Allogeneic Chimeric Antigen Receptor-Invariant Natural Killer T Cells Exert Both Direct and Indirect Antitumor Effects through Host CD8 T Cell Cross-Priming

Chimeric antigen receptor (CAR) T cells have shown impressive results in refractory B-cell malignancies. Unfortunately, to date, commercially available cells, as well as most products tested in clinical trials, are autologous CAR T cells whose widespread use is limited by the logistical and financial burdens related to their ad hoc generation. The development of universal allogeneic CAR T cell products to be used off-the-shelf across MHC-barriers has faced major limitations, namely the risk of Graft-versus-Host-Disease (GvHD) induction and the rejection of the administered cells by the host immune system. Invariant Natural Killer-T (iNKT) cells are innate lymphocytes that are deprived of any GvHD induction potential but that display antitumor effects both directly, through the production of cytotoxic effector molecules, and indirectly, through the enhancement of NK and CD8 T cell-mediated immune responses. Preclinical studies using xenogeneic mouse models have demonstrated the feasibility of using iNKT cells as a platform for CAR-based therapies, and two clinical trials are currently ongoing. In order to study the interaction of CD19-specific iNKT CAR cells with the host immune system, we transduced iNKT cells ex vivo expanded from FVB/N mice with a CAR composed of the variable region cloned from the 1D3 hybridoma recognizing murine CD19 linked to a portion of the murine CD28 molecule and to the cytoplasmic region of the murine CD3-ζ molecule. The cytotoxic potential of CD19-iNKT CAR was confirmed in an in vitro cytotoxic assay against the CD19-expressing A20 lymphoma cell line, revealing a strong, dose dependent cytotoxic effect of CD19-CAR iNKT cells. Accordingly, and similarly to what was previously reported in xenogeneic studies, FVB/N (H-2K^q) derived iNKT CAR (2x10e6 cells iv) significantly improved survival of mice after administration to major histocompatibility complex (MHC)-mismatched, immunodeficient BALB/c (H-2K^d) Rag2^-/- gamma-chain^-/- mice receiving A20 cells (2x10e4 cells iv; Figure 1A) without inducing any signs of GvHD. To test the efficacy of iNKT CAR cells in the presence of host immune cells, we tested the antitumor activity mediated by iNKT CAR against A20 cells in BALB/c mice receiving sublethal irradiation (4.4 Gy), resulting in only a partial and transient lymphopenia. In this model, the antitumor effect of iNKT CAR cells was greatly enhanced, leading to long-term survival of the great majority of treated mice (Figure 1B). Such a difference in iNKT CAR effect between mice that genetically lacked lymphocytes and mice with only partial lymphopenia suggested the participation of host derived lymphocytes in the antitumor effect. To test the hypothesis that host CD8 T cell activation via cross-priming could at least partially mediate the indirect antitumor effect of iNKT CAR cells, we repeated the experiment, employing as recipients BALB/c BATF3^-/- mice, in which CD8 T cell cross-priming is impaired as a result of the absence of BATF3-dependent CD103+ CD8a+ dendritic cells that are thought to present antigens through CD1d to the invariant T cell receptor expressed by iNKT cells. Interestingly, the iNKT CAR effect was partially abrogated in A20-receiving BATF3^-/- mice as compared to WT mice (Figure 1C), supporting the hypothesis that the impact of iNKT CAR cells is mediated at least partially by the activation of host CD8 T cells via their cross-priming. To formally demonstrate the synergistic effect between allogeneic iNKT CAR and autologous CD8 T cells, we employed an autologous bone marrow transplantation model, co-administering allogeneic iNKT CAR with autologous CD8 T cells at the time of transfer of T-cell-depleted autologous bone marrow cells and A20 lymphoma cells into lethally irradiated (8.8 Gy) BALB/c recipients. Co-administration of allogeneic iNKT CAR and autologous CD8 T cells resulted in a synergistic effect, significantly extending animal survival (Figure 1D) compared to mice receiving no treatment, as well as to mice receiving either allogeneic iNKT or autologous CD8 T cells alone. Collectively, these results represent the first demonstration of an immune adjuvant effect exerted by an allogeneic CAR cell product toward the autologous immune system of the host, suggesting that the effect of the administered cells would last longer than the physical persistence of the allogeneic cells after they will be rejected by the host immune system.

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