title: A subtraction tolerization method of immunization allowed for Wilms' tumor protein-1 (WT1) identification in melanoma and discovery of an antitumor peptide sequence.
abstract: On searching for melanoma transcription factors in a project focusing on internal antitumor peptide sequences from transcription factors, we found that a highly immunogenic component emerged upon using a subtraction tolerization method of immunization. While several conventional immunization procedures using whole melanoma cells induced a plethora of low affinity antibodies of various specificities, the subtraction tolerization method efficiently elicited mono-specific antibodies that recognized Wilms' tumor protein 1 (WT1), which is known as an important marker in melanoma prognosis and treatment. For the tolerization step, pre-immunization of Balb/c mice with a membrane-rich preparation of glioblastoma U87 cells was used. The subsequent immunizations with SK-MEL-28 melanoma cells elicited antibodies strongly reacting with 50 and 55 kDa proteins, identified as WT1. Remarkably, this was the only component strongly reactive with these antibodies in a melanoma cell lysate. WT1 was then chosen as a target for selecting internally bioactive peptides. A hydrophilic Trojan peptide containing most of the zinc finger-2 domain of WT1 was synthesized and shown to inhibit SK-MEL-28 melanoma growth in vitro. The peptide WT1-pTj was also protective in vivo in a metastatic melanoma model and peptide-stimulated syngeneic dendritic cells reproduced the anti-melanoma effect of the unprotected peptide. Identification of antitumor peptides derived from major transcription factors represents a new tool to be explored in cancer research aiming at new therapeutic drugs.
authors.affiliation: Experimental Oncology Unit, Department of Microbiology, Immunology and Parasitology, Federal University of São Paulo, São Paulo, Brazil.
authors.forename: Mariana H
authors.initials: MH
authors.lastname: Massaoka
authors.affiliation: Experimental Oncology Unit, Department of Microbiology, Immunology and Parasitology, Federal University of São Paulo, São Paulo, Brazil.
authors.forename: Alisson L
authors.initials: AL
authors.lastname: Matsuo
authors.affiliation: Experimental Oncology Unit, Department of Microbiology, Immunology and Parasitology, Federal University of São Paulo, São Paulo, Brazil.
authors.forename: Carlos R
authors.initials: CR
authors.lastname: Figueiredo
authors.affiliation: Experimental Oncology Unit, Department of Microbiology, Immunology and Parasitology, Federal University of São Paulo, São Paulo, Brazil.
authors.forename: Natalia
authors.initials: N
authors.lastname: Girola
authors.affiliation: Experimental Oncology Unit, Department of Microbiology, Immunology and Parasitology, Federal University of São Paulo, São Paulo, Brazil.
authors.forename: Camyla F
authors.initials: CF
authors.lastname: Farias
authors.affiliation: Biochemistry and Biophysical Laboratory, Butantan Institute, São Paulo, SP, Brazil.
authors.forename: Kerly
authors.initials: K
authors.lastname: Pasqualoto
authors.affiliation: Experimental Oncology Unit, Department of Microbiology, Immunology and Parasitology, Federal University of São Paulo, São Paulo, Brazil. Electronic address: luiztravassos@gmail.com.
authors.forename: Luiz R
authors.initials: LR
authors.lastname: Travassos
mesh_terms: D000818:Animals; D000912:Antibodies, Neoplasm / Q000276:immunology*; D014408:Biomarkers, Tumor / Q000276:immunology; D019496:Cancer Vaccines / Q000276:immunology* / Q000627:therapeutic use; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003713:Dendritic Cells / Q000276:immunology; D005260:Female; D006801:Humans; D007108:Immune Tolerance / Q000276:immunology; D007114:Immunization / Q000379:methods; D008297:Male; D008546:Melanoma, Experimental / Q000235:genetics / Q000276:immunology* / Q000628:therapy; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D010455:Peptides / Q000276:immunology / Q000627:therapeutic use; D025721:WT1 Proteins / Q000276:immunology* / Q000627:therapeutic use
chemical_list: D000912:Antibodies, Neoplasm; D014408:Biomarkers, Tumor; D019496:Cancer Vaccines; D010455:Peptides; D025721:WT1 Proteins
keywords: Antitumor peptide; Immunization protocol; Malignant melanoma; Wilms' tumor protein 1 (WT1)