Antibody-mediated targeting of human single-chain class I MHC with covalently linked peptides induces efficient killing of tumor cells by tumor or viral-specific cytotoxic T lymphocytes.
Soluble forms of human MHC class I HLA-A2 were produced in which the peptide binding groove was uniformly occupied by a single tumor or viral-derived peptides attached via a covalent flexible peptide linker to the N terminus of a single-chain beta-2-microglobulin-HLA-A2 heavy chain fusion protein. A tetravalent version of this molecule with various peptides was found to be functional. It could stimulate T cells specifically as well as bind them with high avidity. The covalently linked single chain peptide-HLA-A2 construct was next fused at its C-terminal end to a scFv antibody fragment derived from the variable domains of an anti-IL-2R alpha subunit-specific humanized antibody, anti-Tac. The scFv-MHC fusion was thus encoded by a single gene and produced in E. coli as a single polypeptide chain. Binding studies revealed its ability to decorate Ag-positive human tumor cells with covalent peptide single-chain HLA-A2 (scHLA-A2) molecules in a manner that was entirely dependent upon the specificity of the targeting Antibody fragment. Most importantly, the covalent scHLA-A2 molecule, when bound to the target tumor cells, could induce efficient and specific HLA-A2-restricted, peptide-specific CTL-mediated lysis. These results demonstrate the ability to generate soluble, stable, and functional single-chain HLA-A2 molecules with covalently linked peptides, which when fused to targeting antibodies, potentiate CTL killing. This new approach may open the way for the development of new immunotherapeutic strategies based on antibody targeting of natural cognate MHC ligands and CTL-based cytotoxic mechanisms.
Oved
Kfir
K
Department of Biology, The Technion-Israel Institute of Technology, Technion City, Room 333, Haifa, 32000, Israel.
Lev
Avital
A
Noy
Roy
R
Segal
Dina
D
Reiter
Yoram
Y
D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism; D000920:Antibody-Dependent Cell Cytotoxicity / Q000235:genetics; D017951:Antigen Presentation; D000951:Antigens, Neoplasm / Q000235:genetics / Q000276:immunology; D003601:Cytotoxicity Tests, Immunologic; D015789:HLA-A2 Antigen / Q000276:immunology*; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D053645:Interleukin-2 Receptor alpha Subunit; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology; D009363:Neoplasm Proteins / Q000235:genetics / Q000276:immunology; D010446:Peptide Fragments / Q000235:genetics; D018123:Receptors, Interleukin / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D001613:beta 2-Microglobulin / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D058951:gp100 Melanoma Antigen
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D015789:HLA-A2 Antigen; C508017:IL2RA protein, human; D007128:Immunoglobulin Fragments; D053645:Interleukin-2 Receptor alpha Subunit; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; C548637:PMEL protein, human; D010446:Peptide Fragments; D018123:Receptors, Interleukin; D011993:Recombinant Fusion Proteins; D001613:beta 2-Microglobulin; D058951:gp100 Melanoma Antigen; C055966:immunoglobulin Fv