DNA Sequencing and FACS Core Facilities

DNA 测序和 FACS 核心设施

基本信息

批准号：
10487218
负责人：
Paul Robbins
金额：
$ 280.58万
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/10487218
关键词：
Adoptive Immunotherapy Adoptive Transfer Algorithms Antigen Presentation Pathway Antigen Targeting Antigens Atypical lymphocyte Autologous Autologous Tumor-Infiltrating Lymphocyte Binding Bioinformatics Biological Models Breast Cancer Patient Cell Line Cell Separation Cell surface Cells Characteristics Cholangiocarcinoma Clinical Clinical Trials Colon Carcinoma Colorectal Neoplasms Copy Number Polymorphism Core Facility Cytotoxic T-Lymphocytes DNA DNA Sequencing Facility DNA analysis DNA sequencing Data Development Differentiation Antigens Elements Epitopes Expression Library Family Future Gene Expression Genes Helper-Inducer T-Lymphocyte High-Throughput Nucleotide Sequencing Histology Human Immunotherapy In Vitro Individual Investigation KRAS2 gene Lead Lesion Malignant Neoplasms Malignant neoplasm of gastrointestinal tract Malignant neoplasm of lung Malignant neoplasm of ovary Mammary Neoplasms Mediating Methods Monophenol Monooxygenase Mus Mutate Mutation Nature Neoplasm Metastasis Oncogenes Operative Surgical Procedures Patients Peptides Phenotype Population Postdoctoral Fellow Proteins RNA Reagent Recurrence Renal carcinoma Research Research Personnel Reverse Transcriptase Polymerase Chain Reaction SILV gene Sampling Series Services Somatic Mutation T-Cell Receptor T-Lymphocyte Testing Tumor Antigens Tumor Escape Tumor-Infiltrating Lymphocytes base clinically significant driver mutation exome sequencing experimental study gene cloning gene product immune function immunotherapy trials malignant breast neoplasm melanocyte melanoma mutant neoantigens neoplastic cell novel patient population peripheral blood resistance mechanism response screening service member therapy resistant tissue culture transcriptome sequencing tumor tumor progression

项目摘要

Current studies have on the identification of novel antigens expressed in melanoma and renal cancers as well as the characterization of T cells that recognize epitopes of previously described antigens. In a series of recent studies, several novel point-mutated genes have been identified as the targets of melanoma-reactive TIL that mediated long terms clinical tumor regression following adoptive transfer. Current investigations are being carried out to develop methods to facilitate the identification of mutated gene products that can be evaluated as targets of tumor-reactive T cells. Recent studies have focused on the use of high throughput sequencing to identify all of the somatic mutations in patient tumors. Minigenes encoding the somatic mutations identified from tumor sequencing have been generated and tested for their ability to be recognized by autologous TIL. Screening of individual tumor infiltrating lymphocyte (TIL) generated from tumor fragments for their ability to recognize candidate mutated antigens or neoantigens has resulted in the identification of one or more mutated antigen targets in 80-90% of patients with melanoma, gastrointestinal, breast, lung and ovarian cancers. In recent studies carried out in a patient with cholangiocarcinoma, analysis of reactivity with a mutated antigen was used to guide the selection of TIL used for treatment of a patient who has demonstrated significant regression of multiple metastatic lesions. Partial tumor regressions have also been observed in a patient with colon cancer treated with autologous TIL that predominantly recognized a hotspot mutation in the KRAS oncogene and a breast cancer patient whose TIL recognized two neoantigens. Ongoing efforts are focused on the optimizing strategies for identifying neoantigen reactive T cells, including studies involving the use of MHC binding algorithms to identify the short peptides or epitopes and HLA restriction element used for antigen recognition. For these studies, the DNA Core facility provides multiple services members of the Surgery Branch that include construction and analysis of whole exome sequencing (WES) and RNA-seq expression libraries, RT-PCR, quantitative RT-PCR, gene cloning and RNA/DNA analysis. In carrying out bioinformatics analysis of WES and RNA-seq data, the DNA Core provides investigators with lists of candidate neoantigens for screening with patient T cells, along with valuable information regarding tumor cell chromosomal copy number variations and data regarding hotspot driver mutations, and evaluate tumor samples for expression of genes involved with tumor antigen processing and presentation. Tumor samples are also being analyzed to identify chromosomal copy number changes and to determine if mutations are shared between different tumor regions in an attempt to identify the optimal targets for immunotherapy. These studies have identified HLA loss as a mechanism of tumor escape in patients who demonstrate mixed responses in which some tumor lesions regress but additional lesions continue to progress following adoptive immunotherapy. Ongoing studies are also being carried out to identify mechanisms of resistance to therapy in additional patients with mixed responses to immunotherapy. The FACS Core Facility is currently being utilized for the analysis of T cell populations that are administered to patients as a part of the analysis of ongoing clinical cancer adoptive immunotherapy trials. In addition, the FACS Core is utilized on a daily basis for the analysis of the results of experiments to analyze the results of in vitro experiments to examine factors that influence the phenotype and function of tumor reactive T cells as well as to carry out the separation of cells based upon their expression of a wide variety of cell surface markers. For these studies phenotypic analyses are being carried out on a BD FACSCalibur, FACSCantoI, FACSCantoII, as well as a recently acquired LSRFortessa, and cell separations were carried out on a BD FACSAriaII. Recent studies have focused on evaluating the reactivity of T cells against autologous tissue culture lines from colorectal and breast tumor samples. These studies have involved sequencing the cell lines and identifying addition candidate mutant antigens that can be then evaluated for their ability to be recognized by autologous T cells. Initial studies carried out in a breast cancer patient have revealed that unique mutant peptides expressed by the patient tumor cells are recognized by T cells, demonstrating the importance of evaluating these responses, which could aid in the development of effective immunotherapies.

当前的研究已经对鉴定在黑色素瘤和肾脏癌中表达的新型抗原以及识别先前描述的抗原表位的T细胞的表征。在一系列最近的研究中，几个新型点突变的基因被鉴定为黑色素瘤反应性直到介导的长期临床肿瘤回归后的靶标。正在进行目前的研究以开发方法以促进可以评估为肿瘤反应性T细胞靶标的突变基因产物的鉴定。最近的研究集中在使用高通量测序以鉴定患者肿瘤中的所有体细胞突变。已经生成并测试了编码从肿瘤测序中鉴定出的体细胞突变的微型元素，并测试了它们的自体TIL识别的能力。筛选由肿瘤片段产生的单个肿瘤浸润淋巴细胞（TIL），以识别候选突变抗原或新抗原的能力，导致在80-90％的患有黑色素瘤，胃肠道，乳房，肺和卵巢癌的患者中鉴定出80-90％的一个或多个突变的抗原靶标。在最近在胆管癌患者中进行的研究中，使用突变抗原的反应性分析用于指导用于治疗患者的TIL的选择，该患者表现出了多种转移性病变的显着消退。在用自体癌症治疗的患者中，也观察到部分肿瘤回归，该患者主要识别KRAS癌基因中的热点突变和一名乳腺癌患者，其TIL识别两个新抗原。持续的努力集中在确定新抗原反应性T细胞的优化策略上，包括涉及使用MHC结合算法来识别用于抗原识别的短肽或表位和HLA限制元件的研究。在这些研究中，DNA核心设施提供了手术分支的多个服务成员，包括整个外显子组测序（WES）和RNA-SEQ表达文库的构建和分析，RT-PCR，定量RT-PCR，基因克隆和RNA/DNA分析。在对WES和RNA-SEQ数据进行生物信息学分析时，DNA Core为研究人员提供了候选新抗原列表，以筛查患者T细胞的筛查，以及有关肿瘤细胞染色体拷贝数变化和有关热点驱动器突变的肿瘤细胞染色体拷贝数变化的宝贵信息，并评估与肿瘤样本有关涉及毒品毒剂的肿瘤样本，以表达涉及粉刺抗原抗菌素的替代和陈述。还正在分析肿瘤样品以识别染色体拷贝数的变化，并确定不同肿瘤区域之间是否共享突变，以识别免疫疗法的最佳靶标。这些研究已经确定HLA丧失是表现出混合反应的患者的肿瘤逃生机制，其中一些肿瘤病变会消退，但在继发性免疫疗法后继续进展。正在进行的研究也正在进行，以确定对免疫疗法反应混合反应的其他患者的耐药机制。目前，FACS核心设施用于分析对患者进行的T细胞群体，作为正在进行的临床癌症收养免疫疗法试验的一部分。此外，每天都使用FACS核心来分析实验结果，以分析体外实验的结果，以检查影响肿瘤反应性T细胞的表型和功能的因素，并基于其表达各种细胞表面标记的表达来进行细胞的分离。对于这些研究，正在BD FACSCALIBUR，FACSCANTOI，FACSCANTOII以及最近获得的LSRFortessa上进行表型分析，并在BD FACSARIAII上进行了细胞分离。最近的研究重点是评估T细胞对结直肠肿瘤和乳腺肿瘤样品的自体组织培养线的反应性。这些研究涉及对细胞系进行测序并鉴定添加候选突变抗原，然后可以评估其自体T细胞识别其能力。在乳腺癌患者中进行的初步研究表明，由患者肿瘤细胞表达的独特突变肽通过T细胞识别，证明了评估这些反应的重要性，这可以有助于开发有效的免疫疗法。