Immune-restoring CAR-T cells display antitumor activity and reverse immunosuppressive TME in a humanized ccRCC mouse model.

One of the major barriers that have restricted successful use of chimeric antigen receptor (CAR) T cells in the treatment of solid tumors is an unfavorable tumor microenvironment (TME). We engineered CAR-T cells targeting carbonic anhydrase IX (CAIX) to secrete anti-PD-L1 monoclonal antibody (mAb), termed immune-restoring (IR) CAR G36-PDL1. We tested CAR-T cells in a humanized clear cell renal cell carcinoma (ccRCC) orthotopic mouse model with reconstituted human leukocyte antigen (HLA) partially matched human leukocytes derived from fetal CD34+ hematopoietic stem cells (HSCs) and bearing human ccRCC skrc-59 cells under the kidney capsule. G36-PDL1 CAR-T cells, haploidentical to the tumor cells, had a potent antitumor effect compared to those without immune-restoring effect. Analysis of the TME revealed that G36-PDL1 CAR-T cells restored active antitumor immunity by promoting tumor-killing cytotoxicity, reducing immunosuppressive cell components such as M2 macrophages and exhausted CD8+ T cells, and enhancing T follicular helper (Tfh)-B cell crosstalk.

Anti-CAIX BBζ CAR4/8 T cells exhibit superior efficacy in a ccRCC mouse model.

Improving CAR-T cell therapy for solid tumors requires a better understanding of CAR design and cellular composition. Here, we compared second-generation (BBζ and 28ζ) with third-generation (28BBζ) carbonic anhydrase IX (CAIX)-targeted CAR constructs and investigated the antitumor effect of CAR-T cells with different CD4/CD8 proportions in vitro and in vivo. The results demonstrated that BBζ exhibited superior efficacy compared with 28ζ and 28BBζ CAR-T cells in a clear-cell renal cell carcinoma (ccRCC) skrc-59 cell bearing NSG-SGM3 mouse model. The mice treated with a single dose of BBζ CD4/CD8 mixture (CAR4/8) showed complete tumor remission and remained tumor-free 72 days after CAR-T cells infusion. In the other CAR-T and control groups, tumor-infiltrating T cells were recovered and profiled. We found that BBζ CAR8 cells upregulated expression of major histocompatibility complex (MHC) class II and cytotoxicity-associated genes, while downregulating inhibitory immune checkpoint receptor genes and diminishing differentiation of regulatory T cells (Treg cells), leading to excellent therapeutic efficacy in vivo. Increased memory phenotype, elevated tumor infiltration, and decreased exhaustion genes were observed in the CD4/8 untransduced T (UNT) cells compared with CD8 alone, indicating that CD4/8 would be the favored cellular composition for CAR-T cell therapy with long-term persistence. In summary, these findings support that BBζ CAR4/8 cells are a highly potent, clinically translatable cell therapy for ccRCC.