Developing Novel Therapies for Neuroblastoma and Rhabdomyosarcoma

开发神经母细胞瘤和横纹肌肉瘤的新疗法

• 批准号: 10014450
• 负责人: Javed Khan
• 金额: $ 24.16万
• 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/10014450
• 关键词: Animal Model; Antigens; Apoptosis; Biological; Biological Assay; Biology; Burkitt Lymphoma; CD8B1 gene; Cell Death; Cell Line; Cell Therapy; Cell surface; Cells; Cetuximab; Clinical Trials; Data; Development; Down-Regulation; Drug Combinations; Drug Compounding; Drug Screening; Enhancers; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor Receptor; Extracellular Domain; FDA approved; FGFR4 gene; FOXO1A gene; Generations; Genes; Genome; Genomic Library; Goals; Growth; Human; Immune Targeting; Immunohistochemistry; Immunotherapeutic agent; Injury; Isotopically-Coded Affinity Tagging; Lead; Libraries; MYCN gene; Malignant Childhood Neoplasm; Malignant Neoplasms; Measurement; Methods; Monoclonal Antibodies; Mutate; Mutation; Myoblasts; Neuroblastoma; Normal tissue morphology; Oncogenes; Organ; PAX3 gene; PAX7 gene; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacology; Phase; Prognostic Marker; Proteins; Publishing; RNA Interference; RNA interference screen; Receptor Protein-Tyrosine Kinases; Reporting; Rhabdomyosarcoma; Screening for Neuroblastoma; System; T-Lymphocyte; Technology; Testing; Tissue Microarray; Toxic effect; Translating; Translational Research; Transmembrane Domain; Work; Xenograft Model; Xenograft procedure; cancer therapy; chimeric antigen receptor; chimeric antigen receptor T cells; design; diagnostic biomarker; differential expression; disorder risk; drug mechanism; fusion gene; gene repression; high risk; in vivo; kinase inhibitor; muscle regeneration; novel therapeutics; peripheral blood; receptor; response; skeletal muscle differentiation; small molecule; synergism; targeted treatment; therapeutic target; transcriptome sequencing

For neuroblastoma are using four cell lines, two MYCN not-amplified and two MYCN amplified cell line that can be grown in a xenograft model. For RMS we are using ten cell lines, tow with PAX3-FOXO1 fusion genes and two with RAS pathway mutated genes. Among other assays we will use the Incutyte system. For the siRNA screen we will use a druggable genome library of over 6000 genes developed by National Center for Advancing Translational Sciences (NCATS). For the drug screen we will use single agent and combination responses of a panel of 1916 drugs (Mechanism Interrogation Plate (MIPE-v4) Library) also developed by NCATS. The content of this library include 765 FDA approved compounds, 49 of which are approved for cancer therapy, 460 in clinical trials (phase 1, 2 or 3), 149 kinase inhibitors. For 1915 of these compounds, the target or mechanism of action is known. The most promising targets and the appropriate siRNA or drug combination will be further evaluated in the xenograft animal models as outlined above. For Rhabdomyosarcoma (RMS), FGFR4 is a rational target given that it is a key regulator of myogenic differentiation and muscle regeneration after injury; it is expressed in myoblasts, but not in differentiated skeletal muscle. We and others have found that FGFR4 is highly expressed in all RMS, and high expression is a diagnostic and prognostic biomarker. It is a direct target and strongly induced by PAX3-FOXO1, PAX3, and PAX7 and we reported that PAX3-FOXO1 established a super-enhancer at the gene's locus. We have reported that approximately 10% of FN-RMS have activating mutations in FGFR4 and that cells harboring FGFR4 mutations are oncogene addicted and sensitive to pharmacological inhibition by small molecules. Therefore, FGFR4 is a key cell surface tyrosine kinase receptor for RMS biology, growth and survival.we are developing monoclonal antibodies and human scFv binders. The majority detect the human FGFR4 protein by both ELISA and by FACS analysis . To mitigate for potential organ toxicity, we are examining FGFR4 expression levels in normal human organs. We are currently performing extensive RNAseq and immunohistochemistry (IHC) analysis of normal organ and rhabdomyosarcoma tissue arrays. We are testing our scFv binders as potential FGFR4 chimeric antigen receptors (CARs) to generate a second-generation CAR receptor lentiviral construct that contains the CD8 transmembrane region, 41BB and CD3zeta intracellular domains and a human EGFR extracellular domain. This design was chosen because of its efficacy in clinical trials and CAR T cell persistence in patient's peripheral blood for several months after therapy. The truncated EGFR in the CAR construct allows for the measurement of transduced T cells as well as therapeutic targeting of CAR T cells with Cetuximab in clinical trials in case of uncontrolled toxicity. Anti-FGFR4 CART cells could lyse RH30 but not RAJI, a FGFR4 negative Burkitt's lymphoma cell line (data not shown). Work is currently underway to validate FGFR4 CAR T cells in-vivo. If successful we anticipate the development of potent immunotherapeutic biologics and cell based therapies for patients with aggressive RMS. All positive hits will be further screened in a wider panel of NB and RMS Xenografts and PDxs.

对于神经母细胞瘤，正在使用四个细胞系，两个MYCN未扩增的细胞系和两个MYCN扩增的细胞系，可以在异种移植模型中生长。对于RMS，我们使用了10个细胞系，其中两个是PAX3-FOXO1融合基因，两个是RAS途径突变基因。在其他分析中，我们将使用Incutyte系统。对于siRNA筛选，我们将使用由国家先进翻译科学中心(NCATS)开发的包含6000多个基因的可药物基因组文库。对于药物筛选，我们将使用一组1916种药物(也是由NCATS开发的机制询问板(MIPE-v4)库)的单一试剂和组合反应。该文库的内容包括765个FDA批准的化合物，其中49个被批准用于癌症治疗，460个正在进行临床试验(1期、2期或3期)，149种激酶抑制剂。对于这些化合物中的1915个，其作用靶标或作用机制是已知的。最有希望的靶点和适当的siRNA或药物组合将在上文概述的异种移植动物模型中进一步评估。对于横纹肌肉瘤(RMS)来说，FGFR4是一个合理的靶点，因为它是肌源性分化和损伤后肌肉再生的关键调节因子；它在成肌细胞中表达，但在分化的骨骼肌中不表达。我们和其他人发现FGFR4在所有的RMS中高表达，高表达是一个诊断和预后的生物标志物。它是PAX3-FOXO1、PAX3和PAX7强烈诱导的直接靶点，我们报道了PAX3-FOXO1在该基因的基因座上建立了一个超级增强子。我们已经报道了大约10%的FN-RMS具有FGFR4的激活突变，并且携带FGFR4突变的细胞是癌基因成瘾的细胞，并且对小分子的药物抑制敏感。因此，FGFR4是RMS生物学、生长和生存的关键细胞表面酪氨酸激酶受体。我们正在开发单抗和人源单链抗体结合子。大多数检测人FGFR4蛋白的方法是通过酶联免疫吸附试验和流式细胞仪分析。为了减轻潜在的器官毒性，我们正在检测FGFR4在正常人体器官中的表达水平。我们目前正在对正常器官和横纹肌肉瘤组织阵列进行广泛的RNAseq和免疫组织化学(IHC)分析。我们正在测试我们的scFv结合子作为潜在的FGFR4嵌合抗原受体(CARS)，以产生第二代CAR受体慢病毒载体，该载体包含CD8跨膜区、41BB和CD3zeta胞内区以及人EGFR胞外区。之所以选择这种设计，是因为它在临床试验中的有效性以及治疗后患者外周血中CAR T细胞的持久性。CAR结构中截短的EGFR允许测量转导的T细胞，以及在毒性不受控制的情况下在临床试验中使用西妥昔单抗进行CAR T细胞的靶向治疗。抗FGFR4CART细胞能杀伤RH30，但不能杀伤FGFR4阴性的Burkitt淋巴瘤细胞系Raji(数据未显示)。目前正在进行体内验证FGFR4 CAR T细胞的工作。如果成功，我们预计将开发出有效的免疫治疗生物制剂和基于细胞的疗法来治疗侵袭性RMS患者。所有积极的点击率将在更广泛的NB和RMS Xenograft和PDX小组中进一步筛选。