Design of a novel NanoCAR construct that compares well with a conventional ScFv-based CAR targeting BCMA on multiple myeloma

Abstract These findings demonstrate Nb17-nanoCAR-T exhibits potent anti-myeloma efficacy comparable to scFv-based CAR-T, supporting its potential as a promising therapeutic alternative. Multiple myeloma (MM) is an incurable hematologic malignancy arising from clonal plasma cells, with poor long-term outcomes due to inevitable relapse after conventional therapies. Chimeric antigen receptor (CAR) T-cell immunotherapy targeting B-cell maturation antigen (BCMA) has shown remarkable efficacy in relapsed patients. Conventional CARs typically employ single-chain variable fragments (scFvs), whereas single-domain antibodies (sdAb or VHHs) offer advantages such as small size, high stability, and potentially reduced immunogenicity. The aim of this research is to design and evaluate the activity of a novel anti-BCMA nanoCAR-T based on the VHH Nb17, and that compares well with a standard one with the conventional CAR-T CT103a. Nb17 was validated for strong BCMA binding on MM cell lines and incorporating into a CAR construct. Both nanoCAR-T and CT103a CAR-T were generated via lentiviral transduction of primary T cells. Their functional properties, including cytotoxicity, cytokine secretion, degranulation, memory phenotype, and gene expression, were assessed in vitro, and their antitumor efficacy was tested in vivo in NSG mice model. Nb17-nanoCAR-T demonstrated specific killing of MM cells, robust cytokine release (IL-2, TFNa, IFNg), and CT107a degranulation comparable to CT103a. Transcriptomic analysis revealed overlapping pathways between the two CAR-T products. Upon rechallenge with MM cells, both showed enhanced proliferation and reduced exhaustion compared with untransduced T cells. In vivo, Nb17-nanoCAR-T and CT103a eradicated MM1.S tumors, although both yet inducing graft-versus-host disease (GVHD)-like toxicity in a subset of mice.