Novel Methods for B-cell Delivery of Tolerogenic Epitop*

简介(申请人提供)：成立于1997年的创业型生物技术公司TOLERGENICS，Inc.正在开发和商业化使用B细胞递送策略诱导和维持表位特异性免疫耐受的新疗法。基于自身免疫球蛋白的B细胞抗原呈递具有高度耐受性的假设，我们采用了一项专利技术(#5,817,308“免疫球蛋白耐受性融合蛋白和诱导和维持耐受性的方法”)，其中我们设计了在小鼠IgG1重(H)链的N端包含多个表位的逆转录病毒构建物。这项技术已被证明通过在体内连续呈现相关表位来实现长期维持免疫耐受。三个实验性自身免疫模型的数据非常有希望，因为已经取得了显著的临床疗效。也就是说，在B细胞中表达Ig融合蛋白既可以预防和逆转葡萄膜炎和EAE的自身免疫反应，又可以显著延迟NOD小鼠合并胰岛素周炎症的糖尿病的发生。然而，最近的人类临床试验已经引起了对使用逆转录病毒载体的安全性的担忧。因此，我们认为，在进一步试验之前，需要开发这些结构的替代表达方法。在这个应用中，我们希望使用两种新的非插入表达基因和/或蛋白质的方法来检验Ig融合蛋白的瞬时表达可以耐受的假设：核转移[TM]和HIV Tat融合蛋白。核转录[TM](NF)是一种非病毒转染法，可有效地将DNA直接输送到未分裂和分裂的细胞的核中。我们发现，核因子可以在人T和B淋巴细胞中高效表达GFP结构。TAT融合蛋白被包括淋巴细胞在内的各种细胞迅速吸收，并可用于运送蛋白质呈递给免疫系统。在我们的第一个目标中，我们将在人B细胞中表达破伤风毒素多肽(p947-967)-Ig作为模式抗原。TT被选为模式抗原是因为来自I型糖尿病患者的未克隆的人类T细胞对糖尿病表位的反应很差。表达这种多肽的B细胞将在体外与自体外周T细胞孵育，以进行耐受性检测。P947-967-Ig的TAT融合蛋白在体外人B细胞中也有类似的表达。在第二个目标中，来自NOD小鼠的小鼠B细胞将与TAT-INS-Ig孵育，然后在体内转移，以测试它们对糖尿病发生的耐受作用。随着Amaxa技术的发展，我们将在AIM 2中对NF进行Beta测试，用于将小鼠B细胞用作NOD接受者的耐受性APC。我们希望这些新的免疫耐受疗法最终将用于自身免疫性糖尿病的治疗。

DESCRIPTION (provided by applicant): TolerGenics, Inc., an entrepreneurial biotechnology company established in 1997, is developing and commercializing novel therapies for inducing and maintaining epitope-specific immune tolerance using a B-cell delivery strategy. Based on the hypothesis that B-cell antigen presentation of self immunoglobulins would be highly tolerogenic, we employ a patented technology (#5,817,308 "Tolerogenic fusion proteins of immunoglobulins and methods for inducing and maintaining tolerance") in which we engineer retroviral constructs containing multiple epitopes in frame at the N-terminus of a murine IgG1 heavy (H) chain. This technology has been shown to achieve long-term maintenance of immune tolerance through continuous in vivo presentation of relevant epitopes. Data in three experimental autoimmune models are highly promising in that significant clinical efficacy has been achieved. That is, expression of Ig fusion proteins in B cells can both prevent and reverse autoimmune responsiveness in uveitis and EAE, and significantly delay the onset of diabetes in NOD mice with peri-insulitis. However, safety concerns about the use of retroviral vectors have arisen from recent human clinical trials. Thus, we believe that alternative expression methods for these constructs need to be developed before further trials can ensue. In this application, we wish to test the hypothesis that transient expression of Ig fusion proteins can be tolerogenic, using two new methods for non-insertional expression of genes and/or proteins: Nucleofection[TM] and HIV TAT fusion proteins. Nucleofection[TM] (Nf) is a non-viral transfection methodology for efficient delivery of DNA directly to the nucleus of non-dividing, as well as dividing, cells. We have found that Nf can be used to express GFP constructs at high efficiency in naive human T and B lymphocytes. TAT fusion proteins are rapidly taken up by a variety of cells, including lymphocytes, and can be used to deliver proteins for presentation to the immune system. In our first aim, we will express a tetanus toxin peptide (p947-967)-Ig as a model antigen in human B cells. TT was chosen as a model antigen because the response of uncloned human T-cells from Type I diabetes patients to diabetogenic epitopes is poor. B cells expressing this peptide will be incubated with autologous peripheral T cells in an in vitro readout for tolerance. TAT fusion proteins of p947-967-Ig will be similarly expressed in human B cells for tolerance in vitro. In the second aim, murine B cells from NOD mice will be incubated with TAT-insulin-Ig and then transferred in vivo to test their tolerogenic effects on development of diabetes. As Amaxa technology develops, we will beta test Nf in aim 2 for use with murine B cells as tolerogenic APC in NOD recipients. We expect these novel immune tolerance therapies will ultimately be utilized in the treatment of autoimmune diabetes.