Background Adoptive cell therapy (ACT) with genetically-modified T cells has shown impressive results against some hematologic cancers, but limited efficacy against tumors with restrictive tumor microenvironments (TMEs). FasL is a particular obstacle for ACT;1 it is expressed in many tumors and TMEs,1 including AML,2 ovarian3 and pancreatic cancers,4 and upregulated on activated T cells, where it can mediate activation-induced cell death (AICD).5
Methods We engineered T cells to boost function with novel immunomodulatory fusion proteins (IFPs) that combine an inhibitory ectodomain with a costimulatory endodomain. Like current checkpoint-blocking therapies, IFPs can abrogate an inhibitory signal, but also provide an often absent costimulatory signal. Additionally, IFP-driven signals are delivered only to the T cells concurrently engineered to be tumor-specific, thereby avoiding systemic T cell activation. For FasL-expressing TMEs, we developed an IFP that replaces the Fas intracellular tail with costimulatory 4-1BB. We tested the the Fas-4-1BB IFP in primary human T cells and in immunocompetent murine models of leukemia and pancreatic cancer.
Results Fas-4-1BB IFP expression enhanced primary human T cell function and enhanced lysis of Panc1 pancreatic tumor cells in vitro. Fas-4-1BB IFP-engineered murine T cells exhibited increased pro-survival signaling, proliferation, antitumor function and altered metabolism in vitro. Notably, the Fas ectodomain is trimeric5 and the 4-1BB intracellular domain requires trimerization to signal.6 In contrast, the CD28 domain is dimeric and did not enhance function when paired with 4-1BB.In vivo, Fas-4-1BB increased T cell persistence and function, and Fas-4-1BB T cell ACT significantly improved survival in a murine AML model. When delivered with a mesothelin-specific TCR, Fas-4-1BB T cells prolonged survival in the autochthonous KPC pancreatic cancer model, increasing median survival to 65 from 37 days (with TCR-only, **P=0.0042). Single-cell RNA sequencing revealed differences in the endogenous tumor-infiltrating immune cells, included changes in cell frequency and programming.
Conclusions We developed an engineering approach to enhance the in vivo persistence and antitumor efficacy of transferred T cells. Our targeted, two-hit strategy uses a single fusion protein to overcome a death signal prevalent in the TME of many cancers and on activated T cells, and to provide a pro-survival costimulatory signal to T cells. Our results suggest that this fusion protein can increase T cell function when combined with murine or human TCRs, and can significantly improve therapeutic efficacy in liquid and solid tumors, supporting clinical translation.
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