Human Monoclonal Fab Fragments Recovered from a Combinatorial Library Bind Specifically to the Platelet HPA-1a Alloantigen on Glycoprotein IIb—IIIa

Abstract

Background and objectives: Certain clinical conditions are related to the presence of platelet-specific alloantibodies in the patient’s serum. We studied the molecular diversity of HPA-1a antibodies to analyze some peculiarities of this antibody response. Materials and methods: Human antibody Fab fragments that bind to the platelet alloantigen HPA-la on glycoprotein Ilb-IIIa (GPIIbllla) were generated by using a recombinant phage display system. We established an immunoglobulin Gl, kappa combinatorial library from the peripheral blood lymphocytes of a person undergoing a severe posttransfusion purpura. Results: Characterization of Fab clones selected from the fifth round of antigen-specific panning of this library demonstrates a highly specific reactivity to the HPA-1a alloantigen. The nucleotide sequence analysis of representative HPA-1a-specific clones reveals at least 3 distinct VL and 3 V(H) gene segments that present an extensive degree of mutation as demonstrated by comparison of gene usage and homologies to the nearest germline genes. Conclusions: These human HPA-laspecific Fab reagents should allow us to better understand the molecular mechanism involved in HPA-1a alloimmunization.

Immunologic and structural analysis of eight novel domain-deletion β3integrin peptides designed for detection of HPA-1 antibodies

“…CamTran007-IgG 1 (IBGRL, Bristol, UK) was derived from the B cells of an FMAIT mother [15], and 19-7 and 23-15 were derived from the B cells of a single patient with post-transfusion purpura (PTP) [16] and were from the Canadian Red Cross.…”

“…The recent crystallographic analysis of the a v b 3 [12] and a IIb b 3 [13] complexes was used to design eight b 3 integrin domain-deletion peptides, four Leu33 and four Pro33, for expression as monomeric proteins in Drosophila S2 cells [14]. Three human HPA-1a V-gene phage antibodies were exploited as prototype type I and II antibodies, and their interaction with b 3 integrin subunit was analyzed by the means of competition studies, and comparison of their binding to canine and human platelets, and further defined by molecular modeling [15,16]. Samples from 140 FMAIT cases from a previously reported clinical series [1] were used to determine the sensitivity and specificity of the peptides for the detection of HPA-1a antibodies and to test whether antibody categorization as type I or type II is predictive of the severity of thrombocytopenia and the risk of intracerebral hemorrhage.…”

HPA-1a phenotype–genotype discrepancy reveals a naturally occurring Arg93Gln substitution in the platelet β3 integrin that disrupts the HPA-1a epitope

“…13,16 Median fluorescence intensity obtained with these mAbs ( Figure 3A) was significantly reduced in comparison to that observed with platelets from control ␤3Leu33Pro33 heterozygous donors. A reduced reactivity of Donor A's platelets with FITClabeled CAMTRAN-007 was also observed in the whole blood, flow cytometry-based phenotyping assay ( Figure 3B).…”

Human IgG Monoclonal Anti-αIIbβ3-Binding Fragments Derived from Immunized Donors Using Phage Display

“…Polyclonal responses have also been reported for patients with posttransfusion purpura where multiple forms of Ab differing in their functional or qualitative characteristics have been demonstrated in the same serum (57). Furthermore, a series of human recombinant mAbs coded by different germline genes but all directed against HPA-1 alloantigen were obtained by phage display (9).…”