Transcriptional signatures of tumor infiltrating CAR-T cells

We compared the second-generation (CD28, 4-1BB) with the third-generation (CD28-4-1BB) carbonic anhydrase IX (CAIX) targeted CAR constructs and investigated the antitumor effect of CAR-T cells with different CD4/CD8 proportion in vivo. The results demonstrated that anti-CAIX G36-4-1BB (BBζ) CAR-T cells exhibited superior efficacy compared to G36-CD28 (28ζ) and G36-CD28-4-1BB (28BBζ) CAR-T cells in a clear cell renal cell carcinoma (ccRCC) skrc-59 cell bearing NSG-SGM3 mouse model. Tumor infiltrating T cells were recovered and profiled via flow cytometry and 10X genomics single cell RNA sequencing (scRNAseq). We found that BBζ CAR-T cells upregulated human leukocyte antigen (HLA) II genes and cytotoxicity associated genes, while, downregulated inhibitory immune checkpoint receptor genes and differentiation of regulatory T cells (Tregs), leading to outstanding therapeutic efficacy in vivo. An increased memory phenotype, an elevated tumor infiltration, and a decrease in exhaustion related genes were observed in the CD4/8 mixture of untransduced T (UNT) cells compared to CD8 only ones, indicating that CD4/8 could be the favored cellular composition for CAR-T cell therapy with long-term persistence. In summary, these findings suggest that anti-CAIX BBζ CAR48 serves as a highly potent clinic translatable cell therapy for ccRCC with enhanced proliferation potential, increased functional capacity, diminished terminal differentiation, as well as durable immune surveillance Profile of anti-CAIX CAR-T cells and untransduced T cells, including BBz, 28z, 28BBz, UNT CD8 and UNT CD4/8.