Humanized biparatopic nanobody-based CAR-T cells overcome antigen-heterogeneity in multiple myeloma

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy targeting B-cell maturation antigen (BCMA) shows activity in multiple myeloma (MM), yet relapse remains common owing to heterogeneous BCMA expression and soluble BCMA (sBCMA). Improving BCMA-directed CAR-T therapy requires persistence, enhanced antitumor activity, tolerance to low antigen density and resistance to sBCMA-mediated inhibition. METHODS: We identified anti-BCMA VHHs from a humanized phage display library, constructed monospecific and biparatopic CARs, and evaluated their function and optimal designs in preclinical models, including patient-derived MM cells and xenografts. RESULTS: Biparatopic Nab5822 CAR-T cells exhibited superior cytotoxicity and cytokine secretion (IL-2, IFN-γ, TNF-α) against antigen-heterogeneous MM cells in vitro, maintained activity under a supraphysiological sBCMA challenge at 120 ng/mL, and retained function under repeated exposure to clinically relevant sBCMA levels. Under repeated antigen challenge, Nab5822 preserved lysis, reduced exhaustion with an increased stem-cell memory T-cell compartment, and achieved durable tumor control in xenograft models without detectable toxicity. Mechanistic analyses indicated that the VHHs engage non-overlapping BCMA epitopes and promote improved immunological synapse organization together with coordinated proximal signaling, features that are not fully explained by equilibrium affinity alone and may contribute to sustained long-term T-cell function. CONCLUSIONS: Our findings support biparatopic CAR design as a strategy to tolerate low antigen density and resist sBCMA-mediated inhibition, providing a rationale for clinical evaluation of next-generation CAR T-cell therapies.