Abstract 1127: Preclinical development of safe and effective T cell receptors specific for mutant KRAS G12D peptide
Authors: Black Cheryl , Parsons James , Thomas Anthony , Drain Allison P. , Narayan Santosh , Francis Joshua , He Xingyue , Gupta Ankit , Webb Jessica , Schmitt Thomas M. , Greenberg Philip D. , Shapiro Gary , Vincent Loïc 
Published: April 2023

Adoptive T cell therapy (ACT) has demonstrated activity in solid tumors but requires further optimization to become a reproducibly effective treatment. T cell receptor (TCR)-engineered T cells recognize peptides derived from intracellular and surface proteins presented in the context of MHC class I. Targeting mutated oncogenic drivers addresses some of the major obstacles of this modality, in that the antigenic epitope is: 1) tumor-specific, 2) essential for tumor survival, and 3) derived from a stably expressed protein. KRAS is the most frequently mutated gene in human cancers with alterations in codon 12 associated with poor clinical outcomes in a high proportion of colon, lung and pancreatic cancers, as well as many others. To isolate TCRs 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, we employed our high-throughput in vitro TCR discovery platform. CD8+ T cells were isolated from healthy donors and co-cultured with autologous antigen-presenting cells exogenously-loaded with mutant KRAS peptides encompassing the G12D mutation. The highest avidity T cells were subsequently identified and enriched by fluorescence-based cell sorting and the corresponding TCR genes isolated by single-cell sequencing and inserted into a lentiviral expression vector. TCR candidates were transduced into primary T cells and prioritized based on functional avidity and specificity (response to titrated peptide-loaded presenting cells and tumor cells endogenously expressing the KRAS G12D antigen) and cytotoxicity (in vitro tumor cell killing assays). X-Scan studies, in which each residue of the reference KRAS G12D peptide was systematically substituted by all other amino acids, revealed a highly restrictive TCR recognition motif, suggesting limited risk of promiscuous off-target activation. We engineered both CD4+ and CD8+ T cells to lentivirally-express candidate TCRs in addition to the genes encoding the CD8αβ co-receptor to enhance TCR-HLA class I avidity in CD4+ T cells, with the aim of creating a coordinated CD4 and CD8 T cell response to the same tumor target to promote increased T cell activity and persistence while minimizing T cell exhaustion. Transduced CD4+ T cells were functional and could be demonstrated to provide help to CD8+ T cells, supporting tumor elimination in several experimental models. In summary, we report a TCR gene therapy approach targeting mutant KRAS G12D-containing peptides with a coordinated CD4 and CD8 T cell response that has a promising efficacy and safety profile. Our work to date supports the planned clinical development of this novel TCR-engineered T cell therapy for treating KRAS-mutant solid tumors.

Citation Format: Cheryl Black, James Parsons, Anthony Thomas, Allison P. Drain, Santosh Narayan, Joshua Francis, Xingyue He, Ankit Gupta, Jessica Webb, Thomas M. Schmitt, Philip D. Greenberg, Gary Shapiro, Loïc Vincent. Preclinical development of safe and effective T cell receptors specific for mutant KRAS G12D peptide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1127.