Alloimmune Defense Receptor Harnesses Host Immune Cell Activation to Potentiate Functional Persistence and Anti-Tumor Activity of Off-the-Shelf, Cell-Based Cancer Therapy

Complexities associated with the production of patient-specific chimeric antigen receptor (CAR) T-cell therapies often limit broad patient accessibility. While many of these challenges can be overcome with an allogeneic cell product, the potential for immune cell-mediated rejection and limited product persistence are significant barriers to its clinical success. Both autologous and allogeneic CAR-T cell therapies currently rely on conditioning chemotherapy to deplete the immune system and create greater access to homeostatic cytokines. However, conditioning chemotherapy has been associated with poor immune reconstitution, protracted cytopenias resulting in an increased susceptibility to opportunistic infections, and secondary malignancies. Therefore, an ideal cell-based cancer therapy would be able to maintain functional persistence and anti-tumor activity while reducing or eliminating the need for patient conditioning.

To address many of these challenges, we thought to combine our alloimmune defense receptor (ADR) that targets 41BB⁺ activated immune cells while providing a CD3z signaling boost upon engagement, with our CAR NK cell construct that has been fine-tuned for NK cell biology and targets B-lymphocyte antigen CD19. These two synthetic modalities were incorporated into a master induced pluripotent stem cell (iPSC) line to serve as a renewable starting material for the derivation of iPSC-derived NK cells uniformly expressing ADR and anti-CD19 CAR (ADR⁺ CAR19 iNK cells).

The ability of ADR⁺ CAR19 iNK cells to resist host-mediated rejection was tested by co-culturing ADR⁺ CAR19 iNK cells with allogeneic PBMCs in a mixed lymphocyte reaction (MLR) assay. Notably, co-cultured ADR⁺ CAR19 iNK cells not only persisted, but also expanded over the duration of the assay, while co-cultured ADR^- CAR19 iNK cells were eliminated (Figure 1A, p<.001). To further increase the stringency of our assessment, we substituted naïve PBMCs with T cells primed and enriched for alloreactivity (primed-T cells) against the iNK cell. As observed with naïve PBMCs, ADR^- CAR19 iNK cells co-cultured with primed-T cells were depleted and expression of 41BB was detected among a large subset of primed-T cells. In contrast, ADR⁺ CAR19 iNK cells persisted, and the primed-T cells in the co-culture were reduced in number and had no detectable 41BB expression. Together, these allogeneic models demonstrate that ADR+ CAR19 iNK cells have the unique ability to target 41BB⁺ alloreactive T cells and to persist in an intact immune system, suggesting the potential to function without conditioning chemotherapy.

To confirm that ADR⁺ CAR19 iNK cells retain potent anti-tumor efficacy, we performed a rigorous tri-culture MLR study that combined daily-dosed CD19⁺ Nalm6 tumor cells and a single dose of primed- T cells with either ADR⁺ CAR19 or ADR^- CAR19 iNK cells (Figure 1B). In co-cultures with repeat restimulation of Nalm6 tumor cells, we found that tumor growth was well controlled with CAR19 iNK cells +/- ADR over the course of the assay. However, when primed-T cells were added, the tri-culture assay with ADR^- CAR19 iNK cells lost tumor control and 41BB⁺ primed-T cells were expanded. In contrast, ADR⁺ CAR19 iNK cells in the tri-culture assay suppressed 41BB⁺ primed-T cell expansion, persisted through the end of the assay, and demonstrated enhanced tumor growth inhibition which was similar to the control culture without primed-T cells (Figure 1B). Allogeneic xenograft tumor models assessing the ability of ADR⁺ CAR19 iNK cells to control lymphoma and leukemia in vivo in the presence of alloreactive T cells are ongoing and will be presented.

Our data suggest that ADR⁺ CAR19 iNK cells have the unique ability to withstand immune cell-mediated attack with uncompromised effector function. In preclinical alloreactivity models, ADR⁺ CAR19 iNK cells expand, functionally persist, and maintain anti-tumor activity in the presence of activated T cells. Collectively, ADR⁺ CAR19 iNK cells represent a promising approach to reduce the intensity of, or eliminate the need for, lymphodepletion. The favorable therapeutic index of cell-based cancer immunotherapies without conditioning potentially facilitates their broad clinical use through multiple lines of therapy, including in patients with newly-diagnosed disease.