A T-cell engager antibody targeting the non-shed site of mesothelin in solid tumors

Mesothelin (MSLN) is a cell surface protein that is overexpressed in various cancers, including mesothelioma, pancreatic, and ovarian cancer. Its expression in normal tissue is limited to the mesothelial cells lining the pleura, peritoneum and pericardium; make it an attractive target for antibody-based therapeutics. Many efforts have been dedicated towards the development of these antibody-based approaches but proteases in the tumor microenvironment promote the cleavage of MSLN from cancer cells. High concentrations of shed MSLN in the tumor microenvironment bind to the antibody inhibiting its activity and preventing the death of cancer cells. To address the presence of shed MSLN, an antibody called 15B6 was designed. It binds the membrane-proximal, protease sensitive region of MSLN that is not shed in the tumor microenvironment. This study investigates the ability of 15B6-targeted bispecific antibodies to eliminate MSLN expressing cancers. We designed humanized and murine versions of a CD3x15B6 bispecific antibody, which binds the CD3 on T cells and the MSLN (15B6) epitope on cancer cells serving as a bridge to promote the activation of T cells to kill MSLN positive cancer cells. These 15B6-based antibodies were compared to SS1-based antibodies that target the distal N-terminal domain of MSLN that is shed by proteases. In-vitro, the SS1 and 15B6 antibodies hold similar cytotoxic activity, but when cocultured with MSLN 296-591, a recombinant protein mimicking shed MSLN, the SS1 antibodies activity is inhibited. In-vivo, in a human mesothelioma model in immunodeficient mice, tumor shrinkage and growth inhibition were observed when treated with the 15B6-based antibody but not those treated with the SS1-based antibody. In an immunocompetent mouse model, the complete regression of colon and breast tumors was observed when treated with the 15B6-based antibody compared to the SS1 antibody and control samples. Transcriptional analysis revealed that 15B6-treated mice had higher levels of activation of both innate and adaptive immune cells, along with significant upregulation of cytokine and STAT5 signaling pathways. Through in-vitro and in-vivo studies, we have demonstrated that this 15B6-targeted antibody binds to the protease-sensitive region, is highly active against MSLN-expressing cancer cell lines in vitro, is not inhibited by shed MSLN, and promotes upregulation of immune cell populations and signaling pathways that support robust anti-tumor activity.