Biology and Therapy of T Cell Depletion

基于我们在T细胞稳态生物学方面的专业知识，我们寻求开发能够在淋巴细胞减少期增强抗肿瘤免疫的疗法，淋巴细胞减少期几乎发生在所有接受化疗的儿童癌症患者中。通过利用淋巴细胞减少引起的T细胞稳态改变所导致的肿瘤负荷低和诱导对肿瘤抗原反应的能力增强的结合，我们寻求使用肿瘤定向免疫疗法来巩固儿童癌症患者的缓解。研究对象包括小鼠，用于模拟淋巴细胞减少期间发生的免疫生理变化，以及由于癌症化疗或HIV感染而导致淋巴细胞减少的儿童，年轻人和成年人，在NIH临床中心接受IRB批准的临床试验治疗。该项目在2006年之前的最新主要成就可以通过两份报告来总结：1)Melchionda等人在2005年的《J . clininvest》中首次证明了il - 7治疗可以有效地增强免疫应答，从而为il - 7应被视为肿瘤疫苗的临床使用提供了坚实的证据。#2) Zhang等人在《自然医学》2005年发表的文章中证明，T细胞的一个特异性抑制性亚群（所谓的CD4+CD25+ Tregs）对淋巴细胞减少的反应增加，在这种情况下给药il - 2进一步扩大了T细胞的抑制性亚群。这一观察结果改变了范式，因为它证明了先前被认为是免疫激活剂的il - 2具有强大的免疫抑制作用。作为这项工作的结果，我们集中了我们的努力，专门操纵淋巴细胞减少宿主中的Treg群体，作为在这种情况下增强免疫治疗有效性的一种方式。在过去的一年中，两项主要成就是完成了rhIL7的前两项临床试验。首次完成试验的结果作为与NCI外科分会合作研究的一部分发表（Rosenberg et al., J of Immunoth, 2006）。它提供了IL-7显著调节人类T细胞稳态的原理证明，并证明与il - 2增加抑制性T细胞不同，IL-7增加CD4+ T细胞数量而不优先扩大抑制性亚群。然而，由于这项研究没有对IL-7发挥其作用的机制提供重要的见解，我们还在难治性癌症患者中进行了第二项I期试验。这项研究纳入了广泛的生物学研究，为IL-7诱导的T细胞稳态的巨大变化提供了显著的基础。最值得注意的是，我们观察到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IL7受体动态调控的严格调控，从而防止了IL7治疗后淋巴细胞的危险增加。由于IL7选择性地扩增了最年轻或最幼稚的T细胞群，因此IL7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，IL7对免疫系统具有抗衰老作用。这种扩大大量细胞池的能力预计对免疫能力非常重要，特别是在T细胞耗竭的情况下。最重要的是，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期试验提供了坚实的基础。这项工作已提交出版。第三个主要成就是完成了一系列研究，通过对不同临床人群进行详细的免疫学评估，证明了在各种临床环境中儿童免疫重建的成功和局限性，因此代表了合作项目。他们建立在我们小组在20世纪90年代发表的先前工作的基础上，该工作表明，接受癌症化疗治疗的儿童和年轻人存在严重的淋巴细胞减少，最近的一项合作研究（Hakim, J clininvest 2005）表明，接受自体干细胞移植治疗的成年女性乳腺癌患者CD4+ T细胞群的不完全恢复时间较长。在第一项研究中，我们研究了完成儿童肉瘤剂量强化治疗后4-30年的青少年和年轻人。[J] .中华血液学杂志，2006;35(2):1 - 7。这与老年人的研究形成了直接对比，在老年人的长期随访中，在淋巴细胞消耗时，40岁以上的患者中没有显示出完全免疫重建的证据。总之，这项工作提供了进一步的证据，证明年龄是决定人类免疫重建程度的核心因素，即使长期随访也是如此。我们还发表了一项针对艾滋病毒感染接受HAART治疗的儿童的合作研究，该研究也显示出非常好的恢复免疫反应的能力（Viral immuno1 . 2007 Spring;20(1):131-41）。最后，我们研究了接受非清髓性骨髓移植治疗的儿童和年轻人的免疫重建，并观察到明显早期的胸腺依赖性免疫重建，比清髓性骨髓移植和接受类似研究的成人患者观察到的更快和更完整。这项研究已在2006年12月的美国血液学学会会议上发表，目前正准备发表。在旨在将收集到的有关免疫重建的原则应用于抗肿瘤免疫治疗的研究中，我们首次发表了免疫重建可以预防骨肉瘤转移性复发的证据。这是用小鼠模型进行的，其中免疫系统对大体积原发肿瘤的不同影响可以与对微观疾病的影响区分开来。值得注意的是，虽然完全恢复的免疫系统不会影响原发肿瘤的生长，但快速的免疫重建显著减少了转移性骨肉瘤的发展（Merchant, 2006, Cancer Immunol Immunoth）。这一观察结果为我们实验室正在进行的多种新肿瘤模型的开发提供了基础，这些模型更准确地反映了自发转移的过程，并使我们能够根据其对转移性疾病的影响来评估和优化免疫疗法，这与临床环境最为相关。最后，在过去的一年里，我们完成了开发一种新的儿童肉瘤免疫治疗临床试验的艰巨过程。这需要NCI多个委员会的广泛审查，以及FDA机构新药申请的批准。该方案已经招募了第一位患者，并将直接应用从我们的动物研究中收集到的原则，以确定联合免疫重建、抑制T细胞的消耗和树突状细胞疫苗的施用是否会改善转移性和复发性尤因肉瘤家族肿瘤、横纹肌肉瘤和神经母细胞瘤患者的预后

Building upon our expertise in the biology of T cell homeostasis, we seek to develop therapies that will enhance antitumor immunity during the phase of lymphopenia, which occurs in nearly all patients who receive chemotherapy for childhood cancer. By exploiting the combination of low tumor burdens and the heightened capacity to induce responses to tumor antigens as a result of the changes in T cell homeostasis induced by lymphopenia, we seek to use tumor directed immunotherapies to consolidate remission in patients with pediatric cancer. Research subjects include mice, used to model the changes in immune physiology which occur during lymphopenia, and children, young adults and adults with lymphopenia as a result of cancer chemotherapy or HIV infection, treated on IRB approved clinical trials in the NIH Clinical Center. Recent major accomplishments of this project prior to 2006 can be summarized by two reports: #1) In Melchionda et al, J Clin Invest 2005, we demonstrated for the first time that IL7 therapy can potently augment responses to immunization, thus providing solid evidence that IL7 should be considered as agent for clinical use in the context of tumor vaccines. #2) In Zhang et al, Nature Medicine, 2005, we demonstrated that a specific suppressive subset of T cells (so-called CD4+CD25+ Tregs) increase in response to lymphopenia and administration of IL2 in this setting further expanded the suppressive subset of T cells. This observation was paradigm changing since it demonstrated that IL2, an agent previously assumed to be immune activating, had potent immunosuppressive effects. As a result of this work, we have focused our efforts of specifically manipulating Treg populations in lymphopenic hosts as a way of augmenting the effectiveness of immunotherapy in this setting. During the past year, two major accomplishments were the completion of the first two clinical trials of rhIL7. Results from the first completed trial were published as part of a collaborative study with the NCI Surgery Branch (Rosenberg et al., J of Immunoth, 2006). It provided proof-of-principle that IL-7 dramatically modulates T cell homeostasis in humans and demonstrated that, unlike IL2, which augments suppressive T cells, IL7 increases CD4+ T cell numbers without preferentially expanding the suppressive subset. However, because this study did not provide significant insight into the mechanisms by IL-7 exerts its effects, we also undertook a second Phase I trial in patients with refractory cancer. This study incorporated extensive biologic studies that provided remarkable insight into the basis for the dramatic changes in T cell homeostasis induced by IL-7. Most notably, we observed 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 is tightly regulated by dynamic regulation of the IL7 receptor, thus preventing dangerous increases in lymphocyte with IL7 therapy. Because IL7 selectively expands the youngest or most nave T cell populations, T cells in IL7 treated patients resembled T cells normally present at young ages. Thus, IL7 has an anti-aging effect on the immune system. This capacity to expand a broad pool of nave cells is predicted to be very important for immune competence, especially in the context of T cell depletion. Most importantly, 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. This work has been submitted for publication. A third major accomplishment was completion of a series of studies demonstrating the successes and limitation of immune reconstitution in children in a variety of clinical settings by conducting detailed immunologic evaluation of varied clinical populations, and therefore represented collaborative projects. They build on previous work by our group published in the 1990s demonstrating profound lymphopenia in children and young adults treated with chemotherapy for cancer and a recent collaborative study (Hakim, J Clin Invest 2005) demonstrating that adult women treated for breast cancer with autologous stem cell transplantation show prolonged incomplete recovery of CD4+ T cell populations. In the first study, we studied adolescents and young adults 4-30 years after completion of dose intensive therapy for pediatric sarcomas. Remarkably, all successfully restored lymphocyte homeostasis (Br J Haematol. 2006 Oct;135(2):270-1). This is in direct contrast to studies in older adults, where prolonged follow-up did not show evidence of full immune reconstitution in substantial percentages of patients over age 40 at the time of lymphocyte depletion. Together, this work provides further evidence that age is the central factor determining the extent of immune reconstitution that occurs in human beings, even with prolonged follow-up. We also published a collaborative study in children undergoing HAART for HIV infection, which also demonstrated remarkably good capacity to restore immune responses: (Viral Immunol. 2007 Spring;20(1):131-41). Finally, we studied immune reconstitution in children and young adults treated with a non-myeloablative bone marrow transplantation and observed remarkably early thymic-dependent immune reconstitution, which was more rapid and complete that observed following myeloablative transplants and following adult patients treated on a similar study. This study, which has been presented at the American Society of Hematology meeting in December 2006, is currently being prepared for publication. In studies aimed at applying the principles gleaned regarding immune reconstitution to antitumor imunotherapy, we published the first evidence that immune reconstitution can prevent metastatic recurrence of osteosarcoma. This was performed using a mouse model wherine differential effects of the immune system on bulky primary tumors could be distinguished from effects on microscopic disease. Remarkably, while a fully recovered immune system did not impact growth of primary tumors, rapid immune reconstitution dramatically reduced the development of metastatic osteosarcoma (Merchant, 2006, Cancer Immunol Immunoth). This observation serves as the basis for ongoing development of multiple new tumor models in our laboratory which more accurately reflect the process of spontaneous metastases and will allow us to evaluate and optimize immunotherapies based upon their effects on metastatic disease, which is most highly relevant to the clinical setting. Finally, during the past year we completed the arduous process of developing a new clinical trial of immunotherapy for pediatric sarcomas. This required extensive reviews multiple committees at the NCI as well as the approval of an Institutional New Drug Application at the FDA. This protocol has already enrolled its first patient and will directly apply the principles gleaned from our animal studies to determine whether combining immune reconstitution, depletion of suppressor T cells and the administration of dendritic cell vaccines will improve outcomes for patients with metastatic and recurrent Ewings sarcoma family of tumors, rhabdomyosarcoma and neuroblastoma