Elimination of a Human T-cell Region in Staphylokinase by T-cell Screening and Computer Modeling

Summary

Staphylokinase is a potent highly fibrin-selective thrombolytic agent, but it induces a humoral immune response in most treated patients. Staphylokinase-specific T-lymphocytes can be found in normal healthy individuals, from whom a large panel of staphylokinasespecific T-cells were cloned. The staphylokinase amino acid sequence 71-87 was widely recognized, as it induced proliferation of T-cell clones isolated from 90% of the donors. Computer modeling of this area, threaded as 11-mer peptides within the peptide-binding groove of the major HLA-DR alleles, indicated two putative partially overlapping binding sequences. The region-(71-87)-specific T-cell clones recognized either one or the other minimal peptide, confirming that both sequences could be functional T-cell epitopes. Furthermore, to guide the mutagenesis to eliminate T-cell reactivity, the contribution of each residue to the HLA-DR-anchoring and T-cell receptor exposure was evaluated for both binding motifs. Computer calculations combined with functional assays resulted in the design of staphylokinasevariants, including 2 to 4 amino acid substitutions in the region 71-87. These variants were no longer recognized by the region-(71-87)specific T-cell clones, and importantly no new staphylokinase-variantspecific cellular immune response could be measured.

Keywords

Staphylokinase - human - T-cell epitope - T-helper lymphocytes - de-immunization

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