Using directed mutagenesis and phage display on a soluble fragment of the human immunoglobulin superfamily receptor ILT2(synonyms:LIR1,MIR7,CD85j),we have selected a range of mutants with binding affinities enhanced b...Using directed mutagenesis and phage display on a soluble fragment of the human immunoglobulin superfamily receptor ILT2(synonyms:LIR1,MIR7,CD85j),we have selected a range of mutants with binding affinities enhanced by up to 168,000-fold towards the conserved region of major histocompatibility complex(MHC)class I molecules.Produced in a dimeric form,either by chemical cross-linking with bivalent polyethylene glycol(PEG)derivatives or as a genetic fusion with human IgG Fc-fragment,the mutants exhibited a further increase in ligand-binding strength due to the avidity effect,with resident half-times(t1/2)on the surface of MHC I-positive cells of many hours.The novel compounds antagonized the interaction of CD8 co-receptor with MHC I in vitro without affecting the peptide-specific binding of T-cell receptors(TCRs).In both cytokine-release assays and cell-killing experiments the engineered receptors inhibited the activation of CD8^(+)cytotoxic T lymphocytes(CTLs)in the presence of their target cells,with subnanomolar potency and in a dose-dependent manner.As a selective inhibitor of CD8^(+)CTL responses,the engineered high affinity ILT2 receptor presents a new tool for studying the activation mechanism of different subsets of CTLs and could have potential for the development of novel autoimmunity therapies.展开更多
基金This work was funded by MediGene AG,Munich,Germany.Authors declare noconflict of interest associated with this work.
文摘Using directed mutagenesis and phage display on a soluble fragment of the human immunoglobulin superfamily receptor ILT2(synonyms:LIR1,MIR7,CD85j),we have selected a range of mutants with binding affinities enhanced by up to 168,000-fold towards the conserved region of major histocompatibility complex(MHC)class I molecules.Produced in a dimeric form,either by chemical cross-linking with bivalent polyethylene glycol(PEG)derivatives or as a genetic fusion with human IgG Fc-fragment,the mutants exhibited a further increase in ligand-binding strength due to the avidity effect,with resident half-times(t1/2)on the surface of MHC I-positive cells of many hours.The novel compounds antagonized the interaction of CD8 co-receptor with MHC I in vitro without affecting the peptide-specific binding of T-cell receptors(TCRs).In both cytokine-release assays and cell-killing experiments the engineered receptors inhibited the activation of CD8^(+)cytotoxic T lymphocytes(CTLs)in the presence of their target cells,with subnanomolar potency and in a dose-dependent manner.As a selective inhibitor of CD8^(+)CTL responses,the engineered high affinity ILT2 receptor presents a new tool for studying the activation mechanism of different subsets of CTLs and could have potential for the development of novel autoimmunity therapies.
