each of the monoclonal antibodies evaluated in this phosphorylation assay. 50 Analysis of the data demonstrated that all monoclonal antibodies inhibited the release of biologically active IGF-1 by neutralizing PAPP-A proteolytic activity from IGFBP-4.

[0237] [Table V]

[0238] Example 5. Non-specific binding of anti-PAPP-A antibodies to HEK293 cells Assessment of nonspecific binding of anti-PAPP-A monoclonal antibodies is a routine part of preclinical evaluation, as nonspecific binding of antibodies can lead to undesirable outcomes in vivo, ranging from poor pharmacokinetics to toxicological findings.

[0239] Nonspecific binding of anti-PAPP-A antibodies to HEK293 cells Human embryonic kidney (HEK293) cells, which do not express PAPP-A, were grown in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum at 37°C and 5% CO and diluted to 1.0 × 10 in FACS buffer (Dulbecco's phosphate-buffered saline plus 10% fetal bovine serum). 6 The cells were suspended at 100 μL / mL and dispensed into a 96-well round-bottom polypropylene plate (Falcon®, Fisher Scientific, Waltham, MA, USA) at 100 μL per well. After centrifugation and removal of the supernatant, 100 μL / well of each anti-PAPP-A monoclonal antibody dissolved in FACS buffer was added, sufficient to resuspend the HEK293 cells. After 30 minutes of incubation on ice, the HEK293 cells were washed with FACS buffer to remove free antibody. A secondary AffiniPure™ goat anti-human IgG, Fcγ fragment-specific APC (allophycocyanin)-conjugated antibody (Jackson Immunoresearch, West Grove, PA, USA) was diluted to 2 μg / mL and added at 100 μL per well. After 30 minutes of incubation on ice, the HEK293 cells were washed with FACS buffer to remove free antibody.

[0240] Flow cytometry analysis of HEK293 cells Flow cytometry of the prepared cells was performed using a BD FACSCanto™ flow cytometry system (Becton Dickinson, Franklin Lakes, NJ, USA). Single live cells were gated using the l / d discriminator and FSC(H) / FSC(A). Addition to the cell type cytometry profile upon incubation with anti-PAPP-A antibody was considered binding.

[0241] Three of the anti-PAPP-A antibodies, Ab4, Ab5, and Ab6, bound nonspecifically to HEK293 cells, whereas none of Ab1, Ab2, or Ab3 bound nonspecifically to HEK293 cells (Table W). The cause of this interaction is not well understood. However, the nonspecific binding of Ab4, Ab5, and Ab6 to HEK293 cells precluded these antibodies from further development.

[0242] [Table W]

[0243] Example 6. Antibody stability under heat stress Antibodies Ab1, Ab2, and Ab3 were subjected to elevated temperatures at increasing concentrations over time in an accelerated stability study. To assess accelerated stability, antibody samples were subjected to storage at 40°C. Samples of Ab1, Ab2, and Ab3 were dissolved in 15 mM histidine, pH 6.0, at concentrations up to 100 mg / mL and stored at elevated temperatures for up to 3 weeks. Samples were then stored at -80°C until analysis by size-exclusion chromatography (SEC). SEC was performed using an Agilent 1260 Infinity II HPLC system equipped with a diode-array UV detector (Agilent Technologies, Palo Alto, CA, USA). Data were analyzed using Agilent ChemStation software. The following chromatographic conditions were used for the analysis: column: Waters™ Acquity UPLC Protein BEH SEC column (200 Å, 1.7 μm, 4.6 × 300 mm; Milford, MA, USA); flow rate: 0.3 mL / min, injection volume: 5 μL; mobile phase: 100 mM disodium phosphate, 100 mM disodium sulfate, 1 mM sodium azide, pH 6.8; detection wavelength: 214 nm, run time: 15 min.

[0244] The analyzed antibody monomer data are shown in Figure 2. Compared to Ab1 and Ab3, a significant decrease in the monomer percentage was observed for Ab2 after 3 weeks of incubation at 40°C. Ab2 was not selected as a clinical candidate due to its lower stability compared to Ab1 and Ab3.

[0245] Example 7. Dependency analysis Forced degradation studies, including heat, acid, base, broad-spectrum UV-visible light stress, and chemical oxidation conditions, were performed to detect sequence dependency. A forced degradation multi-attribute LC-MS automated workflow monitored and confirmed trends in peptide-level post-translational modifications, which indicate changes in key quality attributes due to modifications such as deamidation, oxidation, isomerization, and other peptide-level degradation chemical modifications.

[0246] Sample antibodies were prepared at 2.5 mg / mL in 25 mM phosphate buffer (pH 5.8). Heat stress was performed at 40 °C for 1 and 3 weeks, and pH 9 stress was performed for 7 days. Peptides were separated using a Waters™ Acquity BEH C18 column (300 Å, 1.7 μm, 2.1 mm x 150 mm). A complex gradient of increasing acetonitrile (0–60%) was applied over 28 min at 55 °C, using a mobile phase containing 0.08% formic acid and 0.02% trifluoroacetic acid. A MaXis II TOF mass spectrometer (Bruker, Billerica, MA, USA) was used for peptide analysis. Post-translational modifications of the samples were detected and quantified using Protein Metrics Byonic™ and Byologic® software (Protein Metrics / Dotmics, San Diego, CA, USA). We extensively searched for all possible methionine oxidations, asparagine deamidations, and succinimide formation. All identified positive peptides were verified by tandem mass spectrometry fragmentation patterns and evaluated by XIC for appropriate retention time behavior and window boundaries. Peptide-level phylogenetic analysis after thermal degradation stress showed high levels of oxidation only in Ab3. Ab3 showed elevated oxidation levels of methionine (M102) in the CDR3 of the heavy chain. Methionine oxidation is a common post-translational modification (PTM) that can affect antibody bioactivity and potentially induce immunogenic responses. No oxidation of methionine residues was observed in either Ab1 or Ab2. This data was consistent with the decreased thermal stability of Ab3 and led to the decision not to continue Ab3 as a clinical candidate.

[0247] In summary, the monoclonal antibodies Ab1, Ab2, and Ab3 were examined under heat stress to determine which antibodies were suitable for further development. Only Ab1 showed no loss of stability or protein sequence dependency under the imposed degradation conditions. This developability risk assessment data was consistent with selecting Ab1 as a clinical candidate and moving it forward for development. A mouse chimeric version of Ab1 was generated for in vivo mouse testing. This antibody, Ab7, consisted of the same variable domain as Ab1 (heavy chain: amino acids 1–123, light chain: amino acids 1–107) and mouse antibody constant regions (heavy chain: immunoglobulin heavy constant γ1, light chain: immunoglobulin kappa constant).

[0248] Example 8. Efficacy of anti-PAPP-A (Ab7) in pcy mice The purpose of this study was to evaluate the therapeutic effect of anti-PAPP-A (Ab7) antibodies in suppressing total kidney volume increase and improving renal dysfunction in mice with non-orthologous ADPKD (autosomal dominant polycystic kidney disease) model mice harboring a mutation in the nephrotic gene Nph3, which causes the development and growth of renal cysts, at 3 weeks of age.

[0249] Experimental conditions Mice (pcy) were treated with Ab7 (anti-PAPP-A) 10 mg / kg IP injection once weekly (n=20) or Ab8 (isotype control antibody 10 mg / kg IP injection once weekly (n=22)) starting at approximately 12 weeks of age and continued for approximately 22 weeks. Antibodies were dissolved in phosphate-buffered saline prior to use and administered at a dose volume of 10 mL / kg.

[0250] Total kidney volume (TKV) was measured by magnetic resonance imaging (MRI) at baseline (before treatment began), after 12 weeks, and after 21 weeks of treatment. TKV provided an index of disease progression that was used to evaluate the effectiveness of treatment plans for ADPKD. In this study, T2-weighted (T2W) MRI sequences were used to measure TKV. In vivo MRI was performed using a 38mm transmitter / receiver. 1Mice were anesthetized with a 4.7 Tesla PharmaScan 47 / 16 system (Bruker, Billerica, MA, USA) using an H linear volume coil. T2-weighted (2D multislice Turbo SpinEcho RARE, TR / TE = 2500 / 48 ms, RARE Factor = 8, Average = 15) images were taken on an in-plane 0.2 × 0.2 mm image. 2 The slice thickness was 0.8 mm, and 17–21 sections were acquired to cover the entire kidney volume. Image segmentation was performed manually by iterating through all sections of the image volume and outlining the kidney boundary using the Segment Editor module of the 3D slicer. The results of image segmentation were used to calculate TKV using the following formula: Number of voxels x voxel size

[0251] TKV was estimated longitudinally at baseline, 12 and 21 weeks of treatment (corresponding to mice aged 12, 24 and 33 weeks, respectively).

[0252] Glomerular filtration rate (GFR), an index of renal function, was determined at baseline (before treatment began), 12 weeks after treatment, and 18 weeks after treatment by monitoring transcutaneous GFR (t-GFR) using the FITC-sinistrin clearance method (MediBeacon®). The dorsal side of the animals (from the top of the hind legs to the neck across the ribs) was shaved. A thin layer of depilatory cream (Nair™) was applied to the shaved area and rinsed with warm water after 2 minutes. For t-GFR device implantation, mice were anesthetized with isoflurane (induction 3%, maintenance 1.5%). The shaved area was washed with 70% ethanol, and the t-GFR device was placed on the ribs and secured with silk tape (Cardinal Health, Dublin, OH, USA). A single intravenous injection (0.15 mg / gram body weight) of FITC-sinistrin (MediBeacon®, St. Louis, MO, USA) was administered retroorbitally (5 μL / gram body weight) using a 0.5 mL insulin syringe. The injected mice were allowed to recover in their cages, and data were recorded for 1.0–1.5 h. The clearance of fluorescently labeled sinistrin recorded by the t-GFR device was analyzed using MediBeacon® Studio2 software. This software employed a three-dimensional compartmental modeling technique to calculate the half-life of FITC-sinistrin, which was then used to calculate GFR.

[0253] Effects of anti-PAPP-A (Ab7) or isotype control (Ab8) on kidney size and function in pcy mice In this study, the ability of anti-PAPP-A (Ab7) to reduce total kidney volume increase and ameliorate renal function decline was evaluated in the pcy mouse model of ADPKD. Total kidney volume was measured using MRI at baseline and after 12 and 21 weeks of treatment (corresponding to mice aged 12, 24, and 33 weeks, respectively).

[0254] As shown in Figure 3, mean TKV increased from baseline to week 12 in Ab8 (isotype control)-treated pcy mice (769 mm 3 1753mm 3), and reached a plateau at 21 weeks of administration (1591 mm 3 These data showed a significant increase in TKV in Ab8 (isotype control)-treated mice. Anti-PAPP-A (Ab7) treatment suppressed the increase in TKV at both 12 and 21 weeks of treatment (1200 mmHg, respectively) compared with isotype control (Ab8)-treated pcy mice. 3 and 1159mm 3 Compared with the Ab8 (isotype control) group, the mean increase in TKV from baseline in the Ab7 (anti-PAPP-A) group, adjusted for body weight, was estimated at 482 mm at 12 and 21 weeks of treatment, respectively. 3 and 461mm 3 These data showed that Ab7 (anti-PAPP-A) treatment significantly suppressed TKV proliferation (Table R).

[0255] [Table R]

[0256] Renal function, as measured by the t-GFR method, was assessed at baseline, 12 weeks, and 18 weeks of treatment. At baseline, mean GFR was similar in mice assigned to either the Ab8 (isotype control) or Ab7 (anti-PAPP-A) treatment groups: 1404 μL / min / 100 g body weight and 1395 μL / min / 100 g body weight, respectively (Figure 4). After 12 weeks of treatment, the mean GFR in the Ab8 (isotype control) group was 953 μL / min / 100 g body weight, and the mean GFR in the Ab7 (anti-PAPP-A) group was 1009 μL / min / 100 g body weight. After 18 weeks of treatment, the mean GFR in the Ab8 (isotype control) group decreased to 574 μL / min / 100 g body weight, compared with 963 μL / min / 100 g body weight in the Ab7 (anti-PAPP-A) group (Figure 4). Compared with the isotype control group, the decline in GFR from baseline in the Ab7-treated group was an estimated mean decrease of 399 μL / min / 100 g body weight at 18 weeks of treatment, demonstrating a significant reduction in the decline in renal function with Ab7 (anti-PAPP-A) administration (Table S).

[0257] [Table S]

[0258] Example 9: Administration of PAPP-A antibodies to humans The anti-PAPP-A antibodies described herein are administered intravenously (IV) or subcutaneously (SC) to human subjects. The dosing regimen is as follows: Group 1: Single dose of anti-PAPP-A antibody on day 1 - 30 mg IV injection Group 2: Single dose of anti-PAPP-A antibody on day 1 - up to 100 mg IV injection Group 3: Single dose of anti-PAPP-A antibody on day 1 - up to 100 mg SC injection Group 4: Single dose of anti-PAPP-A antibody on day 1 - up to 300 mg IV injection Group 5: Single dose of anti-PAPP-A antibody on day 1 - up to 300 mg SC injection Group 6: Single dose of anti-PAPP-A antibody on day 1 - up to 900 mg IV injection

[0259] Anti-PAPP-A antibodies can be administered by SC or IV injection every two weeks for up to four doses, or as a single SC or IV injection.

[0260] The antibody has been found to be safe and tolerated by subjects following administration.

[0261] Heavy and light chain antibody sequences The amino acid sequences of the heavy chain (HC) and light chain (LC) of the eight monoclonal antibodies mentioned above: Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, and Ab8 are listed in Table AH below. The first six anti-PAPP-A antibodies share the same heavy and light chain constant regions, with Ab7, also an anti-PAPP-A antibody, having a murine-compatible constant region. Ab1 through Ab6 differ in the variable regions of both the heavy chain (VH) and variable light chain (VL), particularly in the complementarity-determining regions (CDRs). CDRs were determined using the Kabat nomenclature system, and each CDR is indicated in bold and underlined (CDR1, CDR2, and CDR3, respectively) in each variable region sequence. The CDRs are also listed separately in the table. The consensus CDR sequences based on Ab1 through Ab6 are listed in Table I.

[0262] To perform in vivo mouse studies, a mouse chimeric version of Ab1 was generated and characterized. This antibody, Ab7, is composed of the same variable domains as Ab1 (heavy chain amino acids 1-123, light chain amino acids 1-107) and mouse antibody constant regions (heavy chain immunoglobulin heavy constant γ1, light chain immunoglobulin kappa constant). As demonstrated by SPD (see Example 2), Ab7 exhibited similar binding affinity to Ab1 for the PAPP-A substrate. An isotype control antibody (Ab8) was used in all in vivo studies. Ab8 is an anti-tetanus toxoid antibody with mouse immunoglobulin heavy constant γ1 and mouse immunoglobulin kappa constant. The sequences of these two antibodies are shown in Tables G and H.

[0263] [Table A] TIFF2025539937000011.tif84165

[0264] [Table B] TIFF2025539937000013.tif84165

[0265] [Table C] TIFF2025539937000015.tif84165

[0266] Table D TIFF2025539937000017.tif74165

[0267] Table E TIFF2025539937000019.tif84165

[0268] Table F TIFF2025539937000021.tif84165

[0269] Table G TIFF2025539937000023.tif84165

[0270] Table H

[0271] Table I TIFF2025539937000026.tif213165

[0272] Table J TIFF2025539937000028.tif203165TIFF2025539937000029.tif192165TIFF2025539937000030.t if198165TIFF2025539937000031.tif197165TIFF2025539937000032.tif203165TIFF20255399370 00033.tif197165TIFF2025539937000034.tif203165TIFF2025539937000035.tif197165TIFF202 5539937000036.tif203165TIFF2025539937000037.tif197165TIFF2025539937000038.tif203165

[0273] While the present invention has been particularly shown and described with reference to preferred and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

[0274] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

Claims

1. An isolated antibody that binds to pregnancy-associated plasma protein A (PAPP-A) (SEQ ID NO: 79).

2. the antibody comprises a variable heavy chain (VH) sequence comprising three heavy chain CDR sequences, CDR-H1, CDR-H2, and CDR-H3, and a variable light chain (VL) sequence comprising three light chain CDR sequences, CDR-L1, CDR-L2, and CDR-L3; a. CDR-H1 comprises the sequence SYAMH (SEQ ID NO:3); b. CDR-H2 comprises the sequence VISYDGSIKYYADAVKG (SEQ ID NO: 4); c. CDR-H3 comprises the sequence HNRIYSWGWHTFDI (SEQ ID NO:5); d. CDR-L1 comprises the sequence RASQDISIYLN (SEQ ID NO:8); e. CDR-L2 comprises the sequence GASSLQS (SEQ ID NO:9); and f. CDR-L3 comprises the sequence QQADAGPWK (SEQ ID NO: 10); The isolated antibody of claim 1.

3. The isolated antibody of claim 2, wherein the VH sequence comprises the VH sequence shown in SEQ ID NO:

2.

4. The isolated antibody of claim 2 or 3, wherein the VL sequence comprises the VL sequence shown in SEQ ID NO:

7.

5. 5. The isolated antibody of any one of claims 2 to 4, wherein the VH sequence comprises the VH sequence shown in SEQ ID NO: 2 and the VL sequence comprises the VL sequence shown in SEQ ID NO:

7.

6. The isolated antibody of any one of claims 2 to 5, wherein the antibody comprises a heavy chain comprising the sequence shown in SEQ ID NO:

1.

7. The isolated antibody of any one of claims 2 to 6, wherein the antibody comprises a light chain comprising the sequence set forth in SEQ ID NO:

6.

8. 8. The isolated antibody of any one of claims 2 to 7, wherein the antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO: 1 and a light chain comprising the sequence set forth in SEQ ID NO:

6.

9. the antibody comprises a variable heavy chain (VH) sequence comprising three heavy chain CDR sequences, CDR-H1, CDR-H2, and CDR-H3, and a variable light chain (VL) sequence comprising three light chain CDR sequences, CDR-L1, CDR-L2, and CDR-L3, wherein: a. CDR-H1 has the sequence X １ YX ２ MX ３ (SEQ ID NO: 73), wherein X １ is S or T, and X ２ is A or G, and X ３ is H or S; b. CDR-H2 has the sequence X １ IX ２ X ３ X ４ X ５ X ６ X ７ X ８ YYADX ９ VKG (SEQ ID NO: 74), wherein: X １ is V or A, and X ２ is S, Y, or R, and X ３ is Y or M, and X ４ is D or T, and X ５ is G or V, and X ６ is S, R, G, or Q, and X ７ is I, R, N, or E, and X ８ is K or T, and X ９ is A or S; c. CDR-H3 has the sequence HX １ RIX ２ X ３ WGX ４ HTFDI (SEQ ID NO: 75), wherein: X １ is N or E, and X ２ is Y or P, and X ３ is S or P, and X ４ is W or F; or comprises the sequence ADMHRFDV (SEQ ID NO:45), the sequence VWGGVRFDV (SEQ ID NO:55), or the sequence YKPMPFDV (SEQ ID NO:25 or 35); d. CDR-L1 has the sequence RASQX １ IX ２ X ３ YLN (SEQ ID NO: 76), wherein: X １ is D or S, and X ２ is S or I, and X ３ is I, S, T or R; e. CDR-L2 has the sequence X １ ASX ２ LQS (SEQ ID NO: 77), wherein: X １ is G, V, E or A, and X ２ is S or I, and f. CDR-L3 has the sequence X １ QX ２ X ３ X ４ X ５ PX ６ X ７ (SEQ ID NO: 78), wherein: X １ is Q or G, and X ２ is A or S, and X ３ is D, Y, S or H, and X ４ is A, S, G, Y or P, and X ５ is G, P or T, and X ６ is W, Y or F, and X ７ is K, T or P; The isolated antibody of claim 1.

10. 10. The isolated antibody of claim 9, wherein the VH sequence comprises a sequence selected from the sequences set forth in SEQ ID NOs: 2, 12, 22, 32, 42, or 52.

11. 11. The isolated antibody of claim 9 or 10, wherein the VL sequence comprises a sequence selected from the sequences set forth in SEQ ID NO: 7, 17, 27, 37, 47, or 57.

12. 12. The isolated antibody of any one of claims 9 to 11, wherein the VH sequence comprises the VH sequence set forth in SEQ ID NO: 2, 12, 22, 32, 42, or 52, and the VL sequence comprises the VL sequence set forth in SEQ ID NO: 7, 17, 27, 37, 47, or 57.

13. The isolated antibody of any one of claims 9 to 12, wherein the antibody comprises a heavy chain sequence selected from the sequences set forth in SEQ ID NOs: 1, 11, 21, 31, 41, or 51.

14. The isolated antibody of any one of claims 9 to 13, wherein the antibody comprises a light chain sequence selected from the sequences set forth in SEQ ID NO: 6, 16, 26, 36, 46, or 56.

15. 15. The isolated antibody of any one of claims 9 to 14, wherein the antibody comprises a heavy chain sequence selected from the sequences set forth in SEQ ID NOs: 1, 11, 21, 31, 41, or 51, and a light chain sequence selected from the sequences set forth in SEQ ID NOs: 6, 16, 26, 36, 46, or 56.

16. 16. The isolated antibody of any one of claims 9 to 15, wherein the antibody comprises two heavy chain sequences comprising a sequence selected from the sequences set forth in SEQ ID NOs: 1, 11, 21, 31, 41, or 51, and two light chain sequences comprising a sequence selected from the sequences set forth in SEQ ID NOs: 6, 16, 26, 36, 46, or 56.

17. The isolated antibody of any one of claims 9 to 16, wherein the CDR-H3 comprises HNRIYSWGWHTFDI (SEQ ID NO: 5) or HERIPPWGFHTFDI (SEQ ID NO: 15).

18. a. CDR-H1 comprises the sequence set forth in SEQ ID NO: 13; b. CDR-H2 comprises the sequence set forth in SEQ ID NO: 14; c. CDR-H3 comprises the sequence set forth in SEQ ID NO: 15; d. CDR-L1 comprises the sequence set forth in SEQ ID NO: 18; e. CDR-L2 comprises the sequence set forth in SEQ ID NO: 19; and f. CDR-L3 comprises the sequence set forth in SEQ ID NO: 20; The isolated antibody of any one of claims 9 to 17.

19. 19. The isolated antibody of claim 18, wherein the VH sequence comprises the VH sequence shown in SEQ ID NO:

12.

20. 20. The isolated antibody of claim 18 or 19, wherein the VL sequence comprises the VL sequence shown in SEQ ID NO:

17.

21. 21. The isolated antibody of any one of claims 18 to 20, wherein the VH sequence comprises the VH sequence shown in SEQ ID NO: 12 and the VL sequence comprises the VL sequence shown in SEQ ID NO:

17.

22. 22. The isolated antibody of any one of claims 18 to 21, wherein the antibody comprises a heavy chain comprising the sequence shown in SEQ ID NO:

11.

23. 23. The isolated antibody of any one of claims 18 to 22, wherein the antibody comprises a light chain comprising the sequence set forth in SEQ ID NO:

16.

24. 24. The isolated antibody of any one of claims 18 to 23, wherein the antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO:11 and a light chain comprising the sequence set forth in SEQ ID NO:

16.

25. a. CDR-H1 comprises the sequence set forth in SEQ ID NO:23; b. CDR-H2 comprises the sequence set forth in SEQ ID NO:24; c. CDR-H3 comprises the sequence set forth in SEQ ID NO:25; d. CDR-L1 comprises the sequence set forth in SEQ ID NO:28; e. CDR-L2 comprises the sequence set forth in SEQ ID NO:29; and f. CDR-L3 comprises the sequence set forth in SEQ ID NO: 30; The isolated antibody of any one of claims 9 to 16.

26. 26. The isolated antibody of claim 25, wherein the VH sequence comprises the VH sequence shown in SEQ ID NO:

22.

27. 27. The isolated antibody of claim 25 or 26, wherein the VL sequence comprises the VL sequence shown in SEQ ID NO:

27.

28. 28. The isolated antibody of any one of claims 25 to 27, wherein the VH sequence comprises the VH sequence set forth in SEQ ID NO: 22 and the VL sequence comprises the VL sequence set forth in SEQ ID NO:

27.

29. 29. The isolated antibody of any one of claims 25 to 28, wherein the antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO:

21.

30. 30. The isolated antibody of any one of claims 25 to 29, wherein the antibody comprises a light chain comprising the sequence set forth in SEQ ID NO:

26.

31. 31. The isolated antibody of any one of claims 25 to 30, wherein the antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO:21 and a light chain comprising the sequence set forth in SEQ ID NO:

26.

32. a. CDR-H1 comprises the sequence set forth in SEQ ID NO: 33; b. CDR-H2 comprises the sequence set forth in SEQ ID NO:34; c. CDR-H3 comprises the sequence set forth in SEQ ID NO:35; d. CDR-L1 comprises the sequence set forth in SEQ ID NO:38; e. CDR-L2 comprises the sequence set forth in SEQ ID NO:39; and f. CDR-L3 comprises the sequence set forth in SEQ ID NO: 40; The isolated antibody of any one of claims 9 to 16.

33. 33. The isolated antibody of claim 32, wherein the VH sequence comprises the VH sequence set forth in SEQ ID NO:

32.

34. 34. The isolated antibody of claim 32 or 33, wherein the VL sequence comprises the VL sequence shown in SEQ ID NO:

37.

35. 35. The isolated antibody of any one of claims 32 to 34, wherein the VH sequence comprises the VH sequence set forth in SEQ ID NO: 32 and the VL sequence comprises the VL sequence set forth in SEQ ID NO:

37.

36. 36. The isolated antibody of any one of claims 32 to 35, wherein the antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO:

31.

37. 37. The isolated antibody of any one of claims 32 to 36, wherein the antibody comprises a light chain comprising the sequence set forth in SEQ ID NO:

36.

38. 38. The isolated antibody of any one of claims 32 to 37, wherein the antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO: 31 and a light chain comprising the sequence set forth in SEQ ID NO:

36.

39. a. CDR-H1 comprises the sequence set forth in SEQ ID NO:43; b. CDR-H2 comprises the sequence set forth in SEQ ID NO:44; c. CDR-H3 comprises the sequence set forth in SEQ ID NO:45; d. CDR-L1 comprises the sequence set forth in SEQ ID NO:48; e. CDR-L2 comprises the sequence set forth in SEQ ID NO:49; and f. CDR-L3 comprises the sequence set forth in SEQ ID NO: 50; The isolated antibody of any one of claims 9 to 16.

40. 40. The isolated antibody of claim 39, wherein the VH sequence comprises the VH sequence set forth in SEQ ID NO:

42.

41. 41. The isolated antibody of claim 39 or 40, wherein the VL sequence comprises the VL sequence shown in SEQ ID NO:

47.

42. 42. The isolated antibody of any one of claims 39 to 41, wherein the VH sequence comprises the VH sequence set forth in SEQ ID NO: 42 and the VL sequence comprises the VL sequence set forth in SEQ ID NO:

47.

43. 43. The isolated antibody of any one of claims 39 to 42, wherein the antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO:

41.

44. 44. The isolated antibody of any one of claims 39 to 43, wherein the antibody comprises a light chain comprising the sequence set forth in SEQ ID NO:

46.

45. 45. The isolated antibody of any one of claims 39 to 44, wherein the antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO: 41 and a light chain comprising the sequence set forth in SEQ ID NO:

46.

46. a. CDR-H1 comprises the sequence set forth in SEQ ID NO:53; b. CDR-H2 comprises the sequence set forth in SEQ ID NO:54; c. CDR-H3 comprises the sequence set forth in SEQ ID NO:55; d. CDR-L1 comprises the sequence set forth in SEQ ID NO:58; e. CDR-L2 comprises the sequence set forth in SEQ ID NO:59; and f. CDR-L3 comprises the sequence set forth in SEQ ID NO: 60; The isolated antibody of any one of claims 9 to 16.

47. 47. The isolated antibody of claim 46, wherein the VH sequence comprises the VH sequence set forth in SEQ ID NO:

52.

48. 48. The isolated antibody of claim 46 or 47, wherein the VL sequence comprises the VL sequence shown in SEQ ID NO:

57.

49. 49. The isolated antibody of any one of claims 46 to 48, wherein the VH sequence comprises the VH sequence set forth in SEQ ID NO: 52 and the VL sequence comprises the VL sequence set forth in SEQ ID NO:

57.

50. 50. The isolated antibody of any one of claims 46 to 49, wherein the antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO:

51.

51. 51. The isolated antibody of any one of claims 46 to 50, wherein the antibody comprises a light chain comprising the sequence set forth in SEQ ID NO:

56.

52. 52. The isolated antibody of any one of claims 46 to 51, wherein the antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO:51 and a light chain comprising the sequence set forth in SEQ ID NO:

56.

53. 2. The isolated antibody of claim 1, wherein the antibody comprises a variable heavy chain (VH) sequence comprising three heavy chain CDR sequences, CDR-H1, CDR-H2, and CDR-H3, wherein the CDRs comprise the CDRs of one of the variable heavy (VH) chain sequences set forth in SEQ ID NOs: 2, 12, 22, 32, 42, or 52, as defined by the Kabat, AbM, IMGT, or Chothia numbering schemes.

54. 54. The isolated antibody of claim 1 or 53, wherein the antibody comprises a variable light chain (VL) sequence comprising three light chain CDR sequences, CDR-L1, CDR-L2, and CDR-L3, wherein the CDRs comprise the CDRs of one of the variable light chain (VL) sequences set forth in SEQ ID NOs: 7, 17, 27, 37, 47, or 57 as defined by the Kabat, AbM, IMGT, or Chothia numbering schemes.

55. 10. The isolated antibody of any of the preceding claims, wherein the antibody comprises a chimeric, human, or humanized antibody, or an antigen-binding fragment.

56. 10. The isolated antibody of any of the above claims, wherein the antibody is a monoclonal antibody.

57. 10. The isolated antibody of any of the above claims, wherein the antibody is a human antibody.

58. 10. The isolated antibody of claim 1, wherein the antibody comprises an Fc region.

59. An isolated antibody described in any of the above claims, wherein the Fc region comprises a human Fc region.

60. 60. The isolated antibody of claim 59, wherein the human Fc region comprises a human IgGl Fc region.

61. 61. The isolated antibody of any one of claims 59 to 60, wherein the Fc region comprises the L234A / L235A mutation according to the EU numbering system.

62. An isolated human antibody that binds to pregnancy-associated plasma protein A (PAPP-A) (SEQ ID NO: 79), wherein the antibody comprises a human IgG1 Fc region, two heavy chains comprising the sequences set forth in SEQ ID NO: 1, and two light chains comprising the sequences set forth in SEQ ID NO:

6.

63. 1. An isolated human antibody that binds to pregnancy-associated plasma protein A (PAPP-A) (SEQ ID NO: 79), wherein the antibody comprises a human IgG1 Fc region, two heavy chains comprising the sequences set forth in SEQ ID NO: 11, and two light chains comprising the sequences set forth in SEQ ID NO:

16.

64. 1. An isolated human antibody that binds to pregnancy-associated plasma protein A (PAPP-A) (SEQ ID NO: 79), wherein the antibody comprises a human IgG1 Fc region, two heavy chains comprising the sequences set forth in SEQ ID NO: 21, and two light chains comprising the sequences set forth in SEQ ID NO:

26.

65. 1. An isolated human antibody that binds to pregnancy-associated plasma protein A (PAPP-A) (SEQ ID NO: 79), wherein the antibody comprises a human IgG1 Fc region, two heavy chains comprising the sequences set forth in SEQ ID NO: 31, and two light chains comprising the sequences set forth in SEQ ID NO:

36.

66. 1. An isolated human antibody that binds to pregnancy-associated plasma protein A (PAPP-A) (SEQ ID NO: 79), wherein the antibody comprises a human IgG1 Fc region, two heavy chains comprising the sequences set forth in SEQ ID NO: 41, and two light chains comprising the sequences set forth in SEQ ID NO:

46.

67. 1. An isolated human antibody that binds to pregnancy-associated plasma protein A (PAPP-A) (SEQ ID NO: 79), wherein the antibody comprises a human IgG1 Fc region, two heavy chains comprising the sequences set forth in SEQ ID NO: 51, and two light chains comprising the sequences set forth in SEQ ID NO:

56.

68. The antibody has a densitometric activity of about 1, 1.5, 2, 2.5, 10, 25, 50, 75, or 100×10 as measured by a surface plasmon resonance (SPR) assay. －１２ K below M Ｄ 10. The isolated antibody of any of the preceding claims, which binds to human PAPP-A at

69. 10. The isolated antibody of any of the preceding claims, wherein the antibody has enzyme inhibiting or neutralizing activity, and optionally, the antibody has metalloprotease inhibiting activity.

70. 10. The isolated antibody of any of the preceding claims, wherein the antibody blocks PAPP-A cleavage of the IGF binding protein.

71. 10. An isolated antibody according to any of the preceding claims for use as a medicament.

72. 10. The isolated antibody of any of the preceding claims for use in the treatment of a PAPP-A associated disorder.

73. An isolated polynucleotide or set of polynucleotides encoding an antibody, its VH, its VL, its light chain, its heavy chain, or an antigen-binding portion thereof according to any of the above claims, which is optionally cDNA.

74. 74. A vector or set of vectors comprising the polynucleotide or set of polynucleotides of claim 73.

75. 75. A host cell comprising the polynucleotide or set of polynucleotides of claim 73 or the vector or set of vectors of claim 74.

76. 76. A method of producing an antibody, comprising expressing the antibody in a host cell of claim 75 and isolating the expressed antibody.

77. 73. A pharmaceutical composition comprising the isolated antibody of any one of claims 1 to 72 and a pharmaceutically acceptable excipient.

78. A kit comprising the isolated antibody of any one of claims 1 to 72 or the pharmaceutical composition of claim 77, and instructions for use.

79. A method of treating a PAPP-A associated disorder in a subject, comprising administering to the subject a composition comprising an anti-PAPP-A antibody.

80. 80. The method of claim 79, wherein the PAPP-A-associated disorder is a kidney disease.

81. 81. The method of claim 80, wherein the kidney disease is polycystic kidney disease.

82. 81. The method of claim 80, wherein the kidney disease is autosomal dominant polycystic kidney disease (ADPKD).

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