Background Chimeric Antigen Receptor (CAR) T cell therapy is a new type of “living drug” that has proven to be a powerful immunotherapy for hematologic malignancies. To date, there are six CAR-T products approved by FDA, four CD19 targeted CAR-T cells, and two targeting B-cell maturation antigen (BCMA).^1-8 However, this success has not yet been transferred to solid tumors. A major hurdle is the on-target off-tumor toxicities due to the shared expression of target antigen on healthy tissues.

Methods Here, we assessed the in vitro cytotoxicity of carbonic anhydrase IX (CAIX) targeted CAR-T cells generated from a series of single chain fragment variables (scFvs) that have various affinities against CAIX. In addition, we studied the avidity of CAR-T cells using a cell avidity analyzer. We established a tetracycline (Tet)-On inducible CAIX expressing system that provides different CAIX levels on the cell surface covering the range from the density on CAIX-high skrc-59 cells to the one on CAIX-low MMNK-1 cholangiocytes. To assess the therapeutic effect of CAR-T on patient samples, we generated patient derived organotypic spheroids (PDOTS) ex vivo cultures and tested CAR-T cell migration and cytokine release using these miniature tumors.

Results We identified a low affinity, high avidity anti-CAIX CAR G9, which only kills CAIX high tumor cells but not CAIX-low normal tissues in vitro. G9 demonstrates a CAIX density dependent response on Tet-On inducible CAIX expressing cell lines. G9 CAR has a wider therapeutic window compared to G250 that caused serious adverse events in the first anti-CAIX CAR-T clinal trial.^9-11 G9 exhibits superior efficacy ex vivo on ccRCC PDOTS 3D cultures which recapitulates ccRCC patient tumor microenvironment (TME), as well as mitigating toxicity on cholangiectasis spheroids.

Conclusions In summary, affinity fine-tuned CAR-T cell therapy holds the promise to achieve cures of ccRCC by killing ccRCC tumor cells and mitigating on-target off-tumor toxicity on normal tissues.

References

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Ethics Approval The study obtained DFCI Office for Human Research Studies (OHRS) approval (IRB protocol #19-194).