Armored allogeneic Epstein-Barr virus specific T cells expressing a multi-functional B7-H3 chimeric antigen receptor exhibit improved persistence, expansion and performance in solid tumor models

Allogeneic chimeric antigen receptor (CAR)-T cells can address the high costs and poor availability associated with autologous CAR-T therapies. However, the development of these “off-the-shelf” are challenged with risks of graft-versus-host disease (GvHD) and allogeneic rejection by the patient's immune cells. Furthermore, whilst remarkable successes have been achieved in the treatment of hematological malignancies, strategies targeting solid tumors face further challenges including tumor trafficking, antigen heterogeneity, chronic antigen stimulation and an immunosuppressive microenvironment. To address these challenges, we employed allogeneic Epstein-Barr virus specific T cells (EBVSTs) that have been well tolerated in clinical trials. With a narrowed TCR repertoire, EBVSTs have lower GvHD potential. EBVSTs also express higher levels of CD74 and CXCR4 and can better migrate towards tumors cells. Allogeneic rejection by T/NK cells involves the activation of caspases. We show that armoring CAR-EBVSTs with an engineered serine protease inhibitor, SerpinB9(CAS), increases survival in mixed lymphocyte reactions and humanized mice models. Interestingly, we observed that the armored CAR EBVSTs are also resistant to activation induced cell death induced by chronic antigen stimulation. B7-H3 is an attractive CAR target due to its overexpression in multiple solid tumors, and tumor stroma, with limited expression in normal tissues. We expressed an engineered V HH -based B7-H3 CAR in our armored EBVSTs (B7H3.CAR-EBVSTs) and observed potent efficacy against various cell-line and patient derived xenografts in vivo models. Importantly, we showed that B7-H3 CAR also targets B7H3+ myeloid derived suppressor cells (MDSCs) and alloreactive T cells in co-culture experiments, further enhancing survivability in the presence of these immunological challenges. Lymphodepletion is an essential preconditioning step prior to CAR-T infusion to ensure efficient engraftment of the cells. However, adverse effects of lymphodepletion include severe cytopenia and increased risks of opportunistic infections. We show that a constitutive IL-7 receptor (IL-7) synergized with SerpinB9(CAS) to boost proliferation in the presence of allogeneic challenge, potentially reducing or removing the need for lymphodepletion. EBV infection has been associated with epithelial cancers including gastric and has been reported to upregulate B7-H3 in nasopharyngeal cancer (NPC). With partial HLA matching, B7H3.CAR EBVSTs can potentially mediate bispecific targeting of B7-H3+, EBV+ tumors in patients. Thus, our armored B7H3.CAR-EVBSTs present an attractive strategy that can overcome significant challenges and improve efficacy in solid tumors, strongly supporting its future development for evaluation in clinic.