DUAL CAR STRATEGY FOR SPECIFIC AND SAFE TARGETING OF T-CELLS AGAINST OVARIAN CANCER
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Omer Samuel Azar*1,3,4, Einav Hubel Manor*1,4, Naamit Deshet Unger1,4, Galit Horn1,4, Moran Rawet Slobodkin1,4, Kenneth Hollander1,4, Tova Waks1,4, Yael Raz2.3, Ido Laskov2, Ben Zion Katz5,6,7, Dan Grisaru#,2,3, and Anat Globerson Levin#,1,4
1. Immunology and Advanced CAR-T Therapy Laboratory, Tel Aviv Sourasky Medical Center,
2. Department of Gynecologic Oncology, Lis Hospital for Women, Tel Aviv Medical Center, Tel Aviv, Israel.
3. Faculty of Medical and Health Sciences, Department of Gynecology, Tel Aviv University, Tel Aviv, Israel
4. Dotan Center for Advanced Therapies, Tel Aviv Sourasky Medical Center, Tel Aviv University, Tel Aviv 6423906, Israel.
5. Faculty of Medical and Health Sciences, Department of Hematology, Tel Aviv University, Tel Aviv, Israel
6. Division of Hematology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel
7. Division of Clinical Laboratories, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel.
Objectives: Ovarian cancer remains one of the most lethal gynecologic malignancies, mainly due to late diagnosis and limited effectiveness of available treatments in advanced disease. Although chimeric antigen receptor T-cell (CAR-T) therapy has revolutionized the treatment of hematologic malignancies, its application has been hampered in solid tumors such as ovarian cancer, partly due to concerns for safety. Chief among these safety concerns is on-target, off-tumor toxicity, whereby CAR-T cells mistakenly attack normal tissues that express the targeted antigen, even if only weakly. This mode of toxicity stems primarily from the unavailability of completely tumor-specific antigens. To breach this crucial limitation, we designed a dual-receptor CAR-T system to enhance tumor selectivity while suppressing off-tumor activity.
Methods: We engineered a novel dual CAR-T construct on the basis of an "AND-gate" logic, where T-cell activation and cytotoxicity both rely on simultaneous recognition of two distinct tumor-associated antigens—CD24 and CD138. Both antigens are strongly overexpressed in ovarian cancer, and are less concurrently expressed on normal tissues. The dual CAR design thus splits signaling between two receptors: one providing activation (e.g., FcyR) and the other providing costimulation (e.g., CD28 or 4-1BB), requiring both for optimal T-cell activation.
Results: In vitro tests revealed that dual CAR-T cells maintained robust effector functions at non-inferior levels to traditional mono-CAR-T cells, while also showing decreased levels of exhaustion. In vivo ovarian cancer xenograft models exhibited a somewhat decreased tumor control when compared to the mono-CARs, yet still markedly superior tumor control than that of untransduced T-cells, possibly striking a good balance between efficacy and safety.
Conclusions: The dual CAR-T strategy forms a strong path to safer, more selective immunotherapies for ovarian cancer. By requiring binding of two distinct tumor-associated antigens, the strategy reduces off-tumor toxicity and holds promise for use in other solid tumors with distinct antigen combinations.