Year: 2026
Type: conference-abstract
Abstract: Background The current FDA-approved chimeric antigen receptor (CAR) T cells are manufactured by infecting T cells with viral vectors containing the CAR transgene, presenting barriers like lot-to-lot variability in vector multiplicity of infection, random insertion of the CAR into genome, and need for long-term safety surveillance for replication competence. Clinical trials using CAR T cells for solid tumors targeting the disialoganglioside GD2 have also been generated using viral vectors, showing encouraging signals of efficacy but with low event-free survival, raising concerns on durability of responses and lack of T cell memory. To address these limitations, we developed a virus-free CRISPR-engineered CAR T cell process to generate anti-GD2 TRAC-targeted CAR T cells (GD2-TRAC-CAR) at clinical scale using a fully closed cGMP manufacturing platform.Methods Third-generation GD2- TRAC-CD28-41BB-CD3ζ CAR T cells were generated from healthy donor T cells activated with CD3/CD28 beads. A Cas9-CLIPT (Cleaved, Linearized with Protein Template) Nanoplasmid containing the CAR transgene and guide RNAs targeting the TRAC locus were electroporated into T cells using the Cellares Cell Shuttle®. The closed, end-to-end platform automated all manufacturing steps, including enrichment, magnetic selection, activation, electroporation, expansion, and harvest. Cells were expanded for 10 days in IL-7 and IL-15 and evaluated for sterility, identity by flow cytometry, and potency through IFNγ release and Incucyte-based cytotoxicity assays against GD2-positive solid tumor cell lines. Toxicology was assessed following intravenous administration into NSG mice.Results GD2- TRAC-CAR T cells were successfully generated using the Cell Shuttle technology with high editing efficiency, achieving expansion to up to 4 billion cells while maintaining >90% viability. Final products demonstrated CAR knock-in efficiencies of 16–20% and combined TRAC knockout/knock-in frequencies of 62–74%, confirming efficient targeted integration. The cells retained a memory-like phenotype after expansion, exhibited normal metabolic profiles, and showed no cytogenetically high-risk genomic alterations. In vitro, GD2-TRAC-CAR T cells demonstrated antigen-dependent activity, including robust IFNγ secretion and potent cytotoxicity against GD2-expressing tumor cell lines. No signs of pharmacological toxicity were observed following intravenous administration in NSG mice at clinically relevant doses.Conclusions Virus-free CRISPR-engineered GD2- TRAC-CAR T cells can be reproducibly manufactured at clinical scale using a fully closed automated platform. The resulting products exhibit high targeted editing efficiency, favorable phenotypic characteristics, and potent antigen-dependent antitumor activity against GD2-positive solid tumors. Ongoing in vivo studies will evaluate potency of GD2-TRAC-CAR T cells in both xenograft and orthotopic models of GD2-expressing solid tumors.Acknowledgements We acknowledge generous support from the Robert Connor Dawes (RCD) Foundation and the MACC Fund. We thank Christopher Huffman and Dr. Andres Mejia at the Comparative Pathology Laboratory at the Research Animal Resources Center of University of Wisconsin (UW) Madison for diagnostic necropsies. The Flow Cytometry Laboratory, Small Animal Imaging and Radiotherapy Facility, and Data Science Resources are supported by Cancer Center support grant P30CA014520 to the UW Carbone Cancer Center.Ethics Approval All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) at University of Wisconsin-Madison (Protocol Number: M005915-R02-A03).
Authors: Portia R Smith, Anna Tommasi, Dmitry Malkov, Karthik Shastry, Victor Rocha, Jessica Fioravanti, Julie Fang, Rohini Emani, Matthew Forsberg, Anikait Patel, Grace Reimers, Dan Cappabianca, Evan L Dray, Lei Shi, Ross O Meyers, Austin J Stuckert, Quaovi Sodji, Jacques Galipeau, Krishanu Saha, Christian M Capitini
Institution: University of Wisconsin–Madison
Language: English
Topic: CAR-T cell therapy research
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $2,490
OpenAlex ID: W7225149403