CTLA-4-Based Activation Inducible Costimulatory Receptors Endow CAR-T Cells with Safe, Tumor-Restricted, Multi-Targeting and Increased Persistence

Adoptive immunotherapy with chimeric antigen receptor-engineered T cells (CAR T cells) has achieved impressive clinical results in B-cell leukemias/lymphomas and multiple myeloma. Nevertheless, there are still challenges related to heterogeneity in antigen expression, off-tumor/on-target toxicity and functional persistence hindering the widespread application of CAR T cell therapy on the entire cancer landscape. In order to overcome the above challenges, next generation CAR T designs have been proposed, using multi-targeting, Boolean logic-gating concepts and/or synthetic receptor-based transcriptional circuits. Approaches that simultaneously allow multi-targeting, ensure tumor-specificity and improve CAR T cell longevity, could greatly increase the potential of CAR T cell therapy and broaden their therapeutic landscape. Here, we exploited the unique trafficking properties of the CTLA-4 molecule to generate a spatially-controlled, multi-targeting strategy, to safely enhance a tumor-specific CAR T cell lytic capacity, overcoming heterogenous target-antigen expression patterns, improve functional persistence and overall anti-tumor function.

CTLA-4 is a primarily intracellular protein whose surface retention, upon T cell activation, is regulated by the phosphorylation of its intracellular (IC) domain. We generated chimeric costimulatory receptors targeting CD38 that contain the CTLA-4 intracellular (IC) domain. Indeed, the addition of CTLA-4-based protein motifs bestowed inducible characteristics, as the receptor remained in the cytoplasm at steady state, while its surface expression was induced upon in vitro T cell activation with CD3/CD28 beads. We named these constructs CTLA-4-based activation inducible receptors (CAVI-R). Further, characterization of the dynamics of the induced CAVI-R expression, upon activation with beads or through a co-expressed BCMA-CAR, showed rapid ON/OFF kinetics; CAVI-R was detectable 6 hours after activation, peaked at 25 hours post-activation and, after removal of the stimulus, expression began decaying with complete disappearance within 30hr.

As expected, when combining a BCMA-CAR with a CD38-CAVI-R, we found that induction of the CAVI-R expression improved T cell cytotoxicity against BCMA⁺CD38⁺ cell lines. In addition, when both the CAR and the CAVI-R were designed with a CD8α TM domain, tumor lysis was mediated also through the CAVI-R engagement. In an heterogenous tumor cell line model, where BCMA⁺CD38⁺ cells were mixed with BCMA^-CD38⁺ cells, BCMA-CAR+CD38-CAVI-R T cells were able to also eliminate BCMA^-CD38⁺ cells, both in-vitro and in-vivo, since induction of CAVI-R expression could be initiated through in-trans engagement of BCMA expressed on bystander tumor cells. Importantly, this effect was abrogated outside the tumor site, as BCMA^-CD38⁺ cells injected at distal sites of the same mouse were left intact. Also, CAVI-R expression was absent in T cells retrieved from these sites, demonstrating the spatially restricted expression of the CAVI-R. Thus, the CAR+CAVI-R strategy could be used for safe, multi-targeting in tumors with heterogeneity in target expression. Moreover, in the absence of a tumor-specific antigen target, fine-tuning of CAR-binding affinity could achieve tumor-restricted CAVI-R expression and ensure CAR+CAVI-R tumor-specificity. Finally, we observed that CAR+CAVI-R T cells expanded significantly better than CAR+CCR T cells in vitro. In vivo, BCMA-CAR+CD38-CAVI-R T cells more effectively controlled BCMA⁺CD38⁺ tumor growth than BCMA-CAR+CD38-CCR T cells and persisted longer within the tumor sites, indicating that non-continuous costimulation further improves the expansion and persistence capacities of CAR T cells. In conclusion, we, here, show the generation of novel activation-inducible costimulatory receptors by the fusion of the CTLA-4 IC domain, without the need of transcriptional regulation. Our in vitro and in vivo data demonstrate that the CAR+CAVI-R strategy can provide CAR T cells with the potential for simultaneous better cytotoxicity, spatially-controlled and safe multi-targeting of heterogenous tumors, as well as improved expansion and in vivo persistence. The CAR+CAVI-R combination can provide a novel approach to expand CAR T cell applicability and improve its anti-tumor efficacy.