3A, top). the sarcolemma from injury. In vivo, anti-LTBP4 treatment of dystrophic mice guarded muscle against pressure loss induced by eccentric contraction. Anti-LTBP4 treatment also reduced muscle fibrosis and enhanced muscle pressure 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 guarded against injury inmdxmice. These data demonstrate the potential of anti-LTBP4 antibodies to treat muscular dystrophy. == INTRODUCTION == Duchenne muscular dystrophy (DMD) is usually caused by loss Rhoa 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, alternative by fibrosis and fatty infiltrate. Clinically, over time, this process causes loss of ambulation, weakened breathing, and impaired heart function. Latent transforming growth factor (TGF) binding protein 4 (LTBP4) is usually 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, and TGF3, as well as the highly related TGF family member myostatin (growth and differentiation factor 8) (5,6).Ltbp4was 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 Methoxatin disodium salt mice, and most mouse strains have the protective allele. The protective allele ofLtbp4encodes an in-frame insertion of 36 base pairs into exon 12, which encodes the hinge region of LTBP4 protein. The strongestLtbp4genomic signals were linked to sarcolemmal stability, measured as membrane leak, and a reduction in fibrosis (7,8). The protective mouseLtbp4 allele encodes a protein with reduced Methoxatin disodium salt susceptibility to proteolysis and increased sequestration of TGF ligands, reducing their deleterious signaling cascades on dystrophic muscle pathology (5,7,9). LTBP4 is usually broadly expressed but highly increased after muscle injury (9). The deleterious mouseLtbp4allele 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 Methoxatin disodium salt muscle strength (5). Human genetic data also support a role forLTBP4in human muscular dystrophy. Patients with DMD with specificLTBP4single-nucleotide polymorphisms have prolonged ambulation (10). This result was replicated in impartial 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 showed that short-term anti-LTBP4 antibody exposure improved sarcolemmal stability and reduced the susceptibility to eccentric contraction (ECC)induced pressure loss. Long-term 24-week administration of anti-LTBP4 antibody improved muscle performance and reduced muscle fibrosis, including in the diaphragm muscle, one of the most adversely affected muscles in DMD and its mouse models. Together, these data demonstrate that stabilizing LTBP4 through anti-LTBP4 antibody administration is an effective strategy to reduce muscle fibrosis and increase muscle performance in DMD. == RESULTS == == A monoclonal antibody to LTBP4 recognizes human and mouse LTBP4 protein == We investigated the translational potential of an antibody-based strategy to stabilize the hinge of LTBP4 because prior work established the hinge as a critical component of latent TGF stability (Fig. 1A) (7,9). To generate an antibody specific for an epitope within the LTBP4 hinge, screening was performed using peptides corresponding to the conserved amino acid residues.
