Revealing the impact of CD70 expression on the manufacture and functions of CAR-70 T-cells based on single-cell transcriptomics

Abstract

Background: Chimeric antigen receptor-modified T cells (CAR T-cells) have shown exhilarative clinical efficacy for hematological malignancies. However, a shared antigen pool between healthy and malignant T-cells remains a concept to be technically and clinically explored for CAR T-cell therapy in T-cell cancers. No guidelines for engineering CAR T-cells targeting self-expressed antigens are currently available.

Method: Based on anti-CD70 CAR (CAR-70) T-cells, we constructed CD70 knock-out and wild-type CAR (CAR-70^KO and CAR-70^WT) T-cells and evaluated their manufacturing and anti-tumor capability. Single-cell RNA sequencing and TCR sequencing were performed to further reveal the underlying differences between the two groups of CAR T-cells.

Results: Our data showed that the disruption of target genes in T-cells before CAR transduction advantaged the expansion and cell viability of CAR T-cells during manufacturing periods, as well as the degranulation, anti-tumor efficacy, and proliferation potency in response to tumor cells. Meanwhile, more naïve and central memory phenotype CAR⁺ T-cells, with higher TCR clonal diversity, remained in the final products in KO samples. Gene expression profiles revealed a higher activation and exhaustion level of CAR-70^WT T-cells, while signaling transduction pathway analysis identified a higher level of the phosphorylation-related pathway in CAR-70^KO T-cells.

Conclusion: This study evidenced that CD70 stimulation during manufacturing process induced early exhaustion of CAR-70 T-cells. Knocking-out CD70 in T-cells prevented the exhaustion and led to a better-quality CAR-70 T-cell product. Our research will contribute to good engineering CAR T-cells targeting self-expressed antigens.

Keywords: CAR T-cell therapy; CD70; Single-cell RNA sequencing; Single-cell TCR sequencing; T-cell malignancy.