Melittin-MIL-2 fusion protein as a candidate for cancer immunotherapy

Abstract

BackgroundCytokine fusion protein that modulates the immune response holds great potential for cancer immunotherapy. IL-2 is an effective treatment against advanced cancers. However, the therapeutic efficacy of IL-2 is limited by severe systemic toxicity. Several mutants recombinant IL-2 can increase antitumor activity and minimize systemic toxicity. Melittin is an attractive anticancer candidate because of its wide-spectrum lytic properties. We previously generated a bifunctional fusion protein melittin-MIL-2, composed of melittin and a mutant IL-2. The melittin-MIL-2 inhibited the growth of human ovarian cancer SKOV3 cells in vitro and in vivo tumor growth. However, whether this antitumor effect could also be used in cancer immunotherapy was unknown. To assess its cancer immunotherapy potential, we further investigated its more effective antitumor immune response and antitumor effect against cancers of different tissue origins in vitro and in vivo.MethodsThe specific IL-2 activity of the melittin-MIL-2 fusion protein was tested on the cytokine growth dependent cell line CTLL-2. The cytolytic activity was detected by standard 4-h 51Cr-release assays. PBMC stimulation in response to the melittin-MIL-2 was determined by IFN-γ release assay. We observed the cancer cell proliferation of different tissue origins by MTT assay. The ability of melittin-MIL-2 to inhibit tumor growth in vivo was evaluated by using human liver (SMMC-7721 cancer cells), lung (A549 cancer cells) and ovarian (SKOV3 cancer cells) cancer xenograft models. To assess the immunity within the tumor microenvironment, the level of some cytokines including IFN-γ, TNF-α, IL-12 and IL-4 was analyzed by ELISA. We injected the MDA-MB-231 cells and the melittin-MIL-2 into mice, and the anti-metastatic effect was examined by counting nodules in the lung.ResultsThe melittin-MIL-2 was more effective in inducing T cell and NK-cell cytotoxicity. The fusion protein significantly increased IFN-γ production in PBMCs. In vitro, the melittin-MIL-2 mediated immune cells killing or directly killed the cancer cell lines of different tissue origins. In vivo, the fusion protein exhibited stronger inhibition on the growth of transplanted human tumors compared to rIL-2. The melittin-MIL-2 treatment promoted the IFN-γ secretion in tumor tissues and decreased the immunosuppressive cells in vivo. Furthermore, the fusion protein reduced lung metastasis of breast cancer.ConclusionsThis study provides the evidence that the melittin-MIL-2 can produce stronger immune stimulation and antitumor effects, and the fusion protein is a potent candidate for cancer immunotherapy.

Melittin, a major peptide component of bee venom, and its conjugates in cancer therapy

“…The fusion protein directly inhibited the growth of human ovarian cancer SKOV3 cells in vitro and inhibited tumor growth in ovarian cancer mice. Later, in a separate study the authors assessed the antitumor immune response and antitumor effect of the conjugate against cancers of different tissue origins both in vitro and in vivo [76]. The Melittin-MIL-2 was very effective in inducing T cell and NK-cell cytotoxicity and the fusion protein significantly increased IFN-γ production in PBMCs.…”

“…In vivo , the fusion protein exhibited stronger inhibition on the growth of transplanted human tumors compared to rIL-2. Furthermore, the fusion protein reduced lung metastasis of breast cancer [76]. …”

Antimicrobial Activity of Bee Venom and Melittin against Borrelia burgdorferi

“…Another focus in recent publications on bee venom or melittin is the clinical effectiveness of these natural antimicrobials on different diseases. Melittin, for example, was shown to have a very strong immunoregulatory activity, anticancer effect and even shows promise as chemotherapy of human immunodeficiency virus (HIV) infection [ 41 , 43 , 61 , 62 ]. Unfortunately, several issues were raised as to the safe administration of melittin in the clinical setting, due its cytotoxicity to human cells; for example, it has the ability to lyse human erythrocytes, exhibits necrotic activity against gastrointestinal and vaginal epithelial cells and can trigger severe allergic reactions [ 43 , 63 , 64 , 65 ].…”

Carbon nanoparticles as transporters of melittin to glioma grade IV U87 cells in in vitro model

“…Melittin, the major component of bee venom, containing 25 amino acids, is considered to have lytic properties after spontaneous integration into lipid bilayers (Terwilligert et al 1982). This small protein has already been tested as anticancer drug, on ovarian cancer cells and in cancer immunotherapy (Jo et al 2012, Liu et al 2016, giving the promising results. The aim of the work was to determine the effect of five different carbon nanoparticles as nanocarriers of melittin to glioma cells.…”