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295 Affinity fine-tuning anti-CAIX CAR-T cells mitigate on-target off-tumor side effects
  1. Yufei Wang1,
  2. Alicia Buck1,
  3. Michael Lynch2,
  4. Gabriella Kastrunes1,
  5. Jae-Won Cho3,
  6. Marion Grimaud1,
  7. David Braun4,
  8. Cecile Razimbaud1,
  9. Matthew Chang1,
  10. Atef Fayed1,
  11. Audrey Apollon1,
  12. Ze-Hua Li1,
  13. Luann Zerefa1,
  14. Brandon Piel1,
  15. Elena Ivanova1,
  16. Dennis Bonal1,
  17. Kristen Jones1,
  18. Quang-De Nguyen1,
  19. Zhu Zhu3,
  20. Kevin Wei3,
  21. Rebecca Jennings1,
  22. Miriam Ficial1,
  23. Maura Aliezah Sticco-Ivins1,
  24. Sabina Signoretti3,
  25. Catherine Wu1,
  26. Toni Choueiri1,
  27. Jon Wee3,
  28. Cloud Paweletz1,
  29. Martin Hemberg3,
  30. Aedin Culhane2,
  31. David Barbie1,
  32. Gordon Freeman1 and
  33. Wayne Marasco1
  1. 1Dana-Farber Cancer Institute, Boston, MA, USA
  2. 2University of Limerick, Limerick, Ireland
  3. 3Brigham and Women’s Hospital, Boston, MA, USA
  4. 4Yale University, New Haven, CT, USA


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.


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

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