B-cell maturation antigen (BCMA)-targeting chimeric antigen receptor (CAR) T-cell immunotherapy has shown promising results in the treatment of relapsed or refractory multiple myeloma (R/RMM). This study presents the updated long-term outcomes from our center.
Between July 30, 2018, and September 27, 2023, 141 patients with R/RMM who received BCMA CAR-T therapy were enrolled. Patients underwent conditioning chemotherapy with cyclophosphamide and fludarabine, followed by BCMA CAR-T cell infusion at a median dose of 2.36×106 cells/kg. The study evaluated overall response rates, long-term efficacy, safety profiles, and their associations with clinical and disease characteristics.
At a median follow-up of 20.2 months, the safety profile of the therapy was manageable. Grade 3/4 cytokine release syndrome occurred in 36.2% of patients, with no cases of severe neurotoxicity reported. 1-month post-infusion, grade ≥3 anemia persisted in 39.6% of patients, while neutropenia (43.3%) and thrombocytopenia (52.2%) were observed. The objective response rate (ORR) among evaluable patients was 94.8%, with 50.7% achieving a complete response (CR). The 4-year progression-free survival and overall survival rates were 37.4% (95% CI, 29.1% to 48.1%) and 63.2% (95% CI, 54.8% to 72.8%), respectively, with survival curves showing gradual flattening over time. Patients with a history of autologous stem cell transplantation (ASCT) and those with extramedullary disease demonstrated significantly inferior efficacy and survival outcomes. Peak CAR-T cell expansion was positively correlated with ORR (p<0.001) and CR (p<0.001). Notably, patients with prior ASCT exhibited significantly lower CAR-T cell expansion compared with those without prior ASCT (p<0.001). Immunophenotypic analysis of infused CAR-T cells demonstrated impaired fitness in patients who received ASCT in the past year.
BCMA CAR-T therapy in patients with R/RMM results in significant and sustained responses, with a manageable safety profile on a large scale. Prior ASCT and extramedullary disease represent adverse prognostic factors. Patients with a history of ASCT demonstrate limited peak CAR-T cell expansion.
© Author(s) (or their employer(s)) 2025. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ Group.

The non-viral production of CAR-T cells through electroporation of transposon DNA plasmids is an alternative approach to lentiviral/retroviral methods. This method is particularly suitable for early-phase clinical trials involving novel types of CAR-T cells. The primary disadvantage of non-viral methods is the lower production efficiency compared to viral-based methods, which becomes a limiting factor for CAR-T production, especially in chemotherapy-pretreated lymphopenic patients.
We describe a good manufacturing practice (GMP)-compliant protocol for producing CD19 and CD123-specific CAR-T cells based on the electroporation of transposon vectors. The lymphocytes were purified from the blood of patients undergoing chemotherapy for B-NHL or AML and were electroporated with piggyBac transposon encoding CAR19 or CAR123, respectively. Electroporated cells were then polyclonally activated by anti-CD3/CD28 antibodies and a combination of cytokines (IL-4, IL-7, IL-21). The expansion was carried out in the presence of irradiated allogeneic blood-derived mononuclear cells (i.e., the feeder) for up to 21 days.
Expansion in the presence of the feeder enhanced CAR-T production yield (4.5-fold in CAR19 and 9.3-fold in CAR123). Detailed flow-cytometric analysis revealed the persistence of early-memory CAR-T cells and a low vector-copy number after production in the presence of the feeder, with no negative impact on the cytotoxicity of feeder-produced CAR19 and CAR123 T cells. Furthermore, large-scale manufacturing of CAR19 carried out under GMP conditions using PBMCs obtained from B-NHL patients (starting number=200x10e6 cells) enabled the production of >50x10e6 CAR19 in 7 out of 8 cases in the presence of the feeder while only in 2 out of 8 cases without the feeder.
The described approach enables GMP-compatible production of sufficient numbers of CAR19 and CAR123 T cells for clinical application and provides the basis for non-viral manufacturing of novel experimental CAR-T cells that can be tested in early-phase clinical trials. This manufacturing approach can complement and advance novel experimental immunotherapeutic strategies against human hematologic malignancies.
Copyright © 2024 Mucha, Štach, Kaštánková, Rychlá, Vydra, Lesný and Otáhal.

Directly activating CD8+ T cells within the tumor through antigen-presenting cells (APCs) hold promise for tumor elimination. However, M2-like tumor-associated macrophages (TAMs), the most abundant APCs in tumors, hinder CD8+ T cell activation due to inefficient antigen cross-presentation. Here, we demonstrated a personalized nanotherapeutic platform using surgical tumor-derived galactose ligand-modified cancer cell membrane (CM)-coated cysteine protease inhibitor (E64)-loaded mesoporous silica nanoparticles for postsurgical cancer immunotherapy. The platform targeted M2-like TAMs and released E64 within lysosomes, which reshaped antigen cross-presentation and directly activated CD8+ T cells, thus suppressing B16-OVA melanoma growth. Furthermore, this platform, in combination with anti-PD-L1 antibodies, enhanced the therapeutic efficacy and substantially inhibited 4T1 tumor growth. CMs obtained from surgically resected tumors were used to construct a personalized nanotherapeutic platform, which, in synergy with immune checkpoint blockade (ICB), effectively inhibited postsurgical tumor recurrence in 4T1 tumor. Our work offered a robust, safe strategy for cancer immunotherapy and prevention of postsurgical tumor recurrence.

Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs). Over 30 ModPoKI screens across human TCR- and CAR-T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct that enhanced fitness of chronically stimulated CAR-T cells and anti-cancer function in vitro and in vivo. ModPoKI's modularity allowed us to generate an ∼10,000-member library of TF combinations. Non-viral KI of a combined BATF-TFAP4 polycistronic construct enhanced fitness. Overexpressed BATF and TFAP4 co-occupy and regulate key gene targets to reprogram T cell function. ModPoKI facilitates the discovery of complex gene constructs to program cellular functions.
Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

The piggyBac transposon system provides a non-viral alternative for cost-efficient and simple chimeric antigen receptor (CAR) T cell production. The generation of clinical-grade CAR T cells requires strict adherence to current good manufacturing practice (cGMP) standards. Unfortunately, the high costs of commonly used lentiviral or retroviral vectors limit the manufacturing of clinical-grade CAR T cells in many non-commercial academic institutions. Here, we present a manufacturing platform for highly efficient generation of CD19-specific CAR T cells (CAR19 T cells) based on co-electroporation of linear DNA transposon and mRNA encoding the piggyBac transposase. The transposon is prepared enzymatically in vitro by PCR and contains the CAR transgene flanked by piggyBac 3' and 5' arms. The mRNA is similarly prepared via in vitro transcription. CAR19 T cells are expanded in the combination of cytokines interleukin (IL)-4, IL-7, and IL-21 to prevent terminal differentiation of CAR T cells. The accurate control of vector copy number (VCN) is achieved by decreasing the concentration of the transposon DNA, and the procedure yields up to 1 × 108 CAR19 T cells per one electroporation of 1 × 107 peripheral blood mononuclear cells (PBMCs) after 21 days of in vitro culture. Produced cells contain >60% CAR+ cells with VCN < 3. In summary, the described manufacturing platform enables a straightforward cGMP certification, since the transposon and transposase are produced abiotically in vitro via enzymatic synthesis. It is suitable for the cost-effective production of highly experimental, early-phase CAR T cell products.
© 2021 The Authors.