Abstract 1515: Hotspot mutations in KRAS and TP53 targeted by TCR-T cells genetically modified with the Sleeping Beauty transposon/transposase system
Authors: Korngold Ana B. , Yong Lin-Kin , Zhang Ming , Ibekwe Ugochi , Hurton Lenka V. , Shi Yaoyao , Simmons Julissa , Collinson-Pautz Matthew R. , De Groot Eleanor , Cooper Laurence J. , Deniger Drew C. 
Published: July 2021

Abstract
Mutations in critical genes for cell survival and proliferation, e.g., KRAS and TP53, are found as clonal events in multiple tumor types of unrelated people likely due to their importance to the malignant phenotype. T cells recognize products of mutated genes, termed neoantigens, because they are expressed in the tumor but not in the normal tissues; thus, neoantigens are foreign entities from an immunological perspective. T-cell receptors (TCRs) with specificity to the neoantigen in the context of human leukocyte antigen (HLA) on the tumor cell surface can be isolated from the neoantigen-reactive T cell and potentially used for genetically-modified adoptive immunotherapy for any patient with matching mutation and HLA. The purpose of this study was to evaluate the ability of the non-viral Sleeping Beauty transposon/transposase gene transfer system to re-direct the specificity of T cells towards p53 and KRAS neoantigens and to characterize the resultant engineered TCR-T cell populations for specificity and function. Genes encoding the alpha and beta chains of p53 or KRAS neoantigen-specific TCRs were linked by a 2A ribosomal slip site linker and cloned into the clinical Sleeping Beauty transposon. These transposons were co-electroporated into donor peripheral blood leukocytes (PBL) with a DNA plasmid encoding the SB11 transposase and expanded in vitro. Logarithmic proliferation was observed and resulted in large numbers of highly pure TCR-T cells (&gt;80% by introduced TCR expression). The TCR-T cells upregulated 41BB on the T-cell surface and secreted interferon-γ in response to antigen presenting cells with the appropriate HLA molecule pulsed with KRAS or p53 neoantigen peptides but not the cognate wild type peptide, confirming re-directed specificity to mutated KRAS or TP53 genes, respectively. Similarly, TCR-engineered T cells demonstrated cytolysis of tumor cells expressing the neoantigen and appropriate HLA restriction, suggesting that adoptive transfer of these TCR-T cells could mediate anti-tumor responses. In all, we demonstrated that multiple TCRs with unique specificities targeting recurrent p53 and KRAS substitutions in frequent HLA haplotypes could be stably expressed using Sleeping Beauty transposition to re-direct peripheral blood T cells towards tumor cells. Translation of these TCR-T cells into adoptive immunotherapy could result in safe and effective treatments for any cancer patient with matching HLA and KRAS or TP53 hotspot mutations.
Citation Format: Ana B. Korngold, Lin-Kin Yong, Ming Zhang, Ugochi Ibekwe, Lenka V. Hurton, Yaoyao Shi, Julissa Simmons, Matthew R. Collinson-Pautz, Eleanor De Groot, Laurence J. Cooper, Drew C. Deniger. Hotspot mutations in KRAS and TP53 targeted by TCR-T cells genetically modified with the Sleeping Beauty transposon/transposase system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1515.