Abstract C004: TCRs directed to mutant KRAS display distinct antigen recognition motifs and minimal cross-reactivity to non-cognate antigens

Abstract Pancreatic ductal adenocarcinoma (PDAC) is among the most immune-resistant tumor types. Emerging evidence suggests improved clinical outcomes for subsets of PDAC patients with neoantigen-specific T cell immunity. A low-moderate tumor mutational burden limits antigenic targets in PDAC tumors, but recurrent missense mutations within the KRAS proto-oncogene drive tumorigenesis in >90% of cases. We and others have validated mutant KRAS (mKRAS) epitopes as immunological targets of T cell receptor (TCR)-based therapies. TCRs directed against mKRAS offer broad therapeutic potential. However, TCR degeneracy poses a major challenge to the clinical development of therapeutic TCRs given the risk of unforeseen off-target immunotoxicity due to peptide cross-reactivity. Here, we investigate the specificity and cross-reactivity of four TCRs specific for mKRAS G12V restricted to the HLA-A3 superfamily of HLA class I alleles. Of the four TCRs, one is restricted to HLA-A*03:01 (TCRA3V) and three are restricted to HLA-A*11:01 (TCRA11Va, TCRA11Vb, TCRA11Vc). All TCRs exhibit reactivity to the decamer peptide VVVGAVGVGK, while TCRA11Vc also exhibits reactivity to the nonamer peptide VVGAVGVGK. In-depth characterization of each TCR peptide recognition motif using alanine/glycine scanning libraries revealed TCRA3V and TCRA11Va-c demonstrate unique antigen binding motifs with a range of 3-6 critical amino acid residues, suggesting that substantial changes within the peptide:TCR interface may be tolerated for epitope recognition in the context of HLA-A*03:01 or HLA-A*11:01. TCR fingerprinting via combinatorial peptide libraries identified peptide repertoires capable of inducing TCR activation. Candidate cross-reactive peptides encoded in the human proteome were identified using the UnitProtKB database and filtered by computational epitope binding (NetMHC 4.0), setting a predicted peptide binding threshold of ≤500 nM. Using this approach, we identified a range of 2-35 candidate cross-reactive peptides for TCRA3V and TCRA11Va-c. Candidate cross-reactive peptides were synthesized and screened for their ability to induce TCR activation, which identified a single potential off-target peptide recognized by TCRA3V. Evaluation of tumor cell lines expressing high endogenous levels of this candidate cross-reactive antigen failed to elicit TCR activation, suggesting limited natural processing and presentation of this epitope. No potential off-target peptides were identified for TCRA11Va-c. Overall, this study serves as a platform to identify TCRs with high therapeutic potency and limited off-target potential for the clinical development of TCR therapy trials. Citation Format: Rebecca B. Nadler, Adham Bear, Robert H. Vonderheide, Gerald P. Linette, Beatriz M. Carreno. TCRs directed to mutant KRAS display distinct antigen recognition motifs and minimal cross-reactivity to non-cognate antigens [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C004.