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

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

Maude, S. L., Laetsch, T. W., Buechner, J., Rives, S., Boyer, M., Bittencourt, H. et al. Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. N Engl J Med. 2018; Feb 1; 378: 439-448, doi:10.1056/NEJMoa1709866. Schuster, S. J., Bishop, M. R., Tam, C. S., Waller, E. K., Borchmann, P., McGuirk, J. P. et al. Tisagenlecleucel in Adult Relapsed or Refractory Diffuse Large B-Cell Lymphoma.N Engl J Med. 2019; Jan 3; 380: 45-56, doi:10.1056/NEJMoa1804980 Wang, M., Munoz, J., Goy, A., Locke, F. L., Jacobson, C. A., Hill, B. T. et al. KTE-X19 CAR T-Cell Therapy in Relapsed or Refractory Mantle-Cell Lymphoma.N Engl J Med. 2020; Apr 2; 382: 1331-1342, doi:10.1056/NEJMoa1914347. Abramson, J. S., Palomba, M. L., Gordon, L. I., Lunning, M. A., Wang, M., Arnason, J. et al. Lisocabtagene maraleucel for patients with relapsed or refractory large B-cell lymphomas (TRANSCEND NHL 001): a multicentre seamless design study. Lancet. 2020; Sep 19; 396: 839-852, doi:10.1016/s0140-6736(20)31366-0 Neelapu, S. S., Locke, F. L., Bartlett, N. L., Lekakis, L. J., Miklos, D. B., Jacobson, C. A. et al. Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma.N Engl J Med. 2017; Dec 28; 377: 2531-2544, doi:10.1056/NEJMoa1707447. Locke, F. L., Ghobadi, A., Jacobson, C. A., Miklos, D. B., Lekakis, L. J., Oluwole, O. O. et al. Long-term safety and activity of axicabtagene ciloleucel in refractory large B-cell lymphoma (ZUMA-1): a single-arm, multicentre, phase 1-2 trial.Lancet Oncol. 2019; Jan; 20: 31-42, doi:10.1016/s1470-2045(18)30864-7. FDA.FDA Approves First Cell-Based Gene Therapy for Adult Patients With Multiple Myeloma. 2021; Mar 27. Available at: https://www.fda.gov/news-events/press-announcements/fda-approves-first-cell-based-gene-therapy-adult-patients-multiple-myeloma.: Berdeja, J. G., Madduri, D., Usmani, S. Z., Jakubowiak, A., Agha, M., Cohen, A. D. et al. Ciltacabtagene autoleucel, a B-cell maturation antigen-directed chimeric antigen receptor T-cell therapy in patients with relapsed or refractory multiple myeloma (CARTITUDE-1): a phase 1b/2 open-label study.Lancet. 2021; Jul 24; 398: 314-324, doi:10.1016/s0140-6736(21)00933-8 Lamers, C. H., Klaver, Y., Gratama, J. W., Sleijfer, S. & Debets, R. Treatment of metastatic renal cell carcinoma (mRCC) with CAIX CAR-engineered T-cells-a completed study overview. Biochem Soc Trans. 2016; Jun 15; 44: 951-959, doi:10.1042/bst20160037 Lamers, C. H., Sleijfer, S., Vulto, A. G., Kruit, W. H., Kliffen, M., Debets, R. et al. Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience.J Clin Oncol. 2006; May 1; 24: e20-22, doi:10.1200/jco.2006.05.9964 Lamers, C. H., Willemsen, R., van Elzakker, P., van Steenbergen-Langeveld, S., Broertjes, M., Oosterwijk-Wakka, J. et al. Immune responses to transgene and retroviral vector in patients treated with ex vivo-engineered T cells.Blood. 2011; Jan 6; 117: 72-82, doi:10.1182/blood-2010-07-294520

Ethics Approval

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