Human IgG Monoclonal Anti-αIIbβ3-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 αIIbβ3 integrin. However, little is known about the molecular diversity of the humoral immune response to αIIbβ3 due to the paucity of mAbs issuing from these pathologies. We have isolated human IgG anti-αIIbβ3 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 αIIbβ3-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 VH and VL segments of our IgG anti-αIIbβ3 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-αIIbβ3 reactivities and structural variations linked to the anti-platelet human immune response. Human αIIbβ3 Abs preferentially directed against the activated form of the integrin were further characterized because platelet αIIbβ3 inhibitors are potential therapeutic reagents for treating acute coronary syndromes. Currently available αIIbβ3 antagonists do not specifically recognize the activated form of the integrin.

MRI of inducible P‐selectin expression in human activated platelets involved in the early stages of atherosclerosis

“…In an ultimate aim to translate such agents into human clinical use, a 'humanized' version of murine VH10 is envisaged in order to attenuate potential immunogenicity We plan to use phage display technology and directed mutagenesis approaches to develop a human antibody presenting the same specificity and affinity as its murine counterpart on human platelets, but also able to recognize with high affinity murine P-selectin (77,78). Toxicologic and pharmacokinetic in vivo data on ApoE mice will be further evaluated in order to lay the basis for preclinical development.…”

Gene therapy for platelet disorders: studies with Glanzmann's thrombasthenia

“…This is a crucial issue, because gene therapy for GT could lead to the formation of (i) inhibitory antibodies to GPIIb–IIIa that prevent platelet aggregation, and/or (ii) an immune response that results in clearance of transduced platelets and life‐threatening thrombocytopenia as observed with neonatal alloimmune thrombocytopenia or from the transfusion of platelets that are mismatched for human leukocyte and platelet antigens [129]. An immune response to human platelet antigens can be polyclonal in nature, with some antibodies recognizing changes in protein structure as occurs when the integrin GPIIb–IIIa goes from its resting to activated state [130]. There is also variation between patients with platelet defects to tolerate neoantigens on transfused platelets, and therefore there are likely to be differences between patients in their capacity to tolerate a normal GPIIb or GPIIIa transgene product.…”

Effects of IgG and its F(ab′)2 fragments of some patients with idiopathic thrombocytopenic purpura on platelet aggregation

“…It is therefore necessary to humanize the antibody by altering both the variable and constant regions. In recent years, much effort has been devoted to the development of completely humanized mAbs by phage surface display technology (17, 18).…”