Abstract PR019: Mutant KRAS-specific T cell receptors directed against prevalent G12D and G12V variants exhibit potent cytotoxic activity and CD8 co-receptor independence
Authors: Bear Adham S. , Nadler Rebecca B. , Scholler John , Vonderheide Robert H. , Linette Gerald P. , Carreno Beatriz M. 
Published: November 2022

Background: KRAS mutations drive tumorigenesis in &gt;90% of pancreatic ductal adenocarcinomas (PDAC). Recent clinical evidence demonstrates potent and durable anti-tumor activity of adoptively transferred KRAS-specific T cells in select patients with metastatic KRAS-mutated solid tumors including PDAC. We have defined mutant KRAS (mKRAS) epitopes restricted to high prevalence HLA class I alleles and validated this oncoprotein as an immunological target amenable to T cell receptor (TCR)-based therapeutic approaches. TCR therapies utilizing HLA class I-restricted TCRs often fail to harness CD4+ T cell activity vital for sustained anti-tumor immune responses. Here, we characterize HLA class I-restricted TCRs isolated from healthy donors and vaccinated patients targeting KRAS G12D or G12V, the most prevalent mKRAS variants observed in PDAC tumors. Methods: mKRAS-specific TCR sequences were identified from healthy donors or vaccinated PDAC patients enrolled on IRB-approved clinical studies at the University of Pennsylvania. Transgenic expression of synthetic TCRs was attained via lentiviral transduction of reporter cell lines or gene-edited (TCRabnull) primary CD8+ or CD4+ T cells. Limiting peptide dilution and combinatorial peptide library assays were used to assess antigen specificity, affinity and cross-reactivity. TCR reactivity to mKRAS cell lines was assessed by FACS analysis, in vitro cytotoxicity assays (4h 51Cr-release, xCELLigence Real Time Cell Analysis), and mouse xenograft tumor models. Results: TCRs exhibit specificity to the cognate KRAS G12D or G12V epitope without cross-reactivity to wild-type KRAS. TCRs are of high affinity and promote cytolysis of HLA-matched tumor cell lines harboring endogenous KRAS mutations irrespective of histologic origin. The adoptive transfer of gene-edited (TCRabnull) primary CD8+ T cells expressing synthetic TCRs exhibit potent in vivo antitumor activity in mouse xenograft tumor models of metastatic cancer. Select TCRs retain functional avidity independent of the CD8 co-receptor as determined by multimer binding and limiting peptide dilution assays. Disruption of the CD8:HLA binding interface via the introduction of D227A and T228A mutations within the HLA a3 domain has limited to no effect on TCR-mediated CD8+ T cell cytolytic activity. Transgenic expression of co-receptor independent TCRs by CD4+ T cells confers cytolytic activity against KRAS-mutated cell lines in both short- and long-term cytotoxicity assays, and flow cytometric analysis confirms antigen-specific expression of CD107a, IFN-g, TNF-a and IL-2 in a subset of CD4+ T cells. Conclusion: This study identifies TCRs with high therapeutic potency for the clinical development of TCR therapy trials targeting mKRAS in PDAC patients. Select TCRs exhibit CD8 co-receptor independence. CD4+ T cells redirected with co-receptor independent TCRs exhibit cytotoxic activity and express Th1 cytokines which may serve to enhance the persistence and anti-tumor activity of adoptive TCR therapy targeting mKRAS.

Citation Format: Adham S. Bear, Rebecca B. Nadler, John Scholler, Robert H. Vonderheide, Gerald P. Linette, Beatriz M. Carreno. Mutant KRAS-specific T cell receptors directed against prevalent G12D and G12V variants exhibit potent cytotoxic activity and CD8 co-receptor independence [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 PR019.