Engineering Bone Marrow Cells for Immunotherapy

描述(申请人提供)：这项建议的主要目标是使用一种新颖而实用的方法来改造骨髓细胞，使其在其表面展示一种具有强大的凋亡活性的修饰形式的FasL蛋白作为免疫调节剂，并测试工程细胞移植到异基因受者身上的能力，而不会出现移植物抗宿主(GVH)或宿主抗移植物(HVG)反应的并发症。 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。骨髓移植也可以作为一种基于细胞的免疫调节方法，诱导对外来抗原和自身抗原的耐受，以预防和/或治疗外来移植排斥反应和自身免疫性疾病。然而，在临床上常规应用BMT作为一种治疗干预措施，会因两种不同类型的免疫反应而变得复杂：HVG和GVH。移植物抗宿主病(GVHD)是导致异基因骨髓移植排斥反应的主要原因，而移植物抗宿主病(GVHD)是目前缺乏有效治疗手段的BMT的一种危及生命的并发症。针对同种异体抗原的T细胞是GVH和HVG反应的罪魁祸首。从供者骨髓接种或移植受者中清除T细胞可以减少这两种反应，植入不足和免疫功能丧失是观察到的并发症。因此，仅针对致病T细胞的特异性和有效清除的策略可能对BMT在临床治疗各种疾病中的常规应用具有重要意义。我们实验室最近开发了一种名为Protex(Tm)的新技术，它允许在不影响细胞或蛋白质功能的情况下，在任何细胞上快速(~2小时)和高效(100%目标细胞)展示感兴趣的外源蛋白质。在这一应用中，Protex(Tm)将被用来展示一种在骨髓细胞上具有强大的凋亡活性的改良的FasL蛋白，然后工程细胞将被用于移植到受到同种异体致死性照射的小鼠身上，以预防GVHD，并移植到最低限度的条件下的宿主中，以建立混合嵌合体和供体特异性耐受性。FasL作为一种免疫调节剂的使用是基于该分子在免疫动态平衡和建立自身耐受中所起的关键作用。我们推测，在表达FasL的BM接种中，成熟的T细胞会对宿主抗原产生反应，上调死亡受体Fas，并在同一细胞或不同细胞上与FasL结合后发生凋亡，从而预防GVHD。类似地，受体T细胞与显示FasL的供体BM细胞发生反应，预计会发生凋亡，导致植入和建立混合嵌合体。雷帕霉素是一种凋亡诱导剂，双硫丹是一种烷基化药物，它优先消耗早期的造血干细胞，它们将被用作辅助疗法，用于i)加强FasL介导的细胞凋亡，ii)为供体干细胞植入创造“空间”，iii)建立一个临床适用的方案。在致死性照射或最低限度的条件下特异性消除致病T细胞有望导致异基因骨髓移植，而不会出现移植物抗宿主病和免疫功能低下的并发症，具有很强的治疗意义。

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.