Antibodies and methods of use thereof

Abstract

Provided herein is an antibody that specifically binds to a peptide antigen.Methods for detecting, isolating or quantifying peptide antigens are also provided.In some embodiments, peptide antigens are released from dystrophin or micro-dystrophin by protease digestion.Methods for detecting or quantifying micro-dystrophin or dystrophin in a sample using antibodies are also provided.In some embodiments, the methods described herein are used to monitor the expression of micro-dystrophin in subjects receiving nucleic acid-based therapy.

Description

[Technical field]

[0001] The present disclosure relates to antibodies that specifically bind to peptide antigens and can be used to detect, isolate or quantitate the peptide antigens.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 63 / 256,511, filed October 15, 2021, which is incorporated herein by reference in its entirety.

[0003] Reference to an Electronically Submitted Sequence Listing The contents of the sequence listing submitted electronically with this application (Name: 6728_1401_Sequence_Listing.xml, Size: 29,401 bytes, Created: October 13, 2022) are incorporated herein by reference in their entirety. [Background technology]

[0004] background A group of neuromuscular diseases called dystrophinopathies are caused by mutations in the DMD gene. Each dystrophinopathy has a different phenotype, and all patients suffer from muscle weakness and eventually cardiomyopathy of various severity. Duchenne muscular dystrophy (DMD) is caused by a frameshift mutation in the dystrophin gene that results in the loss of expression of the dystrophin protein. The lack of dystrophin protein leads to degeneration of skeletal and ultimately cardiac and respiratory muscles (e.g., intercostal and diaphragm), causing premature death. Progressive muscle weakness and atrophy begin in childhood. Affected individuals experience difficulty breathing, respiratory infections, and swallowing problems. Nearly all DMD patients develop cardiomyopathy. Pneumonia complicated by cardiac involvement is the most frequent cause of death, often occurring by the 20s. Becker muscular dystrophy (BMD) is less severe than DMD, but still leads to premature death.

[0005] Dystrophin is a cytoplasmic protein encoded by the DMD gene, the largest known human gene. Full-length dystrophin is a large (427 kDa) protein that contains multiple subdomains that contribute to its function. In DMD, mutations often cause frameshifts that result in premature stop codons and make the protein truncated, nonfunctional, or unstable. In BMD, patients express a truncated, partially functional dystrophin.

[0006] Adeno-associated virus (AAV)-mediated gene therapy has been developed for the treatment of DMD, BMD, and less severe dystrophinopathies. Because the payload size of AAV vectors is limited, attention has been focused on creating a smaller version of dystrophin, micro- or mini-dystrophin, that maintains at least some of the functions of the full-length protein while eliminating non-essential subdomains. International Patent Application Publication No. WO2021108755 (Patent Document 1).

[0007] There is a need for methods to detect and quantitate transgene-mediated micro- and mini-dystrophin expression in subjects undergoing gene therapy. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Patent Application Publication No. WO2021108755 Summary of the Invention

[0009] Quick Overview In one aspect, provided herein is an isolated antibody or antigen-binding fragment thereof capable of binding to a polypeptide comprising the amino acid sequence of SEQ ID NO:1. In some embodiments, the antibody comprises the six complementarity determining regions (CDRs) of 130D2-1, 133E10-1, or 75A2-1. In some embodiments, the antibody comprises the VH domain and the VL domain of 130D2-1, 133E10-1, or 75A2-1. In some embodiments, the antibody is 130D2-1, 133E10-1, or 75A2-1. In some embodiments, the antibody is 130D2-1. In some embodiments, the CDRs are according to Kabat. In some embodiments, the antibody comprises an antigen-binding antibody fragment.

[0010] In one aspect, provided herein is an isolated antibody or antigen-binding fragment thereof capable of binding to a polypeptide comprising the amino acid sequence of SEQ ID NO:3. In some embodiments, the antibody comprises six complementarity determining regions (CDRs) of 112E4-1, 115G6-1, 119H2-1, 121F10-1, or 133D7-1. In some embodiments, the antibody comprises the VH and VL domains of 112E4-1, 115G6-1, 119H2-1, 121F10-1, or 133D7-1. In some embodiments, the antibody is 112E4-1, 115G6-1, 119H2-1, 121F10-1, or 133D7-1. In some embodiments, the antibody is 133D7-1. In some embodiments, the CDRs are according to Kabat. In some embodiments, the antibody comprises an antigen-binding antibody fragment.

[0011] In one aspect, provided herein is a composition comprising an antibody described herein. In some embodiments, the antibody is 130D2-1. In some embodiments, the antibody is 133D7-1.

[0012] In one aspect, provided herein is an affinity resin comprising an antibody described herein and a solid support. In some embodiments, the antibody is 130D2-1. In some embodiments, the antibody is 133D7-1.

[0013] In one aspect, provided herein is an isolated polynucleotide encoding an antibody described herein. In some embodiments, the antibody is 130D2-1. In some embodiments, the antibody is 133D7-1.

[0014] In one aspect, provided herein is a method of producing an antibody disclosed herein, comprising incubating a host cell comprising a polynucleotide encoding the antibody under conditions suitable for producing the antibody. In some embodiments, the antibody is 130D2-1. In some embodiments, the antibody is 133D7-1.

[0015] In one aspect, provided herein is a method of detecting, isolating, or quantifying a peptide having a sequence of SEQ ID NO: 1 or 3 in a sample, comprising contacting the sample containing the peptide with an antibody described herein under conditions that allow binding of the peptide to the antibody. In some embodiments, the method further comprises recovering the peptide. In some embodiments, the method further comprises determining the amount of peptide recovered. In some embodiments, the amount of peptide is determined by LC / MS or LC-MS / MS. In some embodiments, the amino acid sequence of the peptide comprises SEQ ID NO: 1 and the antibody is 130D2-1. In some embodiments, the amino acid sequence of the peptide comprises SEQ ID NO: 3 and the antibody is 133D7-1.

[0016] In one aspect, provided herein is a method of detecting or quantifying levels of a recombinant polypeptide in a sample comprising contacting the sample with an antibody described herein under conditions that allow binding of a peptide having a sequence of SEQ ID NO: 1 or 3 to the antibody, wherein the sample comprises a protease-digested protein isolate and the amino acid sequence of the recombinant polypeptide comprises SEQ ID NO: 28 and / or 29. In some embodiments, the amino acid sequence of the recombinant polypeptide comprises SEQ ID NO: 1 and the antibody is 130D2-1. In some embodiments, the amino acid sequence of the recombinant polypeptide comprises SEQ ID NO: 3 and the antibody is 133D7-1.

[0017] In one aspect, provided herein is a method of detecting or quantifying levels of dystrophin and / or micro-dystrophin in a sample comprising contacting the sample with an antibody described herein under conditions that allow binding of a peptide having a sequence of SEQ ID NO: 1 or 3 to the antibody, wherein the sample comprises a protease-digested protein isolate, and the amino acid sequence of micro-dystrophin comprises SEQ ID NO: 28 and / or 29. In some embodiments, the dystrophin is human, primate, or mouse dystrophin. In some embodiments, the micro-dystrophin comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the sample comprises a protease-digested protein isolate from a subject administered a recombinant adeno-associated virus comprising a polynucleotide encoding micro-dystrophin. In some embodiments, the protein to be detected or quantified is micro-dystrophin and the antibody is 130D2-1. In some embodiments, the proteins to be detected or quantified are micro-dystrophin and dystrophin and the antibody is 133D7-1.

[0018] In some embodiments, the present disclosure provides: [1.] An isolated antibody or antigen-binding fragment thereof capable of binding to a polypeptide comprising the amino acid sequence of SEQ ID NO:1; [2.] The antibody or antigen-binding fragment thereof according to [1], wherein the amino acid sequence of the polypeptide consists of SEQ ID NO:1; [3.] The antibody or antigen-binding fragment thereof according to [1] or [2], which does not bind to a peptide consisting of the amino acid sequence of SEQ ID NO: 2; [4.] The antibody or antigen-binding fragment thereof according to [1] to [3], wherein the antibody is a polyclonal antibody; [5.] The antibody or antigen-binding fragment thereof according to [1] to [3], wherein the antibody is a monoclonal antibody; [6.] An isolated antibody or antigen-binding fragment thereof comprising a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises complementarity determining region 1 (CDR1), CDR2, and CDR3 of VH, and the VL comprises CDR1, CDR2, and CDR3 of VL, wherein the CDR1, CDR2, and CDR3 of the VH and the CDR1, CDR2, and CDR3 of the VL are (a) CDR1, CDR2, and CDR3 of the VH, and CDR1, CDR2, and CDR3 of the VL of the 130D2-1 antibody, each independently containing 0, 1, 2, 3, 4, or 5 substitutions; (b) the CDR1, CDR2, and CDR3 of the VH, and the CDR1, CDR2, and CDR3 of the VL of the 133E10-1 antibody, each independently containing 0, 1, 2, 3, 4, or 5 substitutions; or (c) CDR1, CDR2, and CDR3 of the VH and CDR1, CDR2, and CDR3 of the VL of the 75A2-1 antibody, each independently containing 0, 1, 2, 3, 4, or 5 substitutions. the isolated antibody or antigen-binding fragment thereof; [7.] An isolated antibody or antigen-binding fragment thereof comprising a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises complementarity determining region 1 (CDR1), CDR2, and CDR3 of VH, and the VL comprises CDR1, CDR2, and CDR3 of VL, wherein the CDR1, CDR2, and CDR3 of the VH and the CDR1, CDR2, and CDR3 of the VL are (a) CDR1, CDR2, and CDR3 of the VH and CDR1, CDR2, and CDR3 of the VL of the 130D2-1 antibody, respectively; (b) CDR1, CDR2, and CDR3 of the VH and CDR1, CDR2, and CDR3 of the VL of the 133E10-1 antibody, respectively; or (c) CDR1, CDR2, and CDR3 of VH and CDR1, CDR2, and CDR3 of VL of the 75A2-1 antibody, respectively. the isolated antibody or antigen-binding fragment thereof; [8.] An isolated antibody or antigen-binding fragment thereof according to [6] or [7], wherein the CDR1, CDR2 and CDR3 of the VH and the CDR1, CDR2 and CDR3 of the VL of 130D2-1, 133E10-1 and 75A2-1 are according to Kabat; [9.] An isolated antibody or antigen-binding fragment thereof comprising a variable heavy domain (VH) and a variable light domain (VL), wherein the VH and the VL (a) an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% identity to the VH and VL of the 130D2-1 antibody, respectively; (b) an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% identity to the VH and VL of the 133E10-1 antibody, respectively; or (c) an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% identity to the VH and VL of the 75A2-1 antibody, respectively; the isolated antibody or antigen-binding fragment thereof; [10.] An isolated antibody or antigen-binding fragment thereof comprising a variable heavy domain (VH) and a variable light domain (VL), wherein the VH and VL (a) VH and VL of the 130D2-1 antibody, respectively; (b) the VH and VL of the 133E10-1 antibody, respectively; or (c) VH and VL of the 75A2-1 antibody, respectively. the isolated antibody or antigen-binding fragment thereof; [11.] An isolated antibody or antigen-binding fragment thereof comprising a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises complementarity determining region 1 (CDR1), CDR2, and CDR3 of VH, and the VL comprises CDR1, CDR2, and CDR3 of VL; (a) CDR1 of said VH comprises the amino acid sequence of SEQ ID NO:8 containing 0, 1, 2, 3, 4 or 5 substitutions; (b) CDR2 of said VH comprises the amino acid sequence of SEQ ID NO:9 containing 0, 1, 2, 3, 4 or 5 substitutions; (c) the CDR3 of said VH comprises the amino acid sequence of SEQ ID NO: 10 containing 0, 1, 2, 3, 4 or 5 substitutions; (d) CDR1 of the VL comprises the amino acid sequence of SEQ ID NO: 11 containing 0, 1, 2, 3, 4 or 5 substitutions; (e) CDR2 of the VL comprises the amino acid sequence of SEQ ID NO: 12 containing 0, 1, 2, 3, 4 or 5 substitutions; and (f) the CDR3 of the VL comprises the amino acid sequence of SEQ ID NO: 13 containing 0, 1, 2, 3, 4 or 5 substitutions; the isolated antibody or antigen-binding fragment thereof; [12.] An isolated antibody or antigen-binding fragment thereof comprising a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises complementarity determining region 1 (CDR1), CDR2, and CDR3 of VH, and the VL comprises CDR1, CDR2, and CDR3 of VL; (a) CDR1 of the VH comprises the amino acid sequence of SEQ ID NO:8; (b) CDR2 of the VH comprises the amino acid sequence of SEQ ID NO:9; (c) the CDR3 of the VH comprises the amino acid sequence of SEQ ID NO: 10; (d) CDR1 of the VL comprises the amino acid sequence of SEQ ID NO:11; (e) CDR2 of the VL comprises the amino acid sequence of SEQ ID NO: 12; and (f) the CDR3 of the VL comprises the amino acid sequence of SEQ ID NO: 13; the isolated antibody or antigen-binding fragment thereof; [13.] An isolated antibody or antigen-binding fragment thereof comprising a variable heavy domain (VH) and a variable light domain (VL), (a) the VH comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% identity to SEQ ID NO:4; and (b) the VL comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% identity to SEQ ID NO:5; the isolated antibody or antigen-binding fragment thereof; [14.] An isolated antibody or antigen-binding fragment thereof comprising a variable heavy domain (VH) and a variable light domain (VL), (a) the VH comprises the amino acid sequence of SEQ ID NO:4; and (b) the VL comprises the amino acid sequence of SEQ ID NO:5; the isolated antibody or antigen-binding fragment thereof; [15.] The antibody or antigen-binding fragment thereof according to any one of [6] to

[14] , which is capable of binding to a polypeptide comprising the amino acid sequence of SEQ ID NO:1; [16.] The antibody or antigen-binding fragment thereof according to

[15] , wherein the amino acid sequence of the polypeptide consists of SEQ ID NO:1; [17.] The antibody or antigen-binding fragment thereof according to any one of [6] to

[16] , which does not bind to a peptide consisting of the amino acid sequence of SEQ ID NO: 2; [18.] An isolated antibody or antigen-binding fragment thereof capable of binding to a polypeptide comprising the amino acid sequence of SEQ ID NO:3; [19.] The antibody or antigen-binding fragment thereof of

[18] , wherein the amino acid sequence of the polypeptide consists of SEQ ID NO:3; [20.] The antibody or antigen-binding fragment thereof according to

[18] or

[19] , wherein the antibody is a polyclonal antibody; [21.] The antibody or antigen-binding fragment thereof according to

[18] or

[19] , wherein the antibody is a monoclonal antibody; [22.] An isolated antibody or antigen-binding fragment thereof comprising a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises complementarity determining region 1 (CDR1), CDR2, and CDR3 of VH, and the VL comprises CDR1, CDR2, and CDR3 of VL, wherein the CDR1, CDR2, and CDR3 of the VH and the CDR1, CDR2, and CDR3 of the VL are (a) CDR1, CDR2, and CDR3 of the VH, and CDR1, CDR2, and CDR3 of the VL of the 112E4-1 antibody, each independently containing 0, 1, 2, 3, 4, or 5 substitutions; (b) the CDR1, CDR2, and CDR3 of the VH and the CDR1, CDR2, and CDR3 of the VL of the 115G6-1 antibody, each independently containing 0, 1, 2, 3, 4, or 5 substitutions; (c) CDR1, CDR2, and CDR3 of the VH and CDR1, CDR2, and CDR3 of the VL of the 119H2-1 antibody, each independently containing 0, 1, 2, 3, 4, or 5 substitutions; (d) the CDR1, CDR2, and CDR3 of the VH, and the CDR1, CDR2, and CDR3 of the VL of the 121F10-1 antibody, each independently containing 0, 1, 2, 3, 4, or 5 substitutions; or (e) CDR1, CDR2, and CDR3 of the VH and CDR1, CDR2, and CDR3 of the VL of the 133D7-1 antibody, each independently containing 0, 1, 2, 3, 4, or 5 substitutions. the isolated antibody or antigen-binding fragment thereof; [23.] An isolated antibody or antigen-binding fragment thereof comprising a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises complementarity determining region 1 (CDR1), CDR2, and CDR3 of VH, and the VL comprises CDR1, CDR2, and CDR3 of VL, wherein the CDR1, CDR2, and CDR3 of the VH and the CDR1, CDR2, and CDR3 of the VL are (a) CDR1, CDR2, and CDR3 of the VH and CDR1, CDR2, and CDR3 of the VL of the 112E4-1 antibody, respectively; (b) CDR1, CDR2, and CDR3 of the VH and CDR1, CDR2, and CDR3 of the VL of the 115G6-1 antibody, respectively; (c) CDR1, CDR2, and CDR3 of the VH and CDR1, CDR2, and CDR3 of the VL of the 119H2-1 antibody, respectively; (d) CDR1, CDR2, and CDR3 of the VH and CDR1, CDR2, and CDR3 of the VL of the 121F10-1 antibody, respectively; or (e) CDR1, CDR2, and CDR3 of VH and CDR1, CDR2, and CDR3 of VL of the 133D7-1 antibody, respectively. the isolated antibody or antigen-binding fragment thereof; [24.] An isolated antibody or antigen-binding fragment thereof according to

[22] or

[23] , wherein the CDR1, CDR2 and CDR3 of the VH and the CDR1, CDR2 and CDR3 of the VL of 112E4-1, 115G6-1, 119H2-1, 121F10-1 and 133D7-1 are according to Kabat; [25.] An isolated antibody or antigen-binding fragment thereof comprising a variable heavy domain (VH) and a variable light domain (VL), wherein the VH and VL (a) an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% identity to the VH and VL of the 112E4-1 antibody, respectively; (b) an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% identity to the VH and VL of the 115G6-1 antibody, respectively; (c) an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% identity to the VH and VL of the 119H2-1 antibody, respectively; (d) an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% identity to the VH and VL of the 121F10-1 antibody, respectively; or (e) an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% identity to the VH and VL of the 133D7-1 antibody, respectively; the isolated antibody or antigen-binding fragment thereof; [26.] An isolated antibody or antigen-binding fragment thereof comprising a variable heavy domain (VH) and a variable light domain (VL), wherein the VH and VL (a) VH and VL of the 112E4-1 antibody, respectively; (b) VH and VL of the 115G6-1 antibody, respectively; (c) VH and VL of the 119H2-1 antibody, respectively; (d) the VH and VL of the 121F10-1 antibody, respectively; or (e) VH and VL of the 133D7-1 antibody, respectively. the isolated antibody or antigen-binding fragment thereof; [27.] An isolated antibody or antigen-binding fragment thereof comprising a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises complementarity determining region 1 (CDR1), CDR2, and CDR3 of VH, and the VL comprises CDR1, CDR2, and CDR3 of VL; (a) CDR1 of said VH comprises the amino acid sequence of SEQ ID NO: 18 containing 0, 1, 2, 3, 4 or 5 substitutions; (b) CDR2 of said VH comprises the amino acid sequence of SEQ ID NO: 19 containing 0, 1, 2, 3, 4 or 5 substitutions; (c) the CDR3 of said VH comprises the amino acid sequence of SEQ ID NO: 20 containing 0, 1, 2, 3, 4 or 5 substitutions; (d) CDR1 of the VL comprises the amino acid sequence of SEQ ID NO: 21 containing 0, 1, 2, 3, 4 or 5 substitutions; (e) CDR2 of the VL comprises the amino acid sequence of SEQ ID NO: 22 containing 0, 1, 2, 3, 4 or 5 substitutions; and (f) the CDR3 of the VL comprises the amino acid sequence of SEQ ID NO: 23 containing 0, 1, 2, 3, 4 or 5 substitutions; the isolated antibody or antigen-binding fragment thereof; [28.] An isolated antibody or antigen-binding fragment thereof comprising a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises complementarity determining regions (CDRs) 1, 2, and 3 of VH, and the VL comprises CDRs 1, 2, and 3 of VL; (a) CDR1 of the VH comprises the amino acid sequence of SEQ ID NO: 18; (b) CDR2 of the VH comprises the amino acid sequence of SEQ ID NO: 19; (c) the CDR3 of the VH comprises the amino acid sequence of SEQ ID NO: 20; (d) CDR1 of the VL comprises the amino acid sequence of SEQ ID NO: 21; (e) CDR2 of the VL comprises the amino acid sequence of SEQ ID NO: 22; and (f) the CDR3 of the VL comprises the amino acid sequence of SEQ ID NO: 23; the isolated antibody or antigen-binding fragment thereof; [29.] An isolated antibody or antigen-binding fragment thereof comprising a variable heavy domain (VH) and a variable light domain (VL), (a) the VH comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% identity to SEQ ID NO: 14; and (b) the VL comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% identity to SEQ ID NO: 15; the isolated antibody or antigen-binding fragment thereof; [30.] An isolated antibody or antigen-binding fragment thereof comprising a variable heavy domain (VH) and a variable light domain (VL), (a) the VH comprises the amino acid sequence of SEQ ID NO: 14; and (b) the VL comprises the amino acid sequence of SEQ ID NO: 15; the isolated antibody or antigen-binding fragment thereof; [31.] The antibody or antigen-binding fragment thereof according to any one of

[22] to

[30] , which is capable of binding to a polypeptide comprising the amino acid sequence of SEQ ID NO: 3. [32.] The antibody or antigen-binding fragment thereof according to

[31] , wherein the amino acid sequence of the polypeptide consists of SEQ ID NO:3; [33.] The antibody or antigen-binding fragment thereof according to [1] to

[32] , wherein the antibody fragment comprises a single-chain Fv (scFv), an F(ab) fragment, an F(ab')2 fragment, or an isolated VH domain; [34.] A composition comprising the antibody or antigen-binding fragment thereof described in [1] to

[33] . [35.] The composition according to

[34] , which is a pharmaceutical composition further comprising a pharma- ceutically acceptable excipient; [36.] An affinity resin comprising the antibody or antigen-binding fragment thereof according to any one of [1] to

[33] and a solid support; [37.] The affinity resin of

[36] , wherein the solid support comprises beads, gelatin, or agarose; [38.] The affinity resin according to

[36] or

[37] , wherein the antibody or antigen-binding fragment thereof is bound to the solid support by a covalent bond; [39.] The affinity resin according to

[36] or

[37] , wherein the antibody or antigen-binding fragment thereof is bound to the solid support by a non-covalent association; [40.] An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of [1] to

[33] ; [41.] A vector comprising the polynucleotide according to

[40] ; [42.] A host cell comprising the polynucleotide according to

[40] or the vector according to

[41] . [43.] The host cell according to

[42] , which is a CHO cell or a HEK293 cell; [44.] A method for producing the antibody or antigen-binding fragment thereof according to any one of [1] to

[33] , comprising incubating a host cell according to

[42] or

[43] under conditions suitable for producing the antibody or antigen-binding fragment thereof; [45.] A method for isolating a peptide from a sample, the method comprising: (a) contacting the sample containing the peptide with a composition comprising the antibody or antigen-binding fragment thereof according to any one of [1] to

[17] , and optionally with a composition comprising the antibody or antigen-binding fragment thereof according to any one of claims

[18] to

[33] , under conditions allowing binding of the peptide to the antibody or antigen-binding fragment thereof; (b) removing the portion of the sample that is not bound to the antibody or antigen-binding fragment thereof; (c) dissociating the peptide from the antibody or antigen-binding fragment thereof; wherein the amino acid sequence of the peptide comprises SEQ ID NO: 1 or 3; [46.] The method according to

[45] , wherein the amino acid sequence of the peptide consists of SEQ ID NO: 1 or 3; [47.] The method of

[45] or

[46] , wherein the composition comprising the antibody or antigen-binding fragment thereof is an affinity resin comprising a solid support selected from the group consisting of beads, gelatin, or agarose; [48.] The method of

[47] , wherein the antibody or antigen-binding fragment thereof is covalently bound to the solid support; [49.] The method of

[47] , wherein the antibody or antigen-binding fragment thereof is bound to the solid support by a non-covalent association; [50.] The method according to

[45] to

[49] , wherein the sample comprises a protease-digested protein isolate obtained from the subject; [51.] The method of

[50] , wherein the sample comprises a protease-digested protein isolate obtained from skeletal muscle tissue of the subject; [52.] The method according to

[50] or

[51] , wherein the protease comprises trypsin; [53.] The method according to

[50] to

[52] , wherein the subject is a human subject, a primate subject, a canine subject, or a murine subject; [54.] The method according to

[50] to

[53] , wherein the subject is administered a recombinant polypeptide comprising the amino acid sequence of SEQ ID NO: 28 and / or 29; [55.] The method according to

[50] to

[53] , wherein the subject is administered a recombinant polynucleotide encoding a recombinant polypeptide comprising the amino acid sequence of SEQ ID NO: 28 and / or 29; [56.] The method according to

[50] to

[53] , wherein the subject is administered a recombinant virus comprising a polynucleotide encoding a recombinant polypeptide comprising the amino acid sequence of SEQ ID NO: 28 and / or 29; [57.] The method according to

[56] , wherein the recombinant virus is a recombinant adeno-associated virus; [58.] (d) recovering the peptide; (e) determining the amount of peptide recovered in step (d); The method according to any one of

[45] to

[57] further comprising the steps of: [59.] The method according to

[58] , wherein the amount of the peptide is determined by LC / MS or LC-MS / MS; [60.] The method according to

[45] to

[59] , wherein the sample further contains a stable isotope-labeled peptide standard comprising the amino acid sequence of SEQ ID NO: 1 or 3; [61.] The method according to any one of

[54] to

[60] , wherein the recombinant polypeptide is microdystrophin; [62.] The method according to

[61] , wherein the microdystrophin comprises the amino acid sequence of SEQ ID NO: 27; [63.] A method for quantifying a level of a recombinant polypeptide in a subject, comprising: (a) providing a sample comprising a protease-digested protein isolate obtained from said subject, said sample comprising one or more peptides having an amino acid sequence of SEQ ID NO: 1 or 3; (b) contacting the sample with a composition comprising the antibody or antigen-binding fragment thereof according to any one of [1] to

[17] , and optionally with a composition comprising the antibody or antigen-binding fragment thereof according to any one of claims

[18] to

[33] , under conditions allowing binding of the antibody or antigen-binding fragment thereof to the peptide; (c) recovering the peptide bound to the antibody or antigen-binding fragment thereof; (d) determining the amount of peptide recovered in step (d); wherein the amino acid sequence of the recombinant polypeptide comprises SEQ ID NO: 28 and / or 29; [64.] The method according to

[63] , wherein the amount of the peptide is determined by LC / MS or LC-MS / MS; [65.] The method of

[63] or

[64] , wherein the sample comprises a protease-digested protein isolate obtained from skeletal muscle tissue of the subject; [66.] The method according to

[63] to

[65] , wherein the subject is a human subject, a primate subject, a canine subject, or a murine subject; [67.] The method according to

[63] to

[66] , wherein the subject is administered a recombinant polypeptide comprising the amino acid sequence of SEQ ID NO: 28 and / or 29; [68.] The method according to

[63] to

[66] , wherein the subject is administered a recombinant polynucleotide encoding a recombinant polypeptide comprising the amino acid sequence of SEQ ID NO: 28 and / or 29; [69.] The method according to

[63] to

[66] , wherein the subject is administered a recombinant virus comprising a polynucleotide encoding a recombinant polypeptide comprising the amino acid sequence of SEQ ID NO: 28 and / or 29; [70.] The method according to

[69] , wherein the recombinant virus is a recombinant adeno-associated virus; [71.] The method according to

[63] to

[70] , wherein the recombinant polypeptide is microdystrophin. [72.] The method described in

[71] , wherein the microdystrophin comprises the amino acid sequence of SEQ ID NO: 27. [73.] The method according to any one of

[63] to

[72] , wherein the sample further contains one or more labeled peptides capable of binding to the antibody or antigen-binding fragment thereof; [74.] The method according to

[73] , wherein the labeled peptide or peptides are stable isotope-labeled peptide or peptides; [75.] A method for quantifying dystrophin and / or microdystrophin expression levels in a subject, comprising: (a) providing a sample comprising a protease-digested protein isolate obtained from said subject, said sample comprising one or more peptides having an amino acid sequence of SEQ ID NO: 1 or 3; (b) contacting the sample with a composition comprising the antibody or antigen-binding fragment thereof according to any one of [1] to

[17] , and optionally with a composition comprising the antibody or antigen-binding fragment thereof according to any one of claims

[18] to

[33] , under conditions allowing binding of the antibody or antigen-binding fragment thereof to the peptide; (c) recovering the peptide bound to the antibody or antigen-binding fragment thereof; (d) determining the amount of peptide recovered in step (d); wherein the amino acid sequence of the microdystrophin comprises SEQ ID NO: 28 and / or 29; [76.] The method according to

[73] , wherein the amount of the peptide is determined by LC / MS or LC-MS / MS; [77.] A method according to

[73] or

[74] , providing absolute quantification of dystrophin and / or microdystrophin expression levels; [78.] A method according to

[73] or

[74] , which provides relative quantification of dystrophin and / or microdystrophin expression levels; [79.] The method of any one of

[73] to

[76] , wherein the sample comprises a protease-digested protein isolate obtained from the subject's skeletal muscle tissue; [80.] The method according to any one of

[73] to

[77] , wherein the protease is trypsin. [81.] The method according to any one of

[75] to

[80] , wherein the sample further contains one or more labeled peptides capable of binding to the antibody or antigen-binding fragment thereof; [82.] The method according to

[81] , wherein the labeled peptide or peptides are stable isotope-labeled peptide or peptides; [83.] The method according to

[81] or

[82] , wherein the labeled peptide or peptides comprise the amino acid sequence of SEQ ID NO: 1 or 3; [84.] The method according to

[75] to

[83] , wherein the subject is a human subject, a primate subject, a canine subject, or a murine subject; [85.] The method according to

[84] , wherein the subject is a human; [86.] The method of

[84] , wherein the subject is a primate; [87.] The method according to

[84] , wherein the subject is a mouse; [88.] The method according to

[75] to

[84] , wherein the subject suffers from Duchenne muscular dystrophy; [89.] The method according to

[75] to

[84] , wherein the subject is a non-human mammal that has been genetically engineered to contain one or more mutations in the dystrophin gene; [90.] The method according to

[75] to

[89] , wherein the subject is administered a recombinant polynucleotide encoding microdystrophin comprising the amino acid sequences of SEQ ID NOs: 28 and 29; [91.] The method according to

[90] , wherein the microdystrophin comprises the amino acid sequence of SEQ ID NO: 27; [92.] The method according to

[90] or

[91] , wherein the recombinant polynucleotide is DNA; [93.] The method according to

[90] or

[91] , wherein the recombinant polynucleotide is RNA; [94.] The method according to

[93] , wherein the RNA is an mRNA containing modified ribonucleotides; [95.] The method according to

[75] to

[89] , wherein the subject is administered a recombinant virus comprising a polynucleotide encoding micro-dystrophin comprising the amino acid sequences of SEQ ID NOs: 28 and 29; [96.] The method according to

[69] , wherein the microdystrophin comprises the amino acid sequence of SEQ ID NO: 27; [97.] The method according to

[95] or

[96] , wherein the recombinant virus is a recombinant adeno-associated virus.

[0019] Further features and advantages of the compositions and methods described herein will become more apparent from the following detailed description, taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 shows candidate tryptic peptides for LC-MS quantification of microdystrophin A. [Diagram 2] FIG. 1 shows binding of rabbit monoclonal and rabbit polyclonal antibodies to the LEM peptide. [Diagram 3] FIG. 1 shows the binding of rabbit monoclonal and rabbit polyclonal antibodies to LLQ peptides. [Figure 4] FIG. 1 shows screening of rabbit anti-LEM peptide monoclonal antibodies by LCMS. [Diagram 5] FIG. 1 shows the screening of rabbit anti-LLQ peptide monoclonal antibodies by LCMS. [Figure 6] FIG. 1 shows an LCMS assay for simultaneous quantification of micro-dystrophin A and dystrophin. [Figure 7] FIG. 1 shows the workflow of the LBA / LC-MS / MS assay. [Figure 8-1] FIG. 1 shows the selectivity of the LEM peptide detection assay. [Figure 8-2] FIG. 1 shows the selectivity of the LEM peptide detection assay. [Figure 9-1] FIG. 1 shows the selectivity of the LLQ peptide detection assay. [Figure 9-2] FIG. 1 shows the selectivity of the LLQ peptide detection assay. [Figure 10] FIG. 1 shows absolute quantification of micro-dystrophin transgene product by LEM peptide detection assay. [Figure 11-1] FIG. 1 shows relative quantification of full-length dystrophin in various species by LLQ peptide detection assay. [Figure 11-2]FIG. 1 shows relative quantification of full-length dystrophin in various species by LLQ peptide detection assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Detailed Description Provided herein are antibodies that specifically bind to a peptide having the sequence of SEQ ID NO: 1 or 3. The antibodies are useful for detecting, isolating and quantitating a peptide having the sequence of SEQ ID NO: 1 or 3. The antibodies can also be used to detect or quantitate a protein, such as dystrophin or microdystrophin, that releases a peptide having the sequence of SEQ ID NO: 1 or 3 upon digestion with a protease, such as trypsin.

[0022] In some embodiments, provided herein are methods for detecting and quantifying dystrophin and / or microdystrophin expression in a subject suffering from muscular dystrophy who has been administered a recombinant polynucleotide encoding microdystrophin. In some embodiments, the subject has been administered a recombinant adeno-associated virus comprising a recombinant polynucleotide encoding microdystrophin. In some embodiments, the method detects and quantifies peptides comprising the amino acid sequence of SEQ ID NO: 1 or 3 using liquid chromatography mass spectrometry (LC-MS) or liquid chromatography tandem mass spectrometry (LC-MS / MS). In some embodiments, the LC-MS detection assays disclosed herein provide highly accurate absolute quantification of microdystrophin levels in muscle samples of non-human primate subjects administered recombinant AAV particles comprising a microdystrophin transgene. In some embodiments, the LC-MS detection assays disclosed herein also provide highly accurate relative quantification of total dystrophin levels in muscle samples from various species, e.g., humans and primates.

[0023] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. To facilitate understanding of the methods of this disclosure, several terms and expressions are defined below.

[0024] The term "antibody" refers to an immunoglobulin molecule (or a group of immunoglobulin molecules) that recognizes and specifically binds to a target, e.g., a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing, via at least one antigen recognition site within the variable region of the immunoglobulin molecule. The terms "antibody" and "antibodies" are terms of art and may be used interchangeably herein to refer to a molecule that has an antigen binding site that specifically binds an antigen.

[0025] Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, human antibodies, humanized antibodies, resurfaced antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFvs), affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), bispecific antibodies, and multispecific antibodies. In certain embodiments, the antibodies described herein refer to polyclonal antibody populations. An antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (isotype) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule based on the identity of the heavy chain constant domains, designated alpha, delta, epsilon, gamma, and mu, respectively. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or can be conjugated or fused to other molecules, such as toxins, radioisotopes, other polypeptides, etc.

[0026] The terms "antigen-binding domain," "antigen-binding region," "antigen-binding site," and similar terms refer to that portion of an antibody molecule that contains the amino acid residues that confer to the antibody molecule its specificity for an antigen (e.g., a peptide having the sequence of SEQ ID NO: 1 or 3). The antigen-binding region can be derived from any animal species, such as mouse or human.

[0027] The terms "variable region" or "variable domain" are used interchangeably and are common in the art. The sequence variability is concentrated in these regions called complementarity determining regions (CDRs), while the more highly conserved regions within the variable domains are called framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen (e.g., a peptide having the sequence of SEQ ID NO: 1 or 3). In certain embodiments, the variable region comprises three CDRs (CDR1, CDR2, and CDR3) and four framework regions (FR1, FR2, FR3, and FR4) in the following order from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises human CDRs and human framework regions (FRs). In certain embodiments, the variable region comprises CDRs and framework regions (FRs), and one or more CDRs have been modified by substitution, deletion, or insertion compared to the CDRs of the parent antibody. In certain embodiments, the variable region comprises CDRs and framework regions (FRs), and one or more FRs have been modified by substitution, deletion, or insertion compared to the FRs of the parent antibody. In certain embodiments, the variable region comprises CDRs and framework regions (FRs), and one or more CDRs and one or more FRs have been modified by substitution, deletion, or insertion compared to the CDRs and FRs of the parent antibody. In certain embodiments, the parent antibody is PGZL1. In certain embodiments, the variable region comprises human CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0028] Those skilled in the art understand that there are several methods for determining CDRs. One approach is based on interspecies sequence variation (i.e., Kabat EA, et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.) ("Kabat"). Another approach is based on crystallographic studies of antigen-antibody complexes (Al-lazikani B., et al, J. Mol. Biol. 273:927-948 (1997)) ("Chothia"). In addition, those in the art may use a combination of these two methods to determine CDRs. In some embodiments, CDR sequences are identified according to Kabat. In some embodiments, CDR sequences are identified according to Chothia. It is understood that the identification of CDRs in a variable region also identifies FRs as sequences adjacent to the CDRs.

[0029] The Kabat numbering system is commonly used to refer to residues within the variable domain (approximately residues 1-107 in the light chain and residues 1-113 in the heavy chain) (e.g., Kabat EA, et al., Sequences of Immunological Interest. (5th Ed., 1991, National Institutes of Health, Bethesda, Md.) ("Kabat").

[0030] The numbering of amino acid positions as in Kabat refers to the numbering system used for the heavy or light chain variable domains of the antibody sequences in Kabat EA, et al. (Sequences of Immunological Interest. (5th Ed., 1991, National Institutes of Health, Bethesda, Md.), "Kabat"). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FR or CDR of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to Kabat). The Kabat numbering of residues may be determined for a given antibody by sequence comparison at the homology regions of the antibody sequence with the "standard" Kabat numbering sequence. Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32, if only 35A is present, the loop ends at 33, and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions are a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software, available, for example, at bioinf.org.uk / abs / software. In some embodiments, the CDR sequences are identified according to Kabat. In some embodiments, the CDR sequences are identified according to Chothia. In some embodiments, the CDR sequences are identified according to AbM. In some embodiments, the CDR3 sequences of the VH are identified according to Kabat.In some embodiments, the CDR3 sequences of the VH are identified according to Chothia. In some embodiments, the CDR3 sequences of the VH are identified according to AbM.

[0031] TIFF2024542939000001.tif69165

[0032] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0033] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0034] The term "antibody fragment" refers to a portion of an intact antibody. An "antigen-binding fragment" refers to a portion of an intact antibody that binds to an antigen. An antigen-binding fragment can contain the antigen-determining variable regions of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single chain antibodies.

[0035] A "monoclonal" antibody or antigen-binding fragment thereof refers to a population of homogeneous antibodies or antigen-binding fragments involved in highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies, which typically contain different antibodies directed against different antigenic determinants. The term "monoclonal" antibody or antigen-binding fragment thereof encompasses both intact and full-length monoclonal antibodies, as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv) variants, fusion proteins containing an antibody portion, and any other modified immunoglobulin molecule that contains an antigen recognition site. Furthermore, a "monoclonal" antibody or antigen-binding fragment thereof refers to such antibodies and antigen-binding fragments thereof produced by any method, including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals.

[0036] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition that is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in the form in which they are found in nature. In some embodiments, an antibody, polynucleotide, vector, cell, or composition that is isolated is substantially pure.

[0037] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also include amino acid polymers that are naturally modified, or modified by intervention, such as, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that because the polypeptides described herein are based on antibodies, in certain embodiments the polypeptides can occur as single chains or linked chains.

[0038] The term "identical" or percent "identity" in the context of two or more nucleic acids or polypeptides refers to two or more sequences or subsequences that are the same or have a certain percentage of nucleotides or amino acid residues that are the same when compared and aligned (and introducing gaps, if necessary) for maximum correspondence, without considering conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain sequence comparisons of amino acid or nucleotide sequences are known in the art. One such non-limiting example of an algorithm for sequence comparison is the algorithm described in Karlin S., et al., Proc. Natl. Acad. Sci., 87:2264-2268 (1990) (modified in Karlin S., et al., Proc. Natl. Acad. Sci., 90:5873-5877 (1993)) and incorporated into the NBLAST and XBLAST programs (Altschul SF, et al., Nucleic Acids Res., 25:3389-3402 (1991)). In certain embodiments, Gapped BLAST can be used as described in Altschul SF, et al., Nucleic Acids Res. 25:3389-3402 (1997). BLAST-2, WU-BLAST-2 (Altschul SF, et al., Methods in Enzymology, 266:460-480 (1996)), ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or Megalign (DNASTAR) are additional publicly available software programs that can be used to align sequences. In certain embodiments, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software (e.g., using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6).In certain alternative embodiments, the percent identity between two amino acid sequences can be determined using the GAP program of the GCG software package incorporating the algorithm of Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) (e.g., using either the Blossum 62 matrix or the PAM250 matrix, with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5). Alternatively, in certain embodiments, the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS, 4: 11-17 (1989)). For example, the percent identity can be determined using the ALIGN program (version 2.0) with PAM120 using a residual table, a gap length penalty of 12, and a gap penalty of 4. Those skilled in the art can determine suitable parameters for maximum sequence comparison with a particular sequence comparison software. In certain embodiments, the default parameters of the sequence comparison software are used. In certain embodiments, the percentage identity "X" of a first amino acid sequence to an amino acid of a second sequence is calculated as 100 x (Y / Z), where Y is the number of amino acid residues scored as identical matches in a sequence comparison of the first and second sequences (aligned by visual inspection or by a sequence comparison program for a particular sequence) and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

[0039] As a non-limiting example, whether any particular polynucleotide has a certain percentage of sequence identity (e.g., at least 80% identical, at least 85% identical, at least 90% identical, and in some embodiments at least 95%, 96%, 97%, 98%, or 99% identical) to a reference sequence can, in certain embodiments, be determined using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics, 2:482 489 (1981)) to find the best segment of homology between two sequences. When using Bestfit or other sequence comparison programs to determine whether a particular sequence is, for example, 95% identical to a reference sequence described herein, parameters are set such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and gaps in identity are allowed up to 5% of the total number of nucleotides in the reference sequence.

[0040] In some embodiments, two nucleic acids or polypeptides described herein are substantially identical means that they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence as determined using a sequence comparison algorithm or visual inspection. Identity can exist over a region of the sequence that is at least about 10, about 20, about 40-60 residues in length, or any integer value therebetween, and can exist over a region longer than 60-80 residues, e.g., at least about 90-100 residues, and in some embodiments the sequences are substantially identical over the entire length of the sequences being compared, such as, for example, the coding regions of the nucleotide sequences.

[0041] "AAV" is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or to modifications, derivatives, or pseudotypes thereof. The term encompasses all subtypes and both natural and recombinant forms, unless otherwise required. The abbreviation "rAAV" refers to recombinant adeno-associated virus. The term "AAV" includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, as well as modifications, derivatives, or pseudotypes thereof.

[0042] "Recombinant" as applied to an AAV particle means that the AAV particle is the product of one or more procedures that result in an AAV particle construct that differs from the AAV particle found in nature.

[0043] Recombinant adeno-associated viral particle "rAAV particle" refers to a viral particle composed of at least one AAV capsid protein and a polynucleotide rAAV vector genome enclosed in a capsid that contains a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome, e.g., a transgene to be delivered to a mammalian cell, e.g., a transgene encoding micro-dystrophin comprising the amino acid sequence of SEQ ID NO: 27). The rAAV particle may be of any AAV serotype (e.g., AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, or AAV-10, or derivatives / modifications / pseudotypes thereof), including any modifications, derivatives, or pseudotypes. Such AAV serotypes and derivatives / modifications / pseudotypes, as well as methods for generating such serotypes / derivatives / modifications / pseudotypes, are known in the art (see, e.g., Asokan et al., Mol. Ther. 20(4):699-708 (2012)). Recombinant AAV particles containing a transgene encoding micro-dystrophin are disclosed in International Publication No. WO2021108755, which is incorporated herein by reference for all purposes.

[0044] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0045] Whenever embodiments are described herein using the term "comprising," it should be understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided. Also, when embodiments are described herein using the term "consisting essentially of," it should be understood that other similar embodiments described in terms of "consisting of" are also provided.

[0046] The term "and / or," when used herein in phrases such as "A and / or B," is intended to include both A and B, A or B, A (alone), and B (alone). Similarly, the term "and / or," when used in phrases such as "A, B, and / or C," is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0047] When embodiments of the present disclosure are described in terms of a Markush group or other alternative group, the methods of the present disclosure include not only the entire group listed as a whole, but also each member of the group individually, all possible subgroups of the main group, and even the main group in which one or more of the group members are absent. The disclosed methods also contemplate the explicit exclusion of any one or more of the group members in the disclosed methods.

[0048] antibody In certain aspects, provided herein is an isolated antibody capable of binding to a polypeptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the polypeptide consists of SEQ ID NO: 1. In some embodiments, the antibody does not bind to a peptide consisting of the amino acid sequence of SEQ ID NO: 2. In some embodiments, the antibody comprises an antigen binding fragment. In some embodiments, the antibody fragment comprises a single chain Fv (scFv), a F(ab) fragment, a F(ab')2 fragment, or an isolated VH domain.

[0049] In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a monoclonal antibody.

[0050] In some embodiments, the antibody is a pAb#7684-A, pAb#7684-B, or pAb#7685 polyclonal antibody. In some embodiments, the antibody is a pAb#7684-A polyclonal antibody.

[0051] In some embodiments, the isolated antibody comprises a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises complementarity determining region 1 (CDR1), CDR2, and CDR3 of the VH, and the VL comprises CDR1, CDR2, and CDR3 of the VL, wherein the CDR1, CDR2, and CDR3 of the VH and the CDR1, CDR2, and CDR3 of the VL are - the CDR1, CDR2, and CDR3 of the VH, and the CDR1, CDR2, and CDR3 of the VL of the 130D2-1 antibody, each independently comprising 0, 1, 2, 3, 4 or 5 substitutions; - the CDR1, CDR2, and CDR3 of the VH, and the CDR1, CDR2, and CDR3 of the VL of the 133E10-1 antibody, each independently containing 0, 1, 2, 3, 4, or 5 substitutions; or - the CDR1, CDR2, and CDR3 of the VH, and the CDR1, CDR2, and CDR3 of the VL of the 75A2-1 antibody, each independently comprising 0, 1, 2, 3, 4, or 5 substitutions; Includes.

[0052] In some embodiments, the isolated antibody comprises a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises complementarity determining region 1 (CDR1), CDR2, and CDR3 of the VH, and the VL comprises CDR1, CDR2, and CDR3 of the VL, wherein the CDR1, CDR2, and CDR3 of the VH and the CDR1, CDR2, and CDR3 of the VL are - CDR1, CDR2, and CDR3 of the VH and CDR1, CDR2, and CDR3 of the VL of the 130D2-1 antibody, respectively; - the CDR1, CDR2, and CDR3 of the VH and the CDR1, CDR2, and CDR3 of the VL of the 133E10-1 antibody, respectively; or - CDR1, CDR2, and CDR3 of the VH and CDR1, CDR2, and CDR3 of the VL of the 75A2-1 antibody, respectively Includes.

[0053] In some embodiments, the CDR1, CDR2, and CDR3 of the VH and the CDR1, CDR2, and CDR3 of the VL of 130D2-1, 133E10-1, and 75A2-1 are according to Kabat.

[0054] In some embodiments, the isolated antibody comprises a variable heavy domain (VH) and a variable light domain (VL), wherein the VH and VL are - an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% identity with the VH and VL of the 130D2-1 antibody, respectively; -An amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% identity with the VH and VL of the 133E10-1 antibody, respectively; or -An amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% identity with the VH and VL of the 75A2-1 antibody, respectively. Includes.

[0055] In some embodiments, the isolated antibody comprises a variable heavy domain (VH) and a variable light domain (VL), wherein the VH and VL are - the VH and VL of the 130D2-1 antibody, respectively; - the VH and VL of the 133E10-1 antibody, respectively; or - the VH and VL of the 75A2-1 antibody, respectively Includes.

[0056] In some embodiments, the isolated antibody comprises a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises complementarity determining region 1 (CDR1), CDR2, and CDR3 of the VH, and the VL comprises CDR1, CDR2, and CDR3 of the VL; - CDR1 of VH comprises the amino acid sequence of SEQ ID NO:8 containing 0, 1, 2, 3, 4 or 5 substitutions; - CDR2 of VH comprises the amino acid sequence of SEQ ID NO: 9 containing 0, 1, 2, 3, 4 or 5 substitutions; - the CDR3 of VH comprises the amino acid sequence of SEQ ID NO: 10 containing 0, 1, 2, 3, 4 or 5 substitutions; - CDR1 of VL comprises the amino acid sequence of SEQ ID NO: 11 containing 0, 1, 2, 3, 4 or 5 substitutions; - CDR2 of VL comprises the amino acid sequence of SEQ ID NO: 12 containing 0, 1, 2, 3, 4 or 5 substitutions; and - the CDR3 of the VL comprises the amino acid sequence of SEQ ID NO: 13 containing 0, 1, 2, 3, 4 or 5 substitutions.

[0057] In some embodiments, the isolated antibody comprises a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises complementarity determining region 1 (CDR1), CDR2, and CDR3 of the VH, and the VL comprises CDR1, CDR2, and CDR3 of the VL; - CDR1 of VH comprises the amino acid sequence of SEQ ID NO:8; - CDR2 of VH comprises the amino acid sequence of SEQ ID NO:9; - CDR3 of VH comprises the amino acid sequence of SEQ ID NO: 10; - CDR1 of VL comprises the amino acid sequence of SEQ ID NO: 11; - CDR2 of the VL comprises the amino acid sequence of SEQ ID NO: 12; and - the CDR3 of the VL comprises the amino acid sequence of SEQ ID NO:13;

[0058] In some embodiments, the isolated antibody comprises a variable heavy domain (VH) and a variable light domain (VL), - VH comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% identity to SEQ ID NO: 4; and - VL comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% identity to SEQ ID NO:5.

[0059] In some embodiments, the isolated antibody comprises a variable heavy domain (VH) and a variable light domain (VL), - VH comprises the amino acid sequence of SEQ ID NO:4; and - VL comprises the amino acid sequence of SEQ ID NO:5.

[0060] In some embodiments, the isolated antibody is capable of binding to a polypeptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the polypeptide consists of SEQ ID NO: 1. In some embodiments, the antibody does not bind to a peptide consisting of the amino acid sequence of SEQ ID NO:2.

[0061] In certain aspects, provided herein is an isolated antibody capable of binding to a polypeptide comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the amino acid sequence of the polypeptide consists of SEQ ID NO: 3. In some embodiments, the antibody comprises an antigen binding. In some embodiments, the antibody fragment comprises a single chain Fv (scFv), a F(ab) fragment, a F(ab')2 fragment, or an isolated VH domain.

[0062] In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a monoclonal antibody.

[0063] In some embodiments, the isolated antibody comprises a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises complementarity determining region 1 (CDR1), CDR2, and CDR3 of the VH, and the VL comprises CDR1, CDR2, and CDR3 of the VL, wherein the CDR1, CDR2, and CDR3 of the VH and the CDR1, CDR2, and CDR3 of the VL are - the CDR1, CDR2, and CDR3 of the VH, and the CDR1, CDR2, and CDR3 of the VL of the 112E4-1 antibody, each independently comprising 0, 1, 2, 3, 4 or 5 substitutions; - the CDR1, CDR2, and CDR3 of the VH, and the CDR1, CDR2, and CDR3 of the VL of the 115G6-1 antibody, each independently comprising 0, 1, 2, 3, 4 or 5 substitutions; - the CDR1, CDR2, and CDR3 of the VH, and the CDR1, CDR2, and CDR3 of the VL of the 119H2-1 antibody, each independently comprising 0, 1, 2, 3, 4 or 5 substitutions; - the CDR1, CDR2, and CDR3 of the VH, and the CDR1, CDR2, and CDR3 of the VL of the 121F10-1 antibody, each independently containing 0, 1, 2, 3, 4, or 5 substitutions; or - the CDR1, CDR2, and CDR3 of the VH and the CDR1, CDR2, and CDR3 of the VL of the 133D7-1 antibody, each independently comprising 0, 1, 2, 3, 4, or 5 substitutions; Includes.

[0064] In some embodiments, the isolated antibody comprises a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises complementarity determining region 1 (CDR1), CDR2, and CDR3 of the VH, and the VL comprises CDR1, CDR2, and CDR3 of the VL, wherein the CDR1, CDR2, and CDR3 of the VH and the CDR1, CDR2, and CDR3 of the VL are - CDR1, CDR2, and CDR3 of the VH and CDR1, CDR2, and CDR3 of the VL of the 112E4-1 antibody, respectively; - the CDR1, CDR2, and CDR3 of the VH and the CDR1, CDR2, and CDR3 of the VL of the 115G6-1 antibody, respectively; - CDR1, CDR2, and CDR3 of the VH and CDR1, CDR2, and CDR3 of the VL of the 119H2-1 antibody, respectively; - CDR1, CDR2, and CDR3 of the VH and CDR1, CDR2, and CDR3 of the VL of the 121F10-1 antibody, respectively; or - CDR1, CDR2, and CDR3 of the VH and CDR1, CDR2, and CDR3 of the VL of the 133D7-1 antibody, respectively Includes.

[0065] In some embodiments, the CDR1, CDR2 and CDR3 of the VH and the CDR1, CDR2 and CDR3 of the VL of 112E4-1, 115G6-1, 119H2-1, 121F10-1, and 133D7-1 are according to Kabat.

[0066] In some embodiments, the isolated antibody comprises a variable heavy domain (VH) and a variable light domain (VL), wherein the VH and VL are - an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% identity with the VH and VL of the 112E4-1 antibody, respectively; - an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% identity with the VH and VL of the 115G6-1 antibody, respectively; - an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% identity with the VH and VL of the 119H2-1 antibody, respectively; - an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% identity with the VH and VL of the 121F10-1 antibody, respectively; or -An amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% identity with the VH and VL of the 133D7-1 antibody, respectively. Includes.

[0067] In some embodiments, the isolated antibody comprises a variable heavy domain (VH) and a variable light domain (VL), wherein the VH and VL are - the VH and VL of the 112E4-1 antibody, respectively; - the VH and VL of the 115G6-1 antibody, respectively; - the VH and VL of the 119H2-1 antibody, respectively; - the VH and VL of the 121F10-1 antibody, respectively; or - VH and VL of the 133D7-1 antibody, respectively Includes.

[0068] In some embodiments, the isolated antibody comprises a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises complementarity determining region 1 (CDR1), CDR2, and CDR3 of the VH, and the VL comprises CDR1, CDR2, and CDR3 of the VL; - CDR1 of VH comprises the amino acid sequence of SEQ ID NO: 18 containing 0, 1, 2, 3, 4 or 5 substitutions; - CDR2 of VH comprises the amino acid sequence of SEQ ID NO: 19 containing 0, 1, 2, 3, 4 or 5 substitutions; - the CDR3 of VH comprises the amino acid sequence of SEQ ID NO: 20 containing 0, 1, 2, 3, 4 or 5 substitutions; - CDR1 of VL comprises the amino acid sequence of SEQ ID NO: 21 containing 0, 1, 2, 3, 4 or 5 substitutions; - CDR2 of VL comprises the amino acid sequence of SEQ ID NO: 22 containing 0, 1, 2, 3, 4 or 5 substitutions; and - the CDR3 of the VL comprises the amino acid sequence of SEQ ID NO: 23 containing 0, 1, 2, 3, 4 or 5 substitutions.

[0069] In some embodiments, the isolated antibody comprises a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises complementarity determining region 1 (CDR1), CDR2, and CDR3 of the VH, and the VL comprises CDR1, CDR2, and CDR3 of the VL; - CDR1 of VH comprises the amino acid sequence of SEQ ID NO: 18; - CDR2 of VH comprises the amino acid sequence of SEQ ID NO: 19; - CDR3 of VH comprises the amino acid sequence of SEQ ID NO: 20; - CDR1 of VL comprises the amino acid sequence of SEQ ID NO: 21; - CDR2 of the VL comprises the amino acid sequence of SEQ ID NO: 22; and - the CDR3 of the VL comprises the amino acid sequence of SEQ ID NO:23;

[0070] In some embodiments, the isolated antibody comprises a variable heavy domain (VH) and a variable light domain (VL), - VH comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% identity to SEQ ID NO: 14; and - VL comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% identity to SEQ ID NO:15.

[0071] 1. An isolated antibody or antigen-binding fragment thereof comprising a variable heavy domain (VH) and a variable light domain (VL), - VH comprises the amino acid sequence of SEQ ID NO: 14; and - VL comprises the amino acid sequence of SEQ ID NO: 15.

[0072] In some embodiments, the isolated antibody is capable of binding to a polypeptide comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the amino acid sequence of the polypeptide consists of SEQ ID NO:3.

[0073] In some embodiments, the antibody comprises an antigen-binding, hi some embodiments, the antibody fragment comprises a single chain Fv (scFv), a F(ab) fragment, a F(ab')2 fragment, or an isolated VH domain.

[0074] In some embodiments, the isolated monoclonal antibodies described herein further comprise a heavy and / or light chain constant region.

[0075] In some embodiments, the isolated monoclonal antibodies described herein further comprise human heavy and / or light chain constant regions.

[0076] In some embodiments, the heavy chain constant region is selected from the group consisting of human immunoglobulin IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0077] In some embodiments, the heavy chain constant region comprises a native amino acid sequence.

[0078] In some embodiments, the heavy chain constant region comprises a non-native variant amino acid sequence.

[0079] In one embodiment, the antibody described herein is a recombinant antibody, a chimeric antibody, a bispecific antibody, a trispecific antibody, or a multispecific antibody. In one embodiment, the antibody fragment comprises a single chain Fv (scFv), a F(ab) fragment, a F(ab')2 fragment, or an isolated VH domain.

[0080] In some embodiments, the antibodies described herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In some embodiments, one of the binding specificities is for a peptide having the sequence of SEQ ID NO:1, and the other is for a peptide having the sequence of SEQ ID NO:3. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0081] Techniques for producing multispecific antibodies, e.g., bispecific antibodies, include, but are not limited to, recombinant coexpression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature, 305:537 (1983)), WO93 / 08829, and Traunecker A., ​​et al., EMBO J. 10:3655 (1991)), and "knobs-in-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be produced by engineering electrostatic steering effects to create antibody Fc-heterodimeric molecules (WO 2009 / 089004 A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to generate bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 1999, 11:1112-1114 (1993)). al., J. Immunol., 152:5368 (1994); and by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991). Engineered antibodies with three or more functional antigen binding sites, including "Octopus antibodies" or dual variable domain (DVD) immunoglobulins, are also included herein (see, for example, US 2006 / 0025576 A1 and U.S. Patent No. 10,093,733). The antibodies or fragments disclosed herein also include "dual acting Fabs" or "DAFs" that contain antigen binding sites that bind different epitopes.

[0082] In one embodiment, the antibodies described herein comprise heavy and / or light chain constant regions. In one embodiment, the antibodies described herein comprise human heavy and / or light chain constant regions. In one embodiment, the heavy chain constant region is the constant region of human immunoglobulin IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. In one embodiment, the heavy chain constant region is the constant region of human immunoglobulin IgG1. In one embodiment, the heavy chain constant region comprises a native amino acid sequence.

[0083] In another aspect, provided herein is an antibody that binds to an epitope of a peptide having a sequence of SEQ ID NO: 1 or 3 that is the same as or overlaps with an antibody described herein (e.g., 133D7-1, or 133D7-1). In certain embodiments, the epitope of the antibody can be determined by, for example, NMR spectroscopy, X-ray crystallography, negative staining and cryo-EM (e.g., Lin M, et al., J Am Soc Mass Spectrom. 5:961-971 (2018); Rantalaien et al., Cell Rep. 23(11); 3249-3261 (2018); Torrents de la Pena A et al., PLoS. Pathog. 15; 15(7): e1007920 (2019)), ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization may be achieved using any of the methods known in the art (e.g., Giege R, et al., (1994), Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A(1990) Eur J Biochem 189:1-23; Chayen NE(1997) Structure 5:1269-1274; McPherson A(1976) J Biol Chem 251:6300-6303).Antibody:antigen crystals may be examined using well-known X-ray diffraction techniques and may be probed using computer software such as Phenix (Adams et al., Acta Crystallogr Biol Crystallogr D66,213-221(2010)) and BUSTER (Bricogne G(1993)Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G(1997)Meth.Enzymol.276A:361-423,ed Carter CW; Roversi P et al.,(2000)Acta Crystallogr.D Biol.Crystallogr.56(Pt 10):1316-1323). Mutagenesis mapping studies may be accomplished using any method known to those skilled in the art. For example, see Champe M et al., (1995), supra, and Cunningham BC & Wells JA (1989), supra, for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques. In a specific embodiment, the epitope of an antibody is determined using alanine scanning mutagenesis. Typically, when residues in the epitope are substituted with alanine, binding to the antigen is reduced or abolished. In one embodiment, when residues in the epitope are substituted with alanine, the K of binding to the antigen is reduced. D is increased by about 5-fold, 10-fold, 20-fold, 10-fold or more. In one embodiment, binding affinity is determined by ELISA. In addition, antibodies that recognize and bind to the same or overlapping epitopes can be identified using routine techniques such as immunoassays, for example, by demonstrating the ability of one antibody to block the binding of another antibody to a target antigen, i.e., competitive binding assays.

[0084] In one embodiment, the antibodies described herein immunospecifically bind to 133D7-1 or an epitope that overlaps with the epitope to which 133D7-1 binds.

[0085] In some embodiments, the antibodies described herein are capable of competing with 133D7-1 or 133D7-1 for binding to a peptide having the sequence of SEQ ID NO: 1 or 3, respectively.

[0086] In certain embodiments, epitopes of the antibodies described herein are used as immunogens to generate antibodies.

[0087] In one aspect, provided herein are methods for generating engineered variants of the antibodies described herein. In some embodiments, methods for generating engineered variants include directed evolution and yeast display. Methods for generating engineered antibodies are known to those of skill in the art, for example, as described in PCT / US2019 / 43578, filed July 26, 2019, which is incorporated by reference in its entirety for all purposes. In some embodiments, the engineered antibody possesses one or more improved properties, e.g., a higher binding affinity for a target antigen compared to the parent antibody.

[0088] In some embodiments, the method of generating an engineered variant of a parent antibody comprises substituting one or more amino acid residues in the VH and / or substituting one or more amino acid residues in the VL to create the engineered variant antibody and producing the engineered variant antibody. In some embodiments, the parent antibody is an antibody described herein. In some embodiments, the parent antibody is 130D2-1 or 133D7-1. In some embodiments, the method further comprises determining that the engineered variant antibody has improved properties, for example, by determining the binding affinity of the engineered variant antibody for a target antigen compared to the parent antibody.

[0089] The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method known in the art, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), biolayer interferometry (BLI) assay, radioimmunoassay (RIA), or kinetic analysis (e.g., BIACORE™ analysis). Competitive binding assay formats can be readily employed, as can direct binding assays. (See, e.g., Berzofsky, et al., "Antibody-Antigen Interactions", In Fundamental Immunology, Paul, WE, Ed., Raven Press: New York, NY (1984); Kuby, Janis Immunology, WH Freeman and Company: New York, NY (1992); and methods described therein. The measured affinity of a particular antibody-antigen interaction may vary when measured under different conditions (e.g., salt concentration, pH, temperature). Thus, affinity and other antigen binding parameters (e.g., K D or Kd, K on , K off Measurements of ) are made using standardized solutions of antibody and antigen, and standardized buffers, such as those described herein, as known in the art.

[0090] In some embodiments, the peptide-specific antibodies described herein are monoclonal antibodies. Monoclonal antibodies can be prepared using hybridoma methods such as those described by Kohler and Milstein (1975) Nature 256:495. Using the hybridoma method, a host (e.g., a mouse) is immunized to induce lymphocyte production of antibodies that specifically bind to the immunizing antigen. Lymphocytes can also be immunized in vitro. After immunization, lymphocytes are isolated and fused with a suitable myeloma cell line, for example, using polyethylene glycol, to form hybridoma cells, which can then be selected from unfused lymphocytes and myeloma cells. Hybridomas that produce monoclonal antibodies specifically directed against the selected antigen, as determined by immunoprecipitation, immunoblotting, or by in vitro binding assays (e.g., radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA)), can then be grown in in vitro culture using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986), or in vivo in animals as ascites tumors. The monoclonal antibodies can then be purified from the culture medium or ascites fluid using any method known in the art.

[0091] In some embodiments, the antibodies described herein are monoclonal antibodies. Monoclonal antibodies can be produced using recombinant DNA methods, for example, as described in U.S. Pat. No. 4,816,567. Polynucleotides encoding monoclonal antibodies can be amplified from a suitable source or chemically synthesized. The isolated polynucleotides encoding the heavy and light chains are then cloned into a suitable expression vector, which is transfected into host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, that do not otherwise produce immunoglobulin proteins, and the host cells produce the monoclonal antibodies.

[0092] The polynucleotide(s) encoding a monoclonal antibody can be modified in many different ways using recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant domains of the light and heavy chains can be replaced with non-immunoglobulin polypeptides to generate fusion antibodies. In some embodiments, the constant regions are truncated or removed to generate desired antibody fragments of the monoclonal antibody. Site-directed or high-density mutagenesis of the variable regions can be used to optimize the specificity, affinity, etc. of the monoclonal antibody.

[0093] Methods for engineering antibodies can also be used and are well known in the art. Engineered antibodies can have one or more amino acid residues substituted, deleted, or inserted. These sequence modifications can be used to reduce, enhance, or modify binding, affinity, on-rate, off-rate, avidity, specificity, or any other suitable property, as known in the art. Antibodies can also be engineered to eliminate developmental disadvantages by changing or removing sequence elements that are targets for post-translational modifications, such as glycosylation sites, oxidation sites, or deamination sites. In general, CDR residues are directly and most substantially involved in influencing antigen binding. Thus, some or all of the CDR sequences can be maintained, while variable framework and constant regions can be engineered by introducing substitutions, insertions, or deletions.

[0094] The antibodies disclosed herein can also be optionally engineered to retain high affinity for the antigen and other favorable biological properties. To achieve this goal, engineered antibodies can be prepared by a process of analyzing the parental sequences and the various conceptual engineered products using three-dimensional models of the parental and engineered sequences. Three-dimensional immunoglobulin models are publicly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display the predicted three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, framework (FR) residues can be selected and combined from the consensus and import sequences such that desired antibody properties, such as high affinity for the target antigen(s), are achieved.

[0095] In certain embodiments, antibody fragments are provided. Various techniques are known for producing antibody fragments. Traditionally, these fragments are obtained via proteolytic digestion of intact antibodies (e.g., Morimoto et al., 1993, Journal of Biochemical and Biophysical Methods 24:107-117; Brennan et al., 1985, Science, 229:81). In certain embodiments, the antibody fragments are produced recombinantly. Fab, Fv, and scFv antibody fragments can all be expressed and secreted in E. coli or other host cells, allowing for the production of large amounts of these fragments. Such antibody fragments can also be isolated from antibody phage libraries. The antibody fragments can be linear antibodies, e.g., as described in U.S. Pat. No. 5,641,870, and can be monospecific or bispecific. Other techniques for producing antibody fragments will be apparent to those skilled in the art.

[0096] In certain embodiments, the variable domains in both the heavy and light chains are modified by at least partial replacement of one or more CDRs and, optionally, partial framework region replacement and sequence changes. The CDRs may be derived from antibodies of the same class or even subclass as the antibody from which the framework regions are derived, but it is envisaged that the CDRs may be derived from antibodies of different classes, and in certain embodiments, from different species. To transfer the antigen binding capacity of one variable domain to another, it may not be necessary to replace all of the CDRs with the complete CDRs from the donor variable region. Rather, it may only be necessary to transfer those residues that are necessary to maintain the activity of the antigen binding site. Given the explanations given in U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, obtaining functional antibodies with reduced immunogenicity will be well within the capabilities of one of ordinary skill in the art by performing routine experimentation or by trial and error testing.

[0097] The present invention further includes variants and equivalents that are substantially homologous to the antibodies or antibody fragments thereof described herein. These can contain, for example, conservative substitution mutations, i.e., the replacement of one or more amino acids with similar amino acids. For example, a conservative substitution refers to the replacement of one amino acid with another amino acid within the same general class, such as, for example, an acidic amino acid with another acidic amino acid, a basic amino acid with another basic amino acid, a neutral amino acid with another neutral amino acid, etc. It is well known in the art what is meant by a conservative amino acid substitution.

[0098] In certain aspects, provided herein are compositions comprising the antibodies disclosed herein. In some embodiments, the compositions are pharmaceutical compositions further comprising a pharma- ceutically acceptable excipient. In some embodiments, the antibodies comprise antigen-binding antibody fragments.

[0099] In certain aspects, provided herein is an affinity resin comprising an antibody disclosed herein and a solid support. In some embodiments, the solid support comprises beads, gelatin, or agarose. In some embodiments, the solid support comprises magnetic beads. In some embodiments, the antibody is attached to the solid support by a covalent bond. In some embodiments, the antibody is attached to the solid support by a non-covalent association. In some embodiments, the antibody comprises an antigen-binding antibody fragment.

[0100] Polynucleotides In certain aspects, provided herein are polynucleotides comprising a nucleotide sequence or sequences encoding an antibody (e.g., a variable light chain and / or a variable heavy chain region) or an antigen-binding fragment thereof described herein, and vectors, e.g., vectors comprising such polynucleotides. In one embodiment, the vectors can be used for recombinant expression of the antibodies described herein in host cells (e.g., E. coli and mammalian cells). In some embodiments, the antibody comprises the six complementarity determining regions (CDRs) of 130D2-1. In some embodiments, the antibody comprises the VH and VL domains of 130D2-1. In some embodiments, the antibody is 130D2-1. In some embodiments, the antibody comprises the six complementarity determining regions (CDRs) of 133D7-1. In some embodiments, the antibody comprises the VH and VL domains of 133D7-1. In some embodiments, the antibody is 133D7-1. In some embodiments, the CDRs are according to Kabat. In some embodiments, the antibody comprises an antigen-binding antibody fragment.

[0101] In one aspect, provided herein is an isolated polynucleotide encoding the heavy chain variable region, or heavy chain, of an antibody described herein.

[0102] In one aspect, provided herein is an isolated polynucleotide encoding the light chain variable region or light chain of an antibody described herein.

[0103] In one aspect, provided herein are isolated polynucleotides encoding the heavy chain variable region, or heavy chain, of an antibody described herein, and the light chain variable region, or light chain, of an antibody described herein.

[0104] In some embodiments, the polynucleotides encoding the VH domain and the VL domain comprise the nucleotide sequences of SEQ ID NOs: 6 and 7, respectively. In some embodiments, the polynucleotides encoding the VH domain and the VL domain comprise the nucleotide sequences of SEQ ID NOs: 16 and 17, respectively.

[0105] In certain aspects, provided herein are polynucleotides comprising nucleotide sequences encoding light and heavy chains, e.g., antibodies comprising separate light and heavy chains. With respect to the light chain, in a specific embodiment, the polynucleotides provided herein comprise a nucleotide sequence encoding a kappa light chain. In another specific embodiment, the polynucleotides provided herein comprise a nucleotide sequence encoding a lambda light chain. In yet another specific embodiment, the polynucleotides provided herein comprise a nucleotide sequence encoding an antibody described herein comprising a human kappa light chain or a human lambda light chain. For example, the human constant region sequence can be one described in U.S. Pat. No. 5,693,780.

[0106] In certain embodiments, the polynucleotides provided herein comprise a nucleotide sequence encoding an antibody described herein, wherein the antibody comprises a heavy chain and the constant region of the heavy chain comprises the amino acid sequence of a human alpha or gamma heavy chain constant region.

[0107] In yet another specific embodiment, a polynucleotide provided herein comprises a nucleotide sequence encoding an antibody described herein, wherein the antibody comprises a VL domain and a VH domain comprising any of the amino acid sequences described herein, and the constant region comprises the amino acid sequence of the constant region of human IgA1, human IgA2, human IgG1 (e.g., allotype 1, 17, or 3), human IgG2, or human IgG4.

[0108] In yet another specific embodiment, the polynucleotides provided herein include nucleotide sequences encoding the antibodies described herein that have been optimized, for example, by codon / RNA optimization, replacement with a heterologous signal sequence, and removal of mRNA instability elements. Methods for generating optimized nucleic acids encoding antibodies or fragments thereof (e.g., light chain, heavy chain, VH domain, or VL domain) that bind to a peptide having the sequence of SEQ ID NO: 1 or 3 by introducing codon changes in the mRNA and / or removing inhibitory regions for recombinant expression can be performed, for example, by adapting the optimization methods described in U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498. For example, potential splice sites and instability elements (e.g., A / T or A / U rich elements) in the RNA can be mutated to increase the stability of the RNA for recombinant expression without changing the amino acids encoded by the nucleic acid sequence. This alteration takes advantage of the degeneracy of the genetic code, e.g., the use of alternative codons for the same amino acid. In some embodiments, conservative variations, e.g., altering one or more codons to encode a similar amino acid having similar chemical structure and properties and / or function as the original amino acid, may be desirable.

[0109] Polynucleotides can be obtained by any method known in the art, and the nucleotide sequence of the polynucleotide can be determined. The nucleotide sequences encoding the antibodies described herein, and modified versions of these antibodies, can be determined using methods well known in the art, i.e., nucleotide codons known to encode specific amino acids are constructed in such a way as to generate nucleic acids encoding the antibodies. Such polynucleotides encoding antibodies can be constructed from chemically synthesized oligonucleotides (e.g., as described in Kutmeier G et al., (1994), BioTechniques 17:242-246), which essentially involves synthesizing overlapping oligonucleotides containing portions of the antibody-encoding sequence, annealing and ligating those oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0110] If a clone containing a nucleic acid encoding a particular antibody or fragment thereof is not available, but the sequence of the antibody molecule or fragment thereof is known, nucleic acid encoding an immunoglobulin or fragment thereof can be chemically synthesized by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for a particular gene sequence to identify, for example, a cDNA clone from a cDNA library encoding the antibody, or can be obtained from a suitable source (e.g., an antibody cDNA library generated from any tissue or cell that expresses the antibody, such as hybridoma cells selected to express an antibody described herein, or a cDNA library, or nucleic acid isolated therefrom, preferably polyA+ RNA). The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector using any method well known in the art.

[0111] In certain aspects, provided herein are cells (e.g., host cells) that express (e.g., recombinantly) the antibodies described herein, and associated polynucleotides and expression vectors. Provided herein are vectors (e.g., expression vectors) that include a polynucleotide that includes a nucleotide sequence encoding an antibody or antigen-binding fragment thereof described herein. In one embodiment, the vector can be used for recombinant expression of an antibody described herein in a host cell (e.g., a mammalian cell). Also provided herein are host cells that include such vectors for recombinantly expressing an antibody described herein. In certain aspects, provided herein are methods for producing an antibody described herein, including expressing such an antibody in a host cell. In some embodiments, the antibody includes the six complementarity determining regions (CDRs) of 130D2-1. In some embodiments, the antibody includes the VH and VL domains of 130D2-1. In some embodiments, the antibody is 130D2-1. In some embodiments, the antibody includes the six complementarity determining regions (CDRs) of 133D7-1. In some embodiments, the antibody comprises the VH and VL domains of 133D7-1. In some embodiments, the antibody is 133D7-1. In some embodiments, the CDRs are according to Kabat. In some embodiments, the antibody comprises an antigen-binding antibody fragment.

[0112] In certain aspects, provided herein is an isolated vector comprising a polynucleotide described herein.

[0113] In certain aspects, provided herein is a host cell comprising a polynucleotide described herein or a vector described herein. In one embodiment, the vector encodes an antibody described herein. In one embodiment, the vector described herein comprises a first vector encoding a VH described herein and a second vector encoding a VL described herein. In one embodiment, the vector described herein comprises a first nucleotide sequence encoding a VH described herein and a second nucleotide sequence encoding a VL described herein. In some embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs: 4 and 5. In some embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs: 14 and 15. In some embodiments, the polynucleotides encoding the VH and VL domains comprise the nucleotide sequences of SEQ ID NOs: 6 and 7, respectively. In some embodiments, the polynucleotides encoding the VH and VL domains comprise the nucleotide sequences of SEQ ID NOs: 16 and 17, respectively.

[0114] In one embodiment, the host cell is selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NS0, PER-C6, HEK-293T, NIH-3T3, Helga, BHK, Hep G2, SP2 / 0, R1.1, BW, LM, COS 1, COS 7, BSC1, BSC40, BMT10 cells in tissue culture, plant cells, insect cells, and human cells. In one embodiment, the host cell is CHO.

[0115] In certain aspects, provided herein are methods of producing an antibody described herein (e.g., 130D2-1 and 133D7-1), comprising culturing a host cell described herein such that a polynucleotide is expressed and an antibody is produced. In one embodiment, the method further comprises recovering the antibody.

[0116] The isolated polypeptides, i.e., antibodies described herein, can be produced by any suitable method known in the art. Such methods range from direct protein synthesis to constructing DNA sequences encoding the isolated polypeptide sequences and expressing those sequences in a suitable transformed host. In some embodiments, DNA sequences are constructed by isolating or synthesizing DNA sequences encoding the wild-type protein of interest using recombinant techniques. Optionally, the sequence can be mutated by site-directed mutagenesis to provide a functional analog thereof. See, e.g., Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81:5662-5066 (1984) and U.S. Pat. No. 4,588,585.

[0117] In some embodiments, the DNA sequence encoding the polypeptide of interest will be constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide, selecting the codons that are preferred in the host cell in which the recombinant polypeptide of interest is produced. Standard methods can be applied to synthesize isolated polynucleotide sequences encoding the isolated polypeptide of interest. For example, the complete amino acid sequence can be used to construct a reverse-translated gene. In addition, DNA oligomers can be synthesized that contain the nucleotide sequence that codes for a particular isolated polypeptide. For example, several small oligonucleotides that code for portions of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides usually contain 5' or 3' overhangs for complementary construction.

[0118] Once constructed (by synthesis, site-directed mutagenesis or otherwise), the polynucleotide sequence encoding the particular isolated polypeptide of interest is inserted into an expression vector and operably linked to appropriate expression control sequences for expression of the protein in a desired host. Proper construction can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene needs to be operably linked to transcriptional and translational expression control sequences that are functional in the selected expression host.

[0119] In certain embodiments, recombinant expression vectors are used to amplify and express DNA encoding an antibody or fragment thereof. A recombinant expression vector is a replicable DNA construct having a synthetic or cDNA-derived DNA fragment encoding a polypeptide chain of an antibody or fragment thereof operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. A transcription unit generally comprises a collection of (1) a genetic element or elements that play a role in regulating gene expression, such as a transcriptional promoter or enhancer, (2) a structural or coding sequence that is transcribed into mRNA and translated into protein, and (3) appropriate transcriptional and translational initiation and termination sequences. Such regulatory elements may include operator sequences to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants may further be incorporated. DNA regions are operably linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if it is expressed as a precursor that participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the coding sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to allow translation. Structural elements intended for use in yeast expression systems include leader sequences that allow extracellular secretion of the translated protein by the host cell. Alternatively, if the recombinant protein is expressed without a leader or transport sequence, the protein can include an N-terminal methionine residue, which can optionally then be cleaved from the expressed recombinant protein to provide the final product.

[0120] The choice of expression control sequence and expression vector will depend on the choice of host. A variety of host-expression vector systems can be utilized to express the antibody molecules described herein (see, e.g., U.S. Pat. No. 5,807,715). Such host-expression systems represent vehicles in which the coding sequence of interest can be produced and subsequently purified, but also represent cells which, when transformed or transfected with the appropriate nucleotide coding sequence, can express the antibody molecules described herein in situ. These include microorganisms, such as bacteria (e.g., Escherichia coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody coding sequences; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing the antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody coding sequences; plant cell systems (e.g., Chlamydomonas reinhardtii) infected with recombinant viral expression vectors (e.g., Cauliflower Mosaic Virus, CaMV; Tobacco Mosaic Virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody coding sequences. reinhardtii); or mammalian cell lines harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoters) or mammalian viruses (e.g., adenovirus late promoters; vaccinia virus 7.5K promoters) (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, Helga, and NIH3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells). In a specific embodiment, the cells for expressing the antibodies described herein are CHO cells, e.g., CHO cells from the CHO GS System™ (Lonza).In certain embodiments, the cells for expressing the antibodies described herein are human cells, e.g., human cell lines. In specific embodiments, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In certain embodiments, bacterial cells, such as E. coli, particularly for the expression of whole recombinant antibody molecules, or eukaryotic cells (e.g., mammalian cells) are used for the expression of recombinant antibody molecules. For example, mammalian cells, such as Chinese hamster ovary (CHO) cells, in combination with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus, are effective expression systems for antibodies (Foecking MK & Hofstetter H (1986) Gene 45:101-105; and Cockett MI et al., (1990) Biotechnology 8:662-667). In certain embodiments, the antibodies described herein are produced by CHO cells or NS0 cells. In certain embodiments, expression of the nucleotide sequence encoding the antibodies described herein is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0121] Suitable host cells for expressing a polypeptide of interest, such as an antibody described herein, include prokaryotic, yeast, insect, or higher eukaryotic cells under the control of a suitable promoter. Prokaryotes include gram-negative or gram-positive organisms, such as E. coli or Bacillus. Higher eukaryotic cells include established cell lines of mammalian origin. Cell-free translation systems may also be employed. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985), the relevant disclosures of which are incorporated herein by reference. Additional information regarding methods of protein production, including antibody production, can be found, for example, in U.S. Patent Publication No. 2008 / 0187954, U.S. Patent Nos. 6,413,746 and 6,660,501, and International Patent Publication No. WO04009823, each of which is incorporated herein by reference in its entirety.

[0122] Various mammalian or insect cell culture systems are also advantageously employed to express recombinant proteins such as the antibodies described herein. Expression of recombinant proteins in mammalian cells is feasible since such proteins are generally correctly folded, appropriately modified, and fully functional. Examples of suitable mammalian host cell lines include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK293, NIH3T3, W138, BT483, Hs578T, HTB2, BT2O, and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells. Mammalian expression vectors can include nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking nontranscribed sequences, as well as 5' or 3' nontranslated sequences such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences. Baculovirus systems for producing heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio / Technology 6:47 (1988).

[0123] The protein produced by the transformed host can be purified according to any suitable method. Such standard methods include chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Affinity tags such as hexahistidine, maltose binding domain, influenza HA peptide sequence, and glutathione-S-transferase can be attached to the protein to allow easy purification by passing through an appropriate affinity column. Techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography can also be used to physically characterize the isolated protein.

[0124] For example, supernatants from systems that secrete recombinant proteins, such as antibodies, into culture media can first be concentrated using commercially available protein concentration filters, such as Amicon or Millipore Pellicon ultrafiltration units. After the concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate with pendant diethylaminoethyl (DEAE) groups. The matrix can be acrylamide, agarose, dextran, cellulose, or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices that contain sulfopropyl or carboxymethyl groups. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, such as silica gel with pendant methyl or other aliphatic groups, can be employed to further purify the agent. Some or all of the aforementioned purification steps can also be employed in various combinations to result in homogeneous recombinant proteins.

[0125] Recombinant proteins produced in bacterial culture can be isolated, for example, by first extracting them from a cell pellet, followed by one or more concentration, salting out, aqueous ion exchange or size exclusion chromatographic steps. A final purification step can employ high performance liquid chromatography (HPLC). Microbial cells employed for expression of recombinant proteins can be disrupted by any convenient method, including freeze-thaw cycles, sonication, mechanical disruption, or the use of cell lysing agents.

[0126] Methods known in the art for purifying antibodies and other proteins include, for example, those described in U.S. Patent Publication Nos. 2008 / 0312425, 2008 / 0177048, and 2009 / 0187005, each of which is incorporated by reference herein in its entirety.

[0127] In specific embodiments, the antibodies described herein are isolated or purified. Generally, an isolated antibody is one that is substantially free of other antibodies having antigen specificity different from the isolated antibody. For example, in certain embodiments, preparations of antibodies described herein are substantially free of cellular material and / or chemical precursors. The language "substantially free of cellular material" includes preparations of antibodies in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes preparations of antibodies having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins") and / or antibody variants (e.g., various post-translationally modified forms of the antibody). When a polypeptide (e.g., an antibody described herein) is recombinantly produced, it is also generally substantially free of culture medium, i.e., culture medium will be less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation. When a polypeptide (e.g., an antibody described herein) is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals involved in the synthesis of the protein. Thus, such preparations of a polypeptide (e.g., an antibody described herein) have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the polypeptide of interest. In one embodiment, the antibodies described herein are isolated or purified.

[0128] Methods for detecting, isolating, or quantifying peptides or proteins In certain aspects, provided herein are methods for detecting, isolating or quantitating a peptide in a sample comprising contacting a peptide with an antibody disclosed herein, wherein the amino acid sequence of the peptide comprises SEQ ID NO:1 or SEQ ID NO:3. In some embodiments, the amino acid sequence of the peptide comprises SEQ ID NO:1. In some embodiments, the amino acid sequence of the peptide comprises SEQ ID NO:3. In some embodiments, the sample comprises a protease-digested protein isolate. In some embodiments, the protein isolate is obtained from a subject, e.g., from skeletal muscle tissue of the subject. In some embodiments, the protease is trypsin. In some embodiments, the sample comprises a peptide comprising SEQ ID NO:1 and a peptide comprising SEQ ID NO:3.

[0129] In certain aspects, provided herein are methods for detecting or quantitating a recombinant polypeptide in a sample comprising a protein isolate digested with a protease, e.g., trypsin, the method comprising contacting the peptide with an antibody disclosed herein, wherein the amino acid sequence of the recombinant polypeptide comprises SEQ ID NO:28 and / or SEQ ID NO:29.

[0130] In certain aspects, provided herein are methods for detecting or quantifying expression of dystrophin and / or micro-dystrophin in a subject. In some embodiments, the method comprises contacting a sample with an antibody disclosed herein, the sample comprising a protein isolate digested with a protease, e.g., trypsin. In some embodiments, the dystrophin is full-length dystrophin. In some embodiments, the dystrophin is endogenous dystrophin. In some embodiments, the dystrophin is recombinant dystrophin. In some embodiments, the dystrophin is mutated or engineered dystrophin. In some embodiments, the dystrophin is mutated or engineered dystrophin that binds to anti-LLQ mAb but not to anti-LEM mAb. In some embodiments, the micro-dystrophin binds to anti-LLQ mAb but not to anti-LEM mAb. In some embodiments, the micro-dystrophin has the amino acid sequence of SEQ ID NO: 27. In some embodiments, the sample comprises a protease, e.g., trypsin-digested protein isolate from a subject administered a recombinant polynucleotide encoding micro-dystrophin, e.g., a recombinant adeno-associated virus comprising a polynucleotide encoding micro-dystrophin. In some embodiments, the sample comprises protease (e.g., trypsin) digested micro-dystrophin and protease-digested dystrophin, where protease digestion of micro-dystrophin releases the LEM peptide of SEQ ID NO: 1. In some embodiments, the methods described herein are used to simultaneously detect or quantitate micro-dystrophin and dystrophin in a sample.

[0131] In some embodiments, provided herein are methods for isolating a peptide comprising the amino acid sequence of SEQ ID NO: 1 or 3 from a sample via immunoaffinity purification, ligand binding assay (LBA), or equivalent assay. In some embodiments, the method for isolating a peptide from a sample comprises (a) contacting the sample comprising the peptide with a composition comprising an antibody described herein under conditions that allow binding of the peptide to the antibody, (b) removing the portion of the sample that is not bound to the antibody, and (c) dissociating the peptide from the antibody. In some embodiments, the composition comprising the antibody comprises an affinity resin comprising a solid support and an antibody described herein. In some embodiments, the solid support is selected from the group consisting of beads, gelatin, or agarose. In some embodiments, the solid support is a magnetic bead. In some embodiments, the antibody is attached to the solid support by a covalent bond. In some embodiments, the antibody is attached to the solid support by a non-covalent association. In some embodiments, the antibody comprises a tag, e.g., biotin, a hexahistidine tag, or a FLAG tag, to facilitate purification of the peptide. In some embodiments, the sample comprises a peptide comprising the amino acid sequence of SEQ ID NO: 1 and a peptide comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the methods described herein are used to simultaneously isolate a peptide comprising the amino acid sequence of SEQ ID NO:1 and a peptide comprising the amino acid sequence of SEQ ID NO:3.

[0132] In some embodiments of the methods of isolating a peptide comprising the amino acid sequence of SEQ ID NO:1 described herein, the antibody comprises six CDRs comprising the amino acid sequences of SEQ ID NOs:8-12 and 13. In some embodiments, the antibody comprises the VH and VL of SEQ ID NOs:4 and 5, respectively.

[0133] In some embodiments of the methods of isolating a peptide comprising the amino acid sequence of SEQ ID NO:3 described herein, the antibody comprises six CDRs comprising the amino acid sequences of SEQ ID NOs:18-22 and 23. In some embodiments, the antibody comprises the VH and VL of SEQ ID NOs:14 and 15, respectively.

[0134] In some embodiments of the methods for isolating a peptide comprising the amino acid sequence of SEQ ID NO: 1 or 3 described herein, the antibody is a polyclonal antibody described herein, such as pAb#7684-A.

[0135] In some embodiments, provided herein is a method of quantifying a peptide comprising the amino acid sequence of SEQ ID NO: 1 or 3 in a sample, comprising contacting the peptide with an antibody described herein. In some embodiments, the method comprises a radioimmunoassay, an immunoaffinity (IA) assay, or a ligand binding assay (LBA). In some embodiments, the method comprises isolating the peptide from the sample and determining the amount of peptide recovered. In some embodiments, the method comprises (a) contacting a sample comprising the peptide with a composition comprising an antibody described herein under conditions that allow binding of the peptide to the antibody, (b) removing a portion of the sample that is not bound to the antibody, (c) recovering the peptide, and (d) determining the amount of peptide recovered in step (c). In some embodiments, the amount of peptide is determined by LC / MS or LC-MS / MS. In some embodiments, the sample further comprises a stable isotope-labeled peptide standard comprising the amino acid sequence of SEQ ID NO: 1 or 3. In some embodiments, the method provides absolute quantification of the peptide. In some embodiments, the method provides relative quantification of the peptide. In some embodiments, the antibody is a monoclonal antibody disclosed herein (e.g., 130D2-1 or 133D7-1). In some embodiments, the antibody is a polyclonal antibody disclosed herein, e.g., pAb#7684-A. In some embodiments, the methods described herein are used to simultaneously quantify a peptide comprising the amino acid sequence of SEQ ID NO:1 and a peptide comprising the amino acid sequence of SEQ ID NO:3 in a sample.

[0136] In some embodiments, the sample comprises a protease-digested protein isolate obtained from a subject. In some embodiments, the sample comprises a protease-digested protein isolate obtained from skeletal muscle tissue of a subject. In some embodiments, the skeletal muscle is gastrocnemius, quadriceps, or diaphragm. In some embodiments, the sample comprises a protease-digested protein isolate obtained from cardiac muscle of a subject. In some embodiments, the protease comprises trypsin. In some embodiments, the subject is a human subject, a primate subject, or a murine subject. In some embodiments, the subject has been administered a recombinant polypeptide comprising the amino acid sequence of SEQ ID NO:28 and / or 29. In some embodiments, the subject has been administered a recombinant polynucleotide encoding a recombinant polypeptide comprising the amino acid sequence of SEQ ID NO:28 and / or 29. In some embodiments, the subject has been administered a recombinant virus comprising a polynucleotide encoding a recombinant polypeptide comprising the amino acid sequence of SEQ ID NO:28 and / or 29. In some embodiments, the recombinant virus is a recombinant adeno-associated virus. In some embodiments, the recombinant polypeptide is microdystrophin. In some embodiments, the microdystrophin comprises the amino acid sequence of SEQ ID NO:27.

[0137] In some embodiments, provided herein are methods for detecting or quantifying a recombinant polypeptide in a sample, wherein the amino acid sequence of the recombinant polypeptide comprises SEQ ID NO:28 and / or 29. One of skill in the art will appreciate that protease digestion, e.g., trypsin digestion, of a polypeptide having an amino acid sequence comprising SEQ ID NO:28 and 29 will release peptides of SEQ ID NO:1 and 3, respectively. One of skill in the art will further appreciate that the methods described herein for detecting or quantifying a peptide having an amino acid sequence of SEQ ID NO:1 or 3 can also be used to detect or quantify a recombinant polypeptide having an amino acid sequence comprising SEQ ID NO:28 and / or 29, based on the fact that protease digestion of the recombinant polypeptide will release peptides of SEQ ID NO:1 and / or 3, respectively. In some embodiments, the recombinant polypeptide is microdystrophin comprising the amino acid sequence of SEQ ID NO:27.

[0138] In some embodiments, provided herein is a method of quantifying a level of a recombinant polypeptide in a subject, comprising: (a) providing a sample comprising a protease-digested protein isolate obtained from a subject, the sample comprising one or more peptides having an amino acid sequence of SEQ ID NO: 1 or 3; (b) contacting the sample with a composition comprising an antibody described herein under conditions that allow binding of the antibody to the peptides; (c) recovering peptides bound to the antibody; and (d) determining the amount of peptide recovered in step (c). In some embodiments, the amount of peptide is determined by LC / MS or LC-MS / MS. In some embodiments, the sample comprises a protease-digested protein isolate obtained from skeletal muscle tissue of the subject. In some embodiments, the subject is a human subject, a primate subject, or a murine subject. In some embodiments, the subject has been administered a recombinant polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and / or 29. In some embodiments, the subject has been administered a recombinant polynucleotide encoding a recombinant polypeptide comprising an amino acid sequence of SEQ ID NO: 28 and / or 29. In some embodiments, the subject has been administered a recombinant virus comprising a polynucleotide encoding a recombinant polypeptide comprising the amino acid sequence of SEQ ID NO:28 and / or 29. In some embodiments, the recombinant virus is a recombinant adeno-associated virus. In some embodiments, the recombinant polypeptide is microdystrophin. In some embodiments, the microdystrophin comprises the amino acid sequence of SEQ ID NO:27. In some embodiments, the antibody is a monoclonal antibody disclosed herein (e.g., 130D2-1 or 133D7-1). In some embodiments, the antibody is a polyclonal antibody disclosed herein, e.g., pAb#7684-A.

[0139] In some embodiments, provided herein are methods for detecting or quantifying the expression of dystrophin and / or micro-dystrophin in a subject. Protease, e.g., trypsin digestion of dystrophin releases a peptide having the sequence of SEQ ID NO:3. Protease, e.g., trypsin digestion of micro-dystrophin, including the amino acid sequence of SEQ ID NO:27, releases a peptide having the sequence of SEQ ID NO:1 or 3. Those skilled in the art will appreciate that the methods described herein for detecting or quantifying a peptide having the amino acid sequence of SEQ ID NO:3 can be used to detect or quantify the levels of dystrophin and micro-dystrophin in a sample after protease, e.g., trypsin digestion of the sample. Those skilled in the art will further appreciate that the methods described herein for detecting or quantifying a peptide having the amino acid sequence of SEQ ID NO:1 can be used to detect or quantify the levels of micro-dystrophin in a sample after protease, e.g., trypsin digestion of the sample. Thus, using the methods described herein, those skilled in the art can monitor the expression of micro-dystrophin in a subject undergoing gene therapy, e.g., AAV-mediated gene therapy, to deliver a recombinant polynucleotide encoding micro-dystrophin. In some embodiments, the methods described herein are used to simultaneously detect or quantitate dystrophin and microdystrophin in a sample.

[0140] In some embodiments, provided herein is a method of quantifying dystrophin and / or micro-dystrophin expression levels in a subject, comprising: (a) providing a sample comprising a protease-digested protein isolate obtained from a subject, the sample comprising one or more peptides having an amino acid sequence of SEQ ID NO: 1 or 3; (b) contacting the sample with a composition comprising an antibody described herein under conditions that allow binding of the antibody to the peptides; (c) recovering peptides bound to the antibody; and (d) determining the amount of peptides recovered in step (c), wherein the amino acid sequence of micro-dystrophin comprises SEQ ID NO: 28 and / or 29. In some embodiments, the amount of peptides is determined by LC / MS or LC-MS / MS. In some embodiments, the method provides absolute quantification of dystrophin and / or micro-dystrophin expression levels. In some embodiments, the method provides relative quantification of dystrophin and / or micro-dystrophin expression levels. In some embodiments, the antibody is a monoclonal antibody disclosed herein (e.g., 130D2-1 or 133D7-1). In some embodiments, the antibody is a polyclonal antibody disclosed herein, e.g., pAb#7684-A. In some embodiments, the methods described herein are used to simultaneously quantify dystrophin and microdystrophin expression levels in a subject.

[0141] In some embodiments, provided herein is a method of quantifying dystrophin and / or microdystrophin expression levels in a subject comprising: (a) providing a sample comprising a protease-digested protein isolate obtained from a subject, the sample comprising one or more peptides having the amino acid sequence of SEQ ID NO:1 or 3; (b) contacting the sample with a composition comprising a first antibody described herein capable of binding to a LEM peptide (SEQ ID NO:1) under conditions that allow binding of the antibody to its peptide target; (c) recovering peptides bound to the antibody; and (d) determining the amount of peptides recovered in step (c). In some embodiments, provided herein is a method of quantifying the expression level of dystrophin and / or micro-dystrophin in a subject, comprising: (a) providing a sample comprising a protease-digested protein isolate obtained from a subject, the sample comprising one or more peptides having an amino acid sequence of SEQ ID NO: 1 or 3; (b) contacting the sample with a composition comprising (i) a first antibody described herein capable of binding to a LEM peptide (SEQ ID NO: 1), and (ii) a composition comprising a second antibody capable of binding to a protease-digested dystrophin peptide, under conditions that allow binding of the first and second antibodies to their respective peptide targets; (c) recovering peptides bound to the antibodies; and (d) determining the amount of each peptide recovered in step (c). In some embodiments, the amino acid sequence of micro-dystrophin comprises SEQ ID NO: 28 and / or 29. In some embodiments, the antibody capable of binding to the LEM peptide (SEQ ID NO: 1) comprises 130D2-1. In some embodiments, the antibody capable of binding to the protease-digested dystrophin peptide is capable of binding to the LLQ peptide (SEQ ID NO: 3). In some embodiments, the antibody capable of binding to a protease-digested dystrophin peptide is an antibody disclosed herein capable of binding to the LLQ peptide (SEQ ID NO: 3).In some embodiments, the antibody capable of binding to protease-digested dystrophin peptides comprises 133D7-1, 112E4-1, 115G6-1, or 121F10-1 antibody, or an antibody that binds to an epitope of a peptide having the same or overlapping sequence of SEQ ID NO:3 as the 133D7-1, 112E4-1, 115G6-1, or 121F10-1 antibody. In some embodiments, the antibody capable of binding to protease-digested dystrophin peptides comprises 133D7-1. In some embodiments, the antibody capable of binding to protease-digested dystrophin peptides also binds to protease-digested micro-dystrophin or engineered dystrophin peptides. Other antibodies or antigen-binding fragments thereof that bind to dystrophin are known in the art. Examples include, but are not limited to, ab275391 (Abcam), ab218198 (Abcam), ab15277 (Abcam), NCL-DYSB (Leica Biosystems). Additional antibodies that bind to dystrophin are disclosed at abcam.com and labome.com. In some embodiments, the amount of peptide is determined by LC / MS or LC-MS / MS. In some embodiments, the method provides absolute quantification of dystrophin and / or micro-dystrophin expression levels. In some embodiments, the method provides relative quantification of dystrophin and / or micro-dystrophin expression levels. In some embodiments, the method described herein is used to simultaneously quantify dystrophin and micro-dystrophin expression levels in a subject.

[0142] In some embodiments, the sample comprises a protease-digested protein isolate obtained from the subject's skeletal muscle tissue, hi some embodiments, the protease is trypsin.

[0143] In some embodiments, the subject is a human subject, a primate subject, or a mouse subject. In some embodiments, the subject is a human. In some embodiments, the subject is a primate. In some embodiments, the subject is a mouse. In some embodiments, the subject suffers from Duchenne muscular dystrophy.

[0144] In some embodiments, the subject is a non-human mammal that has been genetically engineered to contain one or more mutations in the dystrophin gene.

[0145] In some embodiments, the subject is administered a recombinant polynucleotide encoding micro-dystrophin comprising the amino acid sequence of SEQ ID NO:28 and 29. In some embodiments, the micro-dystrophin comprises the amino acid sequence of SEQ ID NO:27. In some embodiments, the recombinant polynucleotide is DNA. In some embodiments, the recombinant polynucleotide is RNA. In some embodiments, the RNA is mRNA comprising modified ribonucleotides.

[0146] In some embodiments, the subject has been administered a recombinant virus comprising a polynucleotide encoding microdystrophin comprising the amino acid sequence of SEQ ID NO: 28 and 29. In some embodiments, the microdystrophin comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the recombinant virus is a recombinant adeno-associated virus.

[0147] The antibodies described herein can be used to detect peptides comprising the amino acid sequence of SEQ ID NO: 1 or 3 in a sample, e.g., a biological sample, using classical immunological methods known to those skilled in the art, including immunoassays such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting. In one embodiment, the antibodies described herein are conjugated to a detectable label. Suitable assay labels are known in the art and include enzyme labels such as glucose oxidase; radioisotopes such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (121In), and technetium (99Tc); luminescent labels such as luminol; and fluorescent labels such as fluorescein, rhodamine, and biotin. Such labels can be used to label the antibodies described herein. Alternatively, a second antibody that recognizes the antibodies described herein can be labeled and used in combination with the antibodies described herein to detect peptides comprising the amino acid sequence of SEQ ID NO: 1 or 3.

[0148] In some aspects, provided herein is a method for detecting a peptide comprising an amino acid sequence of SEQ ID NO: 1 or 3 in a sample, comprising contacting the sample with an antibody described herein. In some embodiments, the antibody described herein can carry a detectable or functional label. The antibody described herein can carry a fluorescent label. Exemplary fluorescent labels include, for example, fluorescein, Texas Red, Alexa Fluor dyes, Cy dyes, and DyLight dyes. The antibody described herein can carry a radioactive label. When a radioactive label is used, currently available counting procedures known in the art may be utilized to identify and quantify specific binding of the antibody described herein. When the label is an enzyme, detection may be achieved by any of the currently available colorimetric, spectrophotometric, fluorospectrophotometric, amperometric, or gas analytical techniques known in the art. This may be achieved by contacting the sample or a control sample with an antibody described herein under conditions that allow for the formation of a complex between the antibody and the peptide comprising the amino acid sequence of SEQ ID NO: 1 or 3. Any complexes formed between the antibody and the peptide are detected and compared in the sample and the control. In some embodiments, the methods described herein are used to simultaneously detect a peptide comprising the amino acid sequence of SEQ ID NO:1 and a peptide comprising the amino acid sequence of SEQ ID NO:3 in a sample.

[0149] kit In certain aspects, provided herein are kits for detecting, isolating or quantitating a peptide in a sample, the kit comprising one or more antibodies disclosed herein capable of binding to a peptide having the amino acid sequence of SEQ ID NO: 1 or 3. In some embodiments, the kit further comprises an isolated peptide having the sequence of SEQ ID NO: 1 and / or an isolated peptide having the sequence of SEQ ID NO: 3. In some embodiments, the isolated peptide or peptides are stable isotope labeled peptides.

[0150] In certain aspects, provided herein are kits for detecting or quantifying a recombinant polypeptide in a sample, the kits comprising one or more antibodies disclosed herein capable of binding to a peptide having an amino acid sequence of SEQ ID NO: 1 or 3, wherein the amino acid sequence of the recombinant polypeptide comprises SEQ ID NO: 28 and / or 29. In some embodiments, the kits further comprise an isolated peptide having a sequence of SEQ ID NO: 1 and / or an isolated peptide having a sequence of SEQ ID NO: 3. In some embodiments, the kits further comprise a composition comprising the recombinant polypeptide, optionally a stable isotope labeled recombinant polypeptide. In some embodiments, the recombinant polypeptide is microdystrophin. In some embodiments, the microdystrophin comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the isolated peptide or peptides are stable isotope labeled peptides.

[0151] In certain aspects, provided herein are kits for detecting or quantifying dystrophin and / or micro-dystrophin expression in a subject, the kits comprising one or more antibodies disclosed herein capable of binding a peptide having an amino acid sequence of SEQ ID NO: 1 or 3, wherein the amino acid sequence of micro-dystrophin comprises SEQ ID NO: 28 and / or 29. In some embodiments, the kit is for detecting or quantifying micro-dystrophin expression in a subject. In some embodiments, the kit further comprises an isolated peptide having a sequence of SEQ ID NO: 1 and / or an isolated peptide having a sequence of SEQ ID NO: 3. In some embodiments, the kit is for detecting or quantifying micro-dystrophin expression in a subject. In some embodiments, the kit further comprises a composition comprising micro-dystrophin, optionally stable isotope labeled micro-dystrophin. In some embodiments, the micro-dystrophin comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the isolated peptide or peptides are stable isotope labeled peptides.

[0152] In some embodiments, the kit comprises an antibody disclosed herein capable of binding a peptide having the amino acid sequence of SEQ ID NO:1 and an isolated peptide having the amino acid sequence of SEQ ID NO:1. In some embodiments, the antibody is 130D2-1. In some embodiments, the antibody comprises the six CDR sequences of 130D2-1. In some embodiments, the antibody comprises the six CDR sequences having the amino acid sequences of SEQ ID NOs:8-12 and 13. In some embodiments, the antibody comprises the VH and VL domains of 130D2-1. In some embodiments, the antibody comprises a VH and VL domain comprising the amino acid sequences of SEQ ID NOs:4 and 5, respectively. In some embodiments, the peptide is a stable isotope labeled peptide.

[0153] In some embodiments, the kit comprises an antibody disclosed herein capable of binding a peptide having the amino acid sequence of SEQ ID NO:3 and an isolated peptide having the amino acid sequence of SEQ ID NO:3. In some embodiments, the antibody is 133D7-1. In some embodiments, the antibody comprises the six CDR sequences of 133D7-1. In some embodiments, the antibody comprises the six CDR sequences having the amino acid sequences of SEQ ID NOs:18-22 and 23. In some embodiments, the antibody comprises the VH domain and the VL domain of 133D7-1. In some embodiments, the antibody comprises a VH and a VL domain comprising the amino acid sequences of SEQ ID NOs:14 and 15, respectively. In some embodiments, the peptide is a stable isotope labeled peptide.

[0154] In some embodiments, the kit comprises an antibody disclosed herein capable of binding a peptide having an amino acid sequence of SEQ ID NO:1, an antibody disclosed herein capable of binding a peptide having an amino acid sequence of SEQ ID NO:3, an isolated peptide having an amino acid sequence of SEQ ID NO:1, and an isolated peptide having an amino acid sequence of SEQ ID NO:3. In some embodiments, the antibody capable of binding a peptide having an amino acid sequence of SEQ ID NO:1 is 130D2-1, and the antibody capable of binding a peptide having an amino acid sequence of SEQ ID NO:3 is 133D7-1. In some embodiments, the antibody capable of binding a peptide having an amino acid sequence of SEQ ID NO:1 comprises the six CDRs of 130D2-1, and the antibody capable of binding a peptide having an amino acid sequence of SEQ ID NO:3 comprises the six CDRs of 133D7-1. In some embodiments, the antibody capable of binding a peptide having an amino acid sequence of SEQ ID NO:1 comprises the VH and VL of 130D2-1, and the antibody capable of binding a peptide having an amino acid sequence of SEQ ID NO:3 comprises the VH and VL of 133D7-1. In some embodiments, the peptide having an amino acid sequence of SEQ ID NO:1 or 3 is a stable isotope labeled peptide. In some embodiments, the kit further comprises a composition comprising micro-dystrophin, optionally stable isotope labeled micro-dystrophin. In some embodiments, the micro-dystrophin comprises the amino acid sequence of SEQ ID NO:27. EXAMPLES

[0155] Example 1 - Candidate tryptic peptides for quantification of microdystrophin A Various microdystrophin constructs, including microdystrophin A (SEQ ID NO: 27), have been developed for use in gene therapy for congenital diseases caused by mutations in the dystrophin gene that result in a reduction or loss of wild-type dystrophin protein, such as Duchenne muscular dystrophy. See, for example, International Application Publication No. 2021108755, incorporated herein by reference for all purposes. Two tryptic peptides were selected for monitoring microdystrophin expression. Figure 1. The LEM peptide (SEQ ID NO: 1) encompasses an artificial junction specific to microdystrophin A, whereas the LLQ peptide (SEQ ID NO: 3) is present in both full-length dystrophin and microdystrophin A. Thus, detection and quantification of the LEM peptide allows for monitoring of microdystrophin A expression. Meanwhile, detection and quantification of the LLQ peptide allows for monitoring of total dystrophin expression, i.e., the sum of full-length dystrophin and microdystrophin A expression. Furthermore, since the LLQ peptide sequence is present in human, primate and mouse dystrophin polypeptides, detection and quantification of the LLQ peptide allows for the monitoring of total dystrophin expression in all of these organisms.

[0156] Those skilled in the art will further understand that the reagents and methods for detecting and quantifying LEM and LLQ peptides disclosed herein can be used to monitor the expression of any polypeptide or protein whose digestion with trypsin releases LEM and / or LLQ peptides.

[0157] Example 2 - LEM peptide specific antibodies Rabbit polyclonal and monoclonal antibodies specific for the LEM peptide were generated using standard laboratory techniques. Antibody binding affinity was assessed using MSD experiments performed according to the manufacturer's protocol. MSD plates were coated with 1 mg / ml peptide in PBS and blocked with casein in PBS. Anti-rabbit sulfo-tagged antibodies were used as detection agents. Antibody binding specificity was assessed using control reactions containing target peptide to inhibit binding. Results are shown in Figure 2. Binding affinity and specificity are shown in Table 1 below. The data show good affinity and specificity binding of antibodies 130D2-1, 133E10-1, and 75A2-1 to the LEM peptide. None of the antibodies examined are able to bind to a fragment of the LEM peptide having the amino acid sequence of LEMPSSLMLEVP (SEQ ID NO:2).

[0158] [Table 1]

[0159] Example 3 - LLQ peptide-specific antibodies Rabbit polyclonal and monoclonal antibodies specific for the LLQ peptide were generated using standard laboratory techniques. The binding affinity of the antibodies was evaluated using MSD experiments performed according to the manufacturer's protocol. MSD plates were coated with 1 mg / ml of peptide in PBS and blocked with casein in PBS. Anti-rabbit sulfo-tagged antibodies were used as detection agents. The binding specificity of the antibodies was evaluated using control reactions containing the target peptide to inhibit binding. The results are shown in Figure 3. The binding affinity and specificity are shown in Table 2 below. The data show that monoclonal antibodies 112E4-1, 115G6-1, 119H2-1, 121F10-1 and 133D7-1 have better binding affinity for the LLQ peptide than the polyclonal antibodies (pAb). Most anti-LLQ monoclonal and polyclonal antibodies (mAbs) bind to the casein blocker, but their binding can be inhibited by the LLQ peptide (data not shown).

[0160] [Table 2]

[0161] Example 4 - Screening of anti-LEM and LLQ peptide antibodies for use in LCMS Anti-LEM and anti-LLQ peptide antibodies were screened for use in LCMS. Briefly, diluted tissue lysates were digested with trypsin overnight. Residual trypsin activity was quenched by adding 1% protease inhibitor. Synthetic LEM or LLQ peptides were added to the diluted tissue lysates at low and high concentration levels. LEM peptide additives were added directly to trypsin-digested non-human primate (NHP) gastrocnemius (GAS) tissue lysates. LLQ peptide additives were added to digested dystrophin-deficient mdx GAS tissue lysates; NHP GAS lysates were used as matrix controls. Each sample was immunocaptured using polyclonal or anti-peptide IgG monoclonal antibodies immobilized on Pierce Protein A / G magnetic beads. Bound peptides were eluted from the beads with acid. Standard amounts of stable isotope-labeled LEM or LLQ peptides were added to the eluted samples for quantification. LCMS results obtained using various anti-LEM and anti-LLQ antibodies are shown in Figures 4 and 5, respectively. The ratio of unlabeled peptides to stable isotope labeled peptides detected in the eluted samples is shown. The 130D2-1 monoclonal anti-LEM antibody gave comparable results to the anti-LEM polyclonal antibody under both high and low loading conditions. All five anti-LLQ mAbs performed similarly to the pAbs, with the 133D7-1 anti-LLQ antibody showing slightly better results.

[0162] Example 5 - LCMS approach for quantification of dystrophin and microdystrophin in tissue samples Anti-LEM and anti-LLQ antibodies were used in an LCMS assay to quantify the expression levels of dystrophin and micro-dystrophin in various tissue samples. The general overview and workflow of the assay are shown in Figure 6 and Figure 7, respectively. The assay allows for simultaneous quantification of both micro-dystrophin A and dystrophin. The assay was performed using 130D2-1 anti-LEM monoclonal antibody and 133D7-1 anti-LLQ monoclonal antibody. A method for quantification of AAV transgene-encoded micro-dystrophin was developed using recombinant micro-dystrophin (SEQ ID NO: 27) as a standard. Beagle skeletal muscle was used as an alternative matrix, and normal human skeletal muscle spiked with micro-dystrophin standard was used as a matrix control. Biotinylated monoclonal anti-LEM and anti-LLQ antibodies immobilized on streptavidin magnetic beads were used as immunocapture reagents. The assay was performed using reversed-phase UHPLC chromatographic separation and a Sciex 7500 Triplequad MS or Thermo Q-Exactive HF-X MS. The assay employed stable isotope-labeled internal standard (SIL-IS) peptides to facilitate target quantification. LEM peptide detection was used for absolute quantification of microdystrophin; the LEM peptide is unique to microdystrophin. LLQ peptide detection was used for quantification of total dystrophin (microdystrophin and dystrophin). The LLQ peptide is present in microdystrophin and full-length dystrophin; it is also conserved in mouse, human, and NHP dystrophin. Relative quantification of total dystrophin was performed relative to full-length dystrophin present in skeletal muscle of normal control subjects. The calibration range for both LEM and LLQ peptides was 12.5–2500 fmol per mg of lysate protein. Precision and accuracy of QC samples spiked with recombinant microdystrophin in alternative and normal human matrices were within 20%. Reproducible recovery of both peptides was observed at low and high levels of microdystrophin spike, indicating efficient trypsin digestion and immunocapture of the peptides.The recovery of LEM peptide was 262% at low spike level and 126% at high spike level (CV<9.2%, n=3; the observed recovery of over 100% was due to non-specific binding loss of LEM peptide in solution after spike), whereas the recovery of LLQ peptide was 92% at low spike level and 75% at high spike level (CV<7.6%, n=3). As shown in Figures 8 and 9, the LEM and LLQ peptide detection assays were highly selective for microdystrophin and total dystrophin. The LBA / LC-MS assay has the capability to quantify both transgene-encoded microdystrophin and full-length dystrophin proteins in various species and various muscle types. This assay has the sensitivity, precision, and accuracy that are suitable for supporting gene therapy for muscular dystrophies, such as Duchenne muscular dystrophy (DMD).

[0163] The LBA / LC-MS LEM detection assay was used for highly accurate absolute quantification of microdystrophin levels in muscle samples from non-human primate subjects administered recombinant AAV8 particles containing the microdystrophin transgene (Figure 10). The LBA / LC-MS LLQ detection assay was used for highly accurate relative quantification of total dystrophin levels in muscle samples from different species (Figure 11).

[0164] While the methods of this disclosure have been described in conjunction with what are presently considered to be the most practical and preferred embodiments, it is to be understood that the methods encompassed by this disclosure are not to be limited to the disclosed embodiments, but on the contrary, are intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0165] All publications, patents, patent applications, internet sites, and accession number / database sequences (including both polynucleotide and polypeptide sequences) cited in this specification are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, internet site, or accession number / database sequence was specifically and individually indicated to be incorporated by reference.

[0166] array SEQ ID NO:1 - LEM peptide LEMPSSLMLEVPTLER SEQ ID NO:2 LEMPSSLMLEVP SEQ ID NO:3-LLQ peptide LLQVAVEDR SEQ ID NO: 4-130D2-1 VH QSLAESGGRLVTPGTPLTLTCTVSGVDLSYYPMTWVRQAPGKGLEYIGIILHNGTSCYARWARGRFTISKTSTTVELRITSPTTEDTATYFCARASVASIVGSSDIWGPGTLVTVSS SEQ ID NO:5-130D2-1 VL LVLTQTPSSVSAAVGGTVTINCQSSQSVYKNSALSWYQQKPGQPPKLLIYGASTLASGVPSRFSGNGSGTQFTLTISGVQCADAATYYCTGAINDEIHAFGGGTEVVVR SEQ ID NO:6-130D2-1 VH CAGTCGCTGGCGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGAGTCGACCTCAGTTACTATCCAATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAATCATTCTTCACAATGGCACCAGTTGCTACG CGCGCTGGGCGAGAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGAGCTGAGAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGCATCTGTTGCTAGTATTGTTGGTCCAGTGATATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA SEQ ID NO: 7-130D2-1 VL CTAGTGCTGACCCAGACTCCATCCTCCGTGTCTGCGGCTGTGGGAGGCACAGTCACCATCAATTGCCAGTCCAGTCAGAGTGTTTATAAGAACAGCGCCTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATGGTGCATCCACTC TGGCATCTGGGGTCCCATCACGGTTCAGCGCAATGGATCTGGGACACAGTTCACTCTCACCATCAGTGGCGTGCAGTGTGCCGATGCTGCCACTTACTACTGTACAGGCGCTATTAATGATGAGATACATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAGA CDR1 of SEQ ID NO:8-130D2-1 VH YYPMT SEQ ID NO: 9-130D2-1 VH CDR2 IILHNGTSCYARWARG SEQ ID NO: 10-130D2-1 VH CDR3 ASVASIVGSSDI CDR1 of SEQ ID NO:11-130D2-1 VL QSSQSVYKNSALS SEQ ID NO: 12-130D2-1 CDR2 of VL GASTLAS SEQ ID NO: 13-130D2-1 VL CDR3 TGAINDEIHA SEQ ID NO:14-133D7-1 VH QSVKESEGGLFKPTDTLTLTCTASGFTISNNAIDWVRQAPGNGLEYIGTIGKSGSAYYASWAKSRSTITRNTNLNTVTLKMTSLTPADTATYFCARLPISKPDTLNLWGPGTLVTVSS SEQ ID NO:15-133D7-1 VL AVLTQTPSPVSAAVGGTVTINCQSSQSVYKNYLSWFQQKPGQPPKLLIYGASTLASGVPSRFKGSGSGTQFTLTISDVQCDDAATYYCLGGYDTSIDIFTFGGGTEVVVK SEQ ID NO:16-133D7-1 VH CAGTCAGTGAAGGAGTCCGAGGGAGGTCTCTTCAAGCCAACGGATACCCTGACACTCACCTGTACAGCCTCCGGATTCACCATCAGTAACAATGCAATAGACTGGGTCCGCCAGGCTCCAGGGAACGGGCTGGAATATATCGGAACCATTGGTAAAAGTGGTAGCGCATACT ACGCGAGCTGGGCGAAAAGCCGATCCACCATCACCAGAAACACCAACCTAAACACGGTGACTCTGAAAATGACCAGTCTGACGCCCGCGGACACGGCCACCTATTTCTGTGCGAGACTTCCTATTTCGAAACCTGATACCCTTAATCTGTGGGCCCAGGCACCCTGGTCACC GTCTCCTCA SEQ ID NO: 17-133D7-1 VL GCCGTGCTGACCCAGACTCCATCTCCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGTCCAGTCAGAGTGTTTATAAGAACTACTTATCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATGGTGCGTCCACTCTGGCA TCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCGGTTATGATACTAGTATTGATATATTTACTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA CDR1 of SEQ ID NO:18-133D7-1 VH NNAID CDR2 of SEQ ID NO:19-133D7-1 VH TIGKSGSAYYASWAKS SEQ ID NO: 20-133D7-1 VH CDR3 LPISKPDTLNL CDR1 of SEQ ID NO:21-133D7-1 VL QSSQSVYKNYLS CDR2 of SEQ ID NO:22-133D7-1 VL GASTLAS SEQ ID NO: 23-133D7-1 VL CDR3 LGGYDTSIDIFT SEQ ID NO:24 PVVTKETAISKLEMPSSLMLEVPALADFNRAWTELTDWLSL SEQ ID NO:25 LEMPSSLMLEVPALADFNR SEQ ID NO:26 PVVTKETAISKLEMPSSLMLEVPTLERLQELQ SEQ ID NO:27 SEQ ID NO:28 XLEMPSSLMLEVPTLER, where X is R or L SEQ ID NO:29 XLLQVAVEDR, where X is R or L

Claims

1. An isolated antibody or antigen-binding fragment thereof capable of binding to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, wherein the antibody or antigen-binding fragment thereof does not bind to a peptide consisting of the amino acid sequence of SEQ ID NO:

2.

2. An isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a monoclonal antibody comprising a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises complementarity-determining region 1 (CDR1), CDR2 and CDR3 of the VH comprising the amino acid sequences of SEQ ID NOs: 8, 9 and 10, respectively, and the VL comprises CDR1, CDR2 and CDR3 of the VL comprising the amino acid sequences of SEQ ID NOs: 11, 12 and 13, respectively. (a) the VH comprises the amino acid sequence of SEQ ID NO: 4; and (b) the VL comprises the amino acid sequence of SEQ ID NO: 5; The isolated antibody or antigen-binding fragment thereof of claim 2.

4. An isolated monoclonal antibody or antigen-binding fragment thereof capable of binding to a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 and comprising a variable heavy domain (VH) and a variable light domain (VL), wherein the VH comprises VH complementarity-determining regions (CDRs) 1, 2 and 3 comprising the amino acid sequences of SEQ ID NOs: 18, 19 and 20, respectively, and the VL comprises VL CDRs 1, 2 and 3 comprising the amino acid sequences of SEQ ID NOs: 21, 22 and 23, respectively. (a) the VH comprises the amino acid sequence of SEQ ID NO: 14; and (b) the VL comprises the amino acid sequence of SEQ ID NO: 15; The isolated antibody or antigen-binding fragment thereof of claim 4.

6. An affinity resin comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 and a solid support.

7. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

8. 6. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, comprising incubating a host cell comprising a polynucleotide encoding the antibody or antigen-binding fragment thereof under conditions suitable for producing the antibody or antigen-binding fragment thereof.

9. 1. A method for isolating a peptide from a sample, the method comprising: (a) contacting the sample containing the peptide with a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 3 under conditions that allow binding of the peptide to the antibody or antigen-binding fragment thereof; (b) removing the portion of the sample that is not bound to the antibody or antigen-binding fragment thereof; (c) dissociating the peptide from the antibody or antigen-binding fragment thereof; wherein the amino acid sequence of the peptide comprises SEQ ID NO:

1.

10. 1. A method for quantifying dystrophin and / or microdystrophin expression levels in a subject, comprising: (a) providing a sample comprising a protease-digested protein isolate obtained from said subject, said sample comprising one or more peptides having the amino acid sequence of SEQ ID NO:1; (b) contacting the sample with a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 3 under conditions that allow binding of the antibody or antigen-binding fragment thereof to the peptide; (c) recovering the peptide bound to the antibody or antigen-binding fragment thereof; (d) determining the amount of peptide recovered in step (c); wherein the amino acid sequence of the microdystrophin comprises SEQ ID NO: 28 and / or 29.

11. The method of claim 10, wherein the amount of the peptide is determined by LC / MS or LC-MS / MS.

12. 11. The method of claim 10, which provides absolute or relative quantification of the expression levels of the dystrophin and / or microdystrophin.

13. 11. The method of claim 10, which provides relative quantification of the expression levels of dystrophin and / or microdystrophin.

14. 11. The method of claim 10, wherein the sample comprises a protease-digested protein isolate obtained from skeletal muscle tissue of the subject.

15. 11. The method of claim 10, wherein the sample further comprises one or more labeled peptides capable of binding to the antibody or antigen-binding fragment thereof.

16. 11. The method of claim 10, wherein the subject is suffering from Duchenne muscular dystrophy or the subject is a non-human mammal that has been genetically engineered to contain one or more mutations in the dystrophin gene.

17. 11. The method of claim 10, wherein the subject has been administered a recombinant polynucleotide encoding micro-dystrophin comprising the amino acid sequence of SEQ ID NOs: 28 and 29.

18. 18. The method of claim 17, wherein the microdystrophin comprises the amino acid sequence of SEQ ID NO:

27.

19. 11. The method of claim 10, wherein the subject has been administered a recombinant adeno-associated virus comprising a polynucleotide encoding micro-dystrophin comprising the amino acid sequence of SEQ ID NO:

27.

20. Contacting the sample with a composition comprising a second antibody or antigen-binding fragment thereof under conditions that allow binding of the second antibody or antigen-binding fragment thereof to the peptide; recovering the peptide bound to the second antibody or antigen-binding fragment thereof; Determining the amount of peptide recovered further comprising the second antibody or antigen-binding fragment thereof is capable of binding to a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, and comprises a variable heavy chain domain (VH) and a variable light chain domain (VL), wherein the VH comprises VH complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences of SEQ ID NOs: 18, 19, and 20, respectively, and the VL comprises VL CDRs 1, 2, and 3 comprising the amino acid sequences of SEQ ID NOs: 21, 22, and 23, respectively; The method of claim 10.

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