Reconstitution of antigen presentation in HLA class I-negative cancer cells with peptide-beta 2m fusion molecules

Engineered MHC-peptide targets capable of inducing recognition by CTL may p rove useful in designing vaccines for infectious disease and cancer. We tes ted whether peptides directly linked to beta2-microglobulin (beta 2m) could complex with human HLA class I heavy chain, and could be recognized by hum an CTL, both as soluble reagents and as cell surface constituents. An HLA-A 2-restricted peptide epitope was physically linked to the N terminus of hum an beta 2m. This fusion protein refolded efficiently in vitro with HLA-A2 h eavy chain, and when multimerized, the resultant complexes ("fusamers") bou nd specifically to appropriate CTL clones. These fused peptide/MHC complexe s were as efficient as standard tetrameric peptide/MHC complexes in recogni zing antigen-specific CTL. When the fusion protein was delivered to target cells using a retroviral vector, these cells were recognized and killed by appropriate CTL clones. Efficient sensitization to CTL lysis was achieved i n TAP-negative and beta 2m-negative cell lines, as well as in unmutated B c ell lines, proving that such constructs may be effective in inducing CTL ev en when the MHC class I pathway has been disrupted. Specific peptides coval ently linked to beta 2m and delivered via retroviral vectors may be useful reagents for in vivo priming of CTL against epitopes of clinical relevance.