Innovative Cell Therapy for Pediatric Acute Myeloid Leukemia

小儿急性髓系白血病的创新细胞疗法

• 批准号: 10044345
• 负责人: Maria Grazia Roncarolo
• 金额: $ 40.55万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10044345
• 关键词: ANXA5 gene; Acute Myelocytic Leukemia; Adult Acute Myeloblastic Leukemia; Age; Allogenic; Antigens; Apoptotic; Binding; Biological Assay; Biological Markers; CD 200; CD4 Positive T Lymphocytes; Cell Line; Cell Therapy; Cell physiology; Cells; Childhood Acute Myeloid Leukemia; Childhood Leukemia; Classification; Clinical; Coculture Techniques; Color; Cytogenetics; Cytolysis; Data; Flow Cytometry; Gender; Gene Expression; Gene Expression Profile; Genes; Genetic; Genotype; Goals; Granzyme; Hematopoietic Stem Cell Transplantation; Heterogeneity; Human; IL10 gene; Immune Targeting; Immune Tolerance; Impairment; In Vitro; Interferon Type II; Interleukin-10; Interleukin-2; Lentivirus Vector; Ligand Binding; Link; Lytic; Malignant - descriptor; Malignant lymphoid neoplasm; Measures; Mediating; Membrane Proteins; Methods; Molecular; Molecular Profiling; Myeloid Cells; Natural Killer Cells; OX40; Outcome; Pathway interactions; Patients; Phenotype; Play; Population Heterogeneity; Process; Proteins; RNA Sequences; Regulatory T-Lymphocyte; Residual Neoplasm; Resistance; Risk stratification; Role; Signal Transduction; Stains; T cell therapy; TNFSF4 gene; Testing; Time; White Blood Cell Count procedure; Work; base; chimeric antigen receptor T cells; cohort; cytotoxic; cytotoxicity; design; differential expression; disorder risk; effective therapy; effector T cell; experimental study; improved; in vivo; innovation; leukemia treatment; neoplastic cell; novel; outcome forecast; perforin; peripheral blood; prevent; protein expression; response; single-cell RNA sequencing; therapeutic target; transcriptome; transcriptome sequencing; tumor necrosis factor ligand superfamily member 4; tumor necrosis factor receptor superfamily member 4

ABSTRACT Survival for pediatric acute myeloid leukemia (pAML) has lagged behind other childhood leukemias due to the lack of effective pAML treatments. We have developed a novel T cell therapy, LV-10, made by transducing normal peripheral blood CD4+ T lymphocytes with a lentiviral vector expressing IL10. We discovered that LV-10 can lyse malignant myeloid cells via perforin and granzyme B. Of 23 primary pAML blast tested, 10 were lysed by all LV-10 lines tested, 6 were resistant , and 7 had a variable killing response depending on the LV-10 line to which they were exposed. Notably, the 6 patients who died had pAML blasts that were resistant or variably resistant to LV-10 lysis. The sensitivity of the pAML blasts to LV-10 lysis did not correlate with their genetic landscape, but RNA-sequencing (RNA-seq) of killing-sensitive and resistant blasts revealed dramatic differences in their transcriptomes. The molecular mechanism underlying the pAML resistance to lysis is yet unknown; resistance could be: 1. an active process impairing the LV-10 function, or 2. due to the absence of an antigen on pAML required for LV-10 recognition of target cells. In support of 1., RNA-seq revealed increased expression of OX40L and CD200 on resistant pAML blasts. Their respective receptors OX40 and CD200R1 are both expressed on LV-10, and may provide an inhibitory downstream signal. Thus, we hypothesize that resistant pAML blasts express protein(s) that mediate resistance to LV-10 lysis. However, the blasts with variable response to killing also suggest that the inherent heterogeneity of the different LV-10 lines may impact the efficiency of lysis. Thus, a second hypothesis states that lysis of variable blasts requires both LV-10 and pAML to have killing-permissive phenotypes. We will test these hypotheses by determining the lysis mechanism and pAML and LV-10 features required for LV-10 killing, paving the way for optimization of LV-10 for clinical use in pAML. To this end, we will test a new cohort of 45 pAML blasts for their sensitivity to killing, and their effect on LV-10 lytic capacity and activation. We will also ask if blocking OX40L and/or CD200 reverses the resistance, and if OX40 and CD200R1 signaling modifies LV-10 phenotype and function (Aim 1). In Aim 2, we will perform single cell RNA-seq on sensitive, resistant and variable pAML and LV-10, at baseline and after their interaction, to identify dynamic changes in both pAML and LV-10 population heterogeneity and gene expression, and uncover genes involved in resistance. More broadly, we note that the killing sensitivity of the tested pAML blasts did not correlate with conventional risk stratification methods such as WHO or FAB classification, cytogenetics, age, gender, leukocyte counts or minimal residual disease (MRD) +/- post-induction. This suggests that sensitivity to cytotoxic killing may offer a new means to stratify pAML patients. Importantly, the constitutive secretion of IL-10 implies allogeneic LV-10 can be tolerated in vivo, and may have wide application as a readily available, 3rd party off-the-shelf product administered as part of a hematopoietic stem cell transplantation, or as a stand-alone cell therapy forpAML.

抽象的 小儿急性髓样白血病（PAML）的生存落后于其他童年白血病 缺乏有效的PAML治疗。我们已经开发了一种新型的T细胞疗法LV-10，通过转导制造 正常的外周血CD4+ T淋巴细胞具有表达IL10的慢病毒载体。我们发现LV-10 可以通过穿孔蛋白和颗粒状B裂解恶性髓样细胞。 在所有测试的LV-10线中，有6条均具有抗性，而7条具有可变的杀伤反应，具体取决于LV-10线对 他们被暴露了。值得注意的是，死亡的6例患者的PAML爆炸具有抗性或可变的 对LV-10裂解的抗性。 PAML对LV-10裂解的敏感性与其遗传无关 景观，但对杀戮敏感和抗性爆炸的RNA测序（RNA-Seq）显示出巨大的差异 在它们的转录组中。 PAML对裂解的抗性的分子机制尚不清楚。 电阻可能是：1。一个主动过程损害LV-10功能，或2。由于没有抗原 在LV-10识别靶细胞所需的PAML上。为了支持1.，RNA-seq揭示了表达增加 在抗性PAML爆炸上的OX40L和CD200。他们各自的受体OX40和CD200R1都是 在LV-10上表达，并可能提供抑制性下游信号。因此，我们假设这种抗药性 PAML爆炸表达介导对LV-10裂解的抗性的蛋白质。但是，爆炸具有可变的 对杀戮的反应还表明，不同LV-10线的固有异质性可能会影响 裂解效率。因此，第二个假设指出，可变爆炸的裂解需要LV-10和 PAML具有杀伤性的表型。 我们将通过确定裂解机制以及PAML和LV-10特征来检验这些假设 LV-10杀戮，为在PAML中优化LV-10铺平了道路。为此，我们将测试一个新的 45个PAML爆炸队的队列对杀伤的敏感性以及对LV-10裂解能力和激活的影响。我们 还会询问阻断OX40L和/或CD200是否会逆转电阻，以及OX40和CD200R1信号传导是否逆转 修改LV-10表型和功能（AIM 1）。在AIM 2中，我们将对敏感的单细胞RNA-seq执行 抗性和可变的PAML和LV-10，在基线和它们相互作用后，以确定动态变化 PAML和LV-10种群的异质性和基因表达都及其抗抗性的基因。 更广泛地，我们注意到，经过测试的PAML爆炸的杀戮灵敏度与常规风险无关 诸如谁或FAB分类，细胞遗传学，年龄，性别，白细胞计数等分层方法或 最小残留疾病（MRD）+/-诱导后。这表明对细胞毒性杀伤的敏感性可能会提供 对PAML患者进行分层的新手段。重要的是，IL-10的本构分泌意味着同种异体LV-10 可以在体内耐受，并且可能具有广泛的应用，作为一个容易获得的第三方现成产品 作为造血干细胞移植的一部分或作为独立细胞疗法的一部分给药。