Immunobiology and Immunotherapy of Pediatric Cancer

免疫疗法在治疗癌症方面取得了越来越大的成功，新兴的临床研究表明，这种新方法将为治疗儿童癌症提供第四大支柱。一些新的类别和类型的免疫疗法正在出现，这为将免疫疗法应用于越来越多的靶点和癌症提供了可能性。该项目利用免疫治疗领域的技术和见解的爆炸式发展，为儿童癌症带来新的免疫治疗药物。我们试图回答关于儿童和成人癌症之间的异同的基本问题，因为它涉及到免疫基础疗法，并扩展我们对限制实体肿瘤与液体肿瘤相比免疫基础疗法有效性的特定因素的理解。2014财年的一项主要成就是创造了一种全新的靶向CD22抗原的嵌合抗原受体，该受体在B细胞白血病（最常见的儿童癌症）上表达。先前用于生成靶向其他抗原的嵌合抗原受体（例如用于白血病的CD19）的最先进技术被用于生成一系列靶向CD22的新受体。CD22靶向嵌合抗原受体显示出与CD19靶向受体相似的效力，因此预计在临床试验中具有活性。这项工作于2013年发表在《Blood》杂志上（Haso等人），并申请了专利，新的受体已获得一家商业实体的许可，并将于2014年在儿童和年轻人中进行早期试验。2014财年的第二个主要成就是发表了一份报告，该报告将骨髓源性抑制细胞定义为小鼠横纹肌肉瘤在使用免疫检查点抑制剂anti-PD1治疗后免疫逃逸的基础。抗pd1是一类新的免疫治疗药物的一部分，它绕过了癌症使用的免疫抑制机制，并允许自然的、内源性的抗肿瘤免疫反应出现。据报道，在恶性黑色素瘤、肺癌和肾细胞癌中，有效率约为10-30%。这项工作将抗pd1用于儿童实体肿瘤模型，即横纹肌肉瘤。简而言之，在肿瘤接种前用抗pd1治疗小鼠完全阻止了癌症的发展，但在肿瘤接种后延迟抗pd1治疗即使7天也收效甚微。我们发现横纹肌肉瘤诱导骨髓来源的抑制细胞通过CXCL1/CXCR1轴向肿瘤运输。因此，我们假设阻断骨髓源性抑制细胞向横纹肌肉瘤的运输可以增强抗pd1治疗的疗效。为了做到这一点，我们在造血细胞上制造了缺乏CXCR2的嵌合体，我们也用抗CXCR2阻断抗体治疗动物。在这两种情况下，我们观察到抗pd1治疗后横纹肌肉瘤的消退。本研究确定了MDSC是儿童实体肿瘤中免疫逃逸的重要介质，并确定了成人肿瘤诱导的细胞与儿童肿瘤诱导的细胞之间的区别。我们还试图确定CXCL/CXCR轴是否可能在人类儿童肉瘤中发挥作用。我们首先证明了人类肉瘤细胞系产生几种CXCR配体，并且进一步发现，接受免疫疗法试验的患者血清中CXCL1和CXCL8的水平升高。此外，在转移性肉瘤患者中，CXCL8水平升高与预后不良相关。因此，我们认为CXCL/CXCR趋化因子轴代表了开发可用于增强儿童癌症免疫治疗疗效的疗法的潜在基础。这项工作发表在2014年《科学转化医学》的封面文章中，并在《自然评论癌症》中得到强调。inFY13的第三个成就是即将完成的一项研究，该研究表明，T细胞通过基因工程识别胸腺中表达的自身抗原，通过T细胞受体本身作为致癌基因的过程，发展为T细胞白血病。我们开发了一种T细胞转基因小鼠，其中绝大多数T细胞对“存活素”具有特异性，这是一种抗凋亡分子，被认为是一种通用的肿瘤抗原。我们假设，由于大多数肿瘤表达高水平的survivin， survivin TCR转基因小鼠将对肿瘤生长免疫。不幸的是，值得注意的是，这些小鼠并没有抵抗植入肿瘤的生长，而是在基本上所有小鼠中都发生了T细胞淋巴母细胞白血病。这不是由于插入突变。相反，胸腺中表达的survivin抗原的自身反应性通过胸腺中转基因TCR的信号传导来响应survivin基因的表达，从而导致早期胸腺祖细胞的扩增。在T细胞发育的早期阶段，TCR信号传导导致祖细胞的扩增，随后在多个克隆中获得NOTCH突变。TCR信号在这一过程中的作用被以下事实所证实：不能在胸腺内呈递来自survivin抗原的肽的小鼠（例如b2m-/-小鼠）白血病的发生率大大降低。这些见解为基因工程的潜在危险提供了新的线索，包括将发展中的胸腺细胞靶向自身抗原。这部作品正在准备出版。第四个成就是在开发靶向儿童实体瘤的嵌合抗原受体方面取得了实质性进展。我们制备了嵌合抗原受体，靶向多种在儿童实体瘤上表达的抗原，包括GD2、B7H3、ALK和CSPG4。这些嵌合抗原受体（也被称为car）被基因改造成逆转录病毒载体，并在人类T细胞中表达。通过这项工作，我们对靶向CD19的强效CAR与靶向GD2的弱效CAR进行了广泛的比较研究，以确定限制GD2 CAR疗效的因素。我们早期的假设是，效果较差的GD2-CAR CAR受到较低水平的抗原结合和较低的T细胞激活的限制，但我们现在已经做出了显著的观察，即与cd19特异性T细胞相比，表达GD2-CAR的T细胞实际上是“超激活”的。这些表达GD2-CAR的T细胞在发育早期表现出耗竭的特征，并假设这种耗竭限制了它们在体内的有效性。目前正在进行的工作是明确描述该模型系统中耗竭所需的信号通路，确定代谢改变在耗竭发展中的作用，并将gd2靶向CAR细胞与其他可能优化其体内功效的药物结合起来。我们目前正在尝试优化使用嵌合抗原受体的实体瘤免疫治疗方法，通过结合调节髓源性抑制细胞和优化CAR信号传导能力的方案。

Immunotherapy has demonstrated increasing success in the treatment of cancer, and emerging clinical studies demonstrate that this new approach will provide a fourth pillar for treatment of childhood cancer. Several new classes and types of immunotherapeutics are emerging that open up the possibility of applying immunotherapy to an ever-increasing array of targets and cancers. This project leverages the explosion of technologies and insights emerging in the immunotherapy domain to bring new immunotherapy agents to bear on childhood cancer. We seek to answer fundamental questions about similarities and differences between childhood vs adult cancers as it relates to immune based therapies and to extend our understanding of specific factors that limit the effectiveness of immune based therapies for solid tumors compared to liquid tumors. One major accomplishment in FY14 was creation of an entirely novel chimeric antigen receptor targeting the CD22 antigen, which is expressed on B cell leukemia, the single most common cancers of childhood. State-of-the-art technology previously employed to generate chimeric antigen receptors targeting other antigens (e.g. CD19 for leukemia) was used to generate an entire series of new receptors targeting CD22. The CD22 targeted chimeric antigen receptor that showed similar potency to a CD19 targeted receptor, and thus is predicted to have activity in clinical trials. This work was published in Blood in 2013 (Haso et al), a patent was filed, and the new receptor has been licensed by a commercial entity and will be tested in children and young adults in an early phase trial in 2014. A second major accomplishment in FY14 was publication of a report that defined myeloid derived suppressor cells as the basis for immune escape in murine rhabdomyosarcoma following treatment with the immune checkpoint inhibitor anti-PD1. Anti-PD1 is part of a new class of immunotherapeutics that circumvent immunosuppressive mechanisms used by cancer and allow natural, endogenous antitumor immune responses to emerge. Response rate of approximately 10-30% have been reported in malignant melanoma, lung cancer and renal cell carcinoma. This work used anti-PD1 in a model of a pediatric solid tumor, namely rhabdomyosarcoma. Briefly, treatment of mice with anti-PD1 prior to tumor inoculation completely prevented development of cancer, but delaying anti-PD1 treatment for even 7 days after tumor inoculation resulted in minimal benefit. We identified that rhabdomyosarcoma-induced expansion of myeloid derived suppressor cells that traffic to tumors via a CXCL1/CXCR1 axis. We therefore hypothesized that interruption of trafficking of myeloid derived suppressor cells to rhabdomyosarcomas could enhance the efficacy of anti-PD1 therapy. To do this, we created chimeras lacking CXCR2 on hematopoietic cells and we also treated animals with anti-CXCR2 blocking antibodies. In both cases, we observed regression of established rhabdomyosarcoma with anti-PD1 therapy. This work establishes MDSC as important mediators of immune escape in pediatric solid tumors and identified distinctions between the cells induced in adult tumors vs those induced in tumors of childhood. We also sought to determine whether the CXCL/CXCR axis was likely to play a role in human pediatric sarcomas. We first demonstrated that human sarcoma cell lines produce several CXCR ligands and furthermore, discovered that serum from patients treated on trials of immunotherapy had elevated levels of CXCL1 and CXCL8. Moreover, among patients with metastatic sarcoma, elevated CXCL8 levels were associated with poor prognosis. We therefore believe that the CXCL/CXCR chemokine axis represents a potential basis for developing therapies that could be used to augment the efficacy of immunotherapy for childhood cancer. This work was published as the cover article in Science Translational Medicine in 2014 and was also highlighted in Nature Reviews Cancer. A third accomplishment inFY13 is near completion of a study demonstrating that T cells genetically engineered to recognize a self antigen expressed in the thymus develop T cell leukemia through a process whereby the T cell receptor itself serves as an oncogene. We developed a T cell transgenic mouse in which the vast majority of T cells have specificity for "survivin" an anti-apoptotic molecule considered to be a universal tumor antigen. We hypothesized that survivin TCR transgenic mice would be immune to tumor growth as most tumors express high levels of survivin. Unfortunately and remarkably, these mice did not resist growth of implanted tumors, but rather T cell lymphoblastic leukemia developed in essentially all mice. This was not due to insertional mutagenesis. Rather, self-reactivity with survivin antigens expressed in the thymus led to expansion of early thymic progenitors via signaling of the transgenic TCR in the thymus in response to thymic expression of the survivin gene. TCR signaling at this early stage of T cell development leads to expansion of progenitors, followed by acquisition of NOTCH mutations in multiple clones. The role for TCR signaling in this process is confirmed by the fact that mice which cannot present peptides derived from the survivin antigen within the thymus (e.g. b2m-/- mice) have a substantially diminished incidence of leukemia. These insights provide novel clues to potential dangers of genetic engineering that involves targeting developing thymocytes toward self-antigens. This work is being prepared for publication. A fourth accomplishment was substantial progress in developing chimeric antigen receptors targeting solid tumors of childhood. We generated chimeric antigen receptors targeting a variety of antigens expressed on pediatric solid tumors, including GD2, B7H3, ALK and CSPG4. These chimeric antigen receptors (also known as CARs) are genetically engineered into a retroviral vector and expressed in human T cells. Through this work, we have conducted extensive comparison studies of a very potent CAR targeting CD19 vs the less potent CAR targeting GD2 to identify those factors limiting the efficacy of the GD2 CAR. Our early hypothesis was that the less effective GD2-CAR CAR was limited by lesser levels of antigen engagement and lesser T cell activation, but we have now made the remarkable observation that GD2-CAR expressing T cells actually are actually "superactivated" compared to their CD19-specific counterparts. These GD2-CAR expressing T cells show hallmark features of exhaustion early during their development and postulate that this exhaustion limits their effectiveness in vivo. Ongoing work is underway to specifically delineate the signaling pathways required for exhaustion in this model system, to identify the role that altered metabolism plays in the development of exhaustion and to combine GD2-targeted CAR cells with other agents that may optimize their efficacy in vivo. We are currently attempting to optimize approaches for solid tumor immunotherapy using chimeric antigen receptors by incorporating regimen that modulate myeloid derived suppressor cells and optimize CAR signaling capacity.