Engineering Bone Marrow Cells for Immunotherapy

描述（由申请人提供）：本提案的主要目的是使用一种新的实用方法来工程化骨髓细胞，以在其表面上展示具有强效凋亡活性的FasL蛋白的修饰形式作为免疫调节剂，并测试工程化细胞在同种异体受体中植入而没有移植物抗宿主（GVH）或宿主抗移植物（HVG）反应并发症的能力。 异基因骨髓移植（BMT）具有治愈一系列遗传性和获得性血液病和恶性肿瘤的潜力。BMT还可以用作基于细胞的免疫调节方法，以诱导对外来抗原和自身抗原的耐受性，用于预防和/或治疗外来移植物排斥和自身免疫性疾病。然而，BMT作为临床治疗干预的常规应用由于两种不同类型的免疫反应而变得复杂; HVG和GVH。HVG反应是同种异体BM移植物排斥的原因，而GVH反应是移植物抗宿主病（GVHD）的主要原因，GVHD是BMT的危及生命的并发症，没有有效的治疗方式。对同种异体抗原特异性的T细胞是GVH和HVG反应的罪魁祸首。从供体BM接种物或移植物受体中消除T细胞可以减少两种类型的反应，缺乏植入和免疫功能不全是观察到的并发症。因此，靶向特异性和有效消除仅致病性T细胞的策略可能对BMT在临床上用于治疗各种疾病的常规应用具有重要意义。我们的实验室最近开发了一种称为ProtEx（tm）的新技术，该技术允许在任何细胞上快速（~ 2小时）和有效（100%的靶细胞）展示感兴趣的外源蛋白，而不损害细胞或蛋白的功能。在本申请中，ProtEx（tm）将用于展示在BM细胞上具有有效凋亡活性的FasL蛋白的修饰形式，然后将工程化的细胞用于移植到同种异体致死辐射小鼠中以防止GVHD，并移植到最低条件宿主中以建立混合嵌合体和供体特异性耐受。FasL作为免疫调节剂的用途是基于该分子在免疫稳态和建立自身耐受中的关键作用。我们假设，成熟的T细胞在骨髓接种展示FasL将响应于宿主抗原，上调死亡受体Fas，并进行细胞凋亡后的FasL与Fas的参与相同或不同的细胞，导致GVHD的预防。类似地，预期与展示FasL的供体BM细胞反应的受体T细胞经历细胞凋亡，导致植入和混合嵌合体的建立。雷帕霉素（一种凋亡诱导剂）和双硫凡（一种优先消耗早期造血干细胞的烷化剂）将用作辅助疗法，以i）加强FasL介导的凋亡，ii）为供体干细胞植入创造“空间”，iii）建立临床适用的方案。在致死性照射或最低条件宿主中特异性消除致病性T细胞预期导致同种异体BM植入，而没有GVHD和免疫功能不全的并发症，具有强烈的治疗意义。

DESCRIPTION (provided by applicant): The main objective of this proposal is to use a novel and practical approach to engineer bone marrow cells to display on their surface a modified form of FasL protein with potent apoptotic activity as an immunomodulatory agent and test the capacity of the engineered cells to engraft in allogeneic recipients without complications of graft-versus-host (GVH) or host-versus-graft (HVG) reactions. Allogeneic bone marrow transplantation (BMT) has the potential to cure a series of inherited and acquired hematological disorders and malignancies. BMT can also be used as a cell-based immunomodulatory approach to induce tolerance to foreign and auto-antigens for the prevention and/or treatment of foreign graft rejection and autoimmune disorders. The routine application of BMT as a therapeutic intervention in the clinic, however, is complicated by two different types of immunological reactions; HVG and GVH. HVG reaction is responsible for rejection of allogeneic BM transplants whereas GVH reaction is the major cause of graft versus- host disease (GVHD), which is a life-threatening complication of BMT without effective treatment modalities. T cells specific for alloantigens are the primary culprit in GVH and HVG reactions. Elimination of T cells from the donor BM inoculum or the graft recipient can curtail both types of reactions, lack of engraftment and immunoincompetence are the observed complications. Thus, strategies targeting specific and effective elimination of only the pathogenic T cells may have important implications for routine application of BMT to the clinic for the treatment of a variety of diseases. A novel technique, designated as ProtEx(tm), has recently been developed in our laboratory that allows for rapid (~ 2 hr) and efficient (100% of the targetted cells) display of exogenous proteins of interest on any cell without compromising the function of the cell or the proteins. In this application, ProtEx(tm) will be used to display a modified form of FasL protein with potent apoptotic activity on BM cells and the engineered cells will then be used for transplantation into allogeneic lethally irradiated mice to prevent GVHD and into minimally conditioned hosts to establish mixed chimerism and donor specific tolerance. The use of FasL as an immunomodulatory agent is based on the critical role this molecule plays in immune homeostasis and establishment of self tolerance. We hypothesize that mature T cells in the BM inoculum displaying FasL will respond to the host antigens, upregulate the death receptor Fas, and undergo apoptosis following the engagement of FasL with Fas on the same or a different cell, resulting in the prevention of GVHD. Similarly, recipient T cells reacting to the donor BM cells displaying FasL are expected to undergo apoptosis, leading to engraftment and establishment of mixed chimerism. Rapamycin, an apoptosis-inducing agent, and bisulfan, an alkylating agent that preferentially depletes early hematopoietic stem cells, will be used as adjuvant therapies to i) accentuate FasL-mediated apoptosis, ii) create "space" for donor stem cell engraftment, and iii) establish a clinically applicable protocol. Specific elimination of pathogenic T cells in lethally irradiated or minimally conditioned hosts is expected to results in allogeneic BM engraftment without complications of GVHD and immunoincompetence with strong therapeutic implications.