Novel Methods for B-cell Delivery of Tolerogenic Epitop*

描述（由申请人提供）：TolerGenics，Inc.，一家创业生物技术公司，成立于1997年，正在开发和商业化的新疗法，用于诱导和维持表位特异性免疫耐受使用B细胞输送策略。基于自身免疫球蛋白的B细胞抗原呈递具有高度致耐受性的假设，我们采用了一项专利技术（#5，817，308“免疫球蛋白的致耐受性融合蛋白和诱导和维持耐受性的方法”），其中我们设计了在鼠IgG 1重（H）链的N-末端含有符合读框的多个表位的逆转录病毒构建体。该技术已被证明通过相关表位的连续体内呈递来实现免疫耐受的长期维持。在三个实验性自身免疫模型中的数据是非常有希望的，因为已经实现了显著的临床疗效。也就是说，IG融合蛋白在B细胞中的表达可以预防和逆转葡萄膜炎和EAE中的自身免疫应答，并且显著延迟具有胰岛周围炎的NOD小鼠的糖尿病发作。然而，最近的人体临床试验引起了对使用逆转录病毒载体的安全性担忧。因此，我们认为，这些构建体的替代表达方法需要在进一步试验之前开发。在本申请中，我们希望使用两种用于基因和/或蛋白质的非插入表达的新方法：核转染[TM]和HIV达特融合蛋白来检验IG融合蛋白的瞬时表达可以是致耐受性的假设。核转染[TM]（Nf）是一种非病毒转染方法，用于将DNA直接有效递送至非分裂细胞以及分裂细胞的细胞核。我们发现Nf可用于在幼稚人T和B淋巴细胞中高效表达GFP构建体。达特融合蛋白被包括淋巴细胞在内的多种细胞迅速摄取，并可用于递送蛋白质以呈递给免疫系统。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肽（p947-967）-IG作为模型抗原。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的人T细胞对致糖尿病表位的应答差。将表达该肽的B细胞与自体外周T细胞一起孵育，以体外读出耐受性。p947-967-IG的达特融合蛋白将类似地在人B细胞中表达以用于体外耐受。在第二个目的中，将来自NOD小鼠的鼠B细胞与TAT-胰岛素-IG一起孵育，然后在体内转移以测试它们对糖尿病发展的致耐受性作用。随着Amaxa技术的发展，我们将在目标2中对Nf进行β测试，以与鼠B细胞一起用作NOD受体中的致耐受性APC。我们希望这些新的免疫耐受疗法最终将用于治疗自身免疫性糖尿病。

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.