Mateusz Broncel, Emilia Morawiec, Ewa Nowak, Agnieszka Polak, Daria Matczyńska, Adam Pudełko, Anna Bednarska-Czerwińska

Manufacturing of dual anti-BCMA/anti-GPRC5D CAR-T cells in a closed, GMP-compatible system – product characterization and in vitro activity in a multiple myeloma model

2026-09-09

Background. Chimeric antigen receptor T (CAR-T) cells are classified as Advanced Therapy Medicinal Products (ATMPs), whose quality is intrinsically dependent on the manufacturing process. For autologous cell-based medicinal products, Critical Process Parameters (CPPs) directly influence Critical Quality Attributes (CQAs), including gene transfer efficiency, cellular composition, and biological activity. Simultaneous targeting of the BCMA and GPRC5D antigens may improve the therapeutic efficacy against multiple myeloma by reducing the risk of antigen escape; however, this approach requires a robust and reproducible manufacturing process enabling efficient co-transduction with two lentiviral vectors.

Objective. The aim of this study was to evaluate the feasibility of a closed, automated manufacturing process for dual-targeted anti-BCMA/anti-GPRC5D CAR-T cells using a platform compatible with GMP requirements, with particular emphasis on the impact of the manufacturing process on selected critical quality attributes of the final product.

Materials and Methods. The manufacturing process was performed using the CliniMACS Prodigy platform and included T-cell activation, simultaneous co-transduction with two lentiviral vectors, and a 14-day expansion in a closed system. The presence of both transgenes was confirmed using molecular methods. Gene transfer efficiency was assessed by GFP expression, while immunophenotypic changes, cytotoxic activity against the NCI-H929 multiple myeloma cell line at different effector-to-target (E:T) ratios, and cytokine secretion profiles were evaluated as indicators of product functionality.

Results. The manufacturing process enabled the generation of a dual-targeted CAR-T cell product fulfilling the predefined functional quality criteria. The proportion of GFP-positive cells reached 30.7% on day 14 of culture, while the percentage of CD8⁺ T cells increased from 22.4% to 50.8%, indicating effective expansion of the cytotoxic T-cell population. Following 48 hours of co-culture with NCI-H929 cells, tumor growth inhibition reached 94.12%, 76.14%, and 46.39% at E:T ratios of 1:1, 1:2, and 1:5, respectively. Cytokine analysis demonstrated a predominantly Th1-oriented immune response, confirming the preservation of effector function throughout the manufacturing process.

Conclusions. The presented results provide preliminary proof of feasibility for manufacturing a dual-targeted CAR-T product in a closed, automated environment compatible with GMP requirements while preserving the evaluated quality attributes and biological activity. Nevertheless, the lower gene-transfer efficiency observed compared with laboratory-scale production indicates that transfer of the process to GMP conditions requires further optimization and formal validation of critical process parameters within a Quality by Design (QbD) framework. These proof-of-concept data should therefore not be interpreted as full process validation.

Keywords: multiple myeloma, CAR-T cells, BCMA, GPRC5D, advanced therapy medicinal products.

© Farm Pol, 2026, 82(3): 159–167