Fc-engineered anti-CD9 antibodies with reducing platelet toxicity and efficacy against acute lymphoblastic leukemia.

e14501 Background: While CD19/CD22-targeted immunotherapies have advanced, treatment outcomes for patients with relapsed or refractory acute lymphoblastic leukemia (ALL) remain unsatisfactory. CD9 is emerging as a key therapeutic and prognostic target in ALL, but severe platelet toxicity caused by CD9 antibodies has hampered clinical applications. This study focuses on engineering a safe and effective CD9-based therapeutic antibody to address these challenges. Methods: Humanized CD9 antibodies were analyzed for their specificity and binding affinity to the CD9 antigen and ALL cell lines. Efficacy was assessed in both cell line-derived and patient-derived xenograft (PDX) models. Platelet toxicity was evaluated through CD62P expression, platelet aggregation assays, and studies in FcγRIIA transgenic mice. Mechanistic investigations included assays for cell proliferation, apoptosis, imaging, and antibody-dependent cellular cytotoxicity (ADCC). Results: Two murine monoclonal antibodies (mAbs) exhibited distinct binding properties, which were preserved after humanization (hAbs). Both mAbs and hAbs significantly delayed leukemia progression in NOD/SCID mice engrafted with the 697 cell line, leading to improved survival. IgG-based hAbs activated platelets, whereas Fab or scFv fragments did not, indicating Fc domain involvement. Introducing the N297A mutation into the Fc domain prevented platelet activation and aggregation, as confirmed by the lack of CD62P expression and optical aggregation. No thrombocytopenia was observed in FcγRIIA transgenic mice. In PDX models of ALL relapsed from CD19-targeted therapies, N297A-modified hAbs reduced leukemia burden by over 98%. Fc-engineered hAbs triggered homotypic aggregation, leading to direct cell death and growth inhibition in CD9+ ALL cells, independent of ADCC. Conclusions: Engineering the Fc domain of CD9 antibodies mitigates platelet toxicity without compromising therapeutic efficacy in refractory ALL, providing a promising avenue for clinical translation. Further studies in large animal models are ongoing.