Depetris M, Casalis P, Kratje R, Etcheverrigaray M, Oggero M
Laboratorio de Cultivos Celulares, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Ciudad Universitaria, Paraje El Pozo-C.C. 242, S3000ZAA, Santa Fe, Argentina.
J Immunol Methods. 2008 May 20;334(1-2):104-13. doi: 10.1016/j.jim.2008.02.003. Epub 2008 Mar 5.
Despite their significant role in maintaining the normal physiology, cytokines may cause pathological conditions when they are overproduced. In this way, the increased expression of human interferon alpha (hIFN-alpha) is associated with acute viral infections, inflammatory disorders and several autoimmune illnesses, where the cytokine may be a factor in either initiating or maintaining the disease. Currently, there are several mAbs marketed for a variety of indications and many more in clinical trials, in which IFN-alpha represents a potential target for antibody-based therapy. A panel of 11 murine mAbs was prepared using recombinant hIFN-alpha2b as immunogen, all of which bound to the native form of the cytokine with affinity constants ranging from 1.7x10(7) M(-1) to 1.4x10(10) M(-1). An epitope mapping protocol demonstrated four spatially distinct areas of the protein recognized by the mAbs. Taking into account the characterization of the antibodies and their ability to inhibit the IFN-alpha biological activity, four mAbs were selected to produce scFv fragments. One of these fragments (CA5E6) was able to neutralize a wide spectrum of subtypes of the IFN-alpha family, including the recombinant cytokines hIFN-alpha2a and hIFN-alpha2b and a heterogeneous collection of IFN-alpha produced by activated leukocytes and Namalwa cells. With the aim of improving the affinity of the selected fragment, a standard error-prone PCR method was carried out. By using this strategy, it was possible to generate a new fragment (EP18) with increased affinity and ability to neutralize a broad diversity of IFN-alpha subtypes. Consequently, the scFv EP18 represents a potential therapeutic agent for those immune and inflammatory diseases which are associated with an increased IFN-alpha expression.
尽管细胞因子在维持正常生理功能中发挥着重要作用，但当它们过度产生时可能会引发病理状况。如此一来，人类干扰素α（hIFN-α）表达的增加与急性病毒感染、炎症性疾病以及多种自身免疫性疾病相关，在这些疾病中，细胞因子可能是引发或维持疾病的一个因素。目前，有几种单克隆抗体（mAbs）已上市用于多种适应症，还有更多处于临床试验阶段，其中IFN-α是基于抗体治疗的潜在靶点。使用重组hIFN-α2b作为免疫原制备了一组11种鼠源单克隆抗体，所有这些抗体均以1.7×10⁷ M⁻¹至1.4×10¹⁰ M⁻¹的亲和常数与细胞因子的天然形式结合。表位作图方案表明单克隆抗体识别该蛋白的四个空间上不同的区域。考虑到抗体的特性及其抑制IFN-α生物活性的能力，选择了四种单克隆抗体制备单链抗体片段（scFv）。其中一个片段（CA5E6）能够中和IFN-α家族的广泛亚型，包括重组细胞因子hIFN-α2a和hIFN-α2b以及活化白细胞和Namalwa细胞产生的异质性IFN-α。为了提高所选片段的亲和力，采用了标准易错PCR方法。通过使用这种策略，有可能产生一种具有更高亲和力和中和多种IFN-α亚型能力的新片段（EP18）。因此，scFv EP18对于那些与IFN-α表达增加相关的免疫和炎症性疾病而言是一种潜在的治疗剂。
