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

Area of Science:

• Immunotherapy
• Oncology
• Molecular Biology

Background:

• Chimeric antigen receptor (CAR) T cell therapies face challenges in solid tumors due to on-target off-tumor (OTOT) toxicity.
• This toxicity arises from shared epitopes between tumor-associated antigens (TAAs) and healthy tissues.
• Developing safer CAR T-cell strategies is crucial for effective cancer treatment.

Purpose of the Study:

• To design and evaluate an affinity/avidity fine-tuned CAR T-cell therapy to mitigate OTOT toxicity.
• To investigate carbonic anhydrase IX (CAIX) as a target for clear cell renal cell carcinoma (ccRCC).
• To establish a safer therapeutic window for CAR T-cell treatment against ccRCC.

Main Methods:

• Utilized direct stochastic optical reconstruction microscopy (dSTORM) and flow cytometry to assess CAIX expression density.
• Developed a Tet-On doxycycline-inducible CAIX expressing cell line for controlled antigen mimicry.
• Assessed CAR T-cell killing, migration, and cytokine release using patient-derived organotypic tumor spheroid (PDOTS) cultures and an orthotopic mouse model.

Main Results:

• Identified high CAIX density on ccRCC and low density on healthy bile duct tissues.
• Demonstrated that low-affinity/high-avidity G9 CAR-T cells exhibit a wider therapeutic window than high-affinity/high-avidity G250 CAR-T cells.
• G9 CAR-T cells showed superior efficacy, migration, and cytokine release in PDOTS and enhanced tumor control in vivo compared to G250.

Conclusions:

• The fine-tuned G9 CAR-T therapy successfully mitigated OTOT side effects against CAIX-expressing ccRCC.
• This approach makes CAIX a viable and druggable immunotherapeutic target for ccRCC.
• G9 CAR-T therapy represents a promising advancement in improving the safety and efficacy of CAR T-cell treatments for solid tumors.