VHH-based chimeric antigen receptor T cells against CD70 to eliminate primary and re-challenge renal cell carcinoma tumors in preclinical study.

Abstract

e14540 Background: CD70 is not expressed in normal tissue, but highly expressed on various hematopoietic and solid tumors, including renal cell carcinoma (RCC). It is an ideal target for cancer therapy and recently was proved by good results in clinical trials of CD70 monoclonal antibody. Methods: We obtained 4 VHH antibody（2A5, 8B4, 11C9 and 9D8）against CD70 by screen nanobody library derived from an antigen-immunized alpaca. Generated a panel of CAR-T cells by transduce retrovirus expressing CAR (chimeric antigen receptor) to human T cells isolated from health donors. These CAR constructs include 4 VHH sequences and a human scFv containing VH and VL derived from cusatuzumab. We evaluated CAR-T cell function in vitro included cell phenotype, proliferation, cytotoxicity, and cytokine release. Furthermore, RCC tumor cell-xenograft NOG mice models were used to test the anti-tumor activity of CAR-T cells in vivo. Results: All anti-CD70 CAR-T cells eliminate CD70-positive tumor cells but not CD70-negative cells in vitro. Although there were no significant differences between the killing efficiency of the CAR-T cells, more abundant IL-2 production in two VHH-based CAR-T cells (11C9 and 2A5) were observed in multi-round co-culture assay. CAR-T cells eradicated RCC tumor cell in our Xenograft model. After infusion, human T cells expanded over 250 folds in 11C9 group and over 30 folds in 2A5 group, but only nearly 6 folds in human scFv CAR-T treated group. In our second in vivo experiment, all mice treated with higher dose CAR-T, the VHH groups maintained tumor free statue much longer time than human scFv group. Cured mice were re-challenged by subcutaneously infuse RCC tumor cells, observed and measured tumor in the next 53 days. For 11C9 CAR-T treated group, 3of 7 mice maintained tumor-free, 1 of 7 mice were tumor-palpable but too small to measurement; For 2A5 treated group only 1 of 7 mice maintained tumor-free. A significantly greater expansion of human T cells in 11C9 group but not 2A5 group after re-challenge. Conclusions: Our results indicate a promising VHH-based CD70 CAR-T cells for RCC therapy, supporting further development for future clinical trial and application.