Immunobiology and Immunotherapy of Pediatric Tumors

免疫疗法在治疗癌症方面取得了越来越大的成功，早期的信号表明，免疫疗法将很快为儿童癌症的治疗提供第四轴。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免疫治疗的相似性和差异的基本问题，并扩展对限制实体肿瘤与液体肿瘤免疫治疗有效性的特定因素的理解。在2013财年，我们的第一个重大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）的最先进技术被应用于生成靶向CD22的整个系列的新受体。测试了多种scFv抗原结合物，以及各种间隔结构域和共刺激结构域。通过反复测试，我们创建了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。这项工作得出了以下结论，这些结论对于优化这种新的治疗方法至关重要，但也为这种新兴的治疗方法提供了更基本的见解。首先，在测试的三个候选物中，结合CD22膜近端表位的scFv比靶向远端表位的结合物更有效。其次，scFv与其靶标之间的高亲和力相互作用并没有转化为更高的疗效。第三，与一个共刺激结构域相比，在嵌合抗原受体中设计两个共刺激结构域导致有效性降低。这项工作于2013年发表在《Blood》杂志上（Haso等人），并申请了专利，这种新的受体已经获得了一家商业实体的许可，并将于2014年在儿童和年轻人身上进行早期试验。该项目的第二个成就是我们实验室在开发针对儿童实体瘤的嵌合抗原受体方面取得了实质性进展。我们生成了嵌合抗原受体，靶向多种在儿童实体瘤上表达的抗原，包括GD2、B7H3、ALK和FGFR4。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受到较小水平的抗原结合和较小的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特异性的T细胞相比，表达GD2-CAR的T细胞实际上似乎是“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。第三项成就是完成了研究，确定了免疫检查点抑制剂抗pd1治疗后小鼠横纹肌肉瘤免疫逃逸的基础。简而言之，在原位模型中，抗pd1治疗完全阻止了小鼠横纹肌肉瘤的发展，但在存在既定疾病的情况下，抗pd1治疗的益处微乎其微。我们发现横纹肌肉瘤能有效诱导骨髓源性抑制细胞的扩增，这些细胞在谱系上是中性粒细胞，并通过CXCL1/CXCR1轴运输到肿瘤。这与在模拟成人肿瘤的肿瘤模型中报道的MDSCs不同，后者主要是单核细胞，并通过CCL/CCR趋化因子轴向肿瘤输送。接下来，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显著消退。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。我们进一步提供了在儿童实体瘤中使用PD1家族检查点抑制剂的基础，并结合减少骨髓源性抑制因子的治疗。这项工作已提交出版，正在修订中。2013财年的第四个成就是一项接近完成的研究，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，因为它发生在3个独立的创始基因中，我们在一只小鼠中绘制了插入图，并注意到它不在致癌位点附近。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得到了证实：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。有趣的是，我们在少量具有gp100和WT1特异性TCR转基因的小鼠中发现了类似的t细胞ALL的发展，这意味着旨在表达早期胸腺发育中肿瘤抗原特异性TCR的基因治疗具有继发性白血病的重大风险。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。

Immunotherapy has demonstrated increasing success in the treatment of cancer, and early signals suggest that immunotherapies will soon provide a fourth axis for the treatment of childhood cancer as well. 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 the similarities and differences that exist when targeting childhood vs adult cancers with immune based therapies and to extend out understanding of specific factors that limit the effectiveness of immune based therapies for solid tumors compared to liquid tumors. In FY13 our first major accomplishment was the 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 applied to generate an entire series of new receptors targeting CD22. A variety of scFv antigen binders were tested as were a variety of spacer domains and costimulatory domains. Through iterative testing, we created a CD22 targeted chimeric antigen receptor that shows similar potency to a CD19 targeted receptor, and thus is predicted to have activity in clinical trials. This work yielded the following conclusions, which were critical for optimizing this new therapeutic but also provide more fundamental insights into this emerging class of therapeutics. First, among three candidates tested, the scFv binding the membrane proximal epitope on CD22 was more potent that binders targeting more distal epitopes. Second, higher affinity interactions between the scFv and its target did not translate into improved efficacy. Third, engineering two costimulatory domains into the chimeric antigen receptor resulted in diminished effectiveness compared to one costimulatory domain. This work was published in Blood in 2013 (Haso et al), a patent was filed, and this new receptor has already been licensed by a commercial entity and will be tested in children and young adults in an early phase trial in 2014. A second accomplishment of this project was substantial progress in our laboratory 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 FGFR4. 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 appear to be "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. A third accomplishment was completion of studies that defined the basis for immune escape in murine rhabdomyosarcoma following treatment with theimmune checkpoint inhibitor anti-PD1. Briefly, treatment with anti-PD1 completely prevented development of murine rhabdomyosarcoma in an orthotopic model, but anti-PD1 treatment in the presence of established disease had minimal benefit. We identified that rhabdomyosarcoma potently induced expansion of myeloid derived suppressor cells that are neutrophilic in lineage and traffic to tumors via a CXCL1/CXCR1 axis. This is distinct from MDSCs reported in tumor models that mimic adult tumors, which are primarily monocytic in lineage and traffic to tumor via the CCL/CCR chemokines axes. We next 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 substantial 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 furthermore provide a basis for using checkpoint inhibitors of the PD1 family in pediatric solid tumors, in conjunction with therapies to diminish myeloid derived suppressors. This work has been submitted for publication and is under revision. A fourth accomplishment in FY13 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 whereby 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 trasngeic 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 because it occurred in 3 separate founders, and we mapped the insertion in one mouse and noted that it was not near an oncogenic site. 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 face that mice that cannot present peptides derived from the survivin antigen within the thymus (e.g. b2m-/- mice) have a substantially diminished incidence of leukemia. Interestingly, we saw similar development of T-cell ALL in small numbers mice with TCR transgenes specific for gp100 and for WT1, implying that gene therapies aimed at expressing TCRs specific for tumor antigens early in thymopoiesis carry a substantial risk for secondary 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.