Biochemical and functional characterization of mutant KRAS epitopes validates this oncoprotein for immunological targeting

We employ a multi-omics approach consisting of bioinformatic, biochemical, proteomic and immunological studies to identify, characterize and validate mutant KRAS neoantigens among high prevalence HLA class I alleles highlighting this oncogenic driver protein as a target of immune-based therapies.

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DNA mutations in cancer cells often result in the expression of aberrant proteins known as mutation-associated neoantigens that may be processed and presented on the surface of tumor cells or antigen presenting cells to elicit anti-tumor immune responses1. T cell responses targeting cancer neoantigens are believed to be the key mediators of effective immunotherapies2. The development of widely applicable immune therapies targeting neoantigens is hindered by the fact that the vast majority of cancer neoantigens are “private”, being derived from passenger mutations that are unique to individual patients. In contrast, neoantigens derived from recurrent mutations in cancer driver genes, such as KRAS, may serve as “public” neoantigens to develop immunotherapeutic strategies pertinent to large numbers of cancer patients3.

Mutations in the oncogene KRAS are among the most prevalent mutations observed in human cancers. In particular, KRAS mutations are highly prevalent in the 3 most lethal tumor types which include adenocarcinomas of the pancreas, colon, and lung. Among these tumor types, KRAS mutations most often involve amino acid substitutions at the G12 position that result in constitutive KRAS activation which drive tumorigenesis, making it a highly attractive drug target. Recently, targeted inhibitors of KRAS G12C have exhibited promising clinical benefits in patients with advanced lung cancer4, leading to the first U.S. Food and Drug Administration approval of a mutant KRAS-targeted therapy. However, resistance mechanisms to this approach have been clearly documented5, and no targeted therapies yet exist for alternative KRAS G12 mutations more prevalent among other tumor types, highlighting the need for further therapeutic development.  

Mutant KRAS has been studied as an immunologic target of cancer vaccines, and T cell responses to mutant KRAS have been documented in cancer patients. In a seminal clinical case report, Tran et al demonstrated the anti-tumor activity of T cells reactive against mutant KRAS in a patient with metastatic colon cancer6; however, the therapeutic implications of this observation to greater numbers of cancer patients remain unclear given the low frequency of patients that express this particular mutation and restricting HLA allele7. To date, mutant KRAS has remained poorly characterized as an immunologic target with limited evidence regarding antigen processing, presentation and immunogenicity.

In a highly collaborative effort, we set out to complete the most comprehensive pre-clinical assessment of mutant KRAS as neoantigenic target which may serve to guide the development of future immunotherapies (see Figure). In our study published in Nature Communications, we demonstrate the following:

  • Using computational epitope prediction, we identify candidate mutant KRAS neoantigens with high predicted binding affinity to HLA class I alleles. This prediction highlights HLA-A*02:01, HLA-A*03:01, HLA-A*11:01 and HLA-B*07:02 as potential binding alleles for epitopes derived from the most prevalent KRAS G12 mutations observed in human cancers (G12C, G12D, G12R, G12V). These HLA class I alleles are highly prevalent in the USA population representing 14-42% of individuals.
  • Using biochemical methods, we measure the binding affinities and stabilities of mutant KRAS neoantigens to HLA class I alleles as these two parameters correlate with neoantigen immunogenicity. These results highlight mutant KRAS neoantigens as ligands for HLA-A*03:01, HLA-A*11:01 and HLA-B*07:02 alleles, but not HLA-A*02:01.
  • We utilize a proteomics-based approach to validate the expression of mutant KRAS neoantigens. Using mass spectrometry performed on peptides eluted from the HLA class I alleles of tumor cells expressing mutant KRAS protein, we identify 11 processed and presented mutant KRAS neoantigens restricted to either HLA-A*03:01, HLA-A*11:01 or HLA-B*07:02. No mutant KRAS neoantigens were identified for HLA-A*02:01.
  • To assess the immunogenicity of candidate mutant KRAS neoantigens, we stimulate CD8+ T cells derived from healthy donor blood samples and observe T cells responses to mutant KRAS in HLA-A*03:01, HLA-A*11:01 and HLA-B*07:02 positive donors, validating our stringent approach to the nomination of mutant KRAS neoantigens. Consistent with biochemical and proteomic studies, no mutant KRAS-specific responses were observed among HLA-A*02:01 positive donors.
  • Using T cell receptors (TCRs) specific for mutant KRAS isolated from HLA-A*03:01, HLA-A*11:01 or HLA-B*07:02 positive donors, we validate mutant KRAS neoantigen expression by tumor cells. We demonstrate that primary CD8+ T cells modified to express mutant KRAS TCRs can recognize and kill tumor cells of pancreatic, colon or lung origin, so long as they express both the KRAS mutation and restricting HLA allele of interest. Importantly, these TCRs are exquisitely sensitive for the cognate mutant KRAS neoantigen, exhibiting no cross-reactivity to wild-type KRAS.
  • By direct quantification of mutant KRAS neoantigens expressed on the tumor cell surface, we demonstrate that mutant KRAS-specific TCRs are capable of recognizing low numbers of peptide-HLA complexes.
  • In a mouse xenograft model of metastatic KRAS-mutated lung cancer, we demonstrate that mutant KRAS-TCR therapy leads to tumor clearance and prolonged survival.

This study has direct implications for the development of cancer vaccines, bispecific engagers, and TCR therapies targeting the most common KRAS mutations in patients expressing highly prevalent HLA class I alleles. Based on these results, we have opened an adjuvant vaccine study targeting mutant KRAS in patients with resected pancreatic cancer at the University of Pennsylvania (NCT03592888), and adoptive cell therapy clinical trials using mutant KRAS-specific TCRs are in development. More importantly, this study establishes a roadmap for the nomination, characterization and validation of cancer neoantigens which may serve to guide the development of future immune therapies targeting mutant KRAS or other public neoantigens for the treatment of large subsets of cancer patients.  

Mutant KRAS epitope discovery and validation pipelinenaption

References:

  1. Carreno, B. M. et al. Cancer immunotherapy. A dendritic cell vaccine increases the breadth and diversity of melanoma neoantigen-specific T cells. Science 348, 803–8 (2015).
  2. Tran, E., Robbins, P. F. & Rosenberg, S. A. ‘Final common pathway’ of human cancer immunotherapy: targeting random somatic mutations. Nat. Immunol. 18, 255–262 (2017).
  3. Pearlman, A. H. et al. Targeting public neoantigens for cancer immunotherapy. Nat. Cancer 2, 487–497 (2021).
  4. Hong, D. S. et al. KRAS G12C Inhibition with Sotorasib in Advanced Solid Tumors. N. Engl. J. Med. 383, 1207–1217 (2020).
  5. Awad, M. M. et al. Acquired Resistance to KRASG12C Inhibition in Cancer. N. Engl. J. Med. 384, 2382–2393 (2021).
  6. Tran, E. et al. T-Cell Transfer Therapy Targeting Mutant KRAS in Cancer. N. Engl. J. Med. 375, 2255–2262 (2016).
  7. Rech, A. J. & Vonderheide, R. H. T-Cell Transfer Therapy Targeting Mutant KRAS. N. Engl. J. Med. 376, e11 (2017).

Adham Bear

Physician, University of Pennsylvania