Engineering a Juxtamembrane-Targeting CAR T-Cell Against Mesothelin: A Novel Binder Resilient to Shed Antigen in Ovarian and Pancreatic Cancer.

Mesothelin is an attractive target for CAR-T therapy on a number of cancer types; however, the efficacy of this therapy is diminished because the bulk of the cell surface-expressed mesothelin is shed through naturally occurring proteolysis leaving behind a short juxtamembrane peptide 'stump'. The two problems this creates are (1) the bulk of the target protein is no longer on the tumor cell and (2), the free soluble shed mesothelin remains in the tumor microenvironment and becomes present in blood/other body fluids binding to the mesothelin-targeted CAR-T and interfering with their ability to target the mesothelin that remains on the surface of the tumor. These issues have likely contributed at least in part to the lack of desired efficacy of CAR-T cells that target membrane distal regions of mesothelin (i.e., the shed domain) such as those utilizing anti-mesothelin scFvs SS1 and M5. Here we describe CAR T cells that utilize novel antibodies specific for the mesothelin stump domain thus being unaffected by the natural process of mesothelin shedding. Mesothelin 'stump' specific CAR T cells (ST4-22) had cytotoxicity and in vivo activity that was comparable to standard anti-mesothelin CAR T cells. Importantly, CAR T cells expressing ST4-22 were effective against tumor cells that were resistant to standard anti-mesothelin CAR T cells.