CAR T Cells As Micropharmacies to Induce Locoregional Tumor Vascular Infarction by Antigen-Specific Delivery of Tissue Factor to the Tumor Microenvironment

CAR T cell therapy of solid tumors is challenged by the heterogeneity of target expression and by mechanical and immune-modulatory barriers in the tumor microenvironment (TME). To combine CAR-retargeted T cell effector functions with a second therapeutic mode of action, we designed an innovative cell-based combination strategy. CAR-engineered antitumor effector T cells are used as micropharmacies to produce and deliver a pro-coagulatory fusion protein, tTF-NGR, in the TME to induce locoregional tumor vascular infarction for combined T-cell mediated and hypoxic tumor cell death. tTF-NGR is a CD13-targeted tissue factor variant with coagulation activity upon relocalization into the phospholipid membranes of the CD13-expressing vascular endothelial cells that invade tumor tissues. Consequent thrombosis in tumor blood vessels induces tumor infarction, growth retardation and regression in preclinical in vitro and in vivo studies and selective reduction of tumor blood flow in a clinical phase I study. Human T cells were co-transduced by retroviral one-vector gene transfer to express genes encoding for a GD2-specific CAR and for tTF-NGR, the latter in an antigen-dependent CAR-mediated manner. The engineered T cells exerted potent GD2 antigen-specific effector functions, including secretion of IFN-γ and TNF-α, upregulation of CD107 and tumor cell lysis, comparable to control CAR T cells producing mutant tTF-NGR lacking pro-coagulatory function. They secreted recombinant tTF-NGR in a strictly antigen-dependent manner upon coincubation with the anti-idiotype antibody ganglidiomab, which selectively engages the extracellular scFv of the CAR, or with GD2-positive tumor cells, shown by ELISA. tTF-NGR produced by human T cells effectively activates the extrinsic coagulation cascade, thus it retains its pro-coagulatory activity. In a murine Ewing sarcoma xenograft model which expresses the CAR target GD2on tumor cells along with CD13 on tumor vascular endothelial cells, GD2-specific CAR T cells with inducible tTF-NGR had noticeably superior therapeutic activity compared with control cells excreting mutant tTF-NGR. Mechanistic evidence hints at hypoxia-induced higher CAR T cell cytolytic activity. We conclude that combined CAR-mediated T cell targeting of cancer cells with CD13-targeted vascular infarction of the TME in a one-vector engineering strategy is a promising approach to overcome limitations of both strategies for effective targeting and eradication of solid cancers. Citation Format: Bianca Altvater, Sareetha Kailayangiri, Christian Spurny, Maike Flügge, Jutta Meltzer, Lea Greune, Christian Schwöppe, Caroline Brand, Christoph Schliemann, Wolfgang Hartmann, Hinrich Abken, Axel Schambach, Nicole Farwick, Wolfgang E. Berdel, Claudia Rossig. CAR T cells as micropharmacies to induce locoregional tumor vascular infarction by antigen-specific delivery of tissue factor to the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3182.