295 Affinity fine-tuning anti-CAIX CAR-T cells mitigate on-target off-tumor side effects (2022)

<h3>Background</h3> 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).<sup>1-8</sup> 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. <h3>Methods</h3> Here, we assessed the <i>in vitro</i> 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) <i>ex vivo</i> cultures and tested CAR-T cell migration and cytokine release using these miniature tumors. <h3>Results</h3> We identified a low affinity, high avidity anti-CAIX CAR G9, which only kills CAIX high tumor cells but not CAIX-low normal tissues <i>in vitro</i>. 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.<sup>9-11</sup> G9 exhibits superior efficacy <i>ex vivo</i> on ccRCC PDOTS 3D cultures which recapitulates ccRCC patient tumor microenvironment (TME), as well as mitigating toxicity on cholangiectasis spheroids. <h3>Conclusions</h3> 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. <h3>References</h3> Maude, S. L., Laetsch, T. W., Buechner, J., Rives, S., Boyer, M., Bittencourt, H. <i>et al</i>. Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. <i>N Engl J Med</i>. 2018; Feb 1; <b>378</b>: 439-448, doi:10.1056/NEJMoa1709866. Schuster, S. J., Bishop, M. R., Tam, C. S., Waller, E. K., Borchmann, P., McGuirk, J. P. <i>et al</i>. 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H., Willemsen, R., van Elzakker, P., van Steenbergen-Langeveld, S., Broertjes, M., Oosterwijk-Wakka, J. <i>et al</i>. Immune responses to transgene and retroviral vector in patients treated with ex vivo-engineered T cells.<i>Blood</i>. 2011; Jan 6; <b>117</b>: 72-82, doi:10.1182/blood-2010-07-294520 <h3>Ethics Approval</h3> The study obtained DFCI Office for Human Research Studies (OHRS) approval (IRB protocol #19-194).