GENETICALLY ENGINEERED T CELLS EXPRESSING HIGH AFFINITY TCR ANTI-NY-ESO-1:HLA-A*02 ARE ABLE TO CONTROL SOLID TUMOR GROWTH IN VIVO

Abstract

Despite the remarkable success in the use of T cells expressing Chimeric Antigen Receptors (CAR) for hematologic malignances, their application to solid tumors, which represents the majority of neoplasms, remains challenging. However, T lymphocytes expressing artificial T Cell Receptors (TCR) with enhanced affinity, refered as TCR-T cells, have been providing encouraging results against solid neoplasms. Unlike conventional CAR, these receptors are able to recognize intracellular proteins presented by Human Leukocyte Antigen (HLA), expanding the options for therapeutic targets. The NY-ESO-1 is a prominent target in this immunotherapeutic approach due to its restricted expression in healthy cells, its capacity to induce an immune response and its strong expression in tumors of different origins. Therefore, our main goal was the generation of TCR-T cells targeting the NY-ESO-1:HLA-A*02 complex with antitumor capacity. Additionally, it is suggested that the co-expression of both endogenous and artificial TCR may result in TCR hybridization, compromising the efficacy and safety of TCR-T cells. Thus, we also generated NY-ESO-1:HLA-A*02 TCR-T cells modified to concomitant expression of a short hairpin RNA (shRNA) for endogenous TCR silencing (TCR + shRNA- T cells). The lentiviral transduction efficiency (3 donors), evaluated 8‒12 days after, was 18.2% (±5.2%) for TCR-T cells and 9.9% (±5.9%) for TCR + shRNA- T cells. The engineered T cells displayed in vitro antitumor activity (1‒2 donors) against NY-ESO-1+/HLA-A*02+ human tumor cells (SW982luc: synovial sarcoma; A375luc: melanoma). The TCR-T cells were able to lyse 67.7% of SW982luc or 22.3% (±7.6%) of A375luc cells in a co-culture at the 1:1 effector-to-target ratio. Similarly, the TCR + shRNA- T cells lysed 73% of SW982luc or 25.6% (±14.2%) of A375luc cells. Also, engineered T cells had no cytotoxic potencial against HCT116luc cells (NY-ESO-1−/ HLA-A*02+), demonstrating target specificity. Next, we generated an in vivo melanoma model through subcutaneous injection of 1×106 A375luc cells in NSG mice and after 6 days the animals (3‒5 per group) were intravenously treated with 7×106 engineered T cells or non-transduced T cells (NT T cells) (1 donor). Twenty days after the treatment, the tumor burden of 80% (4/5) of animals treated with TCR-T cells increased up to 1.5 times (average: 4.5 ± 7.7 times) and in 80% (4/5) of the animals that received TCR+ shRNA- T cells this increase was ≤3.4 times (average: 2.4 ± 2.9 times). However, the average tumor burden increased 39.9 times (±35.3) in animals treated with NT T cells and 824.1 times (±1302.1) in untreated mice. From day 20 to 34 post treatment only untreated animals (5/5) had tumors with volume ≥2cm3 and were euthanized. Collectively, these data demonstrate the successfull generation of T cells expressing high affinity TCR anti-NY-ESO-1:HLA-A*02 with NY-ESO-1 specific and potent antitumor activity. These results lay the groundwork for the development of a new advanced cell therapy product for solid neoplasms and other NY-ESO-1+ malignancies.