A Scalable Lentiviral Workflow for Laboratory-Scale Generation of BCMA/GPRC5D Co-Transduced CAR-T Cells in Multiple Myeloma

Establishment of a scalable engineered cell-line platform for direct, GMP-grade production of eVLP vectors enabling streamlined generation of gene-edited CAR-T/NK cells

An industrial-grade platform based on monoclonal producer cell lines that enables the continuous and scalable generation of engineered virus-like particles (eVLPs) co-packaging Cas9–gRNA ribonucleoproteins (RNPs) and provides a GMP-compliant and broadly adaptable strategy for the streamlined manufacturing of next-generation autologous and allogeneic gene-edited CAR-T/NK therapies.

TcBuster-M™ Enables Efficient Non-Viral Delivery of Multicistronic CD19-CAR Constructs in T and NK Cells 2259709

TcBuster-M-mediated cell engineering achieved high CD19-CAR expression in both T and NK cells while preserving cell viability and growth, and demonstrated potent, target-specific cytotoxicity and favorable cytokine secretion.

Suspension-Adapted HEK293FT Cells Enable High-Density Transfection for Efficient Lentiviral Vector Production in CAR-T Therapy

Lentiviral vector (LV) production for CAR-T therapy remains challenging due to the limited scalability of adherent HEK293 cell cultures. To address this, we adapted HEK293FT cells directly to serum-free FreeStyle 293 Expression Medium, generating a novel suspension cell line, HEK293FT-DS. We characterized growth kinetics, stability over 35 passages, and transient transfection parameters (PEI:DNA ratio, cell density). The suspension-adapted cells grew with >93% viability and a specific growth rate of 0.54 day−1, and maintained stable viable cell density (1.51 ± 0.03 × 106 cells/mL) over 35 passages. The optimal PEI:DNA ratio was 2.5:1, and increasing the transfection cell density to 16 × 106 cells/mL boosted functional titers to approximately 9 × 106 TU/mL in clarified supernatant. Scaled production (1 L culture supernatant per batch) yielded up to 5 × 109 TU. The resulting vectors efficiently transduced primary human T cells (57% CAR-positive cells) in a CliniMACS Prodigy-based process. The platform based on suspension-adapted HEK293FT-DS cells enables high-density transfection and provides a cost-effective, scalable alternative to commercial LV production systems, particularly suited for academic CAR-T cell manufacturing.

Development of a 3-day manufacturing method to generate CD19-CD20-CD22 trispecific CAR T-cells from whole blood

Chimeric antigen receptor (CAR) T-cell therapy has transformed the treatment landscape for many hematological malignancies. However, high relapse rates and limited accessibility remain significant challenges. We developed a 3-day streamlined process to address these limitations. T-cells were isolated from whole blood collected from healthy donors via an automated density gradient separation that included a T-isopure™ antibody cocktail, which isolates T-cells through negative selection. T-cells were activated then transduced with a lentiviral vector encoding a trispecific CAR. T-cells were cultured in G-Rex vessels and harvested at day 3 or day 7 for analysis. CAR expression and T-cell phenotype were assessed by flow cytometry and gene expression analysis. Functional activity was evaluated by measuring cytotoxicity and cytokine secretion following co-culture with target cell lines. The T-isopure isolation enriched CD3+ T-cells in whole blood from 18.9% to 88.5% of CD45+ cells, with a mean recovery of 40.6%. RBCs were depleted with ≥ 99% efficiency, with monocytes and NK cells comprising the bulk of remaining CD45+ cells. The 3-day manufacturing process produced T-cells with > 95% viability, 53% transduction efficiency, and vector copy number < 3 copies/cell. Phenotypic analysis revealed a high proportion of stem/central memory T-cells at both timepoints, with no significant differences observed. Cytotoxicity assays demonstrated strong and sustained killing of NALM6 tumor cells, comparable between both products. Gene expression profiling indicated that day 3 products were less differentiated, exhibiting a memory-like phenotype and reduced inflammatory signaling, further supported by protein analysis of culture supernatants. This study establishes a rapid, GMP-compliant method for manufacturing polyfunctional, CAR T-cells directly from whole blood. The workflow outlined here achieved a potent, phenotypically favorable CAR T-cell product without compromising viability or cytotoxic function. Compared to the standard 7-day method, the 3-day approach resulted in expression of genes associated with a more stem-like phenotype while reducing manufacturing time and cost. This method may provide a practical alternative for decentralized CAR T-cell manufacturing, particularly in resource-limited settings.

Engineering Stable Lentiviral Vector Producer Cells Improves LVV Production.

Stable lentiviral vector producer cell lines represent a promising platform for scalable and cost-efficient vector manufacturing, yet their productivity is often limited by intrinsic host-cell constraints. In this study, we aimed to identify cellular factors restricting LVV production in stable doxycycline-inducible GPRTG producer cell lines and to evaluate whether targeted host-cell engineering can improve vector yield. Comparative transcriptomic analysis of high- and low-producing clones revealed distinct differences in cellular pathways related to transcription, translation, energy metabolism, lipid homeostasis, stress response, and chromatin regulation. Based on differential gene expression, candidate genes were modified by CRISPR/Cas9-mediated knockout or overexpression and functionally analyzed in a low-producing clone. Single-gene KO screening identified H1-2, ADAMTS1, INSIG1, GADD45B, and HSPA1B to increase cell-specific LVV productivity up to 2.6-fold, with H1-2 showing the strongest effect. In addition, combinatorial disruption further improved productivity. In contrast, overexpression of selected candidates did not enhance LVV production. Overall, our results demonstrate that transcriptomics-guided host-cell engineering is an effective strategy to identify and relieve intrinsic bottlenecks in stable LVV producer cell lines.

EF-1α-driven nanobody-based CD19-redirected CAR-T cells retain the antitumor efficacy, antigen-driven expansion, and cytokine secretion of CMV-driven counterparts