Overcoming Heterogeneity in AML with a Novel or-Gated Hitcar-1XX Platform

Abstract

Target heterogeneity limits therapeutic success of conventional CAR therapies in B cell and other malignancies. Therefore, novel concepts for efficient combinatorial targeting are urgently needed. Several OR-gated targeting approaches based on CARs have been described and include dual CARs (T cells co-expressing two CARs), tandem CARs (T cells expressing a single bi-specific CAR) and pooled single CAR-T cells (T cells expressing a single CAR). Clinical efficacy of such OR-gated CAR therapies is still under ongoing investigation, and emerging pre-clinical data suggests that optimization of OR-gated approaches requires fine-tuning of combinatorial CAR signaling to balance effector and memory functions, avoiding premature differentiation and eventual exhaustion. Here, we present a novel OR-gated platform based on co-expression of a CAR and a HIT (HLA-Independent TCR), which we previously described to afford T cells with increased antigen sensitivity (Mansilla-Soto et al., Nature Medicine 2022). Since HIT receptors do not incorporate engineered co-stimulatory domains like conventional CARs, we hypothesized that T cells co-expressing CAR and HIT display limited differentiation in comparison to dual CAR T cells, thus resulting in improved long-term anti-tumor activity. We termed this novel OR-gated therapy HITCAR and evaluated it in the setting of AML which stands out with a high degree of inter- and intra-individual phenotypic heterogeneity. We selected two target antigens which we had previously demonstrated to have a favorable and non-overlapping expression profile in normal hematopoietic cells and other tissues (Perna et al., Cancer Cell 2017). We selected a lower-density target for the HIT, using its increased sensitivity to avoid antigen-low escape, and selected a higher-density target for the CAR. We utilized an NSG xenograft model for target heterogeneity, based on co-engraftment of engineered MOLM13 AML cell line populations endogenously expressing either HIT target alone, CAR target alone, or both. To identify an optimal HITCAR design, we treated target-heterogeneous MOLM13 AML-bearing mice with HITCAR T cells containing different CAR architectures and found that only the 28z1XX CAR allowed for complete AML remission and long-term survival in conjunction with the HIT. All other CAR architectures failed to control AML long-term, highlighting the importance for fine-tuned CAR signaling in combination with HIT. We then selected the superior HITCAR-1XX and compared it to dual CARs containing BBz and 28z1XX architectures. In our NSG AML xenograft heterogeneity model, the HITCAR-1XX induced complete AML remission while the alternative dual CARs failed to control AML. T cell phenotyping demonstrated a less differentiated T cell phenotype over time with HITCAR-1XX as compared to the alternative dual CARs, supporting our hypothesis. Further validation of our HITCAR-1XX platform in clinically relevant AML PDX models is ongoing. Overall, we established a novel OR-gated HITCAR T cell platform based on combinatorial expression of HIT and CAR which can be applied to overcome target heterogeneity in various malignancies. In the setting of AML, we compared different HITCAR configurations and identified HITCAR-1XX as most promising. Furthermore, we demonstrated superiority of HITCAR-1XX over alternative dual CAR designs, highlighting its high potential for clinical translation.