ORCID Profile
0000-0002-5447-5381
Current Organisation
Monash University
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Publisher: Springer US
Date: 2020
Publisher: PeerJ
Date: 20-07-2018
DOI: 10.7717/PEERJ.5056
Abstract: Somatic 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. In this study, the region spanning exon 2 from the 4th to 18th codon within the peptide sequence of wt KRAS 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. In 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. In 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.
Publisher: Bentham Science Publishers Ltd.
Date: 15-11-2019
DOI: 10.2174/1568026619666190904163524
Abstract: The occurrence of somatic substitution mutations of the KRAS proto-oncogene is highly prevalent in certain cancer types, which often leads to constant activation of proliferative pathways and subsequent neoplastic transformation. It is often seen as a gateway mutation in carcinogenesis and has been commonly deemed as a predictive biomarker for poor prognosis and relapse when conventional chemotherapeutics are employed. Additionally, its mutational status also renders EGFR targeted therapies ineffective owing to its downstream location. Efforts to discover new approaches targeting this menacing culprit have been ongoing for years without much success, and with incidences of KRAS positive cancer patients being on the rise, researchers are now turning towards immunotherapies as the way forward. In this scoping review, recent immunotherapeutic developments and advances in both preclinical and clinical studies targeting K-ras directly or indirectly via its downstream signal transduction machinery will be discussed. Additionally, some of the challenges and limitations of various K-ras targeting immunotherapeutic approaches such as vaccines, adoptive T cell therapies, and checkpoint inhibitors against KRAS positive cancers will be deliberated.
Publisher: Malaysian Society for Molecular Biology and Biotechnology
Date: 09-07-2020
DOI: 10.35118/APJMBB.2020.028.3.03
Abstract: Background. KRAS mutations are highly prevalent in pancreatic, lung, and colorectal carcinomas with G12V point substitution being one of the most prevalent mutations. While developments of peptide vaccines for KRAS(+) cancers are usually associated with poor immunogenicity, coupling mutant K-ras vaccines with universal CD4+ carrier molecules may enhance its outcome. Additionally, recent immunotherapeutic advances also suggest the possibility of inducing mucosal immunity against cancers using Lactococcus lactis as a live gastrointestinal delivery vehicle. Methods. A region of wild-type K-ras peptide was previously modified with a V7D substitution flanking the G12V mutation, generating a K-ras peptide (termed 68-V) with improved predicted antigenicity. This peptide was fused with a diphtheria toxoid sequence, and cloned into pNZ8048 vector within Lactococcus lactis NZ9000. BALB/c mice were then immunized orally, and then subjected to T/B cells immunophenotyping, as well as IgG and IgA detection. Results. Modified 68-V K-ras peptide and controls were successfully cloned and detection of His-tagged proteins expressed following induction by nisin was observed. Populations of CD3-CD19+ immune cells increased following immunization of 68-V, while K-ras specific-IgG and -IgA sera titers were elevated compared to wild-type and G12V K-ras controls. Conclusion. 68-V K-ras mimotope was shown to induce humoral-mediated immunity, highlighting the ability of an additional mutation flanking the G12V KRAS mutation to induce B cell activation and production of K-ras specific antibodies, while diphtheria toxoid was unable to stimulate an enhanced response when fused to 68-V. Nevertheless, these findings showed that further assessments are required to understand the role of K-ras specific antibodies within a KRAS(+) environment.
No related grants have been discovered for Winfrey, Pui Yee Hoo.