Mufarrege Eduardo F, Giorgetti Sofía, Etcheverrigaray Marina, Terry Frances, Martin William, De Groot Anne S
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Laboratorio de Cultivos Celulares, FBCB, UNL, Santa Fe, Argentina.
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Laboratorio de Cultivos Celulares, FBCB, UNL, Santa Fe, Argentina.
Clin Immunol. 2017 Mar;176:31-41. doi: 10.1016/j.clim.2017.01.003. Epub 2017 Jan 10.
Interferon α (IFN-α) exerts potent antiviral, immunomodulatory, and antiproliferative activity and have proven clinical utility in chronic hepatitis B and C virus infections. However, repeated IFN-α administration induces neutralizing antibodies (NAb) against the therapeutic in a significant number of patients. Associations between IFN-α immunogenicity and loss of efficacy have been described. So as to improve the in vivo biological efficacy of IFN-α, a long lasting hyperglycosylated protein (4N-IFN) derived from IFN-α2b wild type (WT-IFN) was developed. However, in silico analysis performed using established in silico methods revealed that 4N-IFN had more T cell epitopes than WT-IFN. In order to develop a safer and more efficient IFN therapy, we applied the DeFT (De-immunization of Functional Therapeutics) approach to producing functional, de-immunized versions of 4N-IFN. Using the OptiMatrix in silico tool in ISPRI, the 4N-IFN sequence was modified to reduce HLA binding potential of specific T cell epitopes. Following verification of predictions by HLA binding assays, eight modifications were selected and integrated in three variants: 4N-IFN(VAR1), (VAR2) and (VAR3). Two of the three variants (VAR1 and VAR3) retained anti-viral function and demonstrated reduced T-cell immunogenicity in terms of T-cell proliferation and Th1 and Th2 cytokine levels, when compared to controls (commercial NG-IFN (non-glycosylated), PEG-IFN, WT-IFN and 4N-IFN). It was previously demonstrated that N-glycosylation improved IFN-α pharmacokinetic properties. Here, we further reduce immunogenicity as measured in vitro using T cell assays and cytokine profiling by modifying the T cell epitope content of a protein (de-immunizing). Taking into consideration the present results and previously reported immunogenicity data for commercial IFN-α2b variants, 4N-IFN(VAR1) and 4N-IFN-4N(VAR3) appear to be promising candidates for improved IFN-α therapy of HCV and HBV.
干扰素α（IFN-α）具有强大的抗病毒、免疫调节和抗增殖活性，在慢性乙型和丙型肝炎病毒感染中已证明具有临床应用价值。然而，在大量患者中，重复给予IFN-α会诱导产生针对该治疗药物的中和抗体（NAb）。IFN-α免疫原性与疗效丧失之间的关联已有报道。为了提高IFN-α的体内生物学疗效，开发了一种源自IFN-α2b野生型（WT-IFN）的长效高糖基化蛋白（4N-IFN）。然而，使用既定的计算机方法进行的计算机分析显示，4N-IFN比WT-IFN具有更多的T细胞表位。为了开发更安全、更有效的IFN治疗方法，我们应用了功能性治疗药物去免疫（DeFT）方法来生产功能性、去免疫的4N-IFN版本。使用ISPRI中的OptiMatrix计算机工具，对4N-IFN序列进行了修饰，以降低特定T细胞表位的HLA结合潜力。通过HLA结合试验验证预测结果后，选择了8种修饰并整合到三个变体中：4N-IFN（VAR1）、（VAR2）和（VAR3）。与对照（商业非糖基化NG-IFN、聚乙二醇化IFN、WT-IFN和4N-IFN）相比，三个变体中的两个（VAR1和VAR3）保留了抗病毒功能，并在T细胞增殖以及Th1和Th2细胞因子水平方面表现出降低的T细胞免疫原性。先前已证明N-糖基化改善了IFN-α的药代动力学特性。在此，我们通过修饰蛋白质的T细胞表位含量（去免疫），进一步降低了使用T细胞检测和细胞因子分析在体外测量的免疫原性。考虑到目前的结果以及先前报道的商业IFN-α2b变体的免疫原性数据，4N-IFN（VAR1）和4N-IFN-4N（VAR3）似乎是用于改善HCV和HBV的IFN-α治疗的有前景的候选药物。
