One of the selected variants showed significantly improved cytotoxicity despite lower expression frequency and exhibited higher CD62L within CAR-positive cells, suggesting enhanced intrinsic function with a less differentiated phenotype.
Abstract
Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable therapeutic outcomes in hematological cancers. However, broader clinical use has uncovered substantial challenges arising from intrinsic properties of both T cells and tumor tissues. As the functional phenotype of CAR T cells is affected by the CAR molecular architecture, optimizing CAR constructs continues to be a critical and ongoing task. Here, we present a practical workflow for scalable screening of CAR variants in primary T cells using fitness-guided design and mRNA electroporation. Using a CD19-targeted second-generation CAR, we built a library of point mutants that focused mutagenesis on hinge and costimulatory domains. Amino acid substitutions were prioritized using the sequence-based zero-shot fitness predictor to enrich evolutionarily tolerated variants. From 340 designed variants, we electroporated mRNA encoding 85 constructs into primary human CD8+ T cells and quantified cytotoxicity against CD19-positive Nalm6 cells. Twenty-four variants reproducibly exceeded wild-type cytotoxicity across three runs, and three hits were selected for lentiviral validation. One of the selected variants showed significantly improved cytotoxicity despite lower expression frequency and exhibited higher CD62L within CAR-positive cells, suggesting enhanced intrinsic function with a less differentiated phenotype. This approach enables scalable, rapid discovery of improved CAR domain variants directly in primary T cells.
Chimeric antigen receptor T cell therapy (CAR-T) has demonstrated promising efficacy in hematological malignancies, but translating that success to solid tumors remains challenging. Here, we construct a CAR library comprising approximately 1000 variants targeting prostate-specific membrane antigen by recombining transmembrane (TM), co-stimulatory, and activation domains from Natural Killer (NK) and T cell receptors. Single-cell screening identifies ICOSTM-containing variants with improved T cell activation; NK-derived activation domains, such as DAP10ζ, DAP12ζ, and FcRγζ, further augment the effector capacity. Gene regulatory network analysis reveals that CAR variants with elevated expression of T cell-activation-related transcription factors correlates with enhanced cell function. Overall, our study advances early-stage CAR design by expanding the repertoire of structural components from diverse immune cells, providing a scalable platform for identifying candidates with functional profiles comparable to clinical benchmarks.
Hai-Rong Jing, Dan Yuan, Bangquan Ye et al.· Communications Biology· 0 citations
BACKGROUND
Chimeric antigen receptor T-cell (CAR-T) therapy has achieved remarkable success in hematologic malignancies, yet demonstrates limited efficacy in solid tumors, including hepatic cancers. T-cell exhaustion and insufficient persistence represent major obstacles. We hypothesized that optimizing both manufacturing processes and CAR structural design could reduce exhaustion and enhance therapeutic outcomes in immunocompetent mouse models of primary and metastatic liver malignancies.
METHODS
We systematically compared antibody-based versus bead-based activation methods, evaluating their effects on T-cell exhaustion phenotypes. Retroviral vector (RVV) production was optimized for murine T-cell transduction, assessing vector stability and T-cell phenotypes. Different cytokine conditions were tested for their impact on T-cell expansion, memory phenotypes, and anti-tumor efficacy using GPC3-targeted CAR-T cells in hepatocellular carcinoma models. Through structural prediction and electrostatic field simulation, we identified that high positive charge patches (PCP) in the EpCAM-targeting G8.8 scFv caused CAR clustering and tonic signaling. We generated charge-optimized variants and evaluated their therapeutic efficacy in an immunocompetent colorectal cancer liver metastasis model.
RESULTS
Antibody activation showed superior homogeneity and expansion despite initially higher exhaustion markers, which equilibrated by day 10 without affecting viability. RVV harvested at 72 hours post-transfection yielded optimal titers. RVV remained stable through freeze-thaw cycles. IL-7 supplementation to IL-2 significantly enhanced memory phenotypes, reduced exhaustion, and improved tumor control in GPC3-CAR-T therapy. Electrostatic optimization of G8.8 scFv substantially reduced tonic signaling, decreased T-cell exhaustion, and enhanced anti-tumor efficacy in the MC38-EpCAM model.
CONCLUSIONS
Systematic optimization of manufacturing conditions and structure-based charge engineering of CAR constructs synergistically enhance therapeutic efficacy against liver malignancies, providing a translatable framework for improving solid tumor CAR-T therapy.
Linke Bian, Jiufei Zhu, Hongye Wang et al.· Hepatology Communications· 0 citations
This article provides a comprehensive literature review and compares different approaches on viral, non-viral and precision genome editing technologies, aiming to provide an overview and guidelines for methods selection during clinical CAR-T cell manufacturing.
W. Chan, Marcos de Lima· JOURNAL OF BONE MARROW TRANS...· 0 citations
A three-layer mechanistic framework is presented that reframes manufacturing as biology: integration biology determines genomic risk and transgene stability; clonal fitness shapes persistence, dominance, and exhaustion; and epigenomic imprinting preconfigures functional trajectories.
Duc-Hiep Bach, T. Nguyen· Human Gene Therapy· 0 citations
The pharmacological imperative for transient CAR expression is delineated, including lipid nanoparticles in combination with mRNA, which avoids risks of insertional mutagenesis and enables titratable, short-lived CAR expression, thereby enhancing safety management and suitability for applications beyond oncology.
Yudian Xiao, Ming-Liang Bai, M. Ang et al.· Advances in Pharmacology· 0 citations
This review synthesizes the mechanistic insights, engineering strategies, clinical evidence, and emerging platforms, including in vivo lentiviral CAR-T generation, that define the current landscape, and proposes a tiered framework for next-generation solid tumor CAR-T development, while explicitly acknowledging the limitations and unknowns that persist.
Samuel Obiosa Onyekweli, Gloria Osayamen Omoruyi, C. O. Akintayo et al.· Oncoscience· 0 citations
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