Non-natural and photo-reactive amino acids as biochemical probes of immune function.
Wilms tumor protein (WT1) is a transcription factor selectively overexpressed in leukemias and cancers; clinical trials are underway that use altered WT1 peptide sequences as vaccines. Here we report a strategy to study peptide-MHC interactions by incorporating non-natural and photo-reactive amino acids into the sequence of WT1 peptides. Thirteen WT1 peptides sequences were synthesized with chemically modified amino acids (via fluorination and photo-reactive group additions) at MHC and T cell receptor binding positions. Certain new non-natural peptide analogs could stabilize MHC class I molecules better than the native sequences and were also able to elicit specific T-cell responses and sometimes cytotoxicity to leukemia cells. Two photo-reactive peptides, also modified with a biotin handle for pull-down studies, formed covalent interactions with MHC molecules on live cells and provided kinetic data showing the rapid clearance of the peptide-MHC complex. Despite "infinite affinity" provided by the covalent peptide bonding to the MHC, immunogenicity was not enhanced by these peptides because the peptide presentation on the surface was dominated by catabolism of the complex and only a small percentage of peptide molecules covalently bound to the MHC molecules. This study shows that non-natural amino acids can be successfully incorporated into T cell epitopes to provide novel immunological, biochemical and kinetic information.
Gómez-Nuñez
Marta
M
Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.
Haro
Kurtis J
KJ
Dao
Tao
T
Chau
Deming
D
Won
Annie
A
Escobar-Alvarez
Sindy
S
Zakhaleva
Victoriya
V
Korontsvit
Tatyana
T
Gin
David Y
DY
Scheinberg
David A
DA
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000276:immunology*; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000494:pharmacology; D015234:HLA-A Antigens / Q000276:immunology; D015789:HLA-A2 Antigen; D006649:Histocompatibility Antigens / Q000276:immunology; D006801:Humans; D007109:Immunity / Q000187:drug effects / Q000276:immunology* / Q000528:radiation effects; D007118:Immunoassay; D007371:Interferon-gamma / Q000378:metabolism; D007700:Kinetics; D008027:Light; D008213:Lymphocyte Activation / Q000187:drug effects / Q000528:radiation effects; D015335:Molecular Probes / Q000737:chemistry / Q000276:immunology*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding / Q000187:drug effects / Q000528:radiation effects; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000276:immunology / Q000528:radiation effects; D014019:Tissue Donors; D025721:WT1 Proteins / Q000737:chemistry / Q000276:immunology
D000596:Amino Acids; D003432:Cross-Linking Reagents; D015234:HLA-A Antigens; C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D006649:Histocompatibility Antigens; D015335:Molecular Probes; D010455:Peptides; D025721:WT1 Proteins; D007371:Interferon-gamma