e14521 Background: Chimeric antigen receptor (CAR) T cell immunotherapy has revolutionized treatment for hematological malignancies, where antigen expression is fairly ubiquitous, and the cancer cells are easily accessible. However, translating this success to solid tumors remains challenge due to unique obstacles in the tumor microenvironment (TME) including, a) metabolic insufficiency, where T cells fail to sustain effector functions and persistence within the metabolically hostile and nutrient-deprived TME; and b) T cell exhaustion and anergy, driven by the immunosuppressive milieu that dampens anti-tumor immunity. To overcome these formidable hurdles, CAR T cell therapy must evolve to incorporate the ability to finely tune CAR T cell activity by adjusting its “intensity” with time, and “flexibility” to target multiple antigens either simultaneously or sequentially. Methods: We used AVATAR and AVATAR AI incubators (Xcell Bio) to precisely control oxygen concentration and pressure to simulate TME conditions. Effector T cells including activated T cells (ATC) or bispecific antibody (BiAb) armed ATC (BATs) and bispecific antibody armed headless CAR T cells (ahCART) or unarmed hCART either grown or were acclimated under TME condition before coculturing with target cells. The effect of TME condition compared to normal incubator environment (NE) were determined on T cell proliferation, phenotypic changes and functional activity of hCARTs. Functional readouts were measured by real time cytotoxicity assay, and cytokine profiles under NE or TME-like conditions. Results: The T cells, when transduced with 4-1BB-ζ hCAR lentiviral vector show metabolically enhanced activity and are able to withstand hypoxic TME without compromising T cell effector functions. Survival was differentially affected by the co-stimulatory endodomains, hCARTs with 4-1BB-ζ (BBζ) showed superior survival under stringent hypoxia 0.5% O 2 (13% apoptosis) compared to hCARTs CD28-ζ (61% apoptosis). Specific cytotoxicity of anti-HER2 (HER2 hCARTs) and anti-EGFR BiAb armed hCARTs (EGFR hCARTs) show that MCF-7 cells are killed more effectively (~65-90%) by sequential treatment than by HER2- or EGFR-hCARTs alone (30-60% and 40-50%, respectively, using by real time cell analysis (RTCA) using xCelligence System. Targeting both EGFR and HER2 antigens sequentially induces a stronger immune response, reduces antigen escape, kills more tumor cells, and sustains a robust anti-tumor Th1 environment compared to targeting a single antigen. T cell reprogramming to adapt TME is likely to represent a novel and important aspect to enhance anti-tumor activity in solid tumor microenvironment. Conclusions: Our new metabolically enhanced TME acclimated hCAR T cells offer a new, versatile, immunotherapy platform that can be targeted "at will" to various tumor targets for prolonged killing and controlled toxicity.
Thakur et al. (Thu,) studied this question.