DNA Sequencing and FACS Core Facilities

DNA 测序和 FACS 核心设施

基本信息

批准号：
9154299
负责人：
Paul Robbins
金额：
$ 203.19万
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/9154299
关键词：
Adoptive Immunotherapy Adoptive Transfer Antigen Targeting Antigens Autologous Autologous Tumor-Infiltrating Lymphocyte Binding Sites Biological Models CD4 Positive T Lymphocytes CTAG1 gene Cell Separation Cell physiology Cell surface Cells Characteristics Cholangiocarcinoma Clinical Clinical Trials Collaborations Complementary DNA Core Facility Cytotoxic T-Lymphocytes DNA DNA Sequence DNA Sequencing Facility DNA analysis Development Differentiation Antigens Epitopes Family Gastrointestinal Neoplasms Genes Goals Helper-Inducer T-Lymphocyte High-Throughput Nucleotide Sequencing Human Human Identifications In Vitro Investigation Laboratories Lead Libraries MAGEA3 gene Malignant Neoplasms Malignant neoplasm of lung Malignant neoplasm of ovary Malignant neoplasm of prostate Malignant neoplasm of urinary bladder Mediating Melanoma Cell Metastatic Lesion Methods Modification Monophenol Monooxygenase Mus Mutate Nature Operative Surgical Procedures Patients Peripheral Blood Mononuclear Cell Phase II Clinical Trials Phenotype Population Postdoctoral Fellow Proteins RNA RNA Interference Reagent Renal carcinoma Research Personnel Reverse Transcriptase Polymerase Chain Reaction Sampling Services Small Interfering RNA Somatic Mutation T-Cell Receptor T-Lymphocyte Testing Transcript Transfection Tumor Antigens Tumor-Infiltrating Lymphocytes United States National Institutes of Health base cancer testis antigen clinically significant gene cloning immune function in vitro Assay malignant breast neoplasm melanocyte melanoma member novel novel strategies patient population peripheral blood research study response screening synovial sarcoma tumor

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 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 candidate antigens identified resulted in the identification of one or more mutated antigen targets in 12 of the 15 melanoma TIL, as well as in two of the four gastrointestinal TIL that have been screened to date. 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. Ongoing studies are focused on the development of strategies for identifying T cells that recognized mutated antigens in an attempt to develop effective adoptive immunotherapies for the treatment of patients with a variety of malignancies including lung and bladder cancer. Studies carried out in collaboration with the NIH RNAi consortium to develop a novel approach to the identification of human tumor antigens utilizing the transfection of a library of siRNA that target 20,000 human cDNA transcripts resulted in the identification of potential targets that are being evaluated for their ability to be recognized by tumor-reactive T cells. Additional studies have involved the identification of T cell receptors (TCRs) directed against widely shared antigens. Recent studies have focused on the identification of TCRs that recognize cancer/germline (CG) antigens, molecules that are expressed in melanoma as well as a variety of highly prevalent malignancies such as breast and prostate cancer. A TCR that recognizes the NY-ESO-1 CG antigen was used to transduce autologous PBMC, and results of a Phase II clinical trial indicate that approximately 50% of either melanoma or synovial cell sarcoma patients who received PBMC transduced with an anti-NY-ESO-1 TCR demonstrated objective clinical responses. Ongoing clinical trials are being carried out to evaluate TCRs that target both HLA class I and class II epitopes of the MAGEA3 CG antigen. The DNA core facility provides services to many of the members of the Surgery Branch, which includes expertise and reagents used for RT-PCR, quantatitive RT-PCR, gene cloning and RNA/DNA analysis. 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 were 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.

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。在最近的一项研究中，已经确定了几个新型点突变的基因为黑色素瘤反应性的靶标，直到继产后介导了长期临床肿瘤消退。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。鉴定出候选抗原的筛查导致在15个黑色素瘤TIL中的12个以及迄今已筛选的四个胃肠道TIL中的两个中鉴定出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。正在进行的研究集中在开发识别识别突变抗原的T细胞的策略上，以试图开发有效的收养免疫疗法，以治疗包括肺和膀胱癌在内的各种恶性肿瘤患者。与NIH RNAi联盟合作进行的研究开发了一种新的方法来鉴定人类肿瘤抗原，利用siRNA库的转染靶向20,000个人cDNA转录物，从而鉴定了潜在靶标，这些靶标可鉴定潜在靶标，以通过肿瘤反应T细胞识别其能力。其他研究涉及针对广泛共享抗原的T细胞受体（TCR）的鉴定。最近的研究集中在识别识别癌/种系（CG）抗原的TCR，在黑色素瘤中表达的分子以及多种高度普遍的恶性肿瘤，例如乳腺癌和前列腺癌。识别NY-ESO-1 CG抗原的TCR用于转导自体PBMC，并且II期临床试验的结果表明，大约50％的黑色素瘤或滑膜细胞肉瘤患者接受了用抗INNY-ESO-1 TCR转导PBMC的PBMC患者，表现出客观的临床反应。正在进行的临床试验正在进行评估MAGEA3 CG抗原I类HLA I类和II类表位的TCR。 DNA核心设施为手术分支的许多成员提供服务，其中包括用于RT-PCR，定量RT-PCR，Gene Cloning和RNA/DNA分析的专业知识和试剂。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，在BD FACSCALIBUR，FACSCANTOI，FACSCANTOII以及最近获得的LSRFortessa上进行了表型分析，并在BD Facsariaii上进行了细胞分离。