In silico-guided sequence modifications of K-ras epitopes improve immunological outcome against G12V and G13D mutant KRAS antigens

Abstract

BackgroundSomatic point substitution mutations in the KRAS proto-oncogene primarily affect codons 12/13 where glycine is converted into other amino acids, and are highly prevalent in pancreatic, colorectal, and non-small cell lung cancers. These cohorts are non-responsive to anti-EGFR treatments, and are left with non-specific chemotherapy regimens as their sole treatment options. In the past, the development of peptide vaccines for cancer treatment was reported to have poor AT properties when inducing immune responses. Utilization of bioinformatics tools have since become an interesting approach in improving the design of peptide vaccines based on T- and B-cell epitope predictions.MethodsIn this study, the region spanning exon 2 from the 4th to 18th codon within the peptide sequence of wtKRAS was chosen for sequence manipulation. Mutated G12V and G13D K-ras controls were generated in silico, along with additional single amino acid substitutions flanking the original codon 12/13 mutations. IEDB was used for assessing human and mouse MHC class I/II epitope predictions, as well as linear B-cell epitopes predictions, while RNA secondary structure prediction was performed via CENTROIDFOLD. A scoring and ranking system was established in order to shortlist top mimotopes whereby normalized and reducing weighted scores were assigned to peptide sequences based on seven immunological parameters. Among the top 20 ranked peptide sequences, peptides of three mimotopes were synthesized and subjected to in vitro and in vivo immunoassays. Mice PBMCs were treated in vitro and subjected to cytokine assessment using CBA assay. Thereafter, mice were immunized and sera were subjected to IgG-based ELISA.ResultsIn silico immunogenicity prediction using IEDB tools shortlisted one G12V mimotope (68-V) and two G13D mimotopes (164-D, 224-D) from a total of 1,680 candidates. Shortlisted mimotopes were predicted to promote high MHC-II and -I affinities with optimized B-cell epitopes. CBA assay indicated that: 224-D induced secretions of IL-4, IL-5, IL-10, IL-12p70, and IL-21; 164-D triggered IL-10 and TNF-α; while 68-V showed no immunological responses. Specific-IgG sera titers against mutated K-ras antigens from 164-D immunized Balb/c mice were also elevated post first and second boosters compared to wild-type and G12/G13 controls.DiscussionIn silico-guided predictions of mutated K-ras T- and B-cell epitopes were successful in identifying two immunogens with high predictive scores, Th-bias cytokine induction and IgG-specific stimulation. Developments of such immunogens are potentially useful for future immunotherapeutic and diagnostic applications against KRAS(+) malignancies, monoclonal antibody production, and various other research and development initiatives.

K-Ras Peptide Mimotope Induces Antigen Specific Th1 and B-Cell Immune Responses against G12A-Mutated K-Ras Antigen in Balb/c Mice

“…The poor immunogenicity of cancer vaccines and immunotolerance towards selfantigens such as mutant K-ras has made them inefficient in triggering a strong immune response when administered without further sequence modifications. In order to overcome immunotolerance towards native mutant K-ras found on tumorigenic cells, a more immunogenic form of mutant K-ras epitope which mimics the natural mutant K-ras epitope was designed and optimized through in silico predictions [19]. This modified version, termed a mimotope, possesses an additional single amino acid substitution (G10P) flanking the native G12A mutation.…”

“…Primers used in this study are listed in Table S2. Oligonucleotide 139-A (YKLVVVPAA GVGKSA) which was designed previously [19] and TTD [20] were synthesised separately in pIDT:SMART vectors (Integrated DNA Technologies, Clareville, IA, USA). Recombinant pIDT:SMART plasmid was transformed via heat-shock into the X-10 GOLD E. coli.…”

“…An ideal length of a mimotope vaccine is typically 8-20 amino acids, which is favorable for MHC-II binding and eases production [14]. To date, numerous peptide mimotopes have been developed against different antigen types, including EGFR, KRAS, HER2, CEA, PSA, MG7-Ag, and many others with improved MHC-binding affinity and presentation by antigen presentation cells (APCs) [15][16][17][18][19]. Furthermore, several studies have reported that poor immunogenicity issues affiliated with short peptide mimotope vaccines could be overcome through conjugation of established bacterial carrier molecules such as tetanus toxoid (TTD) and diphtheria toxoid (DTD) that contain universal T-cells epitopes, thus enhancing both humoral and cell-mediated responses [20,21].…”

Development of a single-chain fragment variable fused-mutant HALT-1 recombinant immunotoxin against G12V mutated KRAS colorectal cancer cells

“…Total RNA was extracted from homogenized mice splenocytes immunized with 164-D (YKLVVVGAGDVYKSA) and 68-V (YKLDVVGAVGVGKSA) mimotopes (Ng et al, 2018) using RNeasy Mini kit (Qiagen, Germantown, MD, USA) according to the manufacturer's instructions. Reverse-transcription with the RNA extract was performed with Tetro cDNA synthesis kit (Bioline, London, United Kingdom) following the manufacturer's protocol.…”

Effect of Secretion Efficiency of Mutant KRAS Neoantigen by Lactococcus lactis on the Immune Response of a Mucosal Vaccine Delivery Vehicle Targeting Colorectal Cancer

“…Nevertheless, the peptide vaccine is often perturbed by its small size, making it weakly immunogenic and rendering it incapable of inducing a sufficient immune response. These limitations can be surmounted by modification of the TSA/TAAs through the incorporation of an additional single point mutation (Ng et al, 2018) [14] or by conjugation with a carrier molecule to improve the adjuvanticity and chemical stability for enhanced immune response (Li et al, 2014) [15].…”