514. Targeting the Negative Immune Regulatory Molecule B7-H4 on Both Tumor and Normal Tissue With Anti-B7-H4 CAR T Cells

Abstract

B7-H4 is a negative immune regulatory molecule frequently overexpressed on the surface of cancer cells and immunosuppressive tumor-associated macrophages (TAMs). Ligation of B7-H4 with an unknown receptor on activated T cells results in inhibited effector function via cell cycle arrest, decreased proliferation and reduced IL-2 production. B7-H4 expression levels in ovarian cancer inversely correlate with patient survival, making B7-H4 an attractive candidate for therapeutic intervention. However, trials of B7-H4 targeted therapy have not been conducted and the biodistribution of B7-H4 protein in mice or humans is not well understood.Here, we tested the hypothesis that safe and effective B7-H4 targeting can be achieved using T cells genetically redirected with chimeric antigen receptors (CARs) against B7-H4 in a preclinical model of human ovarian cancer. Four independent anti-B7-H4 CAR lentivirus constructs were generated using anti-B7H4 single chain variable fragments (scFv) with affinities ranging from low to high, which were transduced into primary human T cells. Transduced T cells efficiently expressed CARs on their cell surface, and each CAR was capable of binding both human and mouse recombinant B7-H4 protein with distinct binding patterns; two CARs (3#68 and 3#54) had high capacity for rhB7-H4 protein binding, another had low binding capacity (56), and the last had virtually none (26). In co-culture assays, high binding B7-H4 CAR T cells secreted Th1 cytokines and specifically lysed human B7-H4+ ovarian cancer cells. In an immunodeficient mouse model, transferred B7-H4 CAR T cells persisted after infusion and exerted efficient anti-tumor activity against subcutaneous B7-H4+ human ovarian tumor xenografts.Unexpectedly, B7-H4 CAR T cell treated mice reproducibly showed signs of delayed onset, lethal toxicity emerging 6-8 weeks after therapy. Postmortem analysis revealed widespread immunopathology in nearly all tissue analyzed, with marked infiltration and destruction of peripheral nerve bundles and perivasculature regions that was inconsistent with non-specific graft versus host disease (GVHD). Immunopathology was evident in both tumor-bearing and healthy mice administered B7-H-4 CAR T cells, and mice administered control CAR T cells experienced tumor regression but without concomitant toxicity, ruling out a generalizable GVHD effect.Our results indicate that CAR T cell-based targeting of B7-H4 can mediate control of B7-H4+ cancer outgrowth in vivo, however, long-term engraftment of B7-H4 CAR T cells mediates lethal on-target, off-tumor toxicity that is likely due to wide expression of B7-H4 in healthy mouse organs. Accordingly, B7-H4 CAR T cells represent an important preclinical model system for the evaluation of safety approaches that limit CAR-mediated toxicity whilst maintaining potent anti-tumor activity in vivo.