A practical workflow for CAR screening in primary T cells using fitness-guided design and mRNA electroporation

High-throughput single-cell screening of a cross-lineage CAR library for early-stage CAR design and candidate discovery.

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.

Manufacturing and electrostatic surface optimization of CAR-T cells improve therapeutic efficacy in liver malignancies.

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.

Genetic engineering strategies for cell-based therapy: viral, non-viral, and precision genome editing technologies

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.

CAR-T Cell Therapy: Manufacturing Platforms and Clinical Consequences.

Chimeric antigen receptor (CAR) T-cell therapy has transformed hematological cancer care, yet variability in efficacy, durability, and safety cannot be explained solely by antigen selection or patient factors. We propose that manufacturing platforms are active biological determinants of outcome. Viral vectors, used in all licensed products, provide stable genomic integration and durable expression but are limited by cost, cargo capacity, and centralized production. Nonviral strategies, including transposons, CRISPR knock-ins, and messenger RNA delivery, enable faster, less-expensive manufacturing with larger payloads, while introducing distinct safety and persistence profiles. This review presents a three-layer mechanistic framework that reframes manufacturing as biology: integration biology determines genomic risk and transgene stability; clonal fitness shapes persistence, dominance, and exhaustion; and epigenomic imprinting, influenced by gene transfer method, cytokines, and culture stress, preconfigures functional trajectories. Clinical observations link platform choice to immune recovery, where prolonged B-cell aplasia and delayed T-cell reconstitution contribute to infection-related nonrelapse mortality, and hematopoietic reserve at apheresis emerges as a practical predictor. Finally, manufacturing is positioned as the key to democratizing cell therapy. Decentralized, nonviral production aligned with regulatory standards may enable equitable access and transition CAR-T therapy from innovation to sustainable global care.

Transient T cell therapies.

Engineering CAR-T cells for solid tumors: Overcoming the microenvironment through integrated design and clinical translation

Chimeric antigen receptor T-cell (CAR-T) therapy has produced remarkable therapeutic results in blood cancers, while its application to solid malignancies remains limited by a pooled objective response rate of approximately 9%. This gap stems from core biological obstacles: heterogeneous antigen expression, physical inaccessibility within dense stromal architectures, and immunosuppressive microenvironments that drive T-cell exhaustion through epigenetically fixed transcriptional programs. The period spanning 2024–2025 represents a pivotal turning point. GD2-targeting CAR-T cells delivered intracerebroventricularly achieved durable complete responses (including one sustained beyond 30 months) in H3K27M-mutated diffuse midline gliomas. CLDN18.2-targeting satricabtagene autoleucel demonstrated randomized superiority over physician’s choice in advanced gastric cancer (progression-free survival HR 0.37). GPC3-targeting CAR-T cells armored with a dominant-negative TGF-β receptor achieved objective response rates of 50–57% in hepatocellular carcinoma, representing a three- to four-fold improvement over unarmored predecessors. These breakthroughs reflect a paradigm shift from potency-driven engineering toward resilience-based design: metabolic armoring via autocrine IL-10 and IL-15, epigenetic protection through DNMT3A disruption and c-Jun overexpression, logic-gated targeting via synNotch circuits, and microenvironmental shielding through dominant-negative receptors. Beyond the local microenvironment, emerging recognition of systemic neuroendocrine-immune dysregulation further informs CAR-T persistence and fitness considerations. 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.