Novel Methods for B-cell Delivery of Tolerogenic Epitop*

描述（由申请人提供）：TolerGenics, Inc.是一家成立于1997年的创业型生物技术公司，正在开发和商业化利用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。基于自体免疫球蛋白的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“免疫球蛋白的耐受性融合蛋白和诱导和维持耐受性的方法”），在该技术中，我们设计了在小鼠IgG1重(H)链的n端框架中含有多个表位的逆转录病毒构建物。该技术已被证明通过相关表位在体内的持续呈现来实现免疫耐受的长期维持。三种实验性自身免疫模型的数据非常有希望取得显著的临床疗效。也就是说，在B细胞中表达Ig融合蛋白可以预防和逆转葡萄膜炎和EAE的自身免疫反应，并显著延缓NOD伴有胰岛素周围炎小鼠糖尿病的发生。然而，在最近的人体临床试验中出现了使用逆转录病毒载体的安全性问题。因此，我们认为，在进一步的试验之前，需要开发这些结构的替代表达方法。在这项应用中，我们希望通过两种新的非插入表达基因和/或蛋白质的方法来验证Ig融合蛋白的瞬时表达可以耐受的假设：核感染[TM]和HIV TAT融合蛋白。核转染[TM] （Nf）是一种非病毒转染方法，用于将DNA直接有效地传递到非分裂细胞和分裂细胞的细胞核中。我们发现Nf可以在人T淋巴细胞和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多种细胞迅速吸收，并可用于向免疫系统递送蛋白质。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肽(p947-967)-Ig作为模型抗原。之所以选择TT作为模型抗原，是因为来自I型糖尿病患者的未克隆人类t细胞对致糖尿病表位的反应较差。表达这种肽的B细胞将与自体外周T细胞孵育，在体外读数中观察耐受性。p947-967-Ig的TAT融合蛋白将在人B细胞中类似地表达以获得体外耐受性。在第二个目标中，NOD小鼠的小鼠B细胞将与tat -胰岛素- ig一起孵育，然后转移到体内，以测试它们对糖尿病发展的耐受性影响。随着Amaxa技术的发展，我们将在aim 2中对Nf进行β测试，以用于NOD受体的小鼠B细胞作为耐受性APC。我们期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.