ABSTRACT
Title
Immunological evaluation of peptides derived from Bcr/Abl-Out of Frame fusion protein in HLA A2.1 transgenic mice
Authors
C. Casnici1, K. Crotta1, G. Volpe2, D. Lattuada1, O. Marelli1.
1Department of Pharmacology, School of Medicine, University of Milan, Milan, Italy.
2Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.
1Department of Pharmacology, School of Medicine, University of Milan, Milan, Italy.
2Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.
Abstract
Chronic myelogenous leukaemia and acute lymphocytic leukaemia express, besides the main BCR/ABL hybrid fusion transcript, a small number of transcripts derived from alternative splicing between BCR exons 1,13 and 14 with ABL exons 4 and 5. The transcriptional products present at their C-terminus an amino acid portion derived from out-of-frame (OOF) reading of the ABL gene (Volpe et al. 2007). As these OOF proteins are expressed only by the Philadelphia-positive cells and have no homology with other known human proteins, they could represent a new group of tumor-specific antigens suitable as immunological targets.
In this study we analysed the OOF sequence using the ProPred I, BIMAS and SYFPEITHI databases to individuate epitopes with high affinity for HLA-A2.1 molecule, leading to the design of four new peptides, peptide 1-20, peptide 22-53, peptide 32mer and peptide 29mer, containing the selected epitopes. To evaluate the capability of these peptides to be presented in association with human MHC class 1 molecules and to induce an immune response, we used C57 BL/6 mice transgenic for the HLA class I A2.1 molecule.
We investigated the ability of antigen-presenting cells to present the peptides, thus priming and expanding functional antigen-specific T lymphocytes. Syngeneic bone morrow dendritic cells loaded with the vaccination peptides induced a proliferative response to peptide 22-53 in lymphocytes from mice immunized with this peptide, to indicate that peptide 22-53 could induce cellular immune response.
Peptide 22-53 and peptide 32mer can induce the generation of specific cytotoxic effector cells able to kill human tumor cell lines in a class I HLA A2.1-restricted manner in vitro. For the study we used the human chronic myelogenous leukaemia K562 cell line. These cells are positive for the main BCR/ABL fusion transcript as well as the alternative hybrid transcript BCR/ABL-OOF (Volpe et al. 2007). In addition the cells were transfected with the HLA A2.1 gene, in order to induce MHC class I molecule expression on the cell surface.
Peptide 22-53 was also able to induce humoral immune response in immunized mice, and anti-peptide 22-53 serum recognized the out-of-frame portion of the native Bcr/Abl-OOF protein expressed in HLA A2.1+ K562 cells and bound to specific epitope/epitopes of this antigen presented in association with HLA-A2.1 molecules.
The specific immune response, both cellular and humoral, obtained ex vivo against HLA A2.1+ leukemic cells indicates that it is possible to evoke a multiple immune response toward OOF Abl protein portion and suggests the possibility to develop an immunotherapy based on the use of OOF peptides, in particular peptide 22-53, for leukemic patients in cytogenetic remission.
Volpe et al. (2007). Cancer Res. 67:5300-5307
In this study we analysed the OOF sequence using the ProPred I, BIMAS and SYFPEITHI databases to individuate epitopes with high affinity for HLA-A2.1 molecule, leading to the design of four new peptides, peptide 1-20, peptide 22-53, peptide 32mer and peptide 29mer, containing the selected epitopes. To evaluate the capability of these peptides to be presented in association with human MHC class 1 molecules and to induce an immune response, we used C57 BL/6 mice transgenic for the HLA class I A2.1 molecule.
We investigated the ability of antigen-presenting cells to present the peptides, thus priming and expanding functional antigen-specific T lymphocytes. Syngeneic bone morrow dendritic cells loaded with the vaccination peptides induced a proliferative response to peptide 22-53 in lymphocytes from mice immunized with this peptide, to indicate that peptide 22-53 could induce cellular immune response.
Peptide 22-53 and peptide 32mer can induce the generation of specific cytotoxic effector cells able to kill human tumor cell lines in a class I HLA A2.1-restricted manner in vitro. For the study we used the human chronic myelogenous leukaemia K562 cell line. These cells are positive for the main BCR/ABL fusion transcript as well as the alternative hybrid transcript BCR/ABL-OOF (Volpe et al. 2007). In addition the cells were transfected with the HLA A2.1 gene, in order to induce MHC class I molecule expression on the cell surface.
Peptide 22-53 was also able to induce humoral immune response in immunized mice, and anti-peptide 22-53 serum recognized the out-of-frame portion of the native Bcr/Abl-OOF protein expressed in HLA A2.1+ K562 cells and bound to specific epitope/epitopes of this antigen presented in association with HLA-A2.1 molecules.
The specific immune response, both cellular and humoral, obtained ex vivo against HLA A2.1+ leukemic cells indicates that it is possible to evoke a multiple immune response toward OOF Abl protein portion and suggests the possibility to develop an immunotherapy based on the use of OOF peptides, in particular peptide 22-53, for leukemic patients in cytogenetic remission.
Volpe et al. (2007). Cancer Res. 67:5300-5307