Abstract
KRAS G12A somatic point mutation in adenocarcinomas is categorized clinically as ineligibility criteria for anti-epidermal growth factor receptor (EGFR) monoclonal antibody therapies. In this study, a modified G12A-K-ras epitope (139A) with sequence-specific modifications to improve immunogenicity was developed as a potential vaccine against G12A-mutant KRAS cancers. Additionally, coupling of the 139A epitope with a tetanus toxoid (TTD) universal T-cell epitope to improve antigenicity was also reported. To facilitate convenient oral administration, Lactococcus lactis, which possesses innate immunomodulatory properties, was chosen as a live gastrointestinal delivery vehicle. Recombinant L. lactis strains secreting a G12A mutated K-ras control and 139A with and without TTD fusion were generated for comparative immunogenicity assessment. BALB/c mice were immunized orally, and high survivability of L. lactis passage through the gastrointestinal tract was observed. Elevations in B-cell count with a concomitant titre of antigen-specific IgG and interferon-γ secreting T-cells were observed in the 139A treated mice group. Interestingly, an even higher antigen-specific IgA response and interferon-γ secreting T-cell counts were observed in 139A-TTD mice group upon re-stimulation with the G12A mutated K-ras antigen. Collectively, these results indicated that an antigen-specific immune response was successfully stimulated by 139A-TTD vaccine, and a TTD fusion was successful in further enhancing the immune responses.
Highlights
Kirsten Rat Sarcoma (KRAS) is one of the most frequently mutated human protooncogenes, and is highly prevalent in pancreatic, colorectal (CRC), prostate and non-smallcell lung carcinoma (NSCLC) [1]
Usp45 signal peptide (SP) was fused to the N-terminal of both control (G12A) and therapeutic (139A, 139A-tetanus toxoid (TTD)) K-ras mimotopes
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
Summary
Kirsten Rat Sarcoma (KRAS) is one of the most frequently mutated human protooncogenes, and is highly prevalent in pancreatic, colorectal (CRC), prostate and non-smallcell lung carcinoma (NSCLC) [1]. This aberration predominantly occurs in codons 12 and 13 of the KRAS gene, with a G12A, G12V, G12C, G12D, or G13D substitution in CRC. KRAS belongs to the RAS family, which encodes for a GTP-binding protein. It functions as a reversible plasma membrane-localized molecular switch that controls several downstream effector pathways, such as the Ras-Raf-MEK-ERK, mTOR, and PI3K/AKT pathways, affecting cell differentiation, proliferation, arrest, and apoptosis [1,2]. Oncogenic mutations in K-ras typically alters the GTP binding region, resulting in the inability to hydrolyze GTP, locking K-ras in a perpetually active state
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