A compact, anti-CD38 T-cell engager with a short half-life demonstrates superior anti-myeloma activity over traditional bispecific antibodies

Abstract

Abstract Introduction: Daratumumab (Dara) is a highly effective monoclonal antibody targeting CD38, a receptor that is overexpressed on multiple myeloma (MM) cells. Despite its efficacy, resistance to Dara inevitably develops, and outcomes following resistance are generally poor. Notably, approximately 80% of MM patients retain CD38 expression following relapse from Dara-based therapies, suggesting that immune dysregulation plays a pivotal role in resistance mechanisms rather than solely target-mediated changes. To overcome this immunologic resistance, we created a novel, compact, single-chain CD38-CD3 T cell engager (BN-CD38) designed to promote an effective immunological synapse between CD38pos leukemia cells and T cells, leading to T-cell activation and expansion, as well as augmented CD38 expression on the cancer cells through T-cell‒induced IFN-γ expression. Results: To evaluate the mechanism of action and efficacy of BN-CD38, we compared its cytotoxic activity in an MM cell line (MM.1S) to that of a conventional IgG1-derived anti-CD38 T cell engager (CD38-Bis) and a modified version of BN-CD38 containing an Fc domain (BN-CD38-Fc), which increases the synaptic distance between target (T) and effector (E) cells. Control groups also included human IgG and a mutant form of BN-CD38 that does not bind to CD38 (BN-CD38mut). BN-CD38 demonstrated significantly enhanced cytotoxicity in MM.1S with an IC₅₀ of 1.2×10-13 M at 24 hours (E:T ratio 1:1), representing about 1000-fold greater potency over that of CD38-Bis (IC₅₀ = 1.12×10-10 M) and BN-CD38-Fc (IC₅₀ = 1.66×10-10). This increased activity correlated with dose-dependent T-cell activation and IFN-γ secretion. Specifically, BN-CD38 induced release of IFN-γ levels at a mean of 20 ng/mL, in contrast to approximately 1 ng/mL for other constructs. BN-CD38 also exhibited superior anti-MM activity across multiple MM cell lines (MM.1S, H929, RPMI-8226), compared to effects from the FDA-approved T-cell engagers teclistamab (Tec) (MM.1S IC50 = 3.22×10-10 M) and talquetamab (Talq) (MM.1S IC50 = 1.69×10-10 M). T-cells pre-treated with BN-CD38 retained enhanced cytotoxicity upon rechallenge, outperforming those treated with Tec or Talq. Notably, BN-CD38 upregulated CD38 expression on MM cells via IRF1-IFN-γ–mediated pathways, potentially enhancing its own efficacy relative to engagers targeting other antigens in the MM cell lines we tested (MM.1S, KMS-11, L363, RPMI-8226, and U266). Pharmacokinetic and pharmacodynamic analyses in both immunodeficient NSG mice and immune-competent double transgenic mice (expressing human CD3 and CD38) showed that BN-CD38 has a plasma half-life of ~4 hours and a target-bound half-life of ~16 hours, substantially shorter than the ~14-day half-life reported for conventional IgG1-based engagers. In vivo efficacy was assessed by injecting 5×106 luciferase-labeled CD38⁺ MM.1S cells into NSG mice. On day 9, mice were randomized into three groups and treated weekly with 2.5 mg/kg BN-CD38, IgG, or BN-CD38mut, plus 5×106 human peripheral blood mononuclear cells (PBMCs) in each group. The BN-CD38 group significantly prolonged survival (median = 42 days) compared to the IgG group (median = 28 days, p = 0.005) and the BN-CD38mut group (median = 35 days, p = 0.005). Finally, BN-CD38 demonstrated robust activity in mice xenografted with RPMI-8226 cells, which are resistant to Dara. In this model, mice treated with BN-CD38 + human PBMCs had a significant increase in survival (median = 54 days) compared to mice co-treated with either IgG+PBMCs (median = 31 days, p = 0.02) or Dara+PBMCs (median = 38 days, p = 0.02). It also effectively activated T cells and killed MM cells in autologous settings using patient-derived samples from individuals who had progressed on Dara, CAR-T cells, or T cell engagers. Conclusions: BN-CD38, featuring a novel, compact structure and a short half-life, is an anti-CD38 T cell engager with superior anti-myeloma activity over that of FDA-approved T cell engagers. BN-CD38 exhibited enhanced T cell activation, potent cytotoxicity, and robust IFN-γ secretion even in models resistant to Dara, CAR-T, or other T-cell engager therapies. Despite its short systemic half-life, BN-CD38 achieved durable anti-tumor responses in vivo, supporting its potential as a next-generation immunotherapeutic for MM. Ongoing studies in immune-competent humanized myeloma models aim to further elucidate its therapeutic potential and inform clinical translation.