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.