Abstract 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 the FDA for hematologic malignancies, four targeting CD19, and two targeting B-cell maturation antigen (BCMA). 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 normal tissues. Carbonic anhydrase IX (CAIX) is highly expressed in clear cell renal cell carcinoma (ccRCC); however, it is also expressed on bile duct at a lower physiological level leading to off-tumor toxicity of CAIX targeted therapies. The first anti-CAIX CAR-T studies, using the 1st generation G250 CAR-T cells plus IL-2 to treat patients with metastatic ccRCC, caused severe liver enzyme abnormalities in the treated patients after CAR-T cell infusions. To understand CAIX expression on tumor and normal tissues, we quantified CAIX expression on ccRCC patient samples and healthy bile duct tissues using direct stochastic optical reconstruction microscopy (dSTORM) which provides single-molecule resolution. Tet-On inducible CAIX expressing cell lines were established to mimic various CAIX densities on normal tissue and tumor samples. Using biolayer interferometry (BLI) and avidity analyzer, we identified a low-affinity, high-avidity anti-CAIX CAR G9. G9 CAR-T cells only kill CAIX high ccRCC tumor cells but not CAIX low normal cholangiocytes, and exhibited a CAIX density dependent response to Tet-On inducible CAIX expressing cell lines. Compared to high-affinity G250 CAR-T cells, G9 showed a better safety profile and a wider therapeutic window. G9 demonstrated a superior ex vivo efficacy on ccRCC patient derived organotypic tumor spheroids (PDOTS) 3D cultures which recapitulate ccRCC patient tumor microenvironment (TME), as well as low toxicity on cholangiocyte derived organotypic spheroids (CDOS). In summary, affinity/avidity fine-tuned CAIX targeted CAR-T cell therapy holds promise to achieve cures of ccRCC by efficaciously killing tumor cells and mitigating on-target off-tumor toxicity on normal tissues. Citation Format: Yufei Wang, Alicia Buck, Gabriella Kastrunes, Rabia Abbas, Michael Lynch, Zhou Zhong, Song-My Hoang, Andras Miklosi, Kun Huang, Jae-Won Cho, Marion Grimaud, Cecile Razimbaud, Matthew Chang, Atef Fayed, Audrey Apollon, Nithyassree Murugan, Ze-Hua Li, Tran Thai, Luann Zerefa, Brandon Piel, Elena Ivanova, Amy Cameron, Quang-De Nguyen, Zhu Zhu, Kevin Wei, Yasmin Nabil Laimon, Aseman Bagheri Sheshdeh, Sabina Signoretti, David A. Braun, Catherine J. Wu, Toni K. Choueiri, Jon Wee, Cloud P. Paweletz, Martin Hemberg, Aedin C. Culhane, David A. Barbie, Gordon J. Freeman, Wayne A. Marasco. Fine-tuned CAIX targeted CAR-T cells exhibit superior efficacy and mitigate on-target off-tumor side effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 886.