Potent and durable control of mesothelin-expressing tumors by a novel T cell-secreted bi-specific engager.

Background: The glycosylphosphatidylinositol-anchored cell surface protein mesothelin (MSLN) shows elevated expression in many malignancies and is an established clinical-stage target for antibody-directed therapeutic strategies. Of these, the harnessing of autologous patient T cells via engineered anti-MSLN chimeric antigen receptors (CAR-T) is an approach garnering considerable interest. Although generally shown to target tumor MSLN safely, CAR-T trials have failed to deliver the impressive curative or response metrics achieved for hematological malignancies using the same technology. A need exists, therefore, for improved anti-MSLN molecules and/or more optimal ways to leverage immune effector cells.

Methods: We performed ELISA, label-free kinetic binding assays, FACS, Western blotting, and transient recombinant MSLN expression to characterize the recognition properties of a novel CAR-active human scFv clone, LABC-13F08. To investigate T cell redirection, we conducted kinetic IncuCyte co-culture killing assays using transduced primary T cells and MSLN⁺ target cell lines and assessed levels of activation markers and effector cytokines. The antitumor potential of LABC-13F08 formatted as a bispecific engager (BiTE) was evaluated in vivo using transduced human primary T cells and immunocompromised NSG mice xenografted with ovarian, mesothelioma, and pancreatic MSLN⁺ tumor cell lines.

Results: The LABC-13F08 scFv is highly unusual and distinct from existing (pre)clinical anti-MSLN antibody fragments, exhibiting an absolute requirement for divalent cations to drive MSLN recognition. As a monovalent BiTE, LABC-13F08 demonstrates robust in vitro potency. Additionally, primary human T cells engineered for constitutive secretion of the 13F08 BiTE exhibit strong antitumor activity toward in vivo ovarian and mesothelioma xenograft models and show encouraging levels of monotherapy control in a challenging pancreatic model. LABC-13F08 BiTE secreted from engineered T cells (BiTE-T) can both recruit non-engineered bystander T cells and also induce activation-dependent MSLN-independent bystander killing of cells lacking cognate antigen. To address safety concerns, 13F08 BiTE-T cells can be rapidly targeted for clearance via a molecular "off" switch.

Conclusions: The novel LABC-13F08 scFv exhibits a mode of binding to MSLN which is not observed in typical anti-MSLN antibodies. Efficacious targeting by a T cell secreted engager would represent a clinically differentiated approach for the treatment of MSLN⁺ tumors.