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E - Disease Models and Clinical Applications -> Cancer – Targeted Gene and Cell Therapy

1691: Novel CRISPR-Associated Gene Editing Systems Enable Efficient and Specific TRAC/TRBC Knockout Triggering Robust Expression and Sensitivity of a T Cell Receptor Specific for Mutant KRAS G12D

Type: Poster Session

Poster Board Number: 1691
Presentation Details
Session Title: Friday Poster Session
Start Time: 5/19/2023 12:00
End Time: 5/19/2023 14:00

Adoptive T cell therapy (ACT) has demonstrated activity in solid tumors, but further optimization is needed to establish reproducibly effective treatments. T cells engineered with T cell receptors (TCRs) recognizing intracellular oncogenic drivers like mutant KRAS, the most frequently altered gene in human cancers, have the potential to induce durable responses in patients with solid tumors. TCRs are heterodimeric proteins consisting of a- and b-chains that can activate T cells following recognition of the desired target peptide presented by MHC. Competition between transgenic and endogenous TCRs for the available pool of CD3 proteins in T cells and the potential mispairing of transgenic and endogenous TCR chains can lead to decreased surface expression of the transgenic TCR, and thus diminished sensitivity. Here we employed programmable nucleases to genetically edit the constant regions of endogenous TCRs to eliminate endogenous TCR expression and mispairing with the transgenic TCR. We used a novel CRISPR-associated nuclease originally identified from environmental microbial samples and identified novel lead gRNAs that resulted in TCR knockout in >90% of human primary T cells. Since off-target activity is a potential safety risk, we characterized nuclease specificity using the ‘gold standard’ oligo capture assay followed by validation of any off-target activity using the Amplicon-sequencing assay. We demonstrated, with a sensitivity of detection approaching 0.1%, preclinical high specificity of editing, supporting safety of our gene edited TCR T cell therapy. To evaluate the functional impact of endogenous TCR deletion, we edited primary T cells transduced with a lentivirus carrying CD8ab co-receptor and a TCR specific for the peptide derived from the KRAS G12D mutation presented in the context of HLA-A*11:01, one of the most common HLA alleles worldwide. TRAC/TRBC knockout improved expression of the transgenic TCR and improved the sensitivity of the engineered T cells, with TRAC/TRBC edited cells exhibiting enhanced in vitro cytotoxicity against tumor cells expressing the mutant KRAS G12D peptide. In summary, we describe the importance of genetic knockout of endogenous TRAC and TRBC for increasing the safety and functional potential of TCR-based cell therapy against solid tumors. The functional benefit and specificity of our genetic editing supports utility in clinical development of TCR-engineered T cell therapy for treating KRAS-mutant solid tumors.

Nicholas Rouillard1, Cheryl Black2, James Parsons2, Anthony Thomas2, Allison Drain1, Santosh Narayan1, Nicole Danek1, Nathaniel Swanson1, Vince Nguyen1, Joshua Francis1, Xingyue He1, Ankit Gupta1, Gary Shapiro1, Patrick Browne3, Rebecca Lamothe3, Gregory Cost3, Ashley Thelen4, Jessica Webb4, Thomas Schmitt4, Philip Greenberg4, Loic Vincent1

1Affini-T Therapeutics, Watertown, MA,2Affini-T Therapeutics, Seattle, WA,3Metagenomi, Emeryville, CA,4Program in Immunology and Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA
  A. Gupta: 1; Commercial Interest i.e. Company X; Affini-T Tx. 1; What was received? i.e. Honorarium; Salary and Stocks. 1; For what role? i.e. Speaker; Employee.

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