Recombinant human single-chain MHC-peptide complexes made fromE. coli byin vitro refolding: functional single-chain MHC-peptide complexes and tetramers with tumor associated antigens

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Abstract
Soluble recombinant MHC‐peptide complexes are valuable tools for molecular characterization of immune responses as well as for other functional and structural studies. In this study, soluble recombinant single‐chain human MHC (scMHC)‐peptide complexes were generated by in vitro refolding of inclusion bodies from bacterially expressed engineered HLA‐A2 in the presence of tumor‐associated or viral peptides. The scMHC molecule was composed of β2‐microglobulin connected to the first three domains of the HLA‐A2 heavy chain through a 15‐amino acid flexible linker. Highly purified scMHC‐peptide complexes were obtained in high yield using several peptides derived from the melanoma antigens gp100 and MART‐1 or a viral peptide derived from HTLV‐1. The scMHC complexes were characterized in detail and were found to be correctly folded and able to specifically bind HLA‐A2‐restricted peptides. We also generated scMHC‐peptide tetramers, which were biologically functional; they induced a peptide‐specific CTL clone to be activated and secrete IFN‐γ, and were able to stain specifically CTL lines. Such recombinant soluble scMHC‐peptide complexes and tetramers should prove of great value for characterization of immune responses involving CTL, for visualization of antigen‐specific immune responses, for in vitro primary CTL induction, and for peptide binding assays and structural studies.