Identification and removal of a promiscuous CD4+ T cell epitope from the C1 domain of factor VIII.
BACKGROUND
The development of inhibitors in hemophiliacs is a severe complication of factor VIII (FVIII) replacement therapy and is a process driven by FVIII specific T helper cells.


OBJECTIVE
To finely map T cell epitopes within the whole FVIII protein in order to investigate the possibility of engineering FVIII variants with reduced propensity for inhibitor development.


METHODS
T cell lines were generated from five patients with severe hemophilia who had developed inhibitors, and were screened for T cell proliferation against pools of overlapping peptides spanning the entire B domain deleted (BDD) FVIII sequence. Positive peptide pools were decoded by screening individual peptides against the T cell lines. Positive peptides, and mutants thereof, were tested for their ability to bind major histocompatibility complex (MHC) Class II and stimulate T cell proliferation in a panel of healthy donors. The activities of the corresponding mutant proteins were assessed via chromogenic assay.


RESULTS
One peptide, spanning FVIII amino acids 2098-2112, elicited a vigorous response from one hemophiliac donor, induced strong T cell responses in the panel of healthy donors and bound to a number of HLA-DR alleles. Mutations were made in this peptide that removed its ability to stimulate T cells of healthy donors and to bind to MHC Class II while retaining full activity when incorporated into a mutant BDD-FVIII protein.


CONCLUSIONS
Fine T cell epitope mapping of the entire FVIII protein is feasible, although challenging, and this knowledge may be used to create FVIII variants which potentially have reduced immunogenicity.
Jones
T D
TD
Biovation Ltd, Babraham, Cambridge, UK. timj@tinyworld.ac.uk
Phillips
W J
WJ
Smith
B J
BJ
Bamford
C A
CA
Nayee
P D
PD
Baglin
T P
TP
Gaston
J S H
JS
Baker
M P
MP
D000483:Alleles; D000595:Amino Acid Sequence; D015496:CD4-Positive T-Lymphocytes / Q000737:chemistry*; D049109:Cell Proliferation; D003001:Cloning, Molecular; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry; D005169:Factor VIII / Q000737:chemistry*; D006684:HLA-DR Antigens / Q000276:immunology; D006467:Hemophilia A / Q000097:blood / Q000276:immunology; D000949:Histocompatibility Antigens Class II / Q000737:chemistry; D006801:Humans; D020128:Inhibitory Concentration 50; D007477:Ions; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D013601:T-Lymphocytes / Q000276:immunology; D013997:Time Factors
D000939:Epitopes; D006684:HLA-DR Antigens; D000949:Histocompatibility Antigens Class II; D007477:Ions; D010455:Peptides; D005169:Factor VIII