Optimizing the graft versus leukemia effect for pediatric ALL

优化儿童 ALL 的移植物抗白血病效果

基本信息

批准号：
8763437
负责人：
Terry Fry
金额：
$ 94.24万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8763437
关键词：
Acute Lymphocytic Leukemia Allogeneic Bone Marrow Transplantation Allogenic Antigen Targeting Antigens Apoptosis Area B-Cell Acute Lymphoblastic Leukemia B-Lymphocytes Blood Body Weight decreased Bone Marrow Bone Marrow Transplantation CD44 gene CD8B1 gene Cancer Etiology Cell Line Cell physiology Cells Cessation of life Child Childhood Childhood Acute Lymphocytic Leukemia Chronic Lymphocytic Leukemia Clinical Clinical Trials Complex Data Dendritic Cells Development Effectiveness Electroporation Epitopes Female Gene Expression Genes Genetic Transcription Goals HLA-A2 Antigen Hematologic Neoplasms Hematopoietic Hematopoietic Stem Cell Transplantation Histologic Homologous Transplantation Human Immune Immune response Immune system Immunoglobulins Immunology Impairment In Vitro Incidence Individual Inferior Infusion procedures Injection of therapeutic agent Investigation Journals L-Selectin Laboratories Length Lymphocyte Activation Lymphoid Tissue Malignant Childhood Neoplasm Malignant Neoplasms Manuscripts Marrow Mediating Memory Minor Minor Histocompatibility Antigens Modeling Monoclonal Antibodies Mouse Strains Mucins Mus Myeloid Leukemia Natural Killer Cells Nephroblastoma Non-Malignant Normal tissue morphology Outcome Ovalbumin Patients Pediatric Oncology Peptides Phenotype Physiologic pulse Population Principal Investigator Process Protocols documentation Publishing RNA Reaction Relapse Risk SELL gene Sampling Solid Solid Neoplasm Sorting - Cell Movement Specificity Supportive care System T cell response T-Cell Receptor T-Lymphocyte T-Lymphocyte Subsets Therapeutic Therapeutic Intervention Tissue Grafts Tissues Transgenic Organisms Transplantation Tumor Antigens Universities Vaccination Vaccines WT1 gene Work Y Chromosome attenuation base cancer therapy clinically relevant graft vs host disease graft vs leukemia effect high risk human data immunogenic in vivo leukemia male mortality neoplastic cell overexpression prevent programs receptor research study response senescence subcutaneous tumor vector

Acute Lymphocytic Leukemia Allogeneic Bone Marrow Transplantation Allogenic Antigen Targeting Antigens Apoptosis Area B-Cell Acute Lymphoblastic Leukemia B-Lymphocytes Blood Body Weight decreased Bone Marrow Bone Marrow Transplantation CD44 gene CD8B1 gene Cancer Etiology Cell Line Cell physiology Cells Cessation of life Child Childhood Childhood Acute Lymphocytic Leukemia Chronic Lymphocytic Leukemia Clinical Clinical Trials Complex Data Dendritic Cells Development Effectiveness Electroporation Epitopes Female Gene Expression Genes Genetic Transcription Goals HLA-A2 Antigen Hematologic Neoplasms Hematopoietic Hematopoietic Stem Cell Transplantation Histologic Homologous Transplantation Human Immune Immune response Immune system Immunoglobulins Immunology Impairment In Vitro Incidence Individual Inferior Infusion procedures Injection of therapeutic agent Investigation Journals L-Selectin Laboratories Length Lymphocyte Activation Lymphoid Tissue Malignant Childhood Neoplasm Malignant Neoplasms Manuscripts Marrow Mediating Memory Minor Minor Histocompatibility Antigens Modeling Monoclonal Antibodies Mouse Strains Mucins Mus Myeloid Leukemia Natural Killer Cells Nephroblastoma Non-Malignant Normal tissue morphology Outcome Ovalbumin Patients Pediatric Oncology Peptides Phenotype Physiologic pulse Population Principal Investigator Process Protocols documentation Publishing RNA Reaction Relapse Risk SELL gene Sampling Solid Solid Neoplasm Sorting - Cell Movement Specificity Supportive care System T cell response T-Cell Receptor T-Lymphocyte T-Lymphocyte Subsets Therapeutic Therapeutic Intervention Tissue Grafts Tissues Transgenic Organisms Transplantation Tumor Antigens Universities Vaccination Vaccines WT1 gene Work Y Chromosome attenuation base cancer therapy clinically relevant graft vs host disease graft vs leukemia effect high risk human data immunogenic in vivo leukemia male mortality neoplastic cell overexpression prevent programs receptor research study response senescence subcutaneous tumor vector

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项目摘要

Under the first aim of this project we have generated a precursor B cell leukemia line derived from mice with transgenic expression of E2aPBX1, a recurring translocation present in approximately 5% of pediatric ALL (Bijl et al, Genes and Development, 2005). The cell line was confirmed to be immunogeneic as vaccination with irradiated leukemia cells protects against subsequent challenge with E2aPBX1 but not other tumors. Using monoclonal antibodies to deplete cell subsets, we demonstrated that protection in immunized mice requires both CD4 and CD8 T cells and is impaired following NK cell depletion. Under aim 2 we have performed bone marrow transplantation experiments to assess GVL in this model. Allogeneic transplantation followed by E2aPBX1 leukemia challenge and subsequent transfer of primed allogeneic T cells results in cure of leukemia in all mice. However, the mice develop weight loss and histologic changes consistent with GVHD that results in late mortality. Interestingly, priming T cell donors with recipient (and leukemia) strain non-malignant B cells did not cure the mice indicating that both minor antigens and leukemia-associated antigens are responsible for cure in this model. We next sought to separate the anti-leukemic GVL effect from GVHD by selecting for T cells subsets. Neither CD4 nor CD8 T cells from primed donors alone were sufficient to cure all of the mice. Using flow sorting based on expression of CD44 and CD62L (L-selectin) we have demonstrated that central memory phenotype T cells (CD44+/CD62L+) can cure leukemia without the induction of GVHD whereas nave T cells (CD44-/CD62L+) induce rapidly lethal GVHD. In summary, we have demonstrated that ALL can be effectively targeted by a T cell response in vivo but that this response requires vaccination of the donor T Cell inocula. Second, allogeneic antigens contribute to the cure following T cell infusion but results in GVHD. Finally, sorted populations of T cells from primed donors can mediate selective graft versus leukemia responses. Using this model we have begun studying the early progression of the leukemia in bone marrow and the impact of this progression on T cells. We have identified that a surprisingly large percentage of T cells in leukemia-infiltrated compartments express high levels of the negative regulator of T cell function, programmed death 1 (PD-1) receptor. Addition studies have shown that the percentage of PD-1+ T cells correlates with the extent of leukemic involvement and that PD-1+ T cells also express other markers of a senescent phenotype such as T cell immunoglobulin and mucin domain 3 (Tim-3). Using an E2aPBX1 cell line expressing ovalbumin and ovalbumin-specific T cells we have demonstrated the induction of PD1 occurs only when T cells are able to recognize antigens on the ALL. Interestingly, careful assessment T cells during early leukemia progression have shown that the induction of PD1 occurs early (by day 5 after injection of leukemia) whereas acquisition of other T cells senescent markers such as Tim-3 and Lymphocyte Activation Gene 3 (LAG3) occur later suggesting that these markers may be more functionally relevant in terms of antileukemic potential. Indeed, T cells from irradiated tumor cell primed mice also express PD1 but not Tim-3 or LAG3 and mediate an antileukemic effect. Finally, preliminary a data from human bone marrow samples leukemia samples from patients with ALL (obtained from Dr. Alan Wayne) have shown expression of PD1, Tim-3 and LAG3 on a subset of T cells. In summary, this data suggests that for pediatric ALL blocking Tim-3 or LAG3 may be more effective than targeting PD1. Another area of investigation in the laboratory is focused on understanding the impact of the response to minor histocompatibility antigens expressed in normal tissues (graft versus host) on the anti-tumor immune response following allogeneic HSCT. Under project Project ZIA BC 011320, we have demonstrated and published that even mild GVHD can significantly impair responses to vaccines targeting tumors (Capitini et al, Blood, 2009) and that this attenuation of vaccine responses results from both diminished proliferation and increased apoptosis (manuscript accepted, Journal of Immunology). The tumor antigenic complex used in these studies is the HY system in which a solid tumor that naturally expresses Y chromosome-derived antigens was injected into female mice receiving female allogeneic bone marrow and T cells (where the mouse strain-specific minor allogeneic antigens are distinct from the HY-derived tumor antigens). We have now assessing HY tumor responses in male mice of the same strain from which the tumor is derived. Importantly, in this model, the tumor antigens completely overlap with tumor antigens, a clinically relevant scenario in which tumor-specific antigens may be weak or absent. The use of CD4 and CD8 HY specific T cell receptor transgenic donors allows careful tracking of T cells targeting shared antigens. We have shown that, while HY-specific T cells mediate mild GVHD and expand to a much larger extent in males than in female hosts (despite HY vaccination), these T cells with specificity to both normal tissues and a solid subcutaneous tumor are less potent at inducing tumor regression. We have now generated and HY expressing pre-B cell ALL line. Interestingly, using female recipients of male bone marrow (where HY expression is restricted to the hematopoietic compartment) we have shown that the impairment of T cell responses against malignancy occurs only when the target minor antigen is coexpressed in the same non-malignant tissue compartment as the tumor since HY specific T cells reject solid tumors but not leukemia in this model. Using candidate molecule (assessment of PD1, Tim3, LAG3) and non-candidate (global gene expression) approaches to assess T cells from different compartments (bone marrow vs secondary lymphoid tissues) we are exploring what distinguishes T cells with anti-tumor potential from those unable to reject tumors. Aim 3 is ongoing and involves the extension of work conducted under aim 2 to clinically relevant leukemia targets. We have established that E2aPBX1 overexpresses the Wilm's Tumor 1 gene, also overexpressed on approximately 70-80% of human leukemias and validated as a target in patients. In order to obtain large numbers of WT-1 specific T cells, we have generated mice that express a T cell receptor specific for the dominant class I epitope derived from WT-1 (called Db126). T cells from these mice show robust expansion to Db126 peptide in vitro resulting T cells with the capability of targeting peptide pulsed targets in vitro. While E2aPBX1 cells can also be targeted, the results have been variable. We are in the process of optimizing the use of these T cells in vivo against E2aPBX1 and other hematologic malignancies that overexpress WT-1. An open protocol in the Pediatric Oncology Branch (Dr. Alan Wayne, Principal Investigator) is utilizing dendritic cell vaccination targeting WT-1 peptides as a therapeutic intervention in patients relapsing following allogeneic HSCT. Although this trial involves peptide-pulsed DC vaccination and is, thus, restricted to HLA-A2+ individuals, we are currently evaluating RNA electroporation of DCs with full-length WT1 RNA as a means to generate a non-HLA-A2 restricted platform. We have obtained RNA transcription vectors from Duke University where clinical trials are ongoing using RNA-electroporated DCs targeted antigens other than WT1. These vectors will be used to produce ?clinical-grade? WT1 RNA in the Duke facility. A proposal to use this platform as a post-transplant pre-emptive therapy in children with high risk ALL and AML has been presented to the Pediatric Blood and Marrow Transplant Consortium.

在该项目的第一个目的下，我们产生了一个前体B细胞白血病系，该白细胞系源自具有E2APBX1的转基因表达的小鼠，这是大约5％的儿科全部存在的反复易位（Bijl等人，Genes and Genes and Development，2005年）。由于辐照性白血病细胞的疫苗接种可免受E2APBX1的挑战，但没有其他肿瘤，因此该细胞系被证实为免疫类别。使用单克隆抗体耗尽细胞亚群，我们证明了免疫小鼠的保护需要CD4和CD8 T细胞，并且在NK细胞耗竭后会受到损害。在AIM 2下，我们进行了骨髓移植实验，以评估该模型中的GVL。同种异体移植，然后进行E2APBX1白血病挑战，随后引发同种异体T细胞的转移导致所有小鼠的白血病治愈。但是，小鼠会产生与GVHD一致的体重减轻和组织学变化，从而导致死亡晚期。有趣的是，具有受体（和白血病）菌株非恶性B细胞的启动T细胞供体不能治愈小鼠，表明小鼠均可抗原和与白血病相关的抗原在该模型中均可治愈。接下来，我们试图通过选择T细胞亚群来将抗白血病GVL效应与GVHD分开。单独的引发供体的CD4和CD8 T细胞都不足以治愈所有小鼠。使用基于CD44和CD62L（L-选择蛋白）表达的流量分类我们已经证明了中央记忆表型T细胞（CD44+/CD62L+）可以治愈白血病，而无需诱导GVHD，而Nave T细胞（CD44-/CD62L+）诱导了快速致命的lethal GVHD。总而言之，我们已经证明了所有这些都可以通过体内的T细胞反应有效地靶向，但是这种反应需要接种供体T细胞接种物。其次，同种异体抗原在输注T细胞后有助于治愈，但导致GVHD。最后，从启动供体中分类的T细胞种群可以介导选择性移植物与白血病反应。使用该模型，我们已经开始研究骨髓中白血病的早期进展以及该进展对T细胞的影响。我们已经确定，白血病室中的T细胞中的T细胞中有很大比例的T细胞表达了T细胞功能的负调节剂，即编程死亡1（PD-1）受体。添加研究表明，PD-1+ T细胞的百分比与白血病受累程度相关，并且PD-1+ T细胞还表达了衰老表型的其他标记，例如T细胞免疫球蛋白和粘蛋白结构域3（TIM-3）。使用表达椭圆蛋白和卵蛋白特异性T细胞的E2APBX1细胞系，我们已经证明了PD1的诱导仅当T细胞能够识别全部抗原时才发生。有趣的是，在白血病早期进展过程中仔细评估的T细胞表明，PD1的诱导发生早期（到注射白血病后的第5天），而获得其他T细胞的衰老标记物（如TIM-3和淋巴细胞激活基因3（LAG3））后来提出这些标记可能在这些标记中可能具有更多功能相关的蚂蚁。实际上，来自辐照肿瘤细胞的T细胞还表达PD1，但不表达TIM-3或LAG3，并介导抗白血病作用。最后，来自人类骨髓样品的初步A来自所有患者（从Alan Wayne博士获得的患者）的白血病样品显示了PD1，TIM-3和LAG3在T细胞子集上的表达。总而言之，该数据表明，对于小儿，所有阻断TIM-3或LAG3的阻塞可能比靶向PD1更有效。实验室中的另一个研究领域的重点是理解在同种异体HSCT后在正常组织（移植物与宿主）中表达的对抗肿瘤免疫反应的较小组织相容性抗原的影响。在项目Zia BC 011320项目的项目下，我们证明并发表了，即使是轻度GVHD也会显着损害针对靶向肿瘤的疫苗的反应（Capitini等，Blood，2009），并且疫苗反应的衰减均来自增殖减少和细胞增多症的减少（手稿接受，杂志，免疫学）。这些研究中使用的肿瘤抗原复合物是HY系统，其中将自然表达Y染色体衍生抗原的实体瘤注射到接受女性同种骨髓和T细胞的雌性小鼠中（其中小鼠菌株特异性的小型同种异体同种异体抗原与氢肿瘤抗原不同）。现在，我们已经评估了从肿瘤得出的相同菌株的雄性小鼠中的HY肿瘤反应。重要的是，在该模型中，肿瘤抗原与肿瘤抗原完全重叠，肿瘤抗原是临床相关的情况，其中肿瘤特异性抗原可能弱或不存在。使用CD4和CD8特异性T细胞受体转基因供体可以仔细跟踪靶向共同抗原的T细胞。我们已经表明，虽然Hy Hy Hy Hy tem t细胞介导了轻度GVHD并在男性宿主中（尽管HY疫苗接种）扩展到更大的程度（但这些T细胞对正常组织和固体皮下肿瘤具有特异性，并且在诱导肿瘤回归时效果较小。现在，我们已经生成并表达了所有线路。有趣的是，使用雄性骨髓的女性接受者（其中HY表达仅限于造血室），我们表明，仅当靶标次要抗原与Tumor在TUMOR相同的非恶性组织隔室中表达靶标的较小抗原时，T细胞对恶性肿瘤的损害仅发生，因为HY特异性T细胞拒绝实体肿瘤，但在这种模型中不拒绝这种模型。使用候选分子（评估PD1，TIM3，LAG3）和非候选（全球基因表达）方法，以评估从不同区室（骨髓与次级淋巴组织组织）评估T细胞的T细胞，我们正在探索是什么使T细胞与无法拒绝肿瘤的抗肿瘤电位的T细胞区分开来。 AIM 3正在进行中，涉及AIM 2下进行的工作扩展到临床相关的白血病靶标。我们已经确定，E2APBX1过表达Wilm的肿瘤1基因，在大约70-80％的人类白血病上也过表达，并作为患者的靶标得到了验证。为了获得大量的WT-1特异性T细胞，我们生成的小鼠表达了对源自WT-1的主要I表位特异性的T细胞受体（称为DB126）。来自这些小鼠的T细胞在体外表现出稳健的DB126肽的膨胀，其T细胞具有靶向肽脉冲靶标体外的能力。尽管E2APBX1细胞也可以靶向，但结果是可变的。我们正在优化这些T细胞在体内使用E2APBX1和其他过表达WT-1的血液系统恶性肿瘤的使用。小儿肿瘤学分支机构的开放方案（首席研究员Alan Wayne博士）正在利用针对WT-1肽的树突状细胞疫苗接种作为同种异体HSCT后复发的患者的治疗干预措施。尽管该试验涉及肽脉冲的DC疫苗接种，因此仅限于HLA-A2+个体，但我们目前正在评估具有全长WT1 RNA的DC的RNA电穿孔，以作为生成非HLA-A2限制平台的一种手段。我们已经从杜克大学获得了RNA转录载体，其中使用了除WT1以外的其他靶向抗原的RNA - 电受影DC进行临床试验。这些向量将用于生产？临床级吗？杜克设施中的WT1 RNA。一项提议，将该平台用作具有高风险的儿童的移植后预先疗法，已向小儿血液和骨髓移植财团提出。