de Lalla C, Tamborini E, Longhi R, Tresoldi E, Manoni M, Siccardi A G, Arosio P, Sidoli A
Department of Biological and Technological Research, San Raffaele Scientific Institute, Milano, Italy.
Mol Immunol. 1996 Sep;33(13):1049-58. doi: 10.1016/s0161-5890(96)00061-2.
One of the major allergens from the pollen of perennial rye grass (Lolium perenne), Lol pII, was used to isolate specific antibody fragments from a random combinatorial library displaying a large repertoire of human Fab on filamentous phages. After five panning cycles on recombinant Lol pII immunotubes, phage binders were isolated and the antibody fragments expressed as soluble Fab molecules in the Escherichia coli periplasm. The DNA sequencing of the clones producing antibodies with the highest binding activity showed three of them to be identical, while one differed by two amino acid substitutions in the heavy chain. The antibody fragments were produced in milligram amounts, affinity-purified and further characterized. They bound the natural allergen as well as the recombinant one, with no cross-reactivity with other allergens contained in the pollen extract of L. perenne. One antibody bound the allergen with Kd = 2.63 x 10(-9) M, as demonstrated by the surface plasmon resonance technique, and was able to compete with a fraction of serum IgE. Epitope mapping using synthetic peptides revealed that antigenic domains, located between amino acids 39 and 51 of Lol pII, are recognized by Fab and polyclonal IgE from sera of allergic donors. The Fab fragments inhibited the binding of serum IgE to the allergen. In vitro experiments on whole blood from allergic subjects showed that recombinant Fab fragments had a blocking activity on histamine release from cells challenged with recombinant Lol pII allergen. Thus, serum IgE and recombinant Fab fragments recognize common epitopes, although they represent the outcome of different maturation and/or selection processes. Our molecular and functional findings altogether indicate that allergen-specific human antibodies may be useful for the characterization of the antigenic structure of allergens. We conclude that a phage library is a powerful source of anti-allergen human antibodies with high affinity and high specificity. Moreover, these molecules may be potentially innovative reagents for the treatment of atopic allergy.
多年生黑麦草（Lolium perenne）花粉中的主要过敏原之一Lol pII，被用于从一个在丝状噬菌体上展示大量人源Fab片段文库中分离特异性抗体片段。在重组Lol pII免疫管上进行五个淘选循环后，分离出噬菌体结合物，并将抗体片段表达为大肠杆菌周质中的可溶性Fab分子。对产生具有最高结合活性抗体的克隆进行DNA测序，结果显示其中三个完全相同，而另一个在重链中有两个氨基酸替换。抗体片段的产量达到毫克级，经过亲和纯化并进一步表征。它们能结合天然过敏原和重组过敏原，与多年生黑麦草花粉提取物中包含的其他过敏原无交叉反应。表面等离子体共振技术表明，一种抗体以Kd = 2.63 x 10(-9) M的亲和力结合过敏原，并且能够与一部分血清IgE竞争。使用合成肽进行的表位作图显示，位于Lol pII氨基酸39至51之间的抗原结构域可被过敏供体血清中的Fab和多克隆IgE识别。Fab片段抑制血清IgE与过敏原的结合。对过敏受试者全血进行的体外实验表明，重组Fab片段对重组Lol pII过敏原刺激的细胞释放组胺具有阻断活性。因此，血清IgE和重组Fab片段识别共同的表位，尽管它们代表了不同成熟和/或选择过程的结果。我们的分子和功能研究结果共同表明，过敏原特异性人源抗体可能有助于表征过敏原的抗原结构。我们得出结论，噬菌体文库是高亲和力和高特异性抗过敏原人源抗体的强大来源。此外，这些分子可能是治疗特应性过敏的潜在创新试剂。
