A tandem bispecific nanobody CAR (SL1703) T cells targeting CD19/BCMA for refractory or relapsed multiple myeloma as salvage treatment after BCMA CAR-T therapy failure

Abstract Background: Despite that high response rate induced by BCMA chimeric antigen receptor T-cell therapy (CAR-T) in relapsed or refractory multiple myeloma (r/r MM), relapses still occurred and were resistant to CAR-T re-infusion. Due to the immunogenicity caused by the single-chain variable fragment (scFv) antibody of the previous BCMA CAR,bispecific nanobody-based CD19/BCMA CAR construct might be the solution. Objective The aim of this study is to develop and validate a structural-optimized bispecific nanobody-based CD19/BCMA CAR-T construct. This will be achieved through comprehensive in vitro screening, with the potential application being in r/r MM patients who relapsed or resistant to prior BCMA CAR-T therapy. Methods We engineered a bispecific CAR structure using our proprietary nanobodies and systematically evaluated its functional advantages over FDA-approved monospecific CAR-T therapies. A pilot study was conducted at Shanghai Liquan Hospital (Shanghai, China). The study enrolled r/r MM patients aged 18-75 years between November 2024 and July 2025, and the last follow-up was conducted on 30 July 2025. All included patients had failed prior BCMA-directed CAR-T therapy. The administered dose of bispecific nanobody-based CD19/BCMA CAR-T cells was 1.0×10⁶ CAR T cells per kg. Results Using our alpaca immunization and phage display platform, we identified nanobodies specific to novel epitopes on CD19 and BCMA. Monospecific CAR-T cells, which incorporate optimized CD19 and BCMA targeting VHH domains within a second-generation CAR framework, exhibited enhanced central memory phenotypes and tumor-killing activity compared to FDA-approved CAR-T therapies. The selected nanobodies were further engineered into tandem bispecific CAR constructs (SL1703 and SL0317), with SL1703 showing superior cytotoxic activity relative to SL0317. Between November 2024 and July 2025,5 patients were enrolled and received CAR-T products,3 were male and 2 were female,1 was white and 4 were Asian.The median age was 59.8 years (range,51-74). The median number of previous therapy lines was 4 (range, 2-5), and the median time from diagnosis was 63.4 months (range, 42-94).3 patients had Revised International Staging System II, and 2 had R-ISS III disease. Four patients had high-risk cytogenetic abnormalities, and 2 patients had extramedullary diseases. Two patients had a history of auto-HSCT. The average percentage of myeloma cells in bone marrow before the second CAR-T treatment was 27.6% (range, 1.5%-60%). In prior carT treatment, 3 patients had received BCMA-GPRC5D CAR-T and the other two received BCMA CAR-T. Three patients were primary resistant to the first CAR-T treatment, and the other two patients relapsed after the first CAR-T, with an average interval between two CAR-T treatments was 13.5 months (range,1-45).No dose-limiting toxicities (DLTs) were observed. All patients had cytokine release syndrome with Grade 1(80%) and Grade 2(20%). No neurotoxicity were observed. The most common grade 4 adverse events were hematological toxicities, affecting all 5 (100%) patients. This included 5 (100%) patients with neutropenia, 4 (80%) with anemia, and 4 (80%) with thrombocytopenia. Three patients had severe pancytopenia before CAR-T treatment. One patient was dependent on blood transfusion for 2 months and received allo-HSCT to restore hematopoietic reconstruction. Assessment at 30 days after CAR-T therapy showed that the overall response rate was (5/5) 100%. This included 4 (80%) with very good partial response (VGPR), and 1 (20%) with partial response (PR). Three patients obtained measurable residual disease (MRD) negativity at two weeks. The peak expansion (Cmax) of CAR-T cells was 231732 copies/μg DNA (range,11890-822166 copies/μg DNA), with a median Tmax of 13.5 days (range, 13-14). The median follow-up time was 3.6 months (range, 0.5-6). Two patients relapsed and died at 6 months, while the others remained remission. Conclusion The novel tandem bispecific nanobody-based CD19/BCMA CAR-T cell therapy might rescue the failure of previous BCMA CAR-T therapy.The phase 1 clinical trial has been registered and is recruiting(NCT07003555). Disclosures CAR vector is provided by Heibei Senlang Biotechnology Co., Ltd. CAR-T manufacturing and QC control are conducted in Beijing Gobroad Hospital.