Multi-Antigen Targeting By Novel Combination of CAR-T Cells and hnCD16 Transgene, Yields in Complete Tumor Clearance Via Antibody Dependent Cellular Cytotoxicity

Tumor heterogeneity and antigen escape are primary obstacles to the durable and long-term efficacy of chimeric antigen receptor (CAR) T-cell therapy across multiple tumor indications. Antibody dependent cellular cytotoxicity (ADCC) is a critical innate mechanism of natural killer (NK) cells mediated to target and eliminate cancer cells bound by therapeutic monoclonal antibodies (mAbs). This process is directed through the Fc receptor CD16a, a potent activating receptor found on human NK cells. We previously demonstrated the high-affinity (158VV) variant of CD16 combined with a point mutation that prevents activation-induced surface cleavage, termed high-affinity non-cleavable CD16 (hnCD16), results in the enhancement of ADCC mediated by NK cells. Here we combined hnCD16 with an off-the shelf, allogeneic CAR T cell strategy to enable flexible and robust targeting of secondary tumor associated antigens (TAA) with therapeutic mAbs, a T-cell multi-antigen targeting strategy that has not yet been fully investigated. Induced pluripotent stem cells (iPSCs) were specifically engineered to express (i) MICA/B CAR from the T cell receptor alpha constant chain (TRAC) locus and (ii) a CAG driven hnCD16 from the CD38 locus. In addition to supporting the transgene expression, the knockout of the TRAC removes the risk of graft versus host disease, while CD38 knockout enables compatibility with anti-CD38 mAb targeting strategies. The multiplexed-engineered iPSCs were differentiated into alpha-beta T (iT) cells, demonstrating uniform and high levels of both CAR and hnCD16 (>95% for both). Downstream signaling of hnCD16 activation in iT cells via cross-linking antibodies elicited a rapid activation of both proximal (ZAP70 (2-fold increase) and LAT (2.4-fold increase) with peak at 10min and SLP76 (5.7-fold increase) with peak at 5 min following activation) and distal (ERK (2.5-fold increase) with peak at 10 min following activation) components of the CD3ζ signal transduction pathway, demonstrating hnCD16 functionality within CAR iT cells. Using several liquid and solid tumor lines (Nalm6, MDA-MB231 and CasKi) with varying CAR- and secondary-TAA levels, we demonstrate that hnCD16 complements CAR-mediated efficacy by enhancing tumor control. As an example, targeting MDA-MB231 line with hnCD16⁺ CAR-MICA/B iT cells resulted in increased cytotoxicity from 35% to 77% when combined with anti-PDL1 antibody at a 1:1 effector to target ratio (p<0.05). Additionally, hnCD16 and CAR co-activation resulted in enhanced inflammatory cytokine production, in accordance with secondary TAA levels. For example, the percentage of IFNy+ TNFa+ CAR iT cells increased by approximately 10-fold in a cytokine release assay when hnCD16⁺ CAR-MICA/B iT cells were combined with anti-CD38 antibody, daratumumab, to target Nalm6 cells. We have also demonstrated that hnCD16 and CAR co-activation over multiple rounds of tumor challenge can drive sustained tumor control and improved CAR iT cell expansion (up to 20-fold higher compared to CAR activation alone, p<0.005) without any evidence of phenotypic or functional exhaustion, underscoring the underlying synergy of co-activation of two unique targeting receptors. In an orthotopic ovarian tumor model where CAR iT cells demonstrate potent, but incomplete tumor control, in vivo activation of hnCD16 with therapeutic mAb enhanced tumor control and prevented tumor recrudescence. Together, these preliminary results demonstrate that specific arming of an off-the-shelf, allogeneic CAR iT cell product with a unique Fc receptor, hnCD16, enables a flexible and potent anti-tumor killing strategy to mitigate against tumor heterogeneity and to provide more durable and long-lasting responses in cancer patients.

Shirinbak:Fate Therapeutics: Current Employment. Grant:Fate Therapeutics: Current Employment. Yeh:Fate Therapeutics: Current Employment. Gentile:Fate Therapeutics: Current Employment. Shrestha:Fate Therapeutics: Current Employment. Yang:Fate Therapeutics: Current Employment. Pribadi:Fate Therapeutics: Current Employment. Sikaroodi:Fate Therapeutics: Current Employment. Mehta:Fate Therapeutics, Inc.: Current Employment. Yzaguirre:Fate Therapeutics Inc.: Current Employment. Pan:Fate Therapeutics: Current Employment. Chang:Fate Therapeutics: Current Employment. Lee:Fate Therapeutics: Current Employment. Smith:Fate Therapeutics: Current Employment. Wu:Fate Therapeutics: Research Funding. Walcheck:Fate Therapeutics: Research Funding. Hosking:Fate Therapeutics: Current Employment. Valamehr:Fate Therapeutics: Current Employment.