Immune Restoring (IR) CAR-T Cells Display Superior Antitumor Activity and Reverse Immunosuppressive TME in a Humanized ccRCC Orthotopic Mouse Model.

Chimeric antigen receptor (CAR) T cells are routinely used in the treatment of hematologic malignancies, but there are several barriers that have restricted their successful use in the treatment of solid tumors. It has been hypothesized that one of the major barriers is due to an unfavorable tumor microenvironment (TME). To provide a more favorable TME we engineered CAR-T cells targeting carbonic anhydrase IX (CAIX) G36 to secrete PD-L1 monoclonal antibodies, which we refer to as immune restoring (IR) CAR G36-PDL1. We tested IR CAR-T cells in a humanized clear cell renal cell carcinoma (ccRCC) orthotopic mouse model (hccRCC-NSG-SGM3) with reconstituted human leukocyte antigen (HLA) matched human leukocytes derived from fetal CD34+ hematopoietic stem cells (HSCs) and bearing human ccRCC skrc-59 cells under the kidney capsule. Our results demonstrated that the CD45+ tumor infiltrating leukocytes (TILs) from hccRCC-NSG-SGM3 reconstituted most CD45+ cell types including NK cells, dendritic cells, macrophages, exhausted CD8 T cells, and regulatory T cells that are observed in the TME of advanced ccRCC patients from clinical trials of the anti-PD-1 monoclonal antibody nivolumab. We found that G36-PDL1 CAR-T cells had a potent anti-tumor effect, while those without immune restoring effect had limited ability to control tumor growth. Analysis of the TME revealed that G36-PDL1 CAR-T cells restored active antitumor immunity by promoting tumor killing cytotoxicity, reducing immunosuppression cell components such as M2 macrophages and exhausted CD8 T cells, and enhancing Tfh-B cell crosstalk..