210. High Expression of Second Generation CD19 CAR with a 4-1BB Costimulatory Domain from a Retroviral Vector Impairs CAR T Cell Expansion by Enhancing Fas-Mediated Apoptosis

Abstract

Recent clinical trials in patients with B cell malignancies demonstrated that CD19 CAR T cells could expand in vivo and eliminate target cells. Two main variants of the CD19 CAR have been successful - a retroviral construct with CD28 and CD3 zeta signaling domains (28. zeta) and a lentivirus with 4-1BB. zeta. While infused CD28. zeta CAR T cells rapidly expanded and produced immediate anti-tumor activity, 4-1BB. zeta CAR T cells persisted longer and had sustained anti-tumor activity. Although these differences may represent differences in the costimulation provided by each CAR, the choice of viral vector may also contribute to differences in performance. We compared the in vitro expansion and anti-tumor activity of T cells transduced with either 28. zeta or 4-1BB. zeta CD19 CAR using a standard retroviral vector SFG. We demonstrate that retroviral transduction of T cells with a 4-1BB. zeta CD19 CAR reduced T cell expansion 18 fold after 2 weeks of culture and impaired anti-tumor activity compared to cells transduced with a similar vector encoding CD28. zeta CD19 CAR. High expression of the 4-1BB. zeta CD19 CAR led to upregulation of Fas and relocation of intracellular FasL to the cell surface, resulting in co-localization of both proteins on the plasma membrane. Consistent with this finding, we observed a 5-fold increase in apoptosis of 4-1BB. zeta CAR T cells which was completely reversed by chemical blockade of caspase-8, a key initiator caspase activated by Fas signaling. We also observed a gradual downregulation of 4-1BB. zeta CD19 CAR levels on surviving T cells suggesting that high CAR expression is toxic for T cells. In fact, reducing the level of 4-1BB. zeta CD19 CAR expression by inserting an IRES sequence upstream of the CAR normalized Fas levels on T cells and restored T cell expansion and anti-tumor activity. Similarly, transducing T cells with a self-inactivating lentiviral vector resulted in reduced levels of 4-1BB. zeta CD19 CAR expression allowed excellent expansion, similar to that of T cells with 28. zeta CD19 CAR. We also observed improved cytotoxicity of T cells with lower expression of 4-1BB. zeta CAR in co-culture assays with CD19+ cell lines Daudi (5-fold reduction in remaining tumor cells with lenti-CAR and 22-fold for IRES-CAR) and Raji (18-fold reduction for both lenti- and IRES-CAR constructs) and sustained performance in sequential killing assays.These studies reveal a mechanism that limits expansion and function of T cells expressing high levels of 4-1BB. zeta CD19 CAR and provide a basis for rational choice of expression systems and design of chimeric molecules that incorporate the 4-1BB signaling domain.