Human IgG monoclonal anti-αIIbβ3-binding fragments derived from immunized donors using phage display
Human IgG monoclonal anti-αIIbβ3-binding fragments derived from immunized donors using phage display
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DOI：
10.4049/jimmunol.168.4.2035
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发表时间：
2002-02-15
影响因子：
4.4
通讯作者：
Clofent-Sanchez, G
中科院分区：
文献类型：
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作者：
Jacobin, MJ;Laroche-Traineau, J;Clofent-Sanchez, G
先前对多次输血的Glanzmann血小板无力症（GT）患者和自身免疫性血小板减少性紫癜（AITP）患者的免疫应答的研究依赖于血清分析，并显示针对α（IIb）β（3）整联蛋白的Ab频繁发生。然而，由于缺乏来自这些病理的mAb，对α（IIb）β（3）的体液免疫应答的分子多样性知之甚少。我们使用从GT和AITP患者的B细胞（两者均具有血清Ab）产生的单链IgG的组合文库，分离了人IgG抗α（II B）β（3）结合片段。使用活化的血小板或活化的表达α（IIb）β（3）的中国仓鼠卵巢细胞进行抗体筛选。所选噬菌体抗体的测序显示，已使用了来自几乎所有V基因家族的基因的广泛选择，表明免疫应答的多样性。我们的IgG抗α（IIb）β（3）片段的约一半V-II和V-L片段在互补决定区显示出广泛的超突变，支持Ag驱动的免疫应答在两名患者中发生的观点。噬菌体抗体的H链互补决定区3分析显示了除了众所周知的RGD和KQAGDV整合素结合序列以外的基序。据我们所知，我们的研究是第一个说明与抗血小板人体免疫应答相关的多种人IgG抗α（IIb）β（3）反应性和结构变异。由于血小板α（IIb）β（3）抑制剂是治疗急性冠状动脉综合征的潜在治疗试剂，因此进一步表征了优先针对活化形式的整联蛋白的人α（IIb）β（3）Ab。目前可用的α（IIb）β（3）拮抗剂不特异性识别整合素的活化形式。
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-II 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.