Developing Immune Based Therapies

该项目的第一个主要成就是发表了rhIL-7在人体中的第二次试验结果（Sportes et al, J of Exp Med, 2008; 205;1701）。该试验纳入了广泛的生物学研究，为IL-7诱导的T细胞稳态的急剧变化提供了深入的基础。我们证明了IL-7导致大量T细胞经历细胞周期并抵抗程序性细胞死亡，导致血液和组织T细胞快速、显著扩张。这种扩张受到IL-7受体动态调控的严格调控，从而防止了IL-7治疗后淋巴细胞的危险增加。由于IL-7选择性地扩增最年轻或最幼稚的T细胞群，IL-7治疗的患者体内的T细胞与正常情况下年轻患者体内的T细胞相似。因此，IL-7对免疫系统具有抗衰老作用。在目前正在研究的细胞因子中，这种扩大大量原始细胞池的能力是独一无二的，预计对免疫能力非常重要，特别是在T细胞耗竭的情况下。我们几乎没有看到IL-7毒性的证据，这为该药物与定向免疫治疗联合的II期试验以及IL-7在HIV感染中的研究提供了坚实的基础。该项目在2009财年取得的第二个主要成就是开发了一种新方案，可支持过继性免疫治疗，而不会导致患者淋巴细胞耗竭。许多研究小组目前正在对患者进行淋巴细胞消耗，作为改善过继转移细胞在体内增殖和存活环境的一种手段。这种方法带来了与用于诱导淋巴细胞减少的方案相关的毒性，以及长期发病率或慢性免疫抑制。这项工作还表明，淋巴细胞减少在其支持过继免疫治疗的能力方面有很大的局限性，这与表位扩散的有限免疫库、淋巴细胞减少环境中T细胞功能的减弱以及免疫监视的减弱有关。这项工作表明，提供增加的IL-7和消耗调节性T细胞将为过继性免疫治疗提供同等或更好的环境。我们采用B16黑色素瘤模型，并过继性地将具有免疫优势抗原特异性的T细胞转移到该肿瘤上。我们观察到，与T细胞丰富的小鼠相比，淋巴细胞减少小鼠的过继免疫治疗仅提供适度的益处，但使用单克隆抗体特异性地消耗CD25+ T细胞可显著提高过继免疫治疗的有效性。与淋巴细胞减少的小鼠相比，这些小鼠在过继免疫治疗后的存活率显著提高。我们的结论是，淋巴细胞减少不应该被认为是过过性免疫治疗的必要条件，提供淋巴细胞减少生理改变的特定成分的靶向方法是可取的，从长远来看将更有效。这项工作于2009年7月被接受发表（Cui et al, Blood, 2009）。在2008财年，该项目的第三个主要成就是证明了我们临床合格的人工抗原提呈细胞主细胞库可以有效地扩增细胞溶解NK细胞，因此可以作为开发基于免疫的癌症疗法的关键试剂。我们之前已经证明这些细胞可以有效地扩增细胞溶解性CD8+ T细胞，这项工作将其扩展到NK细胞。用该试剂扩增的NK细胞可以有效地溶解多种组织学的肿瘤，包括基本上所有测试的儿童肉瘤（横纹肌肉瘤、骨肉瘤、尤因斯肉瘤等）以及急性淋巴细胞白血病细胞，包括原代细胞和肿瘤细胞系。我们目前正在完成一项IND和一项临床试验的提交，以研究aAPC激活的NK细胞在非清髓性造血干细胞移植中的使用。第四个主要成就是证明抗原呈递细胞上的干扰素γ信号可防止移植物抗宿主病的发展，并增强肿瘤疫苗根除这种疾病的能力。这是一个新的轴，以前从未涉及GVHD，该模型首次对同种异体环境在调节基于疫苗的反应中的相对影响进行了认真的评估。这项研究发表在《血液》杂志上（Capitini et al, Blood, 2009）。在过去的一年中，我们还与Christoph Raders博士实验室合作开展了一个项目，研究抗nkg2d单克隆抗体的生物学特性，该抗体可用于阻断或激活NK介导的作用（Kwong等人，J Mol Bio 2008）。利用我们在NKG2D生物学方面的专业知识，基于对NKG2D在CD8+ T细胞上表达尤因斯肉瘤细胞裂解活性的研究，我们能够证明Rader实验室开发的抗NKG2D单克隆特异性地与NKG2D结合，并且Fab片段可用于抑制NKG2D与其配体的结合。重要的是，当交联时，相同的分子也能有效地增强NKG2D介导的杀伤，这是一种潜在的临床应用。我们还发表了一篇论文，证明肿瘤凋亡小体和肿瘤裂解物在产生抗肿瘤免疫方面优于肽，这一结果直接影响了我们目前和未来为癌症患者开发基于免疫疗法的方法（Fry等人，cancer Imm and Immunoth, 2009）。最后，我们主持了一个国际会议，基本上所有参与开发儿童癌症免疫疗法的科学团体都参加了会议。这导致了几次合作和摘要手稿的出版（Capitini等人，J of Ped Hem/Onc, 2009）。

The first major accomplishment of this project was publication of the results of the second trial of rhIL-7 in humans (Sportes et al, J of Exp Med, 2008; 205;1701) . This trial incorporated extensive biologic studies that provided insight into the basis for the dramatic changes in T cell homeostasis induced by IL-7. We demonstrated that IL-7 causes a broad repertoire of T cells to undergo cell cycling and to resist programmed cell death, resulting in rapid, significant expansion of both blood and tissue T cells. The expansion was tightly regulated by dynamic regulation of the IL-7 receptor, thus preventing dangerous increases in lymphocytes with IL7 therapy. Because IL-7 selectively expands the youngest or most naive T cell populations, T cells in IL-7 treated patients resembled T cells normally present at young ages. Thus, IL-7 has an anti-aging effect on the immune system. This capacity to expand a broad pool of nave cells is unique among cytokines currently under study and is predicted to be very important for immune competence, especially in the context of T cell depletion. We saw little to no evidence of toxicity with IL-7, providing a solid basis for Phase II trials of this agent in combination with directed immunotherapy and for studies of IL-7 in HIV infection. A second major accomplishment in this project during FY2009 was the development of a new regimen that can support adoptive immunotherapy without inducing lymphodepletion in patients. Many groups are currently subjecting patients to lymphocyte depletion as a means for improving the milieu within which adoptively transferred cells may proliferate and survive in vivo. This approach brings with it toxicities associated with the regimens used to induce lymphopenia, and the long term morbidity or chronic immunosuppression. This work also demonstrated that lymphopenia has substantial limitations in terms of its ability to support adoptive immunotherapy that are related to a limited immune repertoire for epitope spreading, diminished functionality of T cells in the lymphopenic milieu as well as diminished immune surveillance. This work demonstrated that provision of increased IL-7 and depletion of regulatory T cells would provide an equivalent or superior milieu for adoptive immunotherapy. We utilized the B16 melanoma model and adoptively transferred T cells with specificity for the immunodominant antigen on this tumor. We observed that adoptive immunotherapy administered to lymphopenic mice provided only a modest benefit compared to T cell replete mice however specific depletion of CD25+ T cells using a monoclonal antibody substantially improved the effectiveness of adoptive immunotherapy. Such mice showed substantially improved survival following adoptive immunotherapy compared to lymphopenic mice. We conclude that lymphopenia should not be considered a requisite setting for adoptive immunotherapy and that targeted approaches that provide the specific components of the altered physiology of lymphopenia are preferable and will be more effective in the long run. This work was accepted for publication in July 2009 (Cui et al, Blood, 2009). A third major accomplishment of this project during FY2008 involved the demonstration that our clinically qualified master cell bank of artificial antigen presenting cells effectively expand cytolytic NK cells and can therefore serve as a critical reagent for developing immune based therapies for cancer. We had previously demonstrated that these cells can effectively expand cytolytic CD8+ T cells and this work extends that to NK cells. NK cells expanded with this reagent effectiveless lyse tumors of multiple histologies including essentially all pediatric sarcomas tested (rhabdomyosarcoma, osteosarcoma, Ewings sarcoma, etc.) as well as acute lymphoblastic leukemia cells, both primary cells and tumor cell lines. We are currently completing submission of an IND and a clinical trial to investigate the use of aAPC activated NK cells in the context of non-myeloablative hematopoietic stem cell transplantation. A fourth major accomplishment was the demonstration that interferon gamma signaling on antigen presenting cells prevents the development of graft-versus-host disease and enhance the capacity for tumor vaccines to eradicate disease in this setting. This is a novel axis which has not previously been implicated in GVHD and this model provides the first serious assessment of the relative impact of the allogeneic milieu in modulating vaccine based responses. This work was published in Blood (Capitini et al, Blood, 2009). During the past year we also collaborated on a project with Dr. Christoph Raders laboratory to investigate the biology of an anti-NKG2D monoclonal antibody, which can be used to either block or activate NK mediated effects (Kwong et al, J Mol Bio 2008). Using our expertise in NKG2D biology based upon studies of NKG2D expression on CD8+ T cells in lytic activity of Ewings sarcoma cells, we were able to demonstrate that the anti-NKG2D monoclonal developed by the Rader lab was bound specifically to NKG2D and that the Fab fragment could be used to inhibit NKG2D binding to its ligands. Importantly, when crosslinked, this same molecule was also effective at enhancing NKG2D mediated killing, a feature that is potentially exploitable for clinical applications. We also published a manuscript demonstrating that tumor apoptotic bodies and tumor lysates are superior to peptides in generating antitumor immunity, a result which directly impacts our current and future approaches to developing immune based therapies for patients with cancer (Fry et al, Cancer Imm and Immunoth, 2009). Finally, we hosted an international meeting of essentially all of the scientific groups involved in developing immune based therapies for childhood cancer. This resulted in several collaborations and publication of a summary manuscript (Capitini et al, J of Ped Hem/Onc, 2009).