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

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.

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

“…The washed solution was allowed to stand for 10 min before being centrifuged at 3000× g at 4 • C for 10 min, after which the top layer supernatant containing intestinal washes was separated and stored at −20 • C. The extracted whole blood was collected in collection tubes containing anticoagulant K 2 EDTA (BD, Franklin Lakes, NJ, USA) and stored at −20 • C. Sera were collected from whole blood that was allowed to clot for 60 min on ice, followed by centrifugation at 3000× g at 4 • C for 10 min, before the top layer containing serum was obtained and stored at −20 • C. 4.9. Immunoglobulins (sIgA and IgG) Detection by Indirect ELISA For IgA and IgG detection, 100 µL of undiluted mice intestinal wash samples from a small intestine and large intestine (1:1) or 50 µL of diluted mice sera (1:10), respectively, were used for indirect ELISA assay as previously described (Siak et al, 2021) [56]. A volume of 100 µL of 5 µg/mL of each synthetic peptide (68-V and wild-type KRAS) was used for initial coating on microtiter plates, followed by blocking with 0.1% (w/v) Tween 20 and BSA (1-2%, w/v at 4 • C overnight incubation with either the samples or standard (1X PBS)) for 1 h at RT.…”