Abstract 3410: Fc engineering of CD9 antibodies mitigates platelet toxicity and retains potency against acute lymphoblastic leukemia

Abstract Background: Albeit significant progress in CD19/CD22-based immunotherapies, the outcomes of patients with relapse/refractory acute lymphoblastic leukemia (ALL) remains suboptimal. Tetraspanin CD9 endows proven prognostic impact and therapeutic relevance in ALL (Leung et al, 2020/2024). Yet, lethal platelet toxicities incurred by CD9 antibodies have hampered their clinical translation. This study aims to develop a safe and effective CD9 biologics through antibody engineering. Methods: Murine CD9 antibodies were generated by standard immunization/hybridoma technologies and humanized via complementarity-determining region (CDR) grafting. Antibody affinity and specificity were assessed against soluble CD9 antigen and CD9-expressing/depleted ALL cell lines, while efficacy was confirmed with cell line-derived and patient-derived xenograft (PDX) models. Platelet toxicity was evaluated by CD62P expression, optical aggregometry and in FcγRIIA transgenic mice. Mechanisms were explored by cell proliferation, apoptosis, imaging and antibody-dependent cellular cytotoxicity (ADCC) assays. Results: Two isolated murine monoclonal antibodies (mAbs) displayed distinct binding affinity and specificity towards the CD9 antigen, with biophysical properties preserved following humanization (hAbs). Administration of either mAbs or hAbs in 697-transplanted NOD/SCID mice markedly suppressed leukemia progression, alongside with substantial extension of animal survival. Consistent with previous reports, platelets were fully activated within 30 minutes after exposure to hAb IgGs but not their Fab or scFv fragments, indicating that platelet activation is a Fc-dependent event. By introducing a N297A mutation to the antibody Fc domain, platelet activation and aggregation was completely extinguished compared with the non-engineered version as reflected by absence of phenotypic CD62P expression (5.1±0.9% vs. 89.6±4.8%, P<0.0001) and physical aggregation (AUC: 18.9-29.5 vs. 382-395, P=0.0016). Additionally, no thrombocytopenia was observed in FcγRIIA transgenic mice with platelet counts in the hAb arm similar to those in the human IgG control arm (929±84 vs. 968±118, P=0.7935). Notably, N297A-modified hAbs profoundly reduced leukemia burden by >98% in PDXs developed from ALL patients who relapsed from upfront immunotherapies displaying CD19 antigen loss. Underlying its potency, Fc-modified hAbs triggered direct cell death and proliferation inhibition in CD9+ ALL cell lines following homotypic aggregation, a feature associated with type II antibodies. Cytotoxicity was mediated via an ADCC-independent mechanism. Conclusion: Fc engineering mitigates the platelet toxicity of CD9 antibodies but retains potency against refractory ALL, potentially breaking the decade-long hurdle for their clinical translation. Large animal studies are ongoing. Citation Format: Wing Hei Ng, Shengyu Fu, Kathy Yuen Yee Chan, Kin Yi Chung, Yuen Ching Tiffany Hui, Qi Zhao, Kam Tong Leung. Fc engineering of CD9 antibodies mitigates platelet toxicity and retains potency against acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3410.