TNX-100

The antagonistic anti-CD40 antibody, 2C10, and its recombinant primate derivative, 2C10R4, are potent immunosuppressive antibodies whose utility in allo- and xenotransplantation have been demonstrated in nonhuman primate studies. In this study, we defined the 2C10 binding epitope and found only slight differences in affinity of 2C10 for CD40 derived from four primate species. Staining of truncation mutants mapped the 2C10 binding epitope to the N-terminal portion of CD40. Alanine scanning mutagenesis of the first 60 residues in the CD40 ectodomain highlighted key amino acids important for binding of 2C10 and for binding of the noncross-blocking anti-CD40 antibodies 3A8 and 5D12. All four 2C10-binding residues defined by mutagenesis clustered near the membrane-distal tip of CD40 and partially overlap the CD154 binding surface. In contrast, the overlapping 3A8 and 5D12 epitopes map to an opposing surface away from the CD154 binding domain. This biochemical characterization of 2C10 confirms the validity of nonhuman primate studies in the translation of this therapeutic antibody and provides insight its mechanism of action.

Claudin‐4 (CLDN‐4), a tight‐junction protein, is overexpressed in various malignant tumors, including gastric, colorectal, pancreatic, and breast cancers. However, CLDN‐4 is also expressed in normal tissues, including the liver, pancreas, kidney, and small intestine. Whether CLDN‐4 is an effective and safe target for cancer therapy has been unclear owing to the lack of a binder with both CLDN‐4 specificity and cross‐reactivity to human and murine cells. In this study, we successfully generated a rat anti‐CLDN‐4 monoclonal antibody (5D12) that was specific to, and cross‐reactive with, human and mouse CLDN‐4. 5D12 recognized the second extracellular domain of human CLDN‐4 in a conformation‐dependent manner. A human–rat chimeric IgG1 of 5D12 (xi‐5D12) activated the FcγIIIa receptor, indicating the activation of antibody‐dependent cellular cytotoxicity in CLDN‐4‐expressing cells. Moreover, xi‐5D12 significantly suppressed tumor growth in mice bearing human colorectal and gastric tumors without apparent adverse effects, such as weight loss or liver and kidney damage. These results suggest that CLDN‐4 is a potent target for cancer therapy and that an anti‐CLDN‐4 antibody is a promising candidate anticancer agent.