69. TNFR Costimulatory Domains Impair Expansion of CD5 CAR T Cells Due to Enhanced Fas-Mediated Apoptosis

Abstract

CD5 is expressed at high levels on most normal and malignant T cells. We previously demonstrated that T cells transduced with a CD5 CAR harboring signaling domains from CD28 and TCR zeta chain (CD28. zeta) genes produce only limited fratricide against normal CD5+ T cells and expand>10,000-fold in vitro. Although these CAR T cells also have significant anti-tumor activity against CD5+ cell lines, tonic signaling from the CAR enhances terminal differentiation during in vitro expansion. The accumulation of CCR7- effector T cells and reduced frequency of CD4+ CAR T cells limits the in vivo persistence of CAR T cells ultimately allowing resurgence of CD5+ tumor cells. Since strong PI(3)K activation from the CD28 domain likely contributes to the terminal differentiation of CAR T cells, we aimed to improve CD5 CAR T cell persistence by replacing the CD28 domain with a TNFR superfamily domain 4-1BB, which elicits weaker PI(3)K activation and preserves the central memory population.Here, we show that replacing the CD28 signaling domain with one of the TNFR-superfamily genes (4-1BB, OX40, CD27, CD30 or HVEM) allowed CD5 CAR T cells to retain the CCR7+ central memory population. Unexpectedly, however, TNFR signaling domains dramatically increased CAR T cell death and reduced proliferation, resulting in 5- to 15-fold reduction in cell expansion after 2 weeks of culture. These results correlated with a 3-fold upregulation of Fas on the T cell surface and the formation of stable immunologic synapses between CAR T cells. Disrupting a TRAF2 binding site in the 4-1BB domain prevented Fas upregulation and restored the expansion of CD5 CAR T cells, but also compromised costimulation and undermined long-term cytotoxicity.In contrast, CD28. zeta CD5 CAR T cells displayed normal Fas levels and their expansion was comparable to that of ICOS. zeta and zeta-only (first generation) CD5 CAR T cells. Limited fratricide of CD28. zeta CD5 CAR T cells was dependent on PI(3)K activity, as chemical blockade of PI(3)K resulted in dose-dependent enhancement of apoptosis (56% vs 31%) and a 3-fold reduction in CD5 CAR T cell numbers after 72h. Combining CD28 and 4-1BB domains in a third generation CD5 CAR still did not rescue T cell expansion. Therefore, we developed a regulated retroviral expression system that uses a small molecule to reversibly inhibit CAR expression, thereby preventing tonic signaling and the resulting fratricide and differentiation. Withdrawing the inhibitor restores CAR expression and the anti-tumor function of T cells, and preserves the central memory population by the time the CAR is fully expressed. Overall, these studies provide a mechanism regulating T cell fratricide and offer a means of reducing the negative effects of tonic CD5 CAR signaling.