Demonbreun AR, Fallon KS, Oosterbaan CC, Bogdanovic E, Warner JL, Sell JJ, Page PG, Quattrocelli M, Barefield DY, McNally EM, Recombinant annexin A6 promotes membrane repair and protects against muscle injury. alleles. The protective form of LTBP4, which contains an insertion of 12 amino acids in the proteins hinge region, was linked to increased sequestration of latent TGF, enhanced muscle membrane stability, and reduced muscle fibrosis. The deleterious form of LTBP4 protein, lacking 12 amino acids, was more susceptible to proteolysis and promoted release of latent TGF-, and together, these data underscored the functional role of LTBP4s hinge. Here, we generated a monoclonal human anti-LTBP4 antibody directed toward LTBP4s hinge region. In vitro, anti-LTBP4 bound LTBP4 protein and reduced LTBP4 proteolytic cleavage. In isolated myofibers, the LTBP4 antibody stabilized the sarcolemma from injury. In vivo, anti-LTBP4 treatment of dystrophic mice protected muscle against force loss induced by eccentric contraction. Anti-LTBP4 treatment also reduced muscle fibrosis and enhanced muscle force production, including in the diaphragm muscle, where respiratory function was improved. Moreover, the anti-LTBP4 in combination with prednisone, a standard of care for Duchenne muscular dystrophy, further enhanced muscle function and protected against injury in mice. L-779450 These data demonstrate the potential of anti-LTBP4 antibodies to treat muscular dystrophy. INTRODUCTION Duchenne muscular dystrophy (DMD) is caused by loss of the membrane-associated protein dystrophin (1), and mutations in genes encoding dystrophin-associated proteins such as the sarcoglycans elicit a similar phenotype (2, 3). These disorders are characterized by fragile muscle membranes that are highly prone to injury and disruption. Repetitive injury to myofibers produces extensive muscle degeneration, increased tissue inflammation, and, ultimately, replacement by fibrosis and fatty infiltrate. Clinically, over time, this process causes loss of ambulation, L-779450 weakened breathing, and impaired heart function. Latent transforming growth factor (TGF) binding protein 4 (LTBP4) is a 160-kDa matrix-embedded protein that is a member of the fibrillin super gene family. LTBPs bind to the latent forms of TGF, holding these proteins inactive and unavailable to cell surface receptors (4). LTBP4 binds to latent TGF1, TGF2, L-779450 and TGF3, as well as the highly related TGF family member myostatin (growth and differentiation factor 8) (5, 6). was originally identified as a genetic modifier of mouse muscular dystrophy from an unbiased genome-wide search for modifiers (7). There are two major alleles found in mice, and most mouse strains have the protective allele. The protective allele of encodes an in-frame insertion of 36 base pairs into exon 12, which encodes the hinge region of LTBP4 protein. The Mmp13 strongest genomic signals were linked to sarcolemmal stability, measured as membrane leak, and a reduction in fibrosis (7, 8). L-779450 The protective mouse allele is found in the DBA/2J strain and is characterized by a shorter hinge region. Human LTBP4 has an even shorter hinge region than found in the DBA/2J strain, further increasing the susceptibility of LTBP4 to protease cleavage and TGF release (9). Transgenic overexpression of the protective LTBP4 isoform in dystrophic mice was shown to reduce fibrosis, increase muscle mass, and enhance muscle strength (5). Human genetic data also support a role for in human muscular dystrophy. Patients with DMD with specific single-nucleotide polymorphisms have prolonged ambulation (10). This result was replicated in independent cohorts of human DMD subjects, illustrating the strong modifying effect of this pathway (11, 12). The human protective LTBP4 polymorphisms encode a protein that results in tighter binding to latent TGF, leading to reduced TGF signaling (6). We hypothesized that blocking LTBP4s hinge region would stabilize the protective conformation of LTBP4. Here, we describe the development and testing of monoclonal human and mouse antibodies directed against the human LTBP4 hinge region. The anti-LTBP4 antibody localized within muscle in the expected costameric pattern, and a single injection was detected in mice in vivo for at least 21 days. Using mouse models of DMD, we.