efficient-production-chimeric-antigen-receptor-car-t-cells-with

Efficient Production of Chimeric Antigen Receptor (CAR) T Cells with Transgenes Exceeding 10 kb Using Lentiviral Vectors

Engineering multiple receptors into chimeric antigen receptor (CAR) T cells has emerged as a powerful strategy to prevent antigen-negative relapse and ...

University of Pennsylvania

CD19- and B-cell maturation antigen (BCMA)-directed chimeric antigen receptor (CAR) T cells have enabled unprecedented responses in a subset of refractory patients with B-cell and plasma cell malignancies, leading to their approval by the FDA for the treatment of leukemia, lymphoma, and myeloma. These "living drugs" can become part of a synthetic immune system, persisting at least a decade in some patients. However, despite this tremendous impact, significant unmet treatment needs remain for patients with hematologic malignancies and solid cancers. In this perspective, we highlight recent innovations that advance the field toward production of a more potent and universal cellular immunotherapy of the future. Next-generation CAR T cells will incorporate advances in gene engineering and synthetic biology to enhance functionality and persistence, and reduce treatment-associated toxicities. The combination of autologous CAR T cells with various allogeneic cell treatment strategies designed to target the immunosuppressive tumor microenvironment will broaden the impact of future CAR T-cell therapies.

Next-Generation CAR T-cell Therapies.

In the absence of cell surface cancer-specific antigens, immunotherapies such as chimeric antigen receptor (CAR) T cells, monoclonal antibodies, or bispecific T cell engagers typically target lineage antigens. Currently, such immunotherapies are individually designed and tested for each disease. This approach is inefficient and limited to a few lineage antigens for which the on-target/off-tumor toxicities are clinically tolerated. Here, we sought to develop a universal CAR T cell therapy for blood cancers directed against the pan-leukocyte marker CD45. To protect healthy hematopoietic cells, including CAR T cells, from CD45-directed on-target/off-tumor toxicity while preserving the essential functions of CD45, we mapped the epitope on CD45 that is targeted by the CAR and used CRISPR adenine base editing to install a function-preserving mutation sufficient to evade CAR T cell recognition. Epitope-edited CD45 CAR T cells were fratricide resistant and effective against patient-derived acute myeloid leukemia, B cell lymphoma, and acute T cell leukemia. Epitope-edited hematopoietic stem cells (HSCs) were protected from CAR T cells and, unlike CD45 knockout cells, could engraft, persist, and differentiate in vivo. Ex vivo epitope editing in HSCs and T cells enables the safe and effective use of CD45-directed CAR T cells and bispecific T cell engagers for the universal treatment of hematologic malignancies and might be exploited for other diseases requiring intensive hematopoietic ablation.

Epitope base editing CD45 in hematopoietic cells enables universal blood cancer immune therapy.

Human natural killer (NK) cell-based therapies are under assessment for treating various cancers, but cryopreservation reduces both the recovery and function of NK cells, thereby limiting their therapeutic feasibility. Using cryopreservation protocols optimized for T cells, here we find that ~75% of NK cells die within 24 h post-thaw, with the remaining cells displaying reduced cytotoxicity. Using CRISPR-Cas9 gene editing and confocal microscopy, we find that cryopreserved NK cells largely die via apoptosis initiated by leakage of granzyme B from cytotoxic vesicles. Pretreatment of NK cells with a combination of Interleukins-15 (IL-15) and IL-18 prior to cryopreservation improves NK cell recovery to ~90-100% and enables equal tumour control in a xenograft model of disseminated Raji cell lymphoma compared to non-cryopreserved NK cells. The mechanism of IL-15 and IL-18-induced protection incorporates two mechanisms: a transient reduction in intracellular granzyme B levels via degranulation, and the induction of antiapoptotic genes.

Pretreatment with IL-15 and IL-18 rescues natural killer cells from granzyme B-mediated apoptosis after cryopreservation.

Genome editing technologies have seen remarkable progress in recent years, enabling precise regulation of exogenous and endogenous genes. These advances have been extensively applied to the engineering of human T lymphocytes, leading to the development of practice changing therapies for patients with cancer and the promise of synthetic immune cell therapies for a variety of nonmalignant diseases. Many distinct conceptual and technical approaches have been used to edit T-cell genomes, however targeted assessments of which techniques are most effective for manufacturing, gene editing, and transgene expression are rarely reported. Through extensive comparative evaluation, we identified methods that most effectively enhance engineering of research-scale and preclinical T-cell products at critical stages of manufacturing.

Identification of Core Techniques That Enhance Genome Editing of Human T Cells Expressing Synthetic Antigen Receptors.

The efficacy of chimeric antigen receptor (CAR) T cells against solid tumors is limited. The molecular mechanisms underlying CAR T cell resistance are yet to be elucidated and new strategies need to be developed to improve treatment outcomes. Here we report that solid tumors respond to CAR T cells by upregulating the secretion of small extracellular vesicles carrying tumor antigens, which are horizontally transferred to CAR T cells, leading to antigen recognition and CAR T cell fratricide. Engineered CAR T cells armored with Serpin B9, a major granzyme B inhibitor, show decreased fratricide and increased vitality, tumor infiltration, and antitumor activity in female mice. Moreover, Serpin B9-armored CAR T cells show higher efficacy than parental CAR T cells in treating solid tumors when combined with the anti-programmed death 1 antibody. Our study demonstrates a mechanism that limits CAR T cell function and suggests an improved strategy in tumor treatment.

Overcoming extracellular vesicle-mediated fratricide improves CAR T cell treatment against solid tumors.

Genome-editing technologies have enabled the clinical development of allogeneic cellular therapies, yet the optimal gene-editing modality for multiplex editing of therapeutic T cell product manufacturing remains elusive. In this study, we conducted a comprehensive comparison of CRISPR/Cas9 nuclease and adenine base editor (ABE) technologies in generating allogeneic chimeric antigen receptor (CAR) T cells, utilizing extensive in vitro and in vivo analyses. Both methods achieved high editing efficiencies across four target genes, critical for mitigating graft-versus-host disease and allograft rejection:  or , , , and . Notably, ABE demonstrated higher manufacturing yields and distinct off-target profiles compared to Cas9, with translocations observed exclusively in Cas9-edited products. Functionally, ABE-edited CAR T cells exhibited superior in vitro effector functions under continuous antigen stimulation, including enhanced proliferative capacity and increased surface CAR expression. Transcriptomic analysis revealed that ABE editing resulted in reduced activation of p53 and DNA damage response pathways at baseline, along with sustained activation of metabolic pathways during antigen stress. Consistently, Assay for Transposase-Accessible Chromatin using sequencing data indicated that Cas9-edited, but not ABE-edited, CAR T cells showed enrichment of chromatin accessibility peaks associated with double-strand break repair and DNA damage response pathways. In a preclinical leukemia model, ABE-edited CAR T cells demonstrated improved tumor control and extended overall survival compared to their Cas9-edited counterparts. Collectively, these findings position ABE as superior to Cas9 nucleases for multiplex gene editing of therapeutic T cells.

Quadruple adenine base-edited allogeneic CAR T cells outperform CRISPR/Cas9 nuclease-engineered T cells.

Engineering chimeric antigen receptor (CAR) T cells with logic-gated synthetic Notch (synNotch) receptor circuits can enhance specificity and mitigate on-target/off-tumor toxicity. However, the conventional synNotch system uses two lentiviral vectors encoding the synNotch receptor and inducible CAR, requiring dual transduction and cell sorting, which limits clinical translation. Integrating the synNotch-CAR circuit into a single lentiviral vector could overcome this limitation, yet manufacturing CAR T cells with large transgenes remains challenging, as increasing transgene size drastically reduces lentiviral titers and T cell transduction efficiency. Current production workflows compensate for low transduction efficiency by sorting transduced cells, further impeding clinical translation. Consequently, these constraints have limited the broader development of synNotch-CAR T cell therapies.

Article	Year
Next-Generation CAR T-cell Therapies. Cancer discovery\| PubMed ID: 35417527	2022
Epitope base editing CD45 in hematopoietic cells enables universal blood cancer immune therapy. Science translational medicine\| PubMed ID: 37651540	2023
Pretreatment with IL-15 and IL-18 rescues natural killer cells from granzyme B-mediated apoptosis after cryopreservation. Nature communications\| PubMed ID: 38729924	2024
Identification of Core Techniques That Enhance Genome Editing of Human T Cells Expressing Synthetic Antigen Receptors. Cancer immunology research\| PubMed ID: 38869428	2024
Overcoming extracellular vesicle-mediated fratricide improves CAR T cell treatment against solid tumors. Nature cancer\| PubMed ID: 40234680	2025
Quadruple adenine base-edited allogeneic CAR T cells outperform CRISPR/Cas9 nuclease-engineered T cells. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 40324075	2025
Engineering single-vector logic-gated CAR T cells with transgene sizes beyond current limitations. Journal for immunotherapy of cancer\| PubMed ID: 41513407	2026

Nils W. Engel

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