Abstract 3576: Identification of T-cell receptors targeting RAS hotspot mutations using TIL IVS in human cancers for use in cell-based immunotherapy
Authors: Levin Noam , Lowery Frank J. , Parkhurst Maria R. , Yu Zhiya , Vale Nolan R. , Kim Sanghyun P. , Rosenberg Steven A. 
Published: June 2022

Abstract
Immunotherapies mediate the regression of human tumors through the recognition of tumor antigens by immune cells, which triggers an immune response. Mutations in the RAS oncogene occur across various cancer histologies and are found in about 30% of all cancer patients. These mutations play an essential role in tumor establishment and survival and are most commonly found in hotspots - specifically at amino acid positions 12, 13, and 61. We have recently treated a patient who underwent a durable objective response after receiving tumor-infiltrating lymphocytes (TILs) that recognized a KRAS G12D mutation. T cell receptors (TCR) that recognize shared mutated RAS antigens presented on major histocompatibility complex (MHC) class I and class II molecules are thus promising reagents for “off-the-shelf” adoptive cell therapies (ACT) following insertion of the TCR into lymphocytes. We recently published a paper describing the discovery of anti-mutated RAS reactivities by using screening TIL and by performing in-vitro stimulation (IVS) of peripheral blood lymphocytes (PBLs). Interestingly by those two methods, we sometimes found reactivity or TCR in a patient in one method but not with the other method. In this work, we described the discovery of anti-mutated RAS TCRs by TIL IVS, which combines the best of the two described methods: using TILs coming from the tumor and therefore assumed to have a higher proportion of anti-tumor-reactive cells and stimulate them, which could enrich smaller populations. The specific anti-mutated RAS CD4 and CD8 TCRs were isolated by coculture of the reactive T cells with dendritic cells (DC) loaded with peptides or transfected with mRNA encoding RAS mutated antigens. The reactive T cells were subjected to single-cell sequencing to identify TCRs. The TCRs were reconstructed as a viral plasmid and retrovirally transduced into PBLs. The transduced TCRs were tested for avidity, MHC restriction, and specific recognition of the mutation. In the same patients tested by both methods, by TIL IVS, identified 12 anti-mutated RAS TCRs in addition the 9 TCRs that we could find by TIL screening. Overall, we present here the discovery of multiple mutation-specific anti-RAS TCRs and its MHC class I and class II restriction elements involved in tumor recognition. Each of the newly discovered TCRs recognized mutated RAS with high avidity and specificity and low to no reactivity against wild-type RAS. Testing of the TCR transduced cells to recognize tumor cell lines in-vitro and in-vivo demonstrated tumor cell line recognition.
The anti-RAS mutated TCRs we discovered by all of our methods can potentially be used as an “off the shelf” treatment for the evaluation of TCR-based ACT in 45-60% of patients were tumor bears RAS mutations.
Citation Format: Noam Levin, Frank J. Lowery, Maria R. Parkhurst, Zhiya Yu, Nolan R. Vale, Sanghyun P. Kim, Steven A. Rosenberg. Identification of T-cell receptors targeting RAS hotspot mutations using TIL IVS in human cancers for use in cell-based immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3576.