Background B7-Homolog 6 (B7-H6) is a B7 family member and the natural ligand for NK cell-activation receptor, NKp30. B7-H6 is expressed on multiple tumor types but has limited expression in normal tissues. Given this tumor specificity, B7-H6 represents an attractive target for CAR T therapy. CAR T cell therapy is associated with high clinical response rates in hematologic malignancies, but opportunities for improved efficacy in solid tumors remain. γδ T cells, whose solid tumor infiltration has demonstrated a significant correlation with survival, combine innate and adaptive mechanisms to recognize and kill tumors. In addition, γδ T cells engineered with CARs have shown enhanced tumoricidal activity and compelling clinical efficacy. Here, we evaluated the antitumor activity of γδ T cells modified with a set of novel scFv-based CARs targeting B7-H6, potentially applicable against multiple cancer indications for which natural tissue tropism of γδ T cells may offer advantages.
Methods Phage-display libraries were used to identify scFvs against B7-H6 epitopes. To confirm activation upon target engagement, scFvs formatted into CARs were evaluated in a Jurkat-Lucia™ NFAT reporter cell line. PBMCs from healthy donors were used to activate, expand, and engineer Vδ1 T cells to express libraries of CAR constructs representing permutated scFv arrangements. Vδ1 CAR T cells were assessed for phenotype and in vitro activity using flow cytometry and cell-based assays. Tumor xenograft models were further used to evaluate and assess CAR candidates for in vivo efficacy.
Results Phage-display derived scFvs showed a diverse range of affinities against multiple B7-H6 epitopes. Increased NFAT activity post-activation and minimal tonic signaling was observed in the majority of CAR-modified Jurkat-Lucia™ cells. Vδ1 CAR T cells manufactured from healthy PBMC donors demonstrated a predominant naïve-like phenotype with low levels of exhaustion-associated markers. Upon in vitro stimulation with B7-H6+ cell lines, leading Vδ1 CAR T cells inhibited tumor cell growth, demonstrated antigen associated proliferation, and released proinflammatory cytokines. Robust tumor growth inhibition was also observed for a subset of active constructs against tumor xenografts in immunodeficient mice (figure 1).
Conclusions In summary, we present the preclinical discovery, optimization, and generation of allogeneic γδ CAR T cells targeting B7-H6 with potential applications across a broad set of cancer indications. Based on these data, continued preclinical development of a clinical lead candidate is ongoing for future evaluation in the clinic.
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