title: Melittin-MIL-2 fusion protein as a candidate for cancer immunotherapy.
abstract: Cytokine 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.
  
  
  
  The 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 (51)Cr-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.
  
  
  
  The 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.
  
  
  
  This 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.
authors.affiliation: Department of Clinical Laboratory, The Affiliated Hospital of Qingdao University, 16 Jiangsu Road, Qingdao, 266003, China. jocklmj@hotmail.com.
authors.forename: Mingjun
authors.initials: M
authors.lastname: Liu
authors.affiliation: Department of Public Health, Qingdao University Medical College, 38 Dengzhou Road, Qingdao, 266021, China.
authors.forename: Haitao
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Clinical Laboratory, The Affiliated Hospital of Qingdao University, 16 Jiangsu Road, Qingdao, 266003, China.
authors.forename: Linjie
authors.initials: L
authors.lastname: Liu
authors.affiliation: Key Laboratory of Medicine and Biotechnology, Department of Microbiology, Qingdao University Medical College, 308 Ningxia Road, Qingdao, 266071, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Wang
authors.affiliation: Department of Clinical Laboratory, The Affiliated Hospital of Qingdao University, 16 Jiangsu Road, Qingdao, 266003, China. sunguirong@hotmail.com.
authors.forename: Guirong
authors.initials: G
authors.lastname: Sun
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology / Q000627:therapeutic use; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D060833:Cellular Microenvironment / Q000187:drug effects; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D004195:Disease Models, Animal; D005260:Female; D006801:Humans; D056747:Immunomodulation / Q000187:drug effects; D007167:Immunotherapy; D007371:Interferon-gamma / Q000096:biosynthesis; D008175:Lung Neoplasms / Q000473:pathology / Q000556:secondary; D008213:Lymphocyte Activation / Q000187:drug effects; D008555:Melitten / Q000494:pharmacology / Q000627:therapeutic use*; D008807:Mice, Inbred BALB C; D050505:Mutant Proteins / Q000494:pharmacology / Q000627:therapeutic use*; D009369:Neoplasms / Q000188:drug therapy* / Q000276:immunology* / Q000473:pathology; D011993:Recombinant Fusion Proteins / Q000494:pharmacology / Q000627:therapeutic use*
chemical_list: D000970:Antineoplastic Agents; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D008555:Melitten; D007371:Interferon-gamma
keywords: Cancer immunotherapy; Fusion protein; IL-2; Melittin; Mutant