Anti-PAPP-A antibodies and methods of use thereof

Abstract

Provided herein are anti-PAPP-A antibodies. Also provided are methods of using such antibodies to treat PAPP-A related disorders, such as kidney disease.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 383,875, filed November 15, 2022, which is hereby incorporated by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing which is hereby incorporated by reference in its entirety. The XML copy was created on November 6, 2023, is named CLS102WO_ST.26.xml and is 112,848 bytes in size. Background Art

[0005] Autosomal dominant polycystic kidney disease (ADPKD) is a rare disease with unmet medical needs. In the United States alone, there are approximately 166,000 patients with ADPKD. ADPKD is a major cause of morbidity and accounts for approximately 5%-10% of deaths associated with 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-authorized Risk Evaluation and Mitigation Strategy (REMS) to mitigate the risk of severe and potentially fatal liver damage due to administration. Therefore, additional therapeutic agents are needed to treat kidney diseases including ADPKD. Summary of the Invention

[0006] In some aspects, provided herein are isolated human antibodies that bind 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:

[0007] CDR-H1 comprises the sequence SYAMH (SEQ ID NO: 3);

[0008] CDR-H2 comprises the sequence VISYDGSIKYYADAVKG (SEQ ID NO: 4);

[0009] CDR-H3 comprises the sequence HNRIYSWGWHTFDI (SEQ ID NO: 5);

[0010] CDR-L1 comprises the sequence RASQDISIYLN (SEQ ID NO: 8);

[0011] CDR-L2 comprises the sequence GASSLQS (SEQ ID NO: 9); and

[0012] CDR-L3 comprises the sequence QQADAGPWK (SEQ ID NO: 10).

[0013] In some embodiments, the VH sequence comprises the VH sequence shown in SEQ ID NO:2.

[0014] In some embodiments, the VL sequence comprises the VL sequence set forth in SEQ ID NO:7.

[0015] 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.

[0016] In some embodiments, the antibody comprises a heavy chain comprising the sequence shown in SEQ ID NO:1.

[0017] In some embodiments, the antibody comprises a light chain comprising the sequence shown in SEQ ID NO:6.

[0018] In some embodiments, the antibody comprises a heavy chain comprising the sequence shown in SEQ ID NO: 1 and a light chain comprising the sequence shown in SEQ ID NO:6.

[0019] 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 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.

[0020] 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 sequence shown in SEQ ID NO: 1, and two light chains comprising the sequence shown in SEQ ID NO: 6.

[0021] In some aspects, provided herein are isolated antibodies that bind to pregnancy-associated plasma protein A (PAPP-A) (SEQ ID NO: 79).

[0022] 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, wherein:

[0023] CDR-H1 comprises the sequence X1YX2MX3 (SEQ ID NO: 73), wherein X1 is S or T; X2 is A or G; and X3 is H or S;

[0024] CDR-H2 comprises the sequence X1IX2X3X4X5X6X7X8YYADX9VKG (SEQ ID NO: 74), wherein 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;

[0025] CDR-H3 comprises the sequence HX1RIX2X3WGX4HTFDI (SEQ ID NO: 75), wherein X1 is N or E; X2 is Y or P; X3 is S or P; and X4 is W or F; the sequence ADMHRFDV (SEQ ID NO: 45), the sequence VWGGVRFDV (SEQ ID NO: 55), or the sequence YKPMPFDV (SEQ ID NO: 25 or 35);

[0026] 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;

[0027] CDR-L2 comprises the sequence X1ASX2LQS (SEQ ID NO: 77), wherein X1 is G, V, E, or A; and X2 is S or I; and

[0028] CDR-L3 comprises the sequence X1QX2X3X4X5PX6X7 (SEQ ID NO: 78), wherein 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.

[0029] In some embodiments, the VH sequence comprises a sequence selected from the sequence shown in SEQ ID NO: 2, 12, 22, 32, 42, or 52.

[0030] In some embodiments, the VL sequence comprises a sequence selected from the sequence shown in SEQ ID NO: 7, 17, 27, 37, 47, or 57.

[0031] In some embodiments, the VH sequence comprises the VH sequence shown in SEQ ID NO: 2, 12, 22, 32, 42, or 52, and the VL sequence comprises the VL sequence shown in SEQ ID NO: 7, 17, 27, 37, 47, or 57.

[0032] In some embodiments, the antibody comprises a heavy chain sequence selected from the sequence shown in SEQ ID NO: 1, 11, 21, 31, 41, or 51.

[0033] In some embodiments, the antibody comprises a light chain sequence selected from the sequence shown in SEQ ID NO: 6, 16, 26, 36, 46, or 56.

[0034] In some embodiments, the antibody comprises a heavy chain sequence selected from the sequence shown in SEQ ID NO: 1, 11, 21, 31, 41, or 51; and a light chain sequence selected from the sequence shown in SEQ ID NO: 6, 16, 26, 36, 46, or 56.

[0035] In some embodiments, the antibody comprises two heavy chain sequences comprising a sequence selected from the sequence shown in SEQ ID NO: 1, 11, 21, 31, 41 or 51; and two light chain sequences comprising a sequence selected from the sequence shown in SEQ ID NO: 6, 16, 26, 36, 46 or 56.

[0036] In some embodiments, CDR-H3 comprises HNRIYSWGWHTFDI (SEQ ID NO: 5) or HERIPPWGFHTFDI (SEQ ID NO: 15).

[0037] In some embodiments,

[0038] CDR-H1 comprises the sequence shown in SEQ ID NO: 3;

[0039] CDR-H2 comprises the sequence shown in SEQ ID NO:4;

[0040] CDR-H3 comprises the sequence shown in SEQ ID NO:5;

[0041] CDR-L1 comprises the sequence shown in SEQ ID NO:8;

[0042] CDR-L2 comprises the sequence shown in SEQ ID NO:9; and

[0043] CDR-L3 comprises the sequence shown in SEQ ID NO:10.

[0044] In some embodiments, the VH sequence comprises the VH sequence shown in SEQ ID NO:2.

[0045] In some embodiments, the VL sequence comprises the VL sequence set forth in SEQ ID NO:7.

[0046] 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.

[0047] In some embodiments, the antibody comprises a heavy chain comprising the sequence shown in SEQ ID NO:1.

[0048] In some embodiments, the antibody comprises a light chain comprising the sequence shown in SEQ ID NO:6.

[0049] In some embodiments, the antibody comprises a heavy chain comprising the sequence shown in SEQ ID NO: 1 and a light chain comprising the sequence shown in SEQ ID NO:6.

[0050] In some embodiments,

[0051] CDR-H1 comprises the sequence shown in SEQ ID NO: 13;

[0052] CDR-H2 comprises the sequence shown in SEQ ID NO: 14;

[0053] CDR-H3 comprises the sequence shown in SEQ ID NO: 15;

[0054] CDR-L1 comprises the sequence shown in SEQ ID NO: 18;

[0055] CDR-L2 comprises the sequence shown in SEQ ID NO: 19; and

[0056] CDR-L3 comprises the sequence shown in SEQ ID NO:20.

[0057] In some embodiments, the VH sequence comprises the VH sequence shown in SEQ ID NO:12.

[0058] In some embodiments, the VL sequence comprises the VL sequence set forth in SEQ ID NO:17.

[0059] 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.

[0060] In some embodiments, the antibody comprises a heavy chain comprising the sequence shown in SEQ ID NO:11.

[0061] In some embodiments, the antibody comprises a light chain comprising the sequence shown in SEQ ID NO:16.

[0062] In some embodiments, the antibody comprises a heavy chain comprising the sequence shown in SEQ ID NO: 11 and a light chain comprising the sequence shown in SEQ ID NO: 16.

[0063] In some embodiments,

[0064] CDR-H1 comprises the sequence shown in SEQ ID NO:23;

[0065] CDR-H2 comprises the sequence shown in SEQ ID NO:24;

[0066] CDR-H3 comprises the sequence shown in SEQ ID NO:25;

[0067] CDR-L1 comprises the sequence shown in SEQ ID NO:28;

[0068] CDR-L2 comprises the sequence shown in SEQ ID NO: 29; and

[0069] CDR-L3 comprises the sequence shown in SEQ ID NO:30.

[0070] In some embodiments, the VH sequence comprises the VH sequence shown in SEQ ID NO:22.

[0071] In some embodiments, the VL sequence comprises the VL sequence set forth in SEQ ID NO:27.

[0072] 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.

[0073] In some embodiments, the antibody comprises a heavy chain comprising the sequence shown in SEQ ID NO:21.

[0074] In some embodiments, the antibody comprises a light chain comprising the sequence shown in SEQ ID NO:26.

[0075] In some embodiments, the antibody comprises a heavy chain comprising the sequence shown in SEQ ID NO:21 and a light chain comprising the sequence shown in SEQ ID NO:26.

[0076] In some embodiments,

[0077] CDR-H1 comprises the sequence shown in SEQ ID NO:33;

[0078] CDR-H2 comprises the sequence shown in SEQ ID NO:34;

[0079] CDR-H3 comprises the sequence shown in SEQ ID NO:35;

[0080] CDR-L1 comprises the sequence shown in SEQ ID NO:38;

[0081] CDR-L2 comprises the sequence shown in SEQ ID NO: 39; and

[0082] CDR-L3 comprises the sequence shown in SEQ ID NO:40.

[0083] In some embodiments, the VH sequence comprises the VH sequence shown in SEQ ID NO:32.

[0084] In some embodiments, the VL sequence comprises the VL sequence set forth in SEQ ID NO:37.

[0085] 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.

[0086] In some embodiments, the antibody comprises a heavy chain comprising the sequence shown in SEQ ID NO:31.

[0087] In some embodiments, the antibody comprises a light chain comprising the sequence shown in SEQ ID NO:36.

[0088] In some embodiments, the antibody comprises a heavy chain comprising the sequence shown in SEQ ID NO:31 and a light chain comprising the sequence shown in SEQ ID NO:36.

[0089] In some embodiments,

[0090] CDR-H1 comprises the sequence shown in SEQ ID NO:43;

[0091] CDR-H2 comprises the sequence shown in SEQ ID NO:44;

[0092] CDR-H3 comprises the sequence shown in SEQ ID NO:45;

[0093] CDR-L1 comprises the sequence shown in SEQ ID NO:48;

[0094] CDR-L2 comprises the sequence shown in SEQ ID NO:49; and

[0095] CDR-L3 comprises the sequence shown in SEQ ID NO:50.

[0096] In some embodiments, the VH sequence comprises the VH sequence shown in SEQ ID NO:42.

[0097] In some embodiments, the VL sequence comprises the VL sequence set forth in SEQ ID NO:47.

[0098] 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.

[0099] In some embodiments, the antibody comprises a heavy chain comprising the sequence shown in SEQ ID NO:41.

[0100] In some embodiments, the antibody comprises a light chain comprising the sequence shown in SEQ ID NO:46.

[0101] In some embodiments, the antibody comprises a heavy chain comprising the sequence shown in SEQ ID NO:41 and a light chain comprising the sequence shown in SEQ ID NO:46.

[0102] In some embodiments,

[0103] CDR-H1 comprises the sequence shown in SEQ ID NO:53;

[0104] CDR-H2 comprises the sequence shown in SEQ ID NO:54;

[0105] CDR-H3 comprises the sequence shown in SEQ ID NO:55;

[0106] CDR-L1 comprises the sequence shown in SEQ ID NO:58;

[0107] CDR-L2 comprises the sequence shown in SEQ ID NO:59; and

[0108] CDR-L3 comprises the sequence shown in SEQ ID NO:60.

[0109] In some embodiments, the VH sequence comprises the VH sequence shown in SEQ ID NO:52.

[0110] In some embodiments, the VL sequence comprises the VL sequence set forth in SEQ ID NO:57.

[0111] 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.

[0112] In some embodiments, the antibody comprises a heavy chain comprising the sequence shown in SEQ ID NO:51.

[0113] In some embodiments, the antibody comprises a light chain comprising the sequence shown in SEQ ID NO:56.

[0114] In some embodiments, the antibody comprises a heavy chain comprising the sequence shown in SEQ ID NO:51 and a light chain comprising the sequence shown in SEQ ID NO:56.

[0115] 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 the CDR-H1, CDR-H2, and CDR-H3 comprise the CDRs of one of the variable heavy (VH) chain sequences shown in SEQ ID NO: 2, 12, 22, 32, 42, or 52, as defined by the Kabat, AbM, IMGT, or Chothia numbering schemes.

[0116] 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 the CDR-L1, CDR-L2, and CDR-L3 comprise the CDRs of one of the variable light (VL) chain sequences shown in SEQ ID NO: 7, 17, 27, 37, 47, or 57, as defined by the Kabat, AbM, IMGT, or Chothia numbering schemes.

[0117] In some embodiments, the antibody comprises a chimeric antibody, a human antibody, or a humanized antibody.

[0118] In some embodiments, the antibody is a monoclonal antibody.

[0119] In some embodiments, the antibody is a humanized antibody.

[0120] In some embodiments, the antibody is a human antibody.

[0121] In some embodiments, the antibody comprises an Fc region.

[0122] In some embodiments, the Fc region comprises a human Fc region.

[0123] In some embodiments, the human Fc region comprises a human IgG1 Fc region.

[0124] In some embodiments, the Fc region comprises a L234A / L235A mutation according to the EU numbering system.

[0125] 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 sequence shown in SEQ ID NO: 1, and two light chains comprising the sequence shown in SEQ ID NO: 6.

[0126] 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 sequence shown in SEQ ID NO: 11, and two light chains comprising the sequence shown in SEQ ID NO: 16.

[0127] 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 sequence shown in SEQ ID NO: 21, and two light chains comprising the sequence shown in SEQ ID NO: 26.

[0128] 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 sequence shown in SEQ ID NO: 31, and two light chains comprising the sequence shown in SEQ ID NO: 36.

[0129] 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 sequence shown in SEQ ID NO: 41, and two light chains comprising the sequence shown in SEQ ID NO: 46.

[0130] 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 sequence shown in SEQ ID NO: 51, and two light chains comprising the sequence shown in SEQ ID NO: 56.

[0131] In some embodiments, the antibody has a concentration of less than or equal to about 1, 1.5, 2, 2.5, 10, 25, 50, 75, or 100 x 10 -12 M's K D Binds to human PAPP-A.

[0132] In some embodiments, the antibody has enzyme blocking or neutralizing activity, optionally wherein the antibody has metalloprotease blocking activity.

[0133] In some embodiments, the antibody blocks PAPP-A cleavage of the IGF binding protein.

[0134] The isolated antibodies as disclosed herein are useful as medicaments.

[0135] The isolated antibodies as disclosed herein are useful in treating PAPP-A associated disorders.

[0136] In another aspect, provided herein is an isolated polynucleotide or group of polynucleotides encoding the antibody, VH, VL, light chain, heavy chain, or antigen binding portion thereof as described in any one of the above claims; optionally the isolated polynucleotide or group of polynucleotides is a cDNA.

[0137] In another aspect, provided herein is a vector or set of vectors comprising a polynucleotide or set of polynucleotides as disclosed herein.

[0138] In another aspect, provided herein is a host cell comprising a polynucleotide or set of polynucleotides as disclosed herein or a vector or set of vectors as disclosed herein.

[0139] In another aspect, provided herein is a method of producing an antibody, the method comprising expressing the antibody using a host cell as disclosed herein and isolating the expressed antibody.

[0140] In another aspect, provided herein is a pharmaceutical composition comprising an isolated antibody as disclosed herein and a pharmaceutically acceptable excipient.

[0141] In another aspect, provided herein is a kit comprising an isolated antibody as disclosed herein or a pharmaceutical composition as disclosed herein and instructions for use.

[0142] In another aspect, provided herein are methods of treating a PAPP-A-associated disorder in a subject, the methods comprising administering to the subject a composition comprising an anti-PAPP-A antibody.

[0143] In some embodiments, the PAPP-A-associated disorder is renal disease, polycystic kidney disease, or autosomal dominant polycystic kidney disease (ADPKD). BRIEF DESCRIPTION OF THE DRAWINGS

[0144] These and other features, aspects, and advantages of the present disclosure will become better understood with reference to the following description and accompanying drawings, in which:

[0145] Figure 1 Graphical depiction of the method for in vitro analysis of PAPP-A activity as measured by cleavage of IGF binding protein 4 (IGFBP4). The resulting Western blot shows uncleaved and cleaved IGFBP bands and was analyzed using Compass for SW software (Bio- ) for quantification.

[0146] Figure 2 Stability analysis of Ab1, Ab2, and Ab3 at high temperature: A decrease in the monomer percentage was observed after storage at 40°C for three weeks.

[0147] Figure 3 Total kidney volume (TKV) of 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.

[0148] Figure 4 Glomerular filtration rate (GFR) of pcy mice treated with Ab8 (isotype control) or Ab7 (anti-PAPP-A) was measured using the FITC-shallot method at baseline, 12 weeks, and 18 weeks of treatment. Renal function (GFR) was assessed. 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. DETAILED DESCRIPTION

[0149] definition

[0150] Unless otherwise stated, the terms used in the claims and the specification are defined as set forth below.

[0151] The term "amelioration" refers to any therapeutically beneficial result in the treatment of a disease state (eg, a renal state), including prevention, lessening of severity or progression, remission, or cure thereof.

[0152] The term "in situ" refers to processes that occur in living cells grown separately from a living organism, such as in tissue culture.

[0153] The term "in vivo" refers to processes that occur in a living organism.

[0154] The term "mammal" as used herein includes both humans and non-humans, and includes, but is not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0155] In the context of two or more nucleic acids or polypeptide sequences, the term "identity" percentage refers to the number of identical nucleotides or amino acid residues in a sequence or subsequence when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to the skilled person) or by visual inspection. Depending on the application, the "identity" percentage can be present in a region of the sequences being compared, e.g., in a functional domain, or alternatively over the full length of the two sequences to be compared.

[0156] For sequence comparison, typically a sequence serves as a reference sequence to be compared with a test sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are input into a computer, subsequence coordinates are specified (if necessary), and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the sequence identity percentages of one or more test sequences relative to the reference sequence based on the specified program parameters.

[0157] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981); the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970); the search similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988); computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package; Genetics Computer Group, 575 Science Dr., Madison, Wis.); or by visual inspection (see generally, Ausubel et al., infra).

[0158] An example of an algorithm suitable for determining sequence identity and sequence similarity percentages 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 / ).

[0159] The term "sufficient amount" refers to an amount sufficient to produce a desired effect, for example, an amount sufficient to modulate protein aggregation in a cell.

[0160] The term "therapeutically effective amount" is an amount effective to improve the symptoms of a disease. When prevention and treatment can be considered as therapy, the therapeutically effective amount can be a "preventive and therapeutic effective amount."

[0161] It must be noted that, as used in the 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 a single compound as well as one or more of the same or different compounds; reference to "a pharmaceutically acceptable carrier" refers to a single pharmaceutically acceptable carrier as well as one or more pharmaceutically acceptable carriers, etc.

[0162] The term "ADPKD" as used herein defines autosomal dominant polycystic kidney disease.

[0163] The term "APC" as used herein defines allophycocyanin.

[0164] As used herein, the term "scFv" defines a single-chain variable fragment, a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin linked to a short linker peptide. These chimeric proteins are used in yeast display technology.

[0165] The term "Fc region" as used herein defines the part of IgG (IgG Fc) that interacts with effector proteins, including Fcγ receptors.

[0166] The term "GFR" as used herein defines glomerular filtration rate.

[0167] The term "ICH" as used herein defines the International Conference on Harmonisation, in particular the photostability guidelines.

[0168] The term "neutralizing" as used herein defines the inhibition of an enzyme target protein by an antibody.

[0169] The term "NHP" as used herein defines a non-human primate, particularly a cynomolgus monkey or a rhesus monkey.

[0170] The term "PAPP-A" as used herein defines pregnancy-associated plasma protein-A, which is produced by the placenta and is necessary for the implantation process and maintenance of the placenta during pregnancy.

[0171] The term "SPR" as used herein defines surface plasmon resonance, an optical technique for detecting the interaction between two molecules.

[0172] The term "t-GFR" as used herein defines the transcutaneous glomerular filtration rate.

[0173] The term "TKV" as used herein defines total kidney volume.

[0174] The term "WB" as used herein defines Western blot.

[0175] Antibody

[0176] structure

[0177] The present application provides antibodies and compositions comprising antibodies that bind to pregnancy-associated plasma protein A (PAPP-A). Such antibodies include antibodies that block, inhibit, or reduce the activity of the PAPP-A enzyme.

[0178] The term "antibody" is used herein in its broadest sense and includes certain types of immunoglobulin molecules that contain 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.

[0179] Identified immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as tens of thousands of immunoglobulin variable region genes. Light chains are classified as kappa or lambda. The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different immunoglobulin classes are referred to as α, δ, ε, γ, and μ, respectively.

[0180] An exemplary immunoglobulin (antibody) structural unit consists of two pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD), such as 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 about 100 to 110 or more amino acids that is 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. The IgG1 heavy chain comprises the VH, CH1, CH2, and CH3 domains from the N-terminus to the C-terminus, respectively. The light chain comprises the VL and CL domains from the N-terminus to the C-terminus. The IgG1 heavy chain comprises a hinge between the CH1 and CH2 domains. In certain embodiments, the immunoglobulin construct comprises 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 from or derived from immunoglobulin-based constructs, such as bifunctional antibodies 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 mouse antibody, or any chimeric antibody.

[0181] In some embodiments, the antibodies provided herein comprise 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.

[0182] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of the antibody variable domain that are highly variable in sequence and / or form structurally determined loops ("hypervariable loops"). Typically, a natural four-chain antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically include amino acid residues from hypervariable loops and / or from complementary determining regions (CDRs), which have the highest sequence variability and / or participate in antigen recognition. Except for CDR1 in VH, CDRs typically include amino acid residues that form 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 regions that form the antigen binding regions. This specific region has been described by Kabat et al., U. S. Pat. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and by Chothia et al., J Mol Biol 196:901-917 (1987), wherein the definitions include overlapping or subgroups of amino acid residues when compared relative to each other. However, the application of any definition to refer to the CDRs of antibodies or variants thereof is intended to be within the scope of the terms as defined and used herein. The exact number of residues encompassing a specific CDR will vary depending on the sequence and size of the CDR. Given the variable region amino acid sequence of an antibody, one skilled in the art can determine in a conventional manner which residues constitute a specific CDR.

[0183] The amino acid sequence boundaries of a CDR can be determined by one skilled in the art using any of a number of known numbering schemes, including those described by Kabat et al. (supra) ("Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 ("Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 ("Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 ("IMGT" numbering scheme); and Honegge and Plückthun, J. Mol. Biol., 2001, 309:657-70 ("AHo" numbering scheme); each of which is incorporated herein by reference in its entirety.

[0184] Table 1 provides the positions of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 as identified by the Kabat and Chothia schemes. For CDR-H1, residue numbers are provided using both the Kabat and Chothia numbering schemes.

[0185] CDRs can be assigned, for example, using antibody numbering software, such as 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.

[0186] Table 1. Residues in the CDRs according to the Kabat and Chothia numbering schemes.

[0187] CDR Kabat Chothia L1 L24-L34 L24-L34 L2 L50-L56 L50-L56 L3 L89-L97 L89-L97 H1 (Kabat number) H31-H35B H26-H32 or H34* H1 (Chothia number) H31-H35 H26-H32 H2 H50-H66 H52-H56 H3 H99-H112 H95-H102

[0188] *When numbering using the Kabat numbering convention, the C-terminus of CDR-H1 varies between H32 and H34, depending on the length of the CDR.

[0189] When referring to residues in the antibody heavy chain constant region, the "EU numbering scheme" is generally used (e.g., as reported in Kabat et al., supra). Unless otherwise indicated, the EU numbering scheme is used to refer to residues in the antibody heavy chain constant region described herein.

[0190] As used herein, the term "single chain" refers to a molecule comprising amino acid monomers linearly connected by peptide bonds. In a specific embodiment, in a single-chain Fab molecule, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain. As described in more detail herein, scFv has a light chain variable domain (VL) connected to the N-terminus of the variable domain (VH) of the heavy chain from its C-terminus via a polypeptide chain. Alternatively, scFv comprises a polypeptide chain in which the C-terminus of VH is connected to the N-terminus of VL via a polypeptide chain.

[0191] A "Fab fragment" (also known as an antigen-binding fragment) contains the constant domain (CL) of the light chain and the first constant domain (CH1) of the heavy chain, as well as the variable domains VL and VH on the light and heavy chains, respectively. The variable domains contain the complementarity-determining loops (CDRs, also known as hypervariable regions) involved in antigen binding. Fab' fragments differ from Fab fragments by the addition of a small number of residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region.

[0192] A "F(ab')2" fragment contains two Fab' fragments connected by a disulfide bond near the hinge region. F(ab')2 fragments can be produced, for example, by recombinant methods or by pepsin digestion of intact antibodies. F(ab') fragments can be dissociated, for example, by treatment with β-mercaptoethanol.

[0193] "Single-chain Fv" or "scFv" includes the VH domain and VL domain of an antibody, wherein these domains are present in a single polypeptide chain. In one embodiment, the Fv polypeptide further includes a polypeptide linker between the VH domain and the VL domain that enables the scFv to form a structure required for antigen binding. For a review of scFv, see Pluckthun, 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 WO93 / 16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458.

[0194] The terms "full-length antibody," "intact antibody," and "complete antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to a naturally occurring antibody structure and has heavy chains that include an Fc region. For example, a "full-length antibody," when used to refer to an IgG molecule, is an antibody that comprises two heavy chains and two light chains.

[0195] The term "epitope" means the portion of an antigen that specifically binds to an antibody. Epitopes are often composed of surface-accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that binding to the former, but not the latter, may be lost in the presence of denaturing solvents. An epitope may comprise amino acid residues that are directly involved in binding as well as other amino acid residues that are not directly involved in binding. The epitope to which an antibody binds can be determined using known techniques for epitope determination, such as testing the binding of the antibody to PAPP-A variants having different point mutations or to chimeric PAPP-A variants.

[0196] The term "monoclonal antibody" refers to an antibody from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies comprises antibodies that are substantially similar and that bind one or more identical epitopes, with the exception of variants that may typically be produced during the generation of monoclonal antibodies. Such variants typically exist only in small quantities. Monoclonal antibodies are typically obtained by methods including selecting a single antibody from a variety of antibodies. For example, a selection method can be employed to select a unique clone from a confluence of multiple clones such as 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 its production in cell culture, and / or to reduce its immunogenicity in a subject.

[0197] "Effector functions" refer to those biological activities mediated by the Fc region of an antibody, which may vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding for activation of complement-dependent cytotoxicity (CDC), Fc receptor binding for activation of antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), receptor ligand blocking, stimulation, or antagonism. An active Fc region is one that is capable of exerting Fc-based effector functions, such as ADCC, CDC, and / or ADCP.

[0198] Anti-PAPP-A antibodies may include those described herein, such as the clones set forth in the table. In some embodiments, the antibodies comprise alternative scaffolds. In some embodiments, the antibodies consist of alternative scaffolds. In some embodiments, the antibodies consist essentially of alternative scaffolds. In some embodiments, the antibodies comprise antibody fragments. In some embodiments, the antibodies consist of antibody fragments. In some embodiments, the antibodies consist essentially of antibody fragments. "PAPP-A antibodies," "anti-PAPP-A antibodies," or "PAPP-A-specific antibodies" are antibodies as provided herein that specifically bind to the antigen PAPP-A. In some embodiments, the antibodies bind 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 between PAPP-A proteins from different species.

[0199] 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.

[0200] In some embodiments, the antibody comprises a mouse 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.

[0201] In one embodiment, one or more constant domains from a human antibody are fused to one or more variable domains of a non-human species. In another embodiment, one or more amino acid residues in one or more CDR sequences of a non-human antibody are changed so that when it is administered to a human subject, the potential immunogenicity of the non-human antibody is reduced, wherein the changed amino acid residues are not critical for the immunospecific binding of the antibody to its antigen, or the changes made to the amino acid sequence are conservative changes such that the binding of the humanized antibody to the antigen is not significantly worse than the binding of the non-human antibody to the antigen.

[0202] "Human antibodies" are antibodies with an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source, and the non-human source utilizes a human antibody repertoire or human antibody coding sequence (e.g., obtained or redesigned from a human source). Human antibodies explicitly exclude humanized antibodies. In one embodiment, the variable domains and constant domains are all derived from human immunoglobulin sequences (fully human antibodies). These antibodies can be prepared in a variety of ways, including by immunizing mice that are genetically modified to express antibodies produced by human heavy and / or light chain coding genes with the target antigen.

[0203] In some embodiments, the antibodies provided herein comprise antibody fragments. In some embodiments, the antibodies provided herein consist of antibody fragments. In some embodiments, the antibodies provided herein consist essentially of antibody fragments. 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 a scFv (sFv) fragment. In some embodiments, the antibody fragment is a scFv-Fc fragment. In some embodiments, the antibody fragment is a fragment of a single domain antibody.

[0204] In some aspects, provided herein are isolated polynucleotides or sets of polynucleotides encoding an antibody, VH, VL, light chain, heavy chain, or antigen-binding portion thereof, as described in any of the above claims; optionally, the isolated polynucleotides or sets of polynucleotides are cDNAs. The nucleotide sequences of the VH, VL, heavy chain, and light chain sequences of the PAPP-A antibodies are provided in SEQ ID NOs: 80-103. For example, the heavy chain sequences of the PAPP-A antibodies disclosed herein are provided as shown in SEQ ID NOs: 80, 84, 88, 92, 96, and 100. The variable heavy chain sequences of the PAPP-A antibodies disclosed herein are provided as shown in SEQ ID NOs: 81, 85, 89, 93, 97, and 101. The light chain sequences of the PAPP-A antibodies disclosed herein are provided as shown in SEQ ID NOs: 82, 86, 90, 94, 98, and 102. The variable light chain sequences of the PAPP-A antibodies disclosed herein are provided as shown in SEQ ID NOs: 83, 87, 91, 95, 99 and 103.

[0205] 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.

[0206] CDR

[0207] 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), wherein X1 is S or T; X2 is G or A; and X3 is S or H; CDR-H2 comprises the sequence X1IX2X3X4X5X6X7X8YYADX9VKG (SEQ ID NO: NO:74), wherein X1 is V or A; X2 is Y, S or R; X3 is Y or M; X4 is D or T; X5 is G or V; 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), wherein X1 is E or N; X2 is P or Y; X3 is P or S; and X4 is F or W; the sequence ADMHRFDV (SEQ ID NO:45), the sequence VWGGVRFDV (SEQ ID NO:55), or the sequence YKPMPFDV (SEQ ID NO:25 or SEQ ID NO:35); CDR-L1 comprises the sequence RASQX1IX2X3YLN (SEQ ID NO: NO:76), wherein X1 is D or S; X2 is I or S; and X3 is S, T, I or R; CDR-L2 comprises the sequence X1ASX2LQS (SEQ ID NO:77), wherein X1 is V, G, E or A; and X2 is S or I; and CDR-L3 comprises the sequence X1QX2X3X4X5PX6X7 (SEQ ID NO:78), wherein X1 is Q or G; X2 is S or A; X3 is Y, S, D or H; 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.

[0208] 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).

[0209] In some embodiments, an antibody provided herein comprises CDR-H3 of SEQ ID NO:5, CDR-H2 of SEQ ID NO:4, CDR-H1 of SEQ ID NO:3, CDR-L3 of SEQ ID NO:10, CDR-L2 of SEQ ID NO:9, and CDR-L1 of SEQ ID NO:8. In some embodiments, CDR-H3 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H3 of SEQ ID NO:5, CDR-H2 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H2 of SEQ ID NO:4, CDR-H1 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H1 of SEQ ID NO:3, CDR-L3 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-L3 of SEQ ID NO:10, CDR-L2 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-L2 of SEQ ID NO:9, and CDR-L1 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H1 of SEQ ID NO:3. The CDR-L1 of NO:8 is at least about 50%, 75%, 80%, 85%, 90% or 95% identical. In some embodiments, CDR-H3 is CDR-H3 of SEQ ID NO: 5 having at most 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H2 is CDR-H2 of SEQ ID NO: 4 having at most 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H1 is CDR-H1 of SEQ ID NO: 3 having at most 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L3 is CDR-L3 of SEQ ID NO: 10 having at most 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L2 is CDR-L2 of SEQ ID NO: 9 having at most 1, 2, 3, or 4 amino acid substitutions; and CDR-L1 is CDR-L1 of SEQ ID NO: 8 having at most 1, 2, 3, 4, 5, or 6 amino acid substitutions.

[0210] In some embodiments, an antibody provided herein comprises CDR-H3 of SEQ ID NO: 15, CDR-H2 of SEQ ID NO: 14, CDR-H1 of SEQ ID NO: 13, CDR-L3 of SEQ ID NO: 20, CDR-L2 of SEQ ID NO: 19, and CDR-L1 of SEQ ID NO: 18. In some embodiments, CDR-H3 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H3 of SEQ ID NO: 15, CDR-H2 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H2 of SEQ ID NO: 14, CDR-H1 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H1 of SEQ ID NO: 13, CDR-L3 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-L3 of SEQ ID NO: 20, and CDR-L2 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H2 of SEQ ID NO: 14. The CDR-L2 of NO:19 is at least about 50%, 75%, 80%, 85%, 90% or 95% identical, and the CDR-L1 is at least about 50%, 75%, 80%, 85%, 90% or 95% identical to the CDR-L1 of SEQ ID NO:18. In some embodiments, CDR-H3 is CDR-H3 of SEQ ID NO: 15 having at most 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H2 is CDR-H2 of SEQ ID NO: 14 having at most 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H1 is CDR-H1 of SEQ ID NO: 13 having at most 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L3 is CDR-L3 of SEQ ID NO: 20 having at most 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L2 is CDR-L2 of SEQ ID NO: 19 having at most 1, 2, 3, or 4 amino acid substitutions; and CDR-L1 is CDR-L1 of SEQ ID NO: 18 having at most 1, 2, 3, 4, 5, or 6 amino acid substitutions.

[0211] In some embodiments, an antibody provided herein comprises 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, CDR-H3 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H3 of SEQ ID NO: 25, CDR-H2 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H2 of SEQ ID NO: 24, CDR-H1 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H1 of SEQ ID NO: 23, CDR-L3 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-L3 of SEQ ID NO: 30, and CDR-L2 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H2 of SEQ ID NO: 24. The CDR-L2 of NO:29 is at least about 50%, 75%, 80%, 85%, 90% or 95% identical, and the CDR-L1 is at least about 50%, 75%, 80%, 85%, 90% or 95% identical to the CDR-L1 of SEQ ID NO:28. In some embodiments, CDR-H3 is CDR-H3 of SEQ ID NO: 25 having at most 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H2 is CDR-H2 of SEQ ID NO: 24 having at most 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H1 is CDR-H1 of SEQ ID NO: 23 having at most 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L3 is CDR-L3 of SEQ ID NO: 30 having at most 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L2 is CDR-L2 of SEQ ID NO: 29 having at most 1, 2, 3, or 4 amino acid substitutions; and CDR-L1 is CDR-L1 of SEQ ID NO: 28 having at most 1, 2, 3, 4, 5, or 6 amino acid substitutions.

[0212] In some embodiments, an antibody provided herein comprises 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, CDR-H3 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H3 of SEQ ID NO: 35, CDR-H2 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H2 of SEQ ID NO: 34, CDR-H1 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H1 of SEQ ID NO: 33, CDR-L3 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-L3 of SEQ ID NO: 40, and CDR-L2 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H2 of SEQ ID NO: 34. The CDR-L2 of NO:39 is at least about 50%, 75%, 80%, 85%, 90% or 95% identical, and the CDR-L1 is at least about 50%, 75%, 80%, 85%, 90% or 95% identical to the CDR-L1 of SEQ ID NO:38. In some embodiments, CDR-H3 is CDR-H3 of SEQ ID NO: 35 having at most 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H2 is CDR-H2 of SEQ ID NO: 34 having at most 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H1 is CDR-H1 of SEQ ID NO: 33 having at most 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L3 is CDR-L3 of SEQ ID NO: 40 having at most 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L2 is CDR-L2 of SEQ ID NO: 39 having at most 1, 2, 3, or 4 amino acid substitutions; and CDR-L1 is CDR-L1 of SEQ ID NO: 38 having at most 1, 2, 3, 4, 5, or 6 amino acid substitutions.

[0213] In some embodiments, an antibody provided herein comprises CDR-H3 of SEQ ID NO:45, CDR-H2 of SEQ ID NO:44, CDR-H1 of SEQ ID NO:43, CDR-L3 of SEQ ID NO:50, CDR-L2 of SEQ ID NO:49, and CDR-L1 of SEQ ID NO:48. In some embodiments, CDR-H3 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H3 of SEQ ID NO:45, CDR-H2 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H2 of SEQ ID NO:44, CDR-H1 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H1 of SEQ ID NO:43, CDR-L3 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-L3 of SEQ ID NO:50, and CDR-L2 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H2 of SEQ ID NO:44. The CDR-L2 of NO:49 is at least about 50%, 75%, 80%, 85%, 90% or 95% identical, and the CDR-L1 is at least about 50%, 75%, 80%, 85%, 90% or 95% identical to the CDR-L1 of SEQ ID NO:48. In some embodiments, CDR-H3 is CDR-H3 of SEQ ID NO:45 having at most 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H2 is CDR-H2 of SEQ ID NO:44 having at most 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H1 is CDR-H1 of SEQ ID NO:43 having at most 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L3 is CDR-L3 of SEQ ID NO:50 having at most 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L2 is CDR-L2 of SEQ ID NO:49 having at most 1, 2, 3, or 4 amino acid substitutions; and CDR-L1 is CDR-L1 of SEQ ID NO:48 having at most 1, 2, 3, 4, 5, or 6 amino acid substitutions.

[0214] In some embodiments, an antibody provided herein comprises 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, CDR-H3 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H3 of SEQ ID NO: 55, CDR-H2 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H2 of SEQ ID NO: 54, CDR-H1 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H1 of SEQ ID NO: 53, CDR-L3 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-L3 of SEQ ID NO: 60, and CDR-L2 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to CDR-H2 of SEQ ID NO: 54. The CDR-L2 of NO:59 is at least about 50%, 75%, 80%, 85%, 90% or 95% identical, and the CDR-L1 is at least about 50%, 75%, 80%, 85%, 90% or 95% identical to the CDR-L1 of SEQ ID NO:58. In some embodiments, CDR-H3 is CDR-H3 of SEQ ID NO: 55 having at most 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H2 is CDR-H2 of SEQ ID NO: 54 having at most 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H1 is CDR-H1 of SEQ ID NO: 53 having at most 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L3 is CDR-L3 of SEQ ID NO: 60 having at most 1, 2, 3, 4, or 5 amino acid substitutions; CDR-L2 is CDR-L2 of SEQ ID NO: 59 having at most 1, 2, 3, or 4 amino acid substitutions; and CDR-L1 is CDR-L1 of SEQ ID NO: 58 having at most 1, 2, 3, 4, 5, or 6 amino acid substitutions.

[0215] In some aspects, 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 obtained from the sequences provided herein, for example, by 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 obtained from the sequences provided herein, and can be isolated again, for example, according to the methods provided herein for obtaining antibodies.

[0216] In some embodiments, the antibodies provided herein comprise a CDR-H3 of SEQ ID NO: 5, 15, 25, 35, 45, or 55. In some aspects, the CDR-H3 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to a CDR-H3 of SEQ ID NO: 5, 15, 25, 35, 45, or 55. In some embodiments, the CDR-H3 is a CDR-H3 of SEQ ID NO: 5, 15, 25, 35, 45, or 55 with at most 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

[0217] 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 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical 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 of SEQ ID NO: 4, 14, 24, 34, 44, or 54 with at most 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

[0218] 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 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to a 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 at most 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

[0219] 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 is at least about 50%, 75%, 80%, 85%, 90% or 95% identical to CDR-H3 of SEQ ID NO: 5, 15, 25, 35, 45 or 55; CDR-H2 is at least about 50%, 75%, 80%, 85%, 90% or 95% identical to CDR-H2 of SEQ ID NO: 4, 14, 24, 34, 44 or 54; and CDR-H1 is at least about 50%, 75%, 80%, 85%, 90% or 95% identical to CDR-H1 of SEQ ID NO: 3, 13, 23, 33, 43 or 53. In some embodiments, CDR-H3 is CDR-H3 of SEQ ID NO: 5, 15, 25, 35, 45, or 55 having at most 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; CDR-H2 is CDR-H2 of SEQ ID NO: 34, 14, 24, 34, 44, or 54 having at most 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions; and CDR-H1 is CDR-H1 of SEQ ID NO: 3, 13, 23, 33, 43, or 53 having at most 1, 2, 3, 4, or 5 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

[0220] 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 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical 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 at most 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

[0221] 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 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical 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 at most 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

[0222] 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 is at least about 50%, 75%, 80%, 85%, 90%, or 95% identical to the CDR-L1 of SEQ ID NO: 8, 18, 28, 38, 48, or 58. In some embodiments, the CDR-L1 is the CDR-L1 of SEQ ID NO: 8, 18, 28, 38, 48, or 58 with at most 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions.

[0223] 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, the CDR-L3 is at least about 50%, 75%, 80%, 85%, 90% or 95% identical to the CDR-L3 of SEQ ID NO: 10, 20, 30, 40, 50 or 60; the CDR-L2 is at least about 50%, 75%, 80%, 85%, 90% or 95% identical to the CDR-L2 of SEQ ID NO: 9, 19, 29, 39, 49 or 59; and the CDR-L1 is at least about 50%, 75%, 80%, 85%, 90% or 95% identical to the 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 having at most 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 having at most 1, 2, 3, 4, 5, or 6 amino acid substitutions; and CDR-L1 is CDR-L1 of SEQ ID NO: 8, 18, 28, 38, 48, or 58 having at most 1, 2, 3, 4, 5, or 6 amino acid substitutions. In some aspects, the amino acid substitutions are conservative amino acid substitutions.

[0224] In some embodiments, the antibodies provided herein comprise a V sequence selected from SEQ ID NO: 2, 12, 22, 32, 42, or 52. H In some embodiments, the antibodies provided herein comprise a V sequence selected from SEQ ID NO: 2, 12, 22, 32, 42, or 52. HIn 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.

[0225] In some embodiments, CDR-H1 is a CDR-H1 of a VH domain selected from SEQ ID NO: 2, 12, 22, 32, 42 or 52, having at most 1, 2, 3, 4 or 5 amino acid substitutions. In some embodiments, CDR-H2 is a CDR-H2 of a VH domain selected from SEQ ID NO: 2, 12, 22, 32, 42 or 52, having at most 1, 2, 3, 4, 5, 6, 7 or 8 amino acid substitutions. In some embodiments, CDR-H3 is a CDR-H3 of a VH domain selected from SEQ ID NO: 2, 12, 22, 32, 42 or 52, having at most 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 obtained from sequences provided herein, for example, by 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 obtained from sequences provided herein, and can be isolated again, for example, according to the methods provided herein for obtaining antibodies.

[0226] 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.

[0227] In some embodiments, CDR-L1 is a CDR-L1 of a VL domain selected from SEQ ID NO: 7, 17, 27, 37, 47 or 57, having at most 1, 2, 3, 4 or 5 amino acid substitutions. In some embodiments, CDR-L2 is a CDR-L2 of a VL domain selected from SEQ ID NO: 7, 17, 27, 37, 47 or 57, having at most 1, 2, 3, 4, 5, 6 or 7 amino acid substitutions. In some embodiments, CDR-L3 is a CDR-L3 of a VL domain selected from SEQ ID NO: 7, 17, 27, 37, 47 or 57, having at most 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 obtained from sequences provided herein, for example, by 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 obtained from sequences provided herein, and can be isolated again, for example, according to the methods provided herein for obtaining antibodies.

[0228] 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 aspects, the CDRs are Chothia CDRs. In some aspects, the CDR is an AbM CDR. In some aspects, the CDR is a Contact CDR. In some aspects, the CDR is an IMGT CDR.

[0229] V H domain

[0230] In some embodiments, the antibodies provided herein comprise a V sequence selected from SEQ ID NO: 2, 12, 22, 32, 42, or 52. HIn some embodiments, the antibodies provided herein comprise V of SEQ ID NO: 2 H In some embodiments, the antibodies provided herein comprise V sequences of SEQ ID NO: 12. H In some embodiments, the antibodies provided herein comprise V sequences of SEQ ID NO: 22. H In some embodiments, the antibodies provided herein comprise V sequences of SEQ ID NO: 32. H In some embodiments, the antibodies provided herein comprise V sequences of SEQ ID NO: 42. H In some embodiments, the antibodies provided herein comprise V sequences of SEQ ID NO: 52. H sequence.

[0231] In some embodiments, V H The sequence is at least 70%, 80%, or 90% identical to SEQ ID NO: 2, wherein any variation from SEQ ID NO: 2 does not occur within CDR-H1, CDR-H2, or CDR-H3. H The sequence is at least 70%, 80%, or 90% identical to SEQ ID NO: 12, wherein any variation from SEQ ID NO: 12 does not occur within CDR-H1, CDR-H2, or CDR-H3. H The sequence is at least 70%, 80%, or 90% identical to SEQ ID NO: 22, wherein any variation from SEQ ID NO: 22 does not occur within CDR-H1, CDR-H2, or CDR-H3. H The sequence is at least 70%, 80%, or 90% identical to SEQ ID NO: 32, wherein any variation from SEQ ID NO: 32 does not occur within CDR-H1, CDR-H2, or CDR-H3. H The sequence is at least 70%, 80%, or 90% identical to SEQ ID NO: 42, wherein any variation from SEQ ID NO: 42 does not occur within CDR-H1, CDR-H2, or CDR-H3. H The sequence is at least 70%, 80% or 90% identical to SEQ ID NO: 52, wherein any variation from SEQ ID NO: 52 does not occur within CDR-H1, CDR-H2 or CDR-H3.

[0232] In some embodiments, the antibodies provided herein comprise a V sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 2. H In some embodiments, the antibodies provided herein comprise a V sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 12. H In some embodiments, the antibodies provided herein comprise a V sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 22. H In some embodiments, the antibodies provided herein comprise a V sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 32. H In some embodiments, the antibodies provided herein comprise a V sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 42. H In some embodiments, the antibodies provided herein comprise a V sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 52. H sequence.

[0233] In some embodiments, the antibodies provided herein comprise an illustrative V sequence as provided in SEQ ID NO: 2, 12, 22, 32, 42, or 52. H The sequences are at least about 50%, 60%, 70%, 80%, 90%, 95% or 99% identical to each other. H In some embodiments, the antibodies provided herein comprise a V sequence provided in SEQ ID NO: 2, 12, 22, 32, 42, or 52. H Sequence, having at most 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, 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 obtained by affinity maturation, site-directed mutagenesis, random mutagenesis or any other method known in the art or described herein from sequences provided herein. In some embodiments, such variants are not obtained by sequences provided herein, and can be isolated again, for example, according to the method for obtaining antibodies provided herein.

[0234] V L domain

[0235] In some embodiments, the antibodies provided herein comprise a V sequence selected from SEQ ID NO: 7, 17, 27, 37, 47, or 57. L In some embodiments, the antibodies provided herein comprise V sequences of SEQ ID NO: 7. L In some embodiments, the antibodies provided herein comprise V sequences of SEQ ID NO: 17. L In some embodiments, the antibodies provided herein comprise V sequences of SEQ ID NO: 27. L In some embodiments, the antibodies provided herein comprise V sequences of SEQ ID NO: 37. L In some embodiments, the antibodies provided herein comprise a V sequence of SEQ ID NO: 47. L In some embodiments, the antibodies provided herein comprise a V sequence of SEQ ID NO: 57. L sequence.

[0236] In some embodiments, V L The sequence is at least 70%, 80%, or 90% identical to SEQ ID NO: 7, wherein any variation from SEQ ID NO: 7 does not occur within CDR-L1, CDR-L2, or CDR-L3. L The sequence is at least 70%, 80%, or 90% identical to SEQ ID NO: 17, wherein any variation from SEQ ID NO: 17 does not occur within CDR-L1, CDR-L2, or CDR-L3. L The sequence is at least 70%, 80%, or 90% identical to SEQ ID NO: 27, wherein any variation from SEQ ID NO: 27 does not occur within CDR-L1, CDR-L2, or CDR-L3. L The sequence is at least 70%, 80%, or 90% identical to SEQ ID NO: 37, wherein any variation from SEQ ID NO: 37 does not occur within CDR-L1, CDR-L2, or CDR-L3. L The sequence is at least 70%, 80%, or 90% identical to SEQ ID NO: 47, wherein any variation from SEQ ID NO: 47 does not occur within CDR-L1, CDR-L2, or CDR-L3. LThe sequence is at least 70%, 80% or 90% identical to SEQ ID NO: 57, wherein any variation from SEQ ID NO: 57 does not occur within CDR-L1, CDR-L2 or CDR-L3.

[0237] In some embodiments, the antibodies provided herein comprise a V sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 7. L In some embodiments, the antibodies provided herein comprise a V sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 17. L In some embodiments, the antibodies provided herein comprise a V sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 27. L In some embodiments, the antibodies provided herein comprise a V sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 37. L In some embodiments, the antibodies provided herein comprise a V sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 47. L In some embodiments, the antibodies provided herein comprise a V sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 57. L sequence.

[0238] In some embodiments, the antibodies provided herein comprise an illustrative V sequence as provided in SEQ ID NO: 7, 17, 27, 37, 47, or 57. L The sequences are at least about 50%, 60%, 70%, 80%, 90%, 95% or 99% identical to each other. L In some embodiments, the antibodies provided herein comprise a V sequence provided in SEQ ID NO: 7, 17, 27, 37, 47, or 57. LSequence, having at most 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, 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 obtained by affinity maturation, site-directed mutagenesis, random mutagenesis or any other method known in the art or described herein from sequences provided herein. In some embodiments, such variants are not obtained by sequences provided herein, and can be isolated again, for example, according to the method for obtaining antibodies provided herein.

[0239] V H -V L combination

[0240] In some embodiments, the antibodies provided herein comprise a V sequence selected from SEQ ID NO: 2, 12, 22, 32, 42, or 52. H Sequence; and V selected from SEQ ID NO: 7, 17, 27, 37, 47 or 57 L sequence.

[0241] In some embodiments, the antibodies provided herein comprise V of SEQ ID NO: 2 H Sequence and SEQ ID NO: 7 V L In some embodiments, the antibodies provided herein comprise V sequences of SEQ ID NO: 12. H Sequence and SEQ ID NO: 17 V L In some embodiments, the antibodies provided herein comprise V sequences of SEQ ID NO: 22. H Sequence and V of SEQ ID NO: 27 L In some embodiments, the antibodies provided herein comprise V sequences of SEQ ID NO: 32. H Sequence and SEQ ID NO: 37 V L In some embodiments, the antibodies provided herein comprise V sequences of SEQ ID NO: 42. H Sequence and SEQ ID NO: 47 V L In some embodiments, the antibodies provided herein comprise V sequences of SEQ ID NO: 52. H Sequence and SEQ ID NO: 57 V L sequence.

[0242] In some embodiments, V HThe sequence is at least 70%, 80%, or 90% identical to SEQ ID NO: 2, and wherein the variable region of the light chain is at least 70%, 80%, or 90% identical to SEQ ID NO: 7, wherein any variation from SEQ ID NO: 2 does not occur within CDR-H1, CDR-H2, or CDR-H3, and wherein any variation from SEQ ID NO: 7 does not occur within CDR-L1, CDR-L2, or CDR-L3. In some embodiments, V H The sequence is at least 70%, 80%, or 90% identical to SEQ ID NO: 12, and wherein the variable region of the light chain is at least 70%, 80%, or 90% identical to SEQ ID NO: 17, wherein any variation from SEQ ID NO: 12 does not occur within CDR-H1, CDR-H2, or CDR-H3, and wherein any variation from SEQ ID NO: 17 does not occur within CDR-L1, CDR-L2, or CDR-L3. In some embodiments, V H The sequence is at least 70%, 80%, or 90% identical to SEQ ID NO: 22, and wherein the variable region of the light chain is at least 70%, 80%, or 90% identical to SEQ ID NO: 27, wherein any variation from SEQ ID NO: 22 does not occur within CDR-H1, CDR-H2, or CDR-H3, and wherein any variation from SEQ ID NO: 27 does not occur within CDR-L1, CDR-L2, or CDR-L3. In some embodiments, V H The sequence is at least 70%, 80%, or 90% identical to SEQ ID NO: 32, and wherein the variable region of the light chain is at least 70%, 80%, or 90% identical to SEQ ID NO: 37, wherein any variation from SEQ ID NO: 32 does not occur within CDR-H1, CDR-H2, or CDR-H3, and wherein any variation from SEQ ID NO: 37 does not occur within CDR-L1, CDR-L2, or CDR-L3. In some embodiments, V H The sequence is at least 70%, 80%, or 90% identical to SEQ ID NO: 42, and wherein the variable region of the light chain is at least 70%, 80%, or 90% identical to SEQ ID NO: 47, wherein any variation from SEQ ID NO: 42 does not occur within CDR-H1, CDR-H2, or CDR-H3, and wherein any variation from SEQ ID NO: 47 does not occur within CDR-L1, CDR-L2, or CDR-L3. In some embodiments, V HThe sequence is at least 70%, 80% or 90% identical to SEQ ID NO:52, and wherein the variable region of the light chain is at least 70%, 80% or 90% identical to SEQ ID NO:57, wherein any variation from SEQ ID NO:52 does not occur within CDR-H1, CDR-H2 or CDR-H3, and wherein any variation from SEQ ID NO:57 does not occur within CDR-L1, CDR-L2 or CDR-L3.

[0243] In certain aspects, 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.

[0244] In some embodiments, the antibodies provided herein comprise an illustrative V sequence as provided in SEQ ID NO: 2, 12, 22, 32, 42, or 52. H The sequences are at least about 50%, 60%, 70%, 80%, 90%, 95% or 99% identical to each other. H Sequences; and VL sequences having at least about 50%, 60%, 70%, 80%, 90%, 95% or 99% identity to the illustrative VL sequences provided in SEQ ID NO: 7, 17, 27, 37, 47 or 57. LSequence. In some embodiments, the antibodies provided herein comprise a VH sequence provided in SEQ ID NO: 2, 12, 22, 32, 42, or 52, having at most 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 VL sequence provided in SEQ ID NO: 7, 17, 27, 37, 47, or 57, having at most 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 obtained from sequences provided herein, for example, by 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 obtained from sequences provided herein, and can be isolated again, for example, according to the methods provided herein for obtaining antibodies.

[0245] In some embodiments, the percent homology of the variable heavy chain or variable light chain will be calculated outside of the CDRs. For example, the percent homology in the framework regions can be calculated.

[0246] In some embodiments, the antibody comprises a heavy chain provided in SEQ ID NO: 1, 11, 21, 31, 41, or 51.

[0247] In some embodiments, the antibody comprises a light chain provided in SEQ ID NO: 6, 16, 26, 36, 46, or 56.

[0248] In certain aspects, 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.

[0249] In some embodiments, the antibodies provided herein comprise a heavy chain sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95% or 99% identical to an illustrative heavy chain sequence provided in SEQ ID NO: 1, 11, 21, 31, 41 or 51; and a light chain sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95% or 99% identical to an illustrative light chain sequence provided in SEQ ID NO: 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, having at most 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, having at most 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.

[0250] Fc region

[0251] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains 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 the 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 Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, an "Fc polypeptide" of a dimeric Fc refers to one of the two polypeptides that form the dimeric Fc domain, i.e., a polypeptide that comprises the C-terminal constant region of an immunoglobulin heavy chain that is capable of stable self-association. For example, the Fc polypeptide of a dimeric IgG Fc comprises IgG CH2 and IgG CH3 constant domain sequences. Fc can have classes IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), eg, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.

[0252] The terms "Fc receptor" and "FcR" are used to describe receptors that bind to the Fc region of an antibody. For example, an FcR may be a native sequence human FcR. Typically, an FcR is an FcR (gamma receptor) that binds to an IgG antibody and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Other isotypes of immunoglobulins may also be bound by certain FcRs (see, for example, Janeway et al., Immuno Biology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th edition, 1999)). The 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 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 responsible for 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)).

[0253] In some embodiments, the antibody is an IgG1 antibody.

[0254] Modifications in the CH2 domain can affect the binding of FcR to Fc. Many amino acid modifications in the Fc region are known in the art for selectively altering the affinity of Fc for different Fc gamma (Fcγ) receptors. In one embodiment, Fc comprises one or more modifications that promote selective binding to Fcγ receptors.

[0255] In some embodiments, an antibody described herein comprises an Fc region comprising an L234A / L235A mutation according to the EU numbering system.

[0256] In some embodiments, the antibodies are monoclonal antibodies. In some embodiments, the antibodies are produced by hybridomas. In other embodiments, the antibodies are produced by recombinant cells engineered to express the desired variable and constant domains. In some embodiments, the antibodies are specific for surface antigens, such as the PAPP-A protein. In specific embodiments, the therapeutic antibodies may have a human or non-human primate IgG1 Fc portion.

[0257] Combine

[0258] With respect to the binding of an antibody to a target molecule, the terms "bind to" a specific antigen (e.g., a polypeptide target) or an epitope on a specific antigen, "specifically binds to" a specific antigen (e.g., a polypeptide target) or an epitope on a specific antigen, "specifically binds to" a specific antigen (e.g., a polypeptide target) or an epitope on a specific antigen, "specific for a specific antigen (e.g., a polypeptide target) or an epitope on a specific antigen, "selectively binds to" a specific antigen (e.g., a polypeptide target) or an epitope on a specific antigen, and "selective for a specific antigen (e.g., a polypeptide target) or an epitope on a specific antigen" mean that the binding differs to a measurable degree from non-specific or non-selective interactions (e.g., interactions with non-target molecules). Specific binding can be measured, for example, by measuring binding to a 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 such cases, if the binding of the antibody to the target molecule is competitively inhibited by the control molecule, specific binding is indicated. Cross-linking of the antigen target is one type of binding. In some embodiments, the anti-PAPP-A antibody cross-links PAPP-A to PAPP-A on PAPP-A+ cells.

[0259] "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 indicated, as used herein, "affinity" refers to the intrinsic binding affinity that 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 can be expressed by the dissociation equilibrium constant (K D ). The kinetic components 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) techniques (e.g. ) or biolayer interferometry (e.g. ).

[0260] As used herein, the term " d ”(s -1) refers to the dissociation rate constant for a specific antibody-antigen interaction. This value is also called k 解离 value.

[0261] As used herein, the term " a ”(M -1 ×s -1 ) refers to the association rate constant for a specific antibody-antigen interaction. This value is also known as k 缔合 value.

[0262] As used herein, the term "K D "(M) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. K D =k d / k a In some embodiments, the affinity of an antibody is expressed as the K for the interaction between the antibody and its antigen. D For clarity, as is known in the art, the smaller K D A value indicates a higher affinity interaction, while a larger K D Values ​​indicate lower affinity interactions.

[0263] As used herein, the term "K A ”(M -1 ) refers to the association equilibrium constant for a specific antibody-antigen interaction. K A =k a / k d .

[0264] 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 rhesus macaque PAPP-A. In some embodiments, the antibodies provided herein bind to cynomolgus macaque PAPP-A. In some embodiments, the antibodies provided herein bind to human, rhesus macaque, and / or cynomolgus macaque PAPP-A.

[0265] In some embodiments, the antibodies provided herein have an expression of less than or equal to 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 -12 M's K D Binds to human PAPP-A. In some embodiments, the K of an antibody provided herein is as measured by surface plasmon resonance assay. Dat about 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 90-100x10 -12 Between M.

[0266] In some embodiments, the antibodies provided herein have a surface plasmon resonance (SPR) activity of less than or equal to 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 -12 M or smaller K D In some embodiments, the antibodies provided herein bind to human PAPP-A at 2.5-2.3, 2.5-2.0, 2.0-1.9, 1.9-1.8, 1.8-1.7, 1.7-1.6, 1.6-1.5, or 1.9-1.5 x 10 -12 K between M D Binds to human PAPP-A.

[0267] In some embodiments, the antibodies provided herein inhibit PAPP-A proteolytic activity with an IC50 of less than or equal to 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 as measured by flow cytometry or western blot. In some embodiments, the antibodies provided herein inhibit PAPP-A proteolytic activity against IGFBP-2, IGFBP-4 or IGFBP-5.

[0268] To screen for antibodies that bind to an epitope on the target antigen (e.g., PAPP-A) that is bound by the antibody of interest, a conventional cross-blocking assay, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, ed. Harlow and David Lane (1988), can be performed. Alternatively or additionally, epitope mapping can be performed by methods known in the art.

[0269] Function

[0270] In some embodiments, the antibody is an antagonist antibody. The antagonist antibody can block (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 can bind to the PAPP-A enzyme and block the binding of the PAPP-A enzyme to its substrate, thereby preventing the cleavage of the substrate.

[0271] method

[0272] Methods for treating PAPP-A-related conditions

[0273] In another aspect, provided herein are methods of treating a PAPP-A-associated disorder comprising administering to a subject an effective amount of 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).

[0274] In some embodiments, the kidney disease is polycystic kidney disease or autosomal dominant polycystic kidney disease (ADPKD).

[0275] In one embodiment, the subject is a human.

[0276] Pharmaceutical composition

[0277] The present disclosure also encompasses methods for treating PAPP-A-related disorders. The methods comprise administering a therapeutically effective amount of an anti-PAPP-A antibody or antigen-binding fragment. The PAPP-A antibody or antigen-binding fragment can be formulated in a pharmaceutical composition or as a medicament.

[0278] Medicine boxes and products

[0279] The present application provides a kit comprising any one or more of the antibody compositions described herein. In some embodiments, the kit further comprises a component selected from any one of a secondary antibody, reagents for immunohistochemical analysis, a pharmaceutically acceptable excipient, and an instruction manual, 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 and one or more pharmaceutically acceptable excipients.

[0280] The present application also provides an article of manufacture comprising any of the antibody compositions or kits described herein. Examples of articles of manufacture include vials.

[0281] Example

[0282] The following are examples of specific embodiments of the present disclosure. The examples are provided 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 the values ​​used (e.g., amounts, temperatures, etc.), but of course some experimental errors and deviations should be allowed for.

[0283] Unless otherwise indicated, the implementation of the present disclosure will adopt conventional protein chemistry, biochemistry, recombinant DNA technology and pharmacological methods belonging to the skills of this area. Such technology is fully described in the literature. See, for example, T. Creighton, Proteins: Structures and Molecular Properties (W. H. Freeman and Company, 1993); A. Lehninger, Biochemistry (Worth Publishers, Inc., latest edition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan, Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd edition. (Plenum Press) Volumes A and B (1992).

[0284] Example 1: Identification of anti-PAPP-A monoclonal antibodies

[0285] Yeast display technology is used to identify antibodies that specifically bind to PAPP-A. The purpose of this work is to generate fully human antibodies that bind to and neutralize human and cynomolgus monkey (NHP) PAPP-A with high affinity and efficacy. In brief, six human synthetic scFv (single-chain variable fragment) antibody libraries were selected as binders to PAPP-A. Each library consists of a single human VH germline, a highly diverse HCDR3 fragment with a size range of 7 to 18 amino acids, and a diverse mixture of VK germlines. Using biotinylated PAPP-A protein as a target, scFv fragments were selected using magnetic and fluorescence-activated cell sorting techniques. The output was selected by sequencing analysis and analyzed using GeneData (GeneData, Lexington, MA, USA) for classification. Non-redundant clones were converted to IgG, expressed in EXPI293 (HEK293) cells (Gibco / Fisher) and screened for binding to PAPP-A. A total of 111 clones expressing IgG showed specific binding to human PAPP-A.

[0286] Example 2. Inhibition of PAPP-A proteolytic activity by monoclonal antibodies

[0287] By inhibiting the proteolytic activity of PAPP-A, IGF bioavailability and downstream signaling can be modulated. A graphical representation of the assays used to inhibit PAPP-A proteolytic activity, neutralize PAPP-A activity, is shown in Figure 1 middle.

[0288] Protein expression

[0289] Full-length human, NHP, and mouse PAPP-A proteins were expressed in stably transduced HEK293 cell lines 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-agarose column chromatography. Similarly, human IGFBP-2 and human IGFBP-5 proteins were expressed and purified with 6His tags and C-terminal Flag tags. Before initiating studies, full-length PAPP-A protein activity was confirmed in preliminary assays.

[0290] Enzymatic cleavage experiment

[0291] For the enzymatic cleavage reaction, IGFBP-2 and IGFBP-4 proteins were mixed with human IGF-1 (Bio- / R&D Systems, Minneapolis, MN, USA) were preincubated for 30 minutes at 37°C in enzymatic assay buffer consisting of Dulbecco's Modified Eagle's Medium (DMEM; ThermoFisher / Gibco, Waltham, MA, USA) with 1% bovine serum albumin (ThermoFisher / Invitrogen). The IGFBP / IGF-1 protein was then mixed with PAPP-A in enzymatic assay buffer and incubated at 37°C for two to four hours.

[0292] For enzymatic cleavage reactions with IGFBP-5, the proteins were mixed directly with PAPP-A without preincubation with IGF-1, as the addition of IGF-1 inhibits the proteolytic activity of PAPP-A against IGFBP-5. The final concentrations in the IGFBP-4 cleavage reaction were 90 nM for IGFBP-4, 566 nM for IGF-1, and 0.5 nM for PAPP-A. The final concentrations in the IGFBP-5 cleavage reaction were 80 nM for IGFBP-5 and 0.05 nM for PAPP-A. The final concentrations in the IGFBP-2 cleavage reaction were 80 nM for IGFBP-2, 566 nM for IGF-1, and 5 nM for PAPP-A.

[0293] Dilutions of the monoclonal antibody were prepared from the stock solution in enzymatic assay buffer to a 1x working initial concentration of 30 μg / mL (200 nM) and an 8-point 3x dilution series was performed. Each antibody concentration was tested in triplicate. PAPP-A protein was preincubated with the dilution series of anti-PAPP-A antibody and then added to the IGFBP mixture.

[0294] Analysis of PAPP-A activity

[0295] By capillary electrophoresis in Wes TM Instruments (Bio- / ProteinSimple, Minneapolis, MN, USA) using a capillary box kit (Bio- Proteins were analyzed using the ProteinSimple software and probed with a polyclonal mouse anti-His tag antibody (GeneScript, Piscataway, NJ, USA) and an anti-mouse detection module (Bio- / ProteinSimple) visualization.

[0296] Compass for Simple Western software (Bio- The relative percentages of uncleaved and cleaved IGFBP bands were quantified using the PCR amplification assay (PCR amplification with 5% cleavage of the cleaved IGFBP bands) and the following equation was used to evaluate the inhibitory activity of each tested concentration of monoclonal antibody (S = IGFBP + PAPP-A + antibody) by normalizing to the control lane (A = IGFBP only, B = IGFBP + PAPP-A):

[0297]

[0298] 111 antibody clones expressed as IgG that bind to PAPP-A were screened for their ability to inhibit PAPP-A proteolytic activity. 101 clones completely or partially blocked the cleavage of IGFBP4, while 57 clones completely or partially blocked the cleavage of both IGFBP4 and IGFBP2. However, only a subset of 16 clones could 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 represented by 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 provides a means of selecting antibodies with improved substrate binding and better neutralizing activity. From each of the affinity-matured clones, two antibodies were obtained: Ab1 and Ab2 were produced by one clone, while the second clone produced Ab3 and Ab4, as screened below.

[0299] Since the amount of PAPP-A protein required to complete the cleavage of different IGFBPs varies, the IC values ​​of different inhibition assays are 50 The values ​​are not directly comparable but serve as a relative guide to inhibitory activity. However, for most Abs tested, the IC 50 The values ​​were in the sub-nM range (Table U). Compared to the other five antibodies, only Ab5 showed reduced proteolytic activity against IGFBP-4 and IGFBP-5.

[0300] Table U. Antibody-mediated inhibition of PAPP-A proteolytic activity towards substrates as determined by Western blot (IC 50 )

[0301] substrate Ab1 Ab2 Ab3 Ab4 Ab5 Ab6 IGFBP-2 0.37nM 0.34nM 0.26nM 0.31nM 0.11nM 0.18nM IGFBP-4 0.1nM 0.4nM 0.1nM 0.1nM 1.88nM 0.12nM IGFBP-5 0.07nM 0.04nM 0.18nM 0.31nM 3.02nM 0.09nM

[0302] Example 3: Determination of the substrate affinity of anti-PAPP-A antibodies for PAPP-A

[0303] To determine the substrate affinity of the tested anti-PAPP-A antibodies for PAPP-A, surface plasmon resonance (SPR) binding analysis was used. The substrate affinity (K) of the monoclonal antibodies for human, NHP, and mouse PAPP-A proteins was evaluated. D ).

[0304] Preparation of biosensor surfaces

[0305] Monoclonal antibodies were captured using a goat antibody specific for the Fc region of human IgG (Thermo Fisher Scientific, Waltham, MA, USA). Amine coupling kit (Cytiva) and Biacore TM The Fc-specific antibody was covalently immobilized on the Biacore ELISA via the amino group using the Immobilization Wizard option of the Cytiva instrument control software. TM CM5 biosensor chip (Cytiva Life Sciences, Marlborough, MA, USA) carboxymethyl dextran matrix. The carboxyl groups of the dextran matrix on the chip were activated with 100mM N-hydroxysuccinimide and 400mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Goat anti-human IgG Fc (25 μg / mL; ThermoFisher) diluted in 10mM sodium acetate (pH 4.5) was injected onto the activated surface. Once the binding reaction level reached the target value of 10,000 resonance units (RU), the unreacted groups were inactivated by injecting 1M 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 with a similarly conjugated goat anti-human IgG Fc antibody was used as a reference surface.

[0306] Binding of recombinant PAPP-A to immobilized anti-PAPP-A antibodies

[0307] Anti-PAPP-A monoclonal antibody was diluted to a concentration of 1 μg / mL in running buffer (HEPES-buffered saline; HBS-P+, Cytiva) plus 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 to 110 RU. The net difference between the baseline signal and the signal after the antibody injection is complete represents the amount of bound monoclonal antibody.

[0308] Each antigen binding experiment includes an antigen association and an antigen dissociation phase. Aliquots of recombinant PAPP-A protein were injected at various concentrations onto the captured monoclonal antibody and reference surface at a flow rate of 50 μL / min for five minutes to determine 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 consisted of a continuous flow of (HEPES-buffered saline; HBS-EP+, plus 0.1 mg / mL bovine serum albumin) buffer at 50 μL / min at various dissociation times (due to the slow dissociation rate, a dissociation time of 1 hour was allowed for the higher concentrations of 3.33, 10, and 30 nM PAPP-A; and a dissociation time of 5 minutes was allowed for the lower concentrations of 0.04, 0.12, 0.37, and 1.11 nM PAPP-A). The instrument response is measured in RU and is proportional to the mass of bound PAPP-A antigen.

[0309] Before the next sample was injected, the immobilized surface was regenerated with 10 mM glycine (pH 1.5) (two consecutive 25 μL injections at a flow rate of 50 μL / min). Each interaction between the monoclonal antibody and each of the PAPP-A antigens was run in triplicate. Finally, the reference surface response was subtracted from the reactive surface data to eliminate variations in refractive index and injection noise.

[0310] Determination of association and dissociation rates

[0311] Association rate constant (k a , unit M -1 s -1 ) is obtained by kinetic binding measurements at several antigen concentrations. The dissociation rate constant (k d , s -1 ) is determined by measuring the amount of antigen bound to the monoclonal antibody over time after the association phase is complete. The association rate constant and dissociation rate constant are calculated by the instrument evaluation software based on the values ​​extracted from the data using global fitting analysis, which allows for identical values ​​for each parameter in the data set, except for the R which is set locally due to variations in the antibody capture level. max To calculate the overall apparent dissociation constant (K) of the interaction between the mAb and PAPP-A species D ), the apparent dissociation rate constant (k d ) and the apparent association rate constant (k a ):K D =k d / k a .

[0312] Using these parameters, the substrate affinity between each antibody and recombinant PAPP-A from human, NHP, and mouse was calculated. When data are assigned as less than a specific amount, the data is below the resolution of the instrument. Of the six anti-PAPP-A monoclonal antibodies tested, Ab1 and Ab6 showed the most similar and greater affinities for human, NHP, and mouse PAPP-A compared to the other four antibodies (Table T).

[0313] Table T. Substrate affinity (K) of monoclonal antibodies to PAPP-A from three species as determined by surface plasmon resonance (SPR) binding analysis D )

[0314] species Ab1 Ab2 Ab3 Ab4 Ab5 Ab6 people <2.3pM <1.0pM <1.5pM 25pM 93pM <1.2pM NHP 17pM 24pM 7.2pM 35pM 460pM <1.0pM mice <2.9pM 91pM 120pM 860pM 180pM <3.0pM

[0315] Example 4. Antibody-mediated inhibition of AKT phosphorylation

[0316] To evaluate the effect of the monoclonal antibodies to inhibit PAPP-A cleavage of IGFBP-4, phosphorylated AKT in HEK293 cells was performed in an assay using human full-length PAPP-A protein.

[0317] Preparation of cells and anti-PAPP-A antibodies

[0318] HEK293 cells were plated overnight in serum-free Eagle's Minimum Essential Medium. The following day, the enzymatic reaction was established. Anti-PAPP-A monoclonal antibody dilutions were prepared from stock solutions in Dulbecco's phosphate-buffered saline (PBS) containing 0.015% bovine serum albumin to a working concentration of 0.375 μg / mL (2.5 nM). A nine-point 2.5x dilution series was performed. Antibody titrations for human PAPP-A were initiated at 1 nM. All antibody concentrations were tested in triplicate.

[0319] Phosphorylated AKT assay and analysis

[0320] Monoclonal antibodies were added to human PAPP-A and incubated at ambient temperature for 30 minutes. IGFBP-4 protein was mixed with IGF-1 and incubated at ambient temperature for 30 minutes. The antibody / PAPP-A mixture was added to the IGFBP-4 / IGF-1 mixture and incubated at 37°C for 5 hours. The final concentrations in the reaction were 1.05 nM for IGFBP-4, 4.2 nM for IGF-1, and 0.55 nM for human PAPP-A.

[0321] The IGF-1 / IGFBP-4 / PAPP-A / antibody mixture was added to serum-starved HEK293 cells and incubated for 20 minutes at 37° C. The culture medium was removed, and the cells were lysed in MSD Tris lysis buffer (Meso Scale Diagnostics, Rockville, MD, USA) and analyzed by the phospho (Ser473) / total Akt whole cell lysate kit (Meso Scale Diagnostics) according to the manufacturer's protocol.

[0322] The data in Table V below indicate the IC values ​​for each monoclonal antibody evaluated in this phosphorylation assay. 50 Analysis of the data indicated that all mAbs inhibited the release of bioactive IGF-1 by neutralizing the proteolytic activity of PAPP-A from IGFBP-4.

[0323] Table V. Antibody-mediated inhibition of IGF-1-induced AKT phosphorylation in HEK293 cells (IC 50 )

[0324]

[0325] Example 5. Nonspecific binding of anti-PAPP-A antibodies to HEK293 cells

[0326] Assessing nonspecific binding of anti-PAPP-A monoclonal antibodies is a routine part of preclinical evaluation, as nonspecific binding of antibodies can lead to adverse in vivo outcomes, ranging from adverse PK (pharmacokinetics) to toxicology findings.

[0327] Nonspecific binding of anti-PAPP-A antibodies to HEK293 cells

[0328] Human embryonic kidney (HEK293) cells that do not express PAPP-A were grown in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum at 37°C and 5% CO2, and the cells were plated at 1.0×10 6 Cells / mL were suspended in FACS buffer (Dulbecco's phosphate-buffered saline plus 10% fetal bovine serum), and 100 μL per well was aliquoted into 96-well round-bottom polypropylene plates ( Fisher Scientific, Waltham, MA, USA). After centrifugation and removal of the supernatant, 100 μg / mL of each anti-PAPP-A monoclonal antibody in FACS buffer was added at 100 μL / well, which was sufficient to resuspend the HEK293 cells. After incubation on ice for 30 minutes, the HEK293 cells were washed with FACS buffer to remove free antibodies. TMGoat anti-human IgG, Fcγ fragment-specific APC (allophycocyanin)-conjugated antibody (Jackson Immunoresearch, West Grove, PA, USA) was diluted to 2 μg / mL and 100 μL was added to each well. After incubation on ice for 30 minutes, HEK293 cells were washed with FACS buffer to remove free antibody.

[0329] Flow cytometric analysis of HEK293 cells

[0330] Using BD FACSCanto TM Flow cytometry of the prepared cells was performed using a flow cytometry system (Becton Dickinson, Franklin Lakes, NJ, USA). Live singlets were gated using the l / d discriminator and FSC(H) / FSC(A). When incubated in the presence of anti-PAPP-A antibody, an increase in the cell count distribution of the cell type was considered binding.

[0331] Three of the anti-PAPP-A antibodies, Ab4, Ab5, and Ab6, nonspecifically bound to HEK293 cells, while Ab1, Ab2, and Ab3 all nonspecifically bound to HEK293 cells (Table W). The reason for this interaction is unclear. However, the nonspecific binding of Ab4, Ab5, and Ab6 to HEK293 cells excluded these antibodies from further development.

[0332] Table W. Nonspecific binding of anti-PAPP-A monoclonal antibodies to HEK293 cells as assessed by flow cytometry

[0333] Cell type Ab1 Ab2 Ab3 Ab4 Ab5 Ab6 HEK293 - - - + + +

[0334] -: Similar distribution to isoform +: Binds at 100 μg / mL

[0335] Example 6. Antibody stability under thermal stress

[0336] In the accelerated stability study, antibodies Ab1, Ab2 and Ab3 were subjected to high temperatures over time at high concentrations. In order to assess accelerated stability, antibody samples were stored at 40°C. Samples of Ab1, Ab2 and Ab3 were dissolved in 15mM histidine (pH 6.0) at a concentration of up to 100mg / mL and stored at high temperatures for up to three weeks. The samples were subsequently stored at -80°C until analyzed by size exclusion chromatography (SEC). SEC was performed using an Agilent 1260 Infinity II HPLC system (Agilent Technologies, Palo Alto, CA, USA) equipped with a diode array UV detector. Data were analyzed using Agilent ChemStation software. The following chromatographic conditions were used for analysis: Column: Waters TM Acquity UPLC Protein BEH SEC Column ( 1.7 μm, 4.6 x 300 mm; Milford, MA, USA); operated at ambient temperature with a flow rate of 0.3 mL / min and an injection volume of 5 μL; mobile phase 100 mM disodium phosphate, 100 mM disodium sulfate, 1 mM sodium azide, pH 6.8; detection at 214 nm and a run time of 15 minutes.

[0337] The data of the antibody monomers analyzed are shown in Figure 2 Compared to Ab1 or Ab3, a greater decrease in the percentage of monomers was observed in the case of Ab2 after three weeks of incubation at 40°C. The reduced stability of Ab2 compared to Ab1 and Ab3 led to the decision not to select Ab2 as a clinical candidate.

[0338] Example 7. Trend Analysis

[0339] Forced degradation studies, including heat, acid, base, broad-spectrum UV-visible light stress, and chemical oxidation conditions, were performed to detect sequence trends. The forced degradation multi-attribute method LC-MS automated workflow monitors changes in peptide-level post-translational modifications and confirms trends in peptide-level post-translational modifications, indicating changes in critical quality attributes due to modifications such as deamidation, oxidation, isomerization, and other peptide-level resolved chemical modifications.

[0340] 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 one and three weeks and pH 9 stress for 7 days. TM Acquity BEH C18 column ( 1.7 μm, 2.1 mm x 150 mm) to separate peptides. A complex gradient of increasing acetonitrile (0% to 60%) was applied over a period of 28 minutes at 55°C, with the 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. A Protein Metrics Byonic TM and Software (Protein Metrics / Dotmatics, San Diego, CA, USA) detects and quantifies post-translational modifications of samples. An extensive search was performed for all potential methionine oxidation, asparagine deamidation, and succinimide formation. All positively identified peptides were verified by tandem mass spectrometry fragmentation patterns and evaluated for appropriate retention time behavior and window boundaries of the XIC. Systematic analysis of peptide levels after thermally forced degradation stress showed that only Ab3 had high levels of oxidation. In Ab3, elevated levels of methionine (M102) oxidation in the heavy chain CDR3 were noted. Methionine oxidation is a common post-translational modification (PTM) that can affect the biological activity of antibodies and potentially induce immunogenic responses. No oxidation of methionine residues was observed in either Ab1 or Ab2. This data is consistent with the reduced thermal stability of Ab3 and led to the decision to discontinue use of Ab3 as a clinical candidate.

[0341] In summary, the testing of monoclonal antibodies Ab1, Ab2 and Ab3 under heat stress makes it possible to determine which antibody is suitable for further development. Only Ab1 did not show a decrease in stability or protein sequence tendency under forced degradation conditions. This developability risk assessment data is consistent with selecting Ab1 as a clinical candidate and advancing development. In order to conduct in vivo studies in mice, a mouse chimeric version of Ab1 was produced. This antibody Ab7 comprises the same variable domains as Ab1 (amino acids 1-123 of the heavy chain and amino acids 1-107 of the light chain) and mouse antibody constant regions (immunoglobulin heavy chain constant region γ1 of the heavy chain and immunoglobulin kappa constant region of the light chain).

[0342] Example 8. Efficacy of anti-PAPP-A (Ab7) in pcy mice

[0343] The aim of this study was to evaluate the therapeutic efficacy of anti-PAPP-A (Ab7) antibody in reducing the increase in total kidney volume and improving renal dysfunction in mice using a nonorthologous model of ADPKD (autosomal dominant polycystic kidney disease) carrying mutations in Nph3, a renal tuberculosis gene that causes the development and expansion of renal cysts observed as early as 3 weeks of age.

[0344] Experimental conditions

[0345] Mice were treated with Ab7 (10 mg / kg anti-PAPP-A, IP injection, once a week, n=20) or Ab8 (10 mg / kg isotype control antibody, IP injection, once a week, n=22) starting at approximately 12 weeks of age (pcy) and dosed for approximately 22 weeks. Antibodies were dissolved in phosphate-buffered saline prior to use and administered at an administration volume of 10 mL / kg.

[0346] Total kidney volume (TKV) was measured by magnetic resonance imaging (MRI) at baseline (before treatment started), after 12 weeks and 21 weeks of treatment. TKV, which provides an indicator of disease progression, is used to evaluate the efficacy of treatment regimens for ADPKD. In this study, T2-weighted (T2W) MRI sequences were used to measure TKV. In vivo MRI was performed on a 4.7 Tesla PharmaScan 47 / 16 system (Bruker, Billerica, MA, USA) with a 38 mm 1 An H linear volume coil was used as the transmitter-receiver. Mice were anesthetized with isoflurane (2%-2.5%) in an oxygen-air mixture (1:1 ratio). T2-weighted (2D multislice spiral TurboSpinEchoRARE, TR / TE = 2500 / 48 ms; RARE factor = 8, average = 15) images were acquired at 0.2 x 0.2 mm. 2 In-plane, 0.8 mm slice thickness, 17-21 slices were acquired to cover the entire kidney volume. Image segmentation was performed manually by iterating through all slices of the image volume and drawing contours at the kidney boundaries using the Segment Editor module in 3D Slicer. The results of the image segmentation were used to calculate TKV using the following equation:

[0347] Number of segmented voxels × voxel size

[0348] TKV was assessed longitudinally at baseline, 12 weeks, and 21 weeks of treatment (corresponding to 12-, 24-, and 33-week-old mice, respectively).

[0349] Glomerular filtration rate (GFR), a marker of renal function, was assessed by FITC-shallot clearance at baseline (before treatment), after 12 and 18 weeks of treatment. Transdermal GFR (t-GFR) was monitored to determine the animals were shaved to remove the hair on the back (from the top of the hind legs to the neck and across the ribs). A thin layer of depilatory cream (Nair TM) was applied to the shaved area and washed off with warm water after 2 minutes. For t-GFR device implantation, mice were anesthetized with isoflurane (3% for induction and 1.5% for maintenance). The shaved area was cleaned with 70% ethanol, and the t-GFR device was placed on the ribs and fixed with silk tape (Cardinal Health, Dublin, OH, USA). FITC-squill sugar (5 μL / g body weight) was administered retro-orbitally using a 0.5 mL insulin syringe. A single intravenous injection of 0.15 mg / g body weight of 5-nitro-1-oxazolidinone (0.15 mg / g body weight) was administered to the mice. The injected mice were allowed to recover in their cages, and data were recorded for 1.0-1.5 hours. The clearance of fluorescently labeled scilate was analyzed using the t-GFR device using Studio 2 software. The software uses 3D compartmental modeling to calculate the half-life of FITC-scilate, which is then used to calculate GFR.

[0350] Effects of anti-PAPP-A (Ab7) or isotype control (Ab8) on kidney size and function in PCY mice

[0351] In this study, the ability of anti-PAPP-A (Ab7) to reduce the increase in total kidney volume and improve the decline in renal function was evaluated in the pcy mouse model of ADPKD. Total kidney volume was measured using MRI at baseline, 12 weeks, and 21 weeks of treatment (corresponding to 12-, 24-, and 33-week-old mice, respectively).

[0352] like Figure 3 As shown, the mean TKV of pcy mice treated with Ab8 (isotype control) increased from baseline to 12 weeks (769 mm 3 Compared with 1753mm 3 ) and reached a stable level (1591 mm 3 These data indicate that TKV was significantly increased in mice treated with Ab8 (isotype control). Anti-PAPP-A (Ab7) treatment reduced TKV increase at both 12 and 21 weeks of treatment (1200 mm, respectively) compared to isotype control (Ab8) treated pcy mice. 3 and 1159mm 3 Compared with the Ab8 (isotype control) group, and adjusted for body weight, the mean increase in TKV from baseline in the Ab7 (anti-PAPP-A) treatment group was reduced by 482 mm at 12 and 21 weeks of treatment, respectively. 3 and 461mm 3 These data indicate that Ab7 (anti-PAPP-A) treatment significantly inhibited the increase in TKV (Table R).

[0353] Table R. Estimated mean TKV (mm) in the anti-PAPP-A (Ab7) group compared to the isotype control (Ab8) in pcy mice 3 ) change from baseline (adjusted for body weight).

[0354]

[0355] Renal function, as measured by the t-GFR method, was evaluated at baseline, 12 weeks, and 18 weeks of treatment. At baseline, mean GFR was similar for mice assigned to 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 At 12 weeks of treatment, the mean GFR in the Ab8 (isotype control) group was 953 μL / min / 100 g body weight, while that in the Ab7 (anti-PAPP-A) group was 1009 μL / min / 100 g body weight. At 18 weeks of treatment, the mean GFR in the Ab8 (isotype control) group decreased to 574 μL / min / 100 g body weight, while that in the Ab7 (anti-PAPP-A) group was 963 μL / min / 100 g body weight ( Figure 4 Compared to the isotype control group, the decline in GFR from baseline in the Ab7-treated group was reduced by an estimated mean of 399 μL / min / 100 g body weight at 18 weeks of treatment, indicating a significant inhibition of the decline in renal function from Ab7 (anti-PAPP-A) treatment (Table S).

[0356] Table S. Change from baseline in estimated mean GFR (μL / min 100 g body weight) in the anti-PAPP-A (Ab7) group compared to the isotype control (Ab8) in pcy mice.

[0357]

[0358] Example 9: Administration of PAPP-A Antibodies to Humans

[0359] The anti-PAPP-A antibodies described herein are administered intravenously (IV) or subcutaneously (SC) to human subjects. The dosing regimen is as follows:

[0360] Group 1: Single dose of anti-PAPP-A antibody – 30 mg IV infusion on day 1

[0361] Group 2: Single dose of anti-PAPP-A antibody on day 1 – up to 100 mg IV infusion

[0362] Group 3: Single dose of anti-PAPP-A antibody on day 1 – up to 100 mg SC

[0363] Group 4: Single dose of anti-PAPP-A antibody on day 1 – up to 300 mg IV infusion

[0364] Group 5: Single dose of anti-PAPP-A antibody – up to 300 mg SC injection on day 1

[0365] Group 6: Single dose of anti-PAPP-A antibody on day 1 – up to 900 mg IV infusion

[0366] Anti-PAPP-A antibodies may also be administered SC or IV every two weeks for up to four doses, or as a single dose SC or IV.

[0367] The antibody was observed to be safe and well tolerated by the subjects following administration.

[0368] Heavy and light chain antibody sequences

[0369] The amino acid sequences of the heavy chains (HC) and light chains (LC) of the eight monoclonal antibodies Abl, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, and Ab8 are described in Tables AH below. The first six anti-PAPP-A antibodies have identical heavy and light chain constant regions, while Ab7, also an anti-PAPP-A antibody, has a mouse-compatible constant region. For Abl-Ab6, the variable regions of the heavy chain (VH) and variable light chain (VL) differ, particularly with respect to the complementary determining regions (CDRs). CDRs are identified using Kabat nomenclature, with each CDR in each variable region sequence shown in bold text and underlined (CDR1, CDR2, and CDR3, respectively). CDRs are also listed separately in the table. The consensus sequences based on the CDRs of Abl to Ab6 are provided in Table 1.

[0370] To conduct in vivo studies in mice, a mouse chimeric version of Ab1 was generated and characterized. This antibody, Ab7, contains the same variable domains as Ab1 (amino acids 1-123 of the heavy chain and amino acids 1-107 of the light chain) and mouse antibody constant regions (immunoglobulin heavy chain constant region γ1 of the heavy chain and immunoglobulin kappa constant region of the light chain). As demonstrated by SPD, Ab7 exhibited similar binding affinity to the PAPP-A substrate as Ab1 (see Example 2). An isotype control antibody (Ab8) was used in all in vivo studies. Ab8 is an anti-tetanus toxoid antibody with a mouse immunoglobulin heavy chain constant region γ1 and a mouse immunoglobulin kappa constant region. The sequences of these two antibodies are listed in Tables G and H.

[0371] Table A: Heavy and light chain amino acid sequences of anti-PAPP-A antibody 1 (Ab1)

[0372]

[0373] Table B: Heavy and light chain amino acid sequences of anti-PAPP-A antibody 2 (Ab2)

[0374]

[0375]

[0376] Table C: Heavy and light chain amino acid sequences of anti-PAPP-A antibody 3 (Ab3)

[0377]

[0378]

[0379] Table D: Heavy and light chain amino acid sequences of anti-PAPP-A antibody 4 (Ab4)

[0380]

[0381]

[0382]

[0383] Table E: Heavy and light chain amino acid sequences of anti-PAPP-A antibody 5 (Ab5)

[0384]

[0385]

[0386] Table F: Heavy and light chain amino acid sequences of anti-PAPP-A antibody 6 (Ab6)

[0387]

[0388]

[0389] Table G: Heavy and light chain amino acid sequences of anti-PAPP-A antibody 7 (Ab7)

[0390]

[0391]

[0392]

[0393] Table H: Heavy and light chain amino acid sequences of isotype control antibody 8 (Ab8)

[0394]

[0395] Table 1: Consensus sequences and PAPP-A protein sequences

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and details may be made therein without departing from the spirit and scope of the invention.

[0411] 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 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; and a variable light chain (VL) sequence comprising three light chain CDR sequences, CDR-L1, CDR-L2, and CDR-L3, wherein: 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).

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. The isolated antibody of any one of claims 2-4, 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 2-5, 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 2-6, wherein the antibody comprises a light chain comprising the sequence shown in SEQ ID NO:

6.

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

6.

9. 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; and a variable light chain (VL) sequence comprising three light chain CDR sequences, CDR-L1, CDR-L2, and CDR-L3, wherein: a. CDR-H1 comprises the sequence X1YX2MX3 (SEQ ID NO: 73), wherein X1 is S or T; X2 is A or G; and X3 is H or S; b. CDR-H2 comprises the sequence X1IX2X3X4X5X6X7X8YYADX9VKG (SEQ ID NO: 74), wherein 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; c. CDR-H3 comprises the sequence HX1RIX2X3WGX4HTFDI (SEQ ID NO: 75), wherein X1 is N or E; X2 is Y or P; X3 is S or P; and X4 is W or F; 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 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; e. CDR-L2 comprises the sequence X1ASX2LQS (SEQ ID NO: 77), wherein X1 is G, V, E, or A; and X2 is S or I; and f. CDR-L3 comprises the sequence X1QX2X3X4X5PX6X7 (SEQ ID NO: 78), wherein 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.

10. The isolated antibody of claim 9, wherein the VH sequence comprises a sequence selected from the sequence shown in SEQ ID NO: 2, 12, 22, 32, 42 or 52.

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

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

13. The isolated antibody of any one of claims 9-12, wherein the antibody comprises a heavy chain sequence selected from the sequence shown in SEQ ID NO: 1, 11, 21, 31, 41 or 51.

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

15. The isolated antibody of any one of claims 9 to 14, wherein the antibody comprises a heavy chain sequence selected from the sequence shown in SEQ ID NO: 1, 11, 21, 31, 41, or 51; and a light chain sequence selected from the sequence shown in SEQ ID NO: 6, 16, 26, 36, 46, or 56.

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 group consisting of the sequences shown in SEQ ID NO: 1, 11, 21, 31, 41, or 51; and two light chain sequences comprising a sequence selected from the group consisting of the sequences shown in SEQ ID NO: 6, 16, 26, 36, 46, or 56.

17. The isolated antibody of any one of claims 9-16, wherein the CD R-H3 comprises HNRIYSWGWHTFDI (SEQ ID NO: 5) or HERIPPWGFH TFDI (SEQ ID NO: 15).

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

20.

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

12.

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

17.

21. The isolated antibody of any one of claims 18-20, 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. The isolated antibody of any one of claims 18-21, wherein the antibody comprises a heavy chain comprising the sequence shown in SEQ ID NO:

11.

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

16.

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

16.

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

30.

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

22.

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

27.

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

27.

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

21.

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

26.

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

26.

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

40.

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

32.

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

37.

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

37.

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

31.

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

36.

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

36.

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

50.

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

42.

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

47.

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

47.

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

41.

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

46.

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

46.

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

60.

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

52.

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

57.

49. The isolated antibody of any one of claims 46-48, 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. The isolated antibody of any one of claims 46-49, wherein the antibody comprises a heavy chain comprising the sequence shown in SEQ ID NO:

51.

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

56.

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

56.

53. 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 CDR-H1, CDR-H2, and CDR-H3 comprise the CDRs of one of the variable heavy chain (VH) sequences shown in SEQ ID NO: 2, 12, 22, 32, 42, or 52, as defined by the Kabat, AbM, IMGT, or Chothia numbering schemes.

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 CDR-L1, CDR-L2, and CDR-L3 comprise the CDRs of one of the variable light chain (VL) sequences shown in SEQ ID NO: 7, 17, 27, 37, 47, or 57, as defined by the Kabat, AbM, IMGT, or Chothia numbering schemes.

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

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

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

58. The isolated antibody of any one of the preceding claims, wherein the antibody comprises an Fc region.

59. The isolated antibody of any one of the preceding claims, wherein the Fc region comprises a human Fc region.

60. The isolated antibody of claim 59, wherein the human Fc region comprises a human IgG1 Fc region.

61. The isolated antibody of any one of claims 59-60, wherein the Fc region comprises a 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 sequence shown in SEQ ID NO:1, and two light chains comprising the sequence shown in SEQ ID NO:

6.

63. 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 sequence shown in SEQ ID NO: 11, and two light chains comprising the sequence shown in SEQ ID NO:

16.

64. 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 sequence shown in SEQ ID NO:21, and two light chains comprising the sequence shown in SEQ ID NO:

26.

65. 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 sequence shown in SEQ ID NO:31, and two light chains comprising the sequence shown in SEQ ID NO:

36.

66. 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 sequence shown in SEQ ID NO: 41, and two light chains comprising the sequence shown in SEQ ID NO:

46.

67. 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 sequence shown in SEQ ID NO:51, and two light chains comprising the sequence shown in SEQ ID NO:

56.

68. The isolated antibody of any one of the preceding claims, wherein the antibody has a kinase activity of less than or equal to about 1, 1.5, 2, 2.5, 10, 25, 50, 75, or 100 x 10 -12 M's K D Binds to human PAPP-A.

69. The isolated antibody of any one of the above claims, wherein the antibody has enzyme blocking or neutralizing activity, optionally wherein the antibody has metalloproteinase blocking activity.

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

71. The isolated antibody of any one of the preceding claims for use as a medicament.

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

73. An isolated polynucleotide or group of polynucleotides encoding the antibody, VH, VL, light chain, heavy chain or antigen binding portion thereof of any one of the above claims; optionally the isolated polynucleotide or group of polynucleotides is a cDNA.

74. A vector or a vector set comprising the polynucleotide or polynucleotide set according to claim 73.

75. A host cell comprising the polynucleotide or polynucleotide set according to claim 73 or the vector or vector set according to claim 74.

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

77. 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, the method comprising administering to the subject a composition comprising an anti-PAPP-A antibody.

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

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

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

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