A Conceptual Framework: BiFLEX33-276, a Bispecific Antibody Targeting CD33 and B7-H3 for Enhanced Immunogenic Cell Death

Background: Refractory acute myeloid leukemia (AML) and other aggressive hematologic malignancies often evade existing therapies, underscoring the need for innovative treatment strategies. CD33 is a myeloid antigen broadly expressed on AML blasts and has been successfully targeted by antibody-drug conjugates (ADCs) (e.g., gemtuzumab ozogamicin)(Walter et al., 2012), yet relapse due to residual disease and antigen escape remains common (Jen et al., 2018). B7-H3 (CD276) is an immune checkpoint molecule frequently overexpressed in AML blasts (Lichtman et al., 2021) and associated with immune evasion and poor prognosis (Tan and Zhao, 2024). Objective: We propose BiFLEX33-276, a bispecific IgG-like antibody that simultaneously targets CD33 and B7-H3, aiming to induce enhanced immunogenic cell death (ICD) of leukemic cells while modulating the tumor microenvironment to improve anti-tumor immunity. We compare its theoretical efficacy to monospecific agents (anti-CD33 ADC and anti-B7-H3 antibody) in terms of tumor cell killing, T-cell activation, and ICD marker release. Methods: We performed a comprehensive literature-guided design and in silico simulation. Key parameters (antigen expression, cytotoxic payload effects, and immune checkpoint blockade outcomes) were derived from published experimental data. Simulated co-culture assays measured leukemic cell viability, T-cell activation (e.g., interferon-γ release), and ICD hallmarks (surface calreticulin exposure, ATP and HMGB1 release) across treatment conditions. Results: The simulation predicts that BiFLEX33-276 dramatically reduces leukemic cell viability (to ~20% of control) compared to anti-CD33 ADC (~50%) or anti-B7-H3 antibody (~80%). BiFLEX33-276 also elicited greater T-cell activation (approx. 4–5 fold increase over baseline) and higher levels of ICD markers (e.g. calreticulin exposure on ~40% of cells) than either monospecific treatment alone. Figure 1 illustrates these comparative outcomes, demonstrating that dual targeting yields synergistic tumor cell killing and immune stimulation. Conclusion: The dual-targeting strategy of BiFLEX33-276 addresses both tumor-intrinsic and immune-evasive mechanisms in refractory disease. By combining direct cytotoxicity against CD33^+ cells with concurrent blockade of B7-H3-mediated immunosuppression, BiFLEX33-276 promotes robust ICD and may effectively convert the tumor into an endogenous vaccine, thereby engaging T cells to eradicate residual disease. This bispecific antibody approach holds promise to improve outcomes in patients with refractory AML and related malignancies. Further preclinical validation, including in vivo efficacy and safety studies, is warranted to translate these findings into clinical application.