Melanoma antigen family A4 protein produced by transgenic silkworms induces antitumor immune responses

Abstract

Abstract. Recent clinical trials with the aim of developing tumor antigen (TA)-specific cancer vaccines against a number of malignancies have focused on the identification of TAs presented by tumor cells and recognized by T cells. In the present study, the TA melanoma antigen family A4 (MAGE-A4) protein was produced using a transgenic (TG) silkworm system. Using in vitro stimulation, it was subsequently determined whether MAGE-A4 protein induced MAGE-A4-specific T cells from peripheral blood mononuclear cells of healthy donors. TG silkworm lines expressing a MAGE-A4 gene under an upstream activating sequence (UAS) were mated with those expressing a yeast transcription activator protein (GAL4) at the middle silk glands (MSGs) and embryos that harbored both the GAL4 and UAS constructs were selected. Recombinant MAGE-A4 protein was extracted from the MSGs of TG silkworms and evaluated using SDS-PAGE and western blot analysis. It was observed that MAGE-A4 produced by the TG silkworm system successfully induced MAGE-A4-specific CD4 + T cell responses. Furthermore, MAGE-A4-specific CD4 + T cells recognized antigen-presenting cells when pulsed with a MAGE-A4 + tumor cell lysate. The present data suggests that recombinant tumor antigen production using the TG silkworm system may be a novel tool in the preparation of cancer vaccines.

In vitro assessment of antitumor immune responses using tumor antigen proteins produced by transgenic silkworms

“…We previously reported successful secretory expression and purification of MAGE-A4 [ 13 ]. The expression and purification of WT-p53 conjugated with a his-tag at the C-terminal end was performed in the same manner.…”

“…Recombinant MAGE-A4 was purified from TG silkworm MSGs and confirmed by nickel affinity chromatography and SDS-PAGE analysis as described previously (Fig. 1D, E ) [ 13 ]. Although the purified yield of p53 (~40 µg per larva) was lower than that of MAGE-A4 (~170 µg per larva), a sufficient amount of antigen was prepared for the subsequent measurement of immune activity.…”

“…For assessment of TA-specific immune responses, several factors such as type, quantity, and quality of TAs should be considered. We previously demonstrated the production of a TA, MAGE-A4 protein, using a TG silkworm system as well as the induction of MAGE-A4-specific T cells from PBMCs of healthy donors [ 13 ]. In the present study, another TA, WT-p53 protein, was successfully produced using the TG silkworm system and used for the detection of TA-specific T-cell responses in patients with HNC.…”

“…All TG silkworm strains were generated and maintained at the Transgenic Silkworm Research Unit at the National Institute of Agrobiological Sciences (Ibaraki, Japan) as described previously [ 13 ]. Briefly, the silkworm strain w1-pnd, a non-diapausing strain that produces non-pigmented eyes and eggs, was used.…”

Application of mass spectrometry in silkworm research—Review

“…This prominent capability has made possible the mass production of recombinant proteins using genetic transformation technologies such as the baculovirus system and the transposon‐based vector system (Choi et al, ). It was reported in the literature that transgenic silkworms can be used to produce insulin‐like growth factor‐I (Seong et al, ), pheromone binding protein‐1 (Shiota et al, ), fibroblast growth factor (Wang et al, ), anti‐CD20 monoclonal antibody (Aoyama et al, ), enzyme drugs (Itoh, Nishioka, Hidaka, Tsuji, & Maita, ) and cancer vaccines (Motokawa et al, ).…”