Friday, April 19, 2024
4/19/2024
Abstract Ras mutations are highly prevalent in many human tumors but translating this major discovery into effective therapeutics has been difficult. Immunotherapy has emerged as the most promising approach in the field of oncology thus far. Anti-cancer vaccines offer the promise of a simple yet directed approach to cancer immunotherapy. Yet early efforts at anti-Ras vaccines, dating from 30 years ago, were not successful. Clearly, Ras based vaccines would benefit from the significant amount that has recently been learned about tumor immunology. We developed an anti-EGFRvIII peptide vaccine that showed some success in clinical trials for glioblastoma. To improve the sequence, we found enhancing proteasomal processing significantly enhances the anti-tumor effect and results in greater synergy with anti-PD-1 therapy. Moreover, this enhanced proteasome catalyzed peptide splicing (PCPS) leading to the creation of numerous antigenic peptides that increases the efficacy. When the COVID-19 pandemic struck, we applied what we learned towards SARS-CoV-2. We developed methods to accelerate the identification of these epitopes from large proteins and methods to find the PCPS fragments from lengthy sequences. Applying this to KRasG12D vs. wt KRAS, we were surprised to find that while the two proteins had 18 fragments in common, 89 fragments were unique to KRasG12D but wt Ras only had 13 unique peptides that bound to HLA-A2. This indicates the unexpected finding that there are many more CD8+ epitopes present in mutant Ras that are outside the vicinity of codon 12 that could be exploited as the basis for vaccines. Specific Aim #1. Identifying the MHC Class I binding peptides that arise from the KRasG12D mutation. In this aim, we will extend our work to identify those peptides that bind to murine MHC H-2Kb. Proteasome vs. immunoproteasome generation of fragments will be evaluated, as well as whether a tyrosine substitution at codon 12 further enhances mutant specific fragment generation. We expect many peptides will be found, so we will further evaluate which peptides are highly positive in a MHC-I membrane stabilization assay. Further refinement of the peptide set will be done by evaluating which peptides are actively endogenously presented by GL261 cells which bear KRasG12D. Specific Aim #2. Anti-tumor and CTL activity of mutant KRAS specific peptides. Selected peptides from Specific Aim #1 will be used in tumor vaccination experiments using GL261 cells and tumor regression and survival will be assessed. Immunologic studies to assay serum titers, and the number of CD8+, CD4+, dendritic and NK cells will be determined. We will also analyze for the upregulation of several checkpoint molecules. If indicated, we will perform vaccination experiments plus checkpoint inhibitors to see if this potentiates the anti-tumor effect. CTL assays will be performed to confirm induction of CD8+ T cells. Finally, toxicology studies will be performed to verify that these peptides do not induce autoimmunity against normal tissues.
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