Abstract Title CD84-CD3 Bispecific Antibody Achieves Potent Anti-Leukemic Activity with Favorable Safety Profile in Acute Myeloid Leukemia Background Bispecific T-cell engagers (BiTEs) activate T cells independently of MHC/costimulatory receptors, providing an off-the-shelf approach. However, AML therapy is challenged by lack of antigens specific to leukemia stem cells and toxicity from targeting broadly expressed myeloid cell antigens. Notably, CD84 is highly expressed on AML blasts but minimal expression on normal HSCs -an advantage over CD33 and CD123 that are expressed on HSCs. Our previous studies identified CD84 as a key regulator of AML survival, with high expression promoting leukemogenesis and correlating with shorter overall survival. In this study, we report for the first time that a CD84-CD3 bispecific antibody achieves potent anti-leukemic activity with favorable safety in AML. Methods Nine CD84-CD3 bispecific antibodies were generated; three candidates (#03, #05, #07) were selected by expression yield and purity, with #03 and #05 prioritized for superior T-cell activation and AML cytotoxicity. #05 showed potent in vivo efficacy, and two high-affinity BiTEs (#24, #26) were constructed. In vitro, redirected T-cell cytotoxicity against AML cell lines/patient blasts was tested at varying effector-to-target (E: T) ratios, and antibody concentrations (0.01 to 100nM) via CFSE/7-AAD flow cytometry. At E: T ratio of 3:1, IC50, colony forming cell assays, T cell activation (CD25/CD9), and cytokine release (IFN-γ, TNF-α, IL-2, IL-8) were analyzed. In NCG mice xenografted with AML cell line (THP1) and patient cells, #24 or isotype control was administered post T-cell transfer. Leukemic engraftment (bone marrow, spleen, blood), spleen weight, and survival were evaluated. Results To comprehensively characterize the expression pattern of CD84 in AML, we first analyzed the correlation between CD84 expression and clinical subtypes based on age, FAB classification, and mutational status. Regardless of age or cytogenetics stratification, the proportion of CD84 expression in AML blasts was significantly higher than that in healthy donors (p0.0001). Elevated CD84 levels were observed in M1, M2, M4, and M5 subtypes, compared with healthy controls. Longitudinal analysis showed dynamic changes: decreased levels at complete remission versus initial diagnosis (p=0.01) and re-elevation at relapse (p=0.03), highlighting potential clinical relevance. Standard AML cell lines (HL-60, Molm13, MV4-11, OCI-3, THP-1, Kasumi-1) uniformly exhibited high CD84 expression (80%). Primary AML cells (both bulk blasts and CD34+ populations) showed significantly higher CD84 expression compared to healthy donor PBSCs (p0.0001). Compared to other AML surface antigens, CD84 showed similar levels in AML blasts, suggesting its potential as a selective therapeutic target. In vitro, treatment with serial concentrations of #24/#26 BiTEs (0.01 nM to 100 nM) in the presence of healthy donor T cells and AML cell lines (U937, THP-1) or primary AML blasts led to significant T cell activation, cytokine release (e.g., IFN-γ, TNF-α, IL-2, IL-8), impaired colony formation, and dose-dependent antileukemic activity, indicating effective T cell-mediated cytotoxicity. Importantly, these treatments had no significant effect on the viability or colony formation of healthy donor PBSCs, suggesting a selective action against AML cells. In vivo, THP-1 Luci xenograft NSG mice treated with #03 or #05 in combination with T cells showed significantly reduced tumor burden and prolonged survival (median 61 and 76 days, respectively) compared to the isotype control group (median 34 days, p0.0001 and p=0.0007). Additionally, in a PDX model generated from a CD84-high AML patient, treatment with #24 BiTE + T cells resulted in significantly longer survival (median 107 days vs. 78 days; p=0.0003) and reduced AML burden in bone marrow (p=0.0005), spleen (p=0.04), and peripheral blood (p=0.03). The #24 BiTE-treated group also showed a significant reduction in spleen weight at sacrifice (p=0.03). These findings confirm the potential of #24 BiTE as an effective therapeutic strategy for targeting CD84-high AML, with promising preclinical efficacy in both xenograft and patient-derived models. Conclusion This study establishes a strong foundation for the clinical translation of CD84-CD3 bispecific antibodies, demonstrating enhanced targeting specificity and favorable safety profiles for AML treatment.
Zhuo et al. (Mon,) studied this question.