T-cell engager activity of etentamig (BCMA x CD3) is potentiated by daratumumab in preclinical models of multiple myeloma

Abstract Introduction: Etentamig is a differentiated, 2nd generation, BCMA x CD3 bispecific T-cell engager composed of a high avidity bivalent BCMA-binding domain, a low-affinity CD3-binding domain designed to reduce cytokine release syndrome, and a silenced Fc tail for extended half-life, enabling convenient dosing. Recent findings from a Phase 1b, open-label, dose-escalation and expansion trial (NCT05259839) showed that etentamig in combination with daratumumab, a CD38 monoclonal antibody, and dexamethasone demonstrated promising activity in heavily pre-treated relapsed/refractory multiple myeloma (RRMM) (Blood 2024;144(Supplement 1):496). The current study aims to further elucidate the mechanisms associated with daratumumab enhancement of etentamig activity through ex vivo studies. Methods: Peripheral blood mononuclear cells (PBMCs) and T cells, isolated from either healthy donors or myeloma patients, were co-cultured with MM cell lines (RPMI-8226, NCI-H929, LP-1, and U266B1). The co-cultures were treated with either etentamig alone or in combination with daratumumab for 4 days prior to assessment of tumor cell lysis and T-cell activation, immune modulation, and cytokine secretion through flow cytometric analysis and multiplex cytokine quantification (Luminex FLEXMAP 3D technology), respectively. Additionally, immunosuppressive T cell populations were quantified via multiparameter flow cytometric data from samples derived from newly diagnosed, daratumumab-naïve, and post daratumumab therapy MM patients. Results: Etentamig and daratumumab effectively induced the lysis of MM cell lines in co-culture with healthy donor and MM PBMCs. The amount of tumor cell lysis was significantly enhanced by the combination compared with either therapeutic molecule alone. Interestingly, etentamig-mediated MM lysis was enhanced with PBMCs from RRMM patients who had been exposed to daratumumab compared with daratumumab-naïve RRMM patients. In co-culture assays, etentamig in combination with daratumumab had reduced levels of forkhead box P3 (FOXP3)+ regulatory T cells (Treg) compared with etentamig treatment alone, as well as reduced levels of indoleamine 2,3-dioxygenase (IDO), an enzyme that plays an immunosuppressive role in the bone marrow microenvironment. Detailed immunophenotypic analysis through multiparameter flow cytometry highlighted the impact of daratumumab treatment on several Treg subsets characterized by CD38 expression. CD38+ Treg cells were previously shown to be more suppressive in vitro than CD38-negative Tregs and expressed additional markers indicative of their heightened immunosuppressive activity, such as inducible T-cell costimulator, HLA-DR, CD39, and Ki67. In parallel, etentamig in combination with daratumumab resulted in enriched programmed cell death protein 1 (PD1)-negative CD8+ T cells, with minimal impact on etentamig-induced T cell activation (CD8+CD25+) and proliferation markers (CD8+Ki67+). Furthermore, daratumumab treatment reduced levels of CD8+ T cells expressing PD1 and thymocyte selection-associated HMG-box (TOX), a gene that plays a crucial role in the induction of T cell dysfunction. Conclusions: We show that daratumumab enhances anti-tumor activity of etentamig by i) augmenting direct anti-tumor activity, ii) depleting immune regulatory and dysfunctional T cell populations, and iii) modulating the immunosuppressive bone marrow microenvironment (eg, IDO reduction). Collectively, our findings support the continued investigation of etentamig in combination with daratumumab in patients with MM.