Abstract Details

G2 - Immune Targeting and Approaches with Genetically-Modified Cells and Cell Therapies (Including CAR-T, CAR-NK, TCR editing)

836: THRIVE^TM Non-Viral Targeted Transgene Knock-In Platform Generates Specific and Potent TCR T Cell Products for the Treatment of Solid Cancers

Type: Poster Session

Poster Board Number: 836

Presentation Details

Session Title: Wednesday Posters: Immune Targeting and Approaches with Genetically-Modified Cells and Cell Therapies






Introduction: Selective and homogenous expression of oncogenic driver mutations such as KRAS^G12D and p53^R175H make them ideal targets for cancer therapies. Adoptive T cell therapies with engineered T cell receptors (TCRs) recognizing mutated driver proteins have led to durable responses in patients with solid tumors. Lentiviral vector (LVV)-based T cell manufacturing suffers from limited cargo size, heterogeneous transgene expression, high manufacturing cost and complexity, and the potential for oncogenesis due to random genomic insertions. To overcome these challenges, we have developed THRIVE^TM, a non-viral, gene-editing-based T cell manufacturing platform that achieves high rates of targeted transgene insertion and robust cell expansion, and yields potent TCR T cell therapies. Methods and Results: An efficient and specific novel type V CRISPR-Cas system was used for targeted transgene knock-in (KI) at the TRAC locus via homology directed repair (HDR) in primary human CD4⁺ and CD8⁺ T cells. The optimized HDR template and engineering process achieved efficient KI and expression of multi-cistronic constructs up to 7 kb in size that included: 1) a TCR specific for an oncogenic driver mutation, 2) a CD8αβ coreceptor allowing for effective CD4⁺ T cell target recognition and stimulation, and 3) T cell function-enhancing synthetic fusion proteins. Unlike LVV-mediated random insertions, genotypic analysis of THRIVE-produced T cells showed targeted transgene insertion at the TRAC locus that also disrupted the endogenous TCRα chain, resulting in increased surface expression of the transgenic oncogene-specific TCR and enhanced antigen sensitivity. Thus, THRIVE gene-edited T cells demonstrated improved efficacy as compared to LVV-engineered T cells both in vitro during repeat tumor cell challenges and in vivo after single administration in mice harboring tumor xenografts. Gene-editing safety evaluation demonstrated specificity with no off-target activity in high sensitivity (~0.1%) NGS-based analyses or any gene-editing-related chromosomal rearrangements. When evaluated at expected clinical scale, the THRIVE platform yielded up to 50% transgene-positive T cells that showed a favorable T stem cell memory/ T central memory phenotype and >50-fold expansion achieving anticipated dose levels for clinical trials. Conclusion: The non-viral targeted knock-in platform, THRIVE, enables safe and cost-effective T cell engineering with high efficiency. THRIVE-engineered oncogene driver-specific TCR T cells demonstrated superior cytotoxicity against tumor cells relative to LVV-engineered cells both in vitro and in vivo. These data support the clinical readiness and advancement of the THRIVE platform for engineering TCR T cell therapy for the treatment of advanced solid malignancies.

Plain Language Summary

We are developing therapies to treat difficult to cure solid cancers such as pancreatic and colorectal cancer. We utilize body’s own fighter immune cells and program them to identify the cancer cells that bear cancer-causing genes. These programmed fighter cells can find and kill cancer cells in the body. We describe our approach, THRIVE^TM, to manufacture these programmed fighter cells. Within the THRIVE platform, we insert the therapeutic genes in the fighter cells in an efficient and safe way and grow these cells. In lab setting, we show that our programmed cells kill cancer cells efficiently and spare the normal non-cancer cells. As next steps, we show that we can manufacture these programmed fighter cells in sufficient numbers to potentially allow us to treat patients in clinic. Excited by our results, we plan to test our therapy in patients suffering from devastating solid cancers.

Santosh Narayan¹, Nathaniel Swanson¹, Allison Drain¹, Ken Gareau¹, Nicholas Rouillard¹, Jinsheng Liang¹, Hui-Wen Liu¹, Lauren Brown¹, Rebecca Lamothe², Philip D. Greenberg³, Christopher A. Klebanoff⁴, Ankit Gupta¹, Damien Hallet¹, Gary Shapiro¹, Kim Nguyen¹, Loic Vincent<sup>1

1</sup>Affini-T Therapeutics, Watertown, MA,²Metagenomi Inc., Emeryville, CA,³Program in Immunology and Translational Science and Therapeutics Division, Fred Hutchinson Cancer Research Center, Seattle, WA,⁴Memorial Sloan Kettering Cancer Center, New York, NY"