BCMA-BBZ-OX40 CAR-T Therapy Using an Instant Manufacturing Platform in Multiple Myeloma

Abstract

Background: Chimeric antigen receptor (CAR)-T cell has revolutionary efficacy against relapsed/refractory multiple myeloma (R/R MM). However, current CAR-T cell therapy has several limitations including long vein-to-vein time and limited viability.

Methods: A 4-1BB-costimulated B-cell maturation antigen (BCMA) CAR-T integrating an independently-expressed OX40 (BCMA-BBZ-OX40) was designed and generated by a traditional manufacturing process (TraditionCART) or instant manufacturing platform (named InstanCART). The tumor-killing efficiency, differentiation, exhaustion, and expansion level were investigated in vitro and in tumor-bearing mice. An investigator-initiated clinical trial was performed in patients with R/R MM to evaluate the outcomes of both TraditionCART and InstanCART. The primary objective was safety within 1 month after CAR-T cell infusion. The secondary objective was the best overall response rate.

Results: Preclinical studies revealed that integrated OX40 conferred BCMA CAR-T cells with superior cytotoxicity and reduced exhaustion levels. InstanCART process further enhanced the proliferation and T-cell stemness of BCMA-BBZ-OX40 CAR-T cells. BCMA-BBZ-OX40 CAR-T cells were successfully administered in 22 patients with R/R MM, including 15 patients with TraditionCART and 7 patients with InstanCART. Up to 50% (11/22) patients had a high-risk cytogenetic profile and 36% (8/22) had extramedullary disease. CAR-T therapy caused grade 1-2 cytokine release syndrome in 19/22 (80%) patients, grade 1 neurotoxicity in 2/22 (9%) patients and led to ≥grade 3 adverse events including neutropenia (20/22, 91%), thrombocytopenia (15/22, 68%), anemia (12/22, 55%), creatinine increased (1/22, 5%), hepatic enzymes increased (5/22, 23%), and sepsis (1/22, 5%). The best overall response rate was 100%, and 64% (14/22) of the patients had a complete response or better. The median manufacturing time was shorter for InstanCART therapy (3 days) than for TraditionCART therapy (10 days). Expansion and duration were dramatically higher for InstanCART cells than for TraditionCART cells.

Conclusions: BCMA-BBZ-OX40 CAR-T cells were well tolerated and exhibited potent responses in patients with R/R MM. InstanCART shortened the manufacturing period compared to TraditionCART, and improved the cellular kinetics. Our results demonstrated the potency and feasibility of OX40-modified BCMA CAR-T cells using InstanCART technology for R/R MM therapy.

Trial registration number: This trial was registered at www.

Clinicaltrials: gov as #NCT04537442.

Keywords: Chimeric Antigen Receptor - CAR; Hematologic Malignancies; Immunotherapy; Multiple Myeloma; T cell.

Figure 1. Trial profile, patient response and survival to CAR-T-cell infusion. (A) Trial profile; (B) patient response after CAR-T infusion; (C) Kaplan-Meier plot of progression-free survival; (D) Kaplan-Meier plot of overall survival. CAR-T-cell, chimeric antigen receptor T cell; InstanCART, instant manufacturing platform; TraditionCART, traditional manufacturing process.

Figure 2. CAR-T-cell expansion, persistence, and cytokine analysis in patients. (A) Schematic of BCMA-BBZ-OX40 and BCMA-BBZ CAR constructs; (B) comparison of the AUC_0–28d between InstanCART and TraditionCART group. AUC_0–28d, area under the curve of CAR-T cell expansion from d0 to d28; bars indicate mean of AUC_0–28d of different patients, *p<0.05; (C) CAR-T-cell pharmacokinetics; (D) heatmaps indicating peak values of cytokines grouped by individual patients; (E) boxplots depicting fold changes in proinflammatory cytokine production from pre-infusion to the peak of CAR-T-cell expansion; boxplots indicate the range of the central 50% of the data, with the central line marking the median value; whiskers extend from each box to show the range of the remaining data. *p<0.05 by unpaired t-test. AUC, area under the curve; BCMA, B-cell maturation antigen; CAR-T-cell, chimeric antigen receptor T cell; CRS, cytokine release syndrome; IL, interleukin; InstanCART, instant manufacturing platform; scFv, single-chain fragment variable; TNF, tumor necrosis factor; TraditionCART, traditional manufacturing process.

Figure 3. Reduction of the manufacturing time improved CAR-T-cell function in vitro and in vivo. (A) The general procedure for manufacturing InstanCART and TraditionCART; (B) Flux (p/s) values of tumor burden assessed by IVIS imaging; (C) CAR⁺ T-cell counts of CAR-T cells in the PB of mice at different time points after CAR-T treatment. LD indicated 2×10⁶ CAR⁺ T cells in the TraditionCART group and 2×10⁶ total T cells in the InstanCART group. HD indicated 5×10⁶ CAR⁺ T cells in the TraditionCART group and 5×10⁶ total T cells in the InstanCART group. *p<0.05, **p<0.01, not significant by unpaired Mann-Whitney test. CAR-T-cell, Chimeric antigen receptor T cell; CM, cryopreservation medium; InstanCART, instant manufacturing platform; PB, peripheral blood; TraditionCART, traditional manufacturing process.