Design and production of human leukocyte antigen (HLA)-Fc fusion proteins. (A) Schematic representation of HLA-Fc fusion proteins. The N-terminus of the fusion protein is a single-chain trimer (SCT) of a 9-mer peptide (red), β2-microglobulin (β2m, yellow), and extracellular domains of HLA class I (α1-α3, blue). The C-terminus consists of the constant domains 2 and 3 of the IgG heavy chain (CH2 and CH3, gray) or a fragment crystallizable (Fc). Disulfide bonds at the hinge region of Fc allow the homodimerization of HLA-Fc with bilateral symmetry. (B) The proposed mechanism of action for HLA-Fc. The HLA portion binds to cognate surface immunoglobulin (Ig, purple) and guides the fusion protein to corresponding antibody-producing cells; the Fc portion mediates cytotoxicity to enable target cell killing. (C) Components of HLA-Fc proteins used in the study. All variants start with a signal sequence, followed by a 9-mer peptide, flexible linker #1, human β2m (hβ2m), flexible linker #2, extracellular domains of the indicated HLA allele, and Fc of mouse IgG2a. The broken lines between linker #1 and the α2 domain represent disulfide bonds that secure the 9-mer peptides within the peptide-presenting domains (α1-α2). The A2Fc−LALAPG mutant contains 3 point mutations within the Fc portion (arrowheads). (D) Characterization of HLA-Fc proteins by polyacrylamide gel electrophoresis and Coomassie stain. HLA-Fc variants were purified with protein A from the supernatant of transiently transfected Expi293 cells and analyzed in nonreduced and reduced conditions.