title: Human IgG monoclonal anti-alpha(IIb)beta(3)-binding fragments derived from immunized donors using phage display.
abstract: Previous studies of the immune response in polytransfused Glanzmann thrombasthenia (GT) patients and in autoimmune thrombocytopenic purpura (AITP) have relied on serum analysis and have shown the frequent development of Abs directed against the alpha(IIb)beta(3) integrin. However, little is known about the molecular diversity of the humoral immune response to alpha(IIb)beta(3) due to the paucity of mAbs issuing from these pathologies. We have isolated human IgG anti-alpha(IIb)beta(3) binding fragments using combinatorial libraries of single-chain IgG created from the B cells of a GT and an AITP patient, both with serum Abs. Ab screening was performed using activated platelets or activated alpha(IIb)beta(3)-expressing Chinese hamster ovary cells. Sequencing of selected phage Abs showed that a broad selection of genes from virtually all V gene families had been used, indicating the diversity of the immune response. About one-half of the V(H) and V(L) segments of our IgG anti-alpha(IIb)beta(3) fragments displayed extensive hypermutations in the complementarity-determining region, supporting the idea that an Ag-driven immune response was occurring in both patients. The H chain complementarity-determining region 3 analysis of phage Abs revealed motifs other than the well-known RGD and KQAGDV integrin-binding sequences. To our knowledge, our study is the first to illustrate multiple human IgG anti-alpha(IIb)beta(3) reactivities and structural variations linked to the anti-platelet human immune response. Human alpha(IIb)beta(3) Abs preferentially directed against the activated form of the integrin were further characterized because platelet alpha(IIb)beta(3) inhibitors are potential therapeutic reagents for treating acute coronary syndromes. Currently available alpha(IIb)beta(3) antagonists do not specifically recognize the activated form of the integrin.
authors.affiliation: Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5533, Hopital Cardiologique, Pessac, France.
authors.forename: Marie-Josée
authors.initials: MJ
authors.lastname: Jacobin
authors.forename: Jeanny
authors.initials: J
authors.lastname: Laroche-Traineau
authors.forename: Melvyn
authors.initials: M
authors.lastname: Little
authors.forename: Armin
authors.initials: A
authors.lastname: Keller
authors.forename: Karlheinz
authors.initials: K
authors.lastname: Peter
authors.forename: Martin
authors.initials: M
authors.lastname: Welschof
authors.forename: Alan
authors.initials: A
authors.lastname: Nurden
authors.forename: Gisèle
authors.initials: G
authors.lastname: Clofent-Sanchez
mesh_terms: D000368:Aged; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D000915:Antibody Affinity; D000918:Antibody Specificity; D001323:Autoantibodies / Q000235:genetics / Q000276:immunology*; D016466:CHO Cells; D022801:Complementarity Determining Regions / Q000235:genetics; D006224:Cricetinae; D000939:Epitopes / Q000276:immunology; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D007114:Immunization; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D019151:Peptide Library; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000276:immunology*; D016553:Purpura, Thrombocytopenic, Idiopathic / Q000276:immunology*; D017386:Sequence Homology, Amino Acid; D013915:Thrombasthenia / Q000276:immunology*; D014019:Tissue Donors
chemical_list: D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D022801:Complementarity Determining Regions; D000939:Epitopes; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D019151:Peptide Library; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D005340:Fibrinogen