One of the major hurdles that has hindered the success of chimeric antigen receptor (CAR) T cell therapies against solid tumors is on-target off-tumor (OTOT) toxicity due to sharing of the same epitopes on normal tissues. To elevate the safety profile of CAR-T cells, an affinity/avidity fine-tuned CAR was designed enabling CAR-T cell activation only in the presence of a highly expressed tumor associated antigen (TAA) but not when recognizing the same antigen at a physiological level on healthy cells. Using direct stochastic optical reconstruction microscopy (dSTORM) which provides single-molecule resolution, and flow cytometry, we identified high carbonic anhydrase IX (CAIX) density on clear cell renal cell carcinoma (ccRCC) patient samples and low-density expression on healthy bile duct tissues. A Tet-On doxycycline-inducible CAIX expressing cell line was established to mimic various CAIX densities, providing coverage from CAIX-high skrc-59 tumor cells to CAIX-low MMNK-1 cholangiocytes. Assessing the killing of CAR-T cells, we demonstrated that low-affinity/high-avidity fine-tuned G9 CAR-T has a wider therapeutic window compared to high-affinity/high-avidity G250 that was used in the first anti-CAIX CAR-T clinical trial but displayed serious OTOT effects. To assess the therapeutic effect of G9 on patient samples, we generated ccRCC patient derived organotypic tumor spheroid (PDOTS) ex vivo cultures and demonstrated that G9 CAR-T cells exhibited superior efficacy, migration and cytokine release in these miniature tumors. Moreover, in an RCC orthotopic mouse model, G9 CAR-T cells showed enhanced tumor control compared to G250. In summary, G9 has successfully mitigated OTOT side effects and in doing so has made CAIX a druggable immunotherapeutic target.
Molecular cancer. 2024 Mar 16*** epublish ***
Yufei Wang, Alicia Buck, Brandon Piel, Luann Zerefa, Nithyassree Murugan, Christian D Coherd, Andras G Miklosi, Haraman Johal, Ricardo Nunes Bastos, Kun Huang, Miriam Ficial, Yasmin Nabil Laimon, Sabina Signoretti, Zhou Zhong, Song-My Hoang, Gabriella M Kastrunes, Marion Grimaud, Atef Fayed, Hsien-Chi Yuan, Quang-De Nguyen, Tran Thai, Elena V Ivanova, Cloud P Paweletz, Ming-Ru Wu, Toni K Choueiri, Jon O Wee, Gordon J Freeman, David A Barbie, Wayne A Marasco
Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA., Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA., ONI (Oxford Nanoimaging Ltd.), Oxford, UK., Molecular Imaging Core, Dana-Farber Cancer Institute, Boston, MA, 02215, USA., Department of Pathology, Brigham and Women's Hospital, Boston, MA, 02115, USA., Harvard Medical School, Boston, MA, 02115, USA., LUMICKS, Waltham, MA, 02453, USA., Lurie Family Imaging Center, Center for Biomedical Imaging in Oncology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA., Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA., Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA. .