Optimizing the graft versus leukemia effect for pediatric ALL

优化儿童 ALL 的移植物抗白血病效果

• 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
• 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

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标下，我们从转基因表达E2aPBX1的小鼠中获得了前体B细胞白血病系，E2aPBX1是儿科ALL中约5%的反复易位（Bijl等人，基因与发育，2005）。该细胞系被证实具有免疫基因性，因为接种辐照白血病细胞可保护其免受随后的E2aPBX1侵袭，但对其他肿瘤无效。使用单克隆抗体来消耗细胞亚群，我们证明免疫小鼠的保护需要CD4和CD8 T细胞，并且在NK细胞消耗后受损。在目的2下，我们进行了骨髓移植实验来评估该模型的GVL。同种异体移植后，E2aPBX1白血病攻击，随后转移引物的同种异体T细胞，结果在所有小鼠中治愈白血病。然而，小鼠出现体重减轻和与GVHD一致的组织学变化，导致晚期死亡。有趣的是，用受体（和白血病）非恶性B细胞注入T细胞供体并不能治愈小鼠，这表明在该模型中，次要抗原和白血病相关抗原都能治愈小鼠。接下来，我们试图通过选择T细胞亚群来分离抗白血病GVL和GVHD的作用。单独来自供体的CD4和CD8 T细胞都不足以治愈所有小鼠。利用基于CD44和CD62L （l -选择素）表达的流式分选，我们已经证明，中枢记忆表型T细胞（CD44+/CD62L+）可以在不诱导GVHD的情况下治愈白血病，而中性T细胞（CD44-/CD62L+）可以诱导快速致死性GVHD。总之，我们已经证明，ALL可以通过体内T细胞反应有效靶向，但这种反应需要供体T细胞接种疫苗。其次，同种异体抗原有助于T细胞输注后的治愈，但会导致GVHD。最后，来自供体的T细胞群可以介导选择性移植物对抗白血病反应。利用这个模型，我们已经开始研究骨髓中白血病的早期进展以及这种进展对T细胞的影响。我们已经发现，在白血病浸润的细胞室中，有相当大比例的T细胞表达高水平的T细胞功能负调节因子，程序性死亡1 （PD-1）受体。此外，研究表明，PD-1+ T细胞的百分比与白血病的侵袭程度相关，并且PD-1+ T细胞还表达衰老表型的其他标记物，如T细胞免疫球蛋白和粘蛋白结构域3 （Tim-3）。利用表达卵清蛋白和卵清蛋白特异性T细胞的E2aPBX1细胞系，我们已经证明，只有当T细胞能够识别ALL上的抗原时，PD1才会被诱导。有趣的是，对早期白血病进展过程中T细胞的仔细评估表明，PD1的诱导发生得较早（注射白血病后第5天），而其他T细胞衰老标记物，如Tim-3和淋巴细胞激活基因3 （LAG3）的获得发生得较晚，这表明这些标记物在抗白血病潜能方面可能在功能上更相关。事实上，来自辐照肿瘤细胞启动小鼠的T细胞也表达PD1，但不表达Tim-3或LAG3，并介导抗白血病作用。最后，来自人类骨髓样本的初步数据显示，来自ALL患者的白血病样本（来自Alan Wayne博士）在一个T细胞亚群上表达了PD1， Tim-3和LAG3。总之，这些数据表明阻断Tim-3或LAG3可能比靶向PD1更有效。实验室研究的另一个领域是了解对正常组织中表达的次要组织相容性抗原（移植物对宿主）的反应对同种异体造血干细胞移植后抗肿瘤免疫反应的影响。在project ZIA BC 011320项目中，我们已经证明并发表了即使是轻微的GVHD也会显著损害针对肿瘤的疫苗的反应（Capitini等人，Blood, 2009），并且这种疫苗反应的衰减是由于增殖减少和细胞凋亡增加（论文已被接受，免疫学杂志）。在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肿瘤注射到接受雌性同种异体骨髓和T细胞的雌性小鼠中（其中小鼠株特异性次要同种异体抗原与HY衍生的肿瘤抗原不同）。我们现在已经评估了HY肿瘤在同一品系的雄性小鼠中的反应。重要的是，在这个模型中，肿瘤抗原与肿瘤抗原完全重叠，这是一种临床相关的情况，肿瘤特异性抗原可能很弱或不存在。使用CD4和CD8 HY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。我们已经证明，尽管HY特异性T细胞介导轻度GVHD，并且在雄性宿主中比在雌性宿主中扩展得更大（尽管接种了HY疫苗），但这些对正常组织和实体皮下肿瘤都具有特异性的T细胞在诱导肿瘤消退方面的作用较弱。我们现在产生了表达前b细胞的ALL细胞系。有趣的是，使用男性骨髓的女性受体（其中HY表达仅限于造血室），我们已经表明，只有当目标次要抗原在与肿瘤相同的非恶性组织室中共表达时，才会发生T细胞对恶性肿瘤反应的损害，因为在该模型中，HY特异性T细胞排斥实体瘤，而不是白血病。使用候选分子（评估PD1， Tim3, LAG3）和非候选（全局基因表达）方法来评估来自不同区室（骨髓与次级淋巴组织）的T细胞，我们正在探索具有抗肿瘤潜力的T细胞与无法排斥肿瘤的T细胞的区别。Aim 3正在进行中，涉及将Aim 2下开展的工作扩展到临床相关的白血病靶点。我们已经确定E2aPBX1过表达Wilm's Tumor 1基因，该基因在大约70-80%的人类白血病中也过表达，并被证实为患者的靶标。为了获得大量WT-1特异性T细胞，我们培养了表达WT-1显性I类表位特异性T细胞受体的小鼠（称为Db126）。这些小鼠的T细胞在体外表现出对Db126肽的强大扩增，从而使T细胞在体外具有靶向肽脉冲靶标的能力。虽然E2aPBX1细胞也可以作为靶标，但结果却不尽相同。我们正在优化这些T细胞在体内对抗过表达WT-1的E2aPBX1和其他血液学恶性肿瘤的使用。儿科肿瘤科的一项开放协议（Alan Wayne博士，首席研究员）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造血干细胞移植后复发患者的治疗干预措施。虽然该试验涉及肽脉冲DC疫苗接种，因此仅限于HLA-A2+个体，但我们目前正在评估用全长WT1 RNA电穿孔DC作为产生非HLA-A2限制性平台的手段。我们从杜克大学获得了RNA转录载体，该大学正在进行临床试验，使用RNA电穿孔dc靶向抗原而不是WT1。这些载体将用于生产临床级？杜克大学实验室的WT1 RNA。儿科血液和骨髓移植协会（Pediatric Blood and Marrow transplantation Consortium）提出了一项建议，将该平台作为高风险ALL和AML儿童移植后的先发制人治疗。