ISOLATION & CHARACTERIZATION OF PEPTIDE VESICLES FROM HUMAN OPTIC NERVE

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• 批准号: 6206408
• 负责人: RICHARD E FINE
• 金额: $ 0.25万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6206408
• 关键词: biomedical resource; clinical research; human tissue; neoplasm /cancer; proteins; reproductive system; spectrometry; structural biology; technology /technique; women's health

We have developed a method for isolation, expansion, and selection of tumor-reactive T lymphocytes from either solid ovarian cancer or malignant ascites. Routinely, freshly isolated T cells are placed in anti-CD3 monoclonal antibody-coated tissue culture flasks for 48 hours. Subsequently, T cells are reactivated every 7-10 days for three times with irradiated antologous tumor cells to select for tumor-reactive T cells. After the final reactivation, T cells are tested for recognition of autologous and allogeneic tumor cells. These studies have shown that tumor-reactive T cells can indeed be expanded from almost all specimens. They also showed that therecognition of tumor cells is HLA class I-restricted and T cell receptor mediated. Further studies showed that in particular HLA-A2 is a dominant molecule in presenting tumor antigens. Since HLA-A2 is expressed by about 50% of the population, we decided to focus on HLA-A2-restricted tumor antigens. A more recent finding was that HLA-A2-matched tumors are better recognized by BLA-A2-restricted T cells than HLA-A2-negative tumors. This suggested the presence of shared tumor antigens. One of these was recently identified by us as a nine amino acid peptide derived from Her2/neu, a protooncogene that is overexpressed in 20-30% of ovarian and breast cancers. Using the now commonly used method of acid elution of HLA-bound peptides on tumor ceRs and HPLC fractionation, we have preliminary evidence that multiple shared tumor antigens exist in both ovarian and breast cancer. Thus the methodology that was earlier applied to melanoma appeared also effective in other malignancies. Therefore, we have selected tumor cell lines that express these antigens, but do not overexpress Her2/neu, to identify the nature of these common antigens. We hope that with mass spectrometry we will be able to do so. We are presently studying peptide fractions from both an ovarian and a breast cancer cell line.

我们已经开发出一种分离、扩增和选择的方法 肿瘤反应性T淋巴细胞来自实体卵巢癌或 恶性腹水 随后，将新鲜分离的T细胞置于 48个抗CD 3单克隆抗体包被的组织培养瓶 小时 随后，T细胞每7-10天重新活化， 三次与辐射的抗肿瘤细胞，以选择 肿瘤反应性T细胞 在最后一次重新激活后，T细胞 测试对自体和同种异体肿瘤细胞的识别。 这些研究表明，肿瘤反应性T细胞确实可以被 从几乎所有标本中扩增出来。 他们还表明 肿瘤细胞的识别是HLA I类限制性的，T细胞 受体介导的 进一步的研究表明，特别是HLA-A2， 是呈递肿瘤抗原的主要分子。 由于HLA-A2是 大约50%的人表示，我们决定把重点放在 HLA-A2限制性肿瘤抗原。 最近的一个发现是， BLA-A2限制性T细胞更好地识别HLA-A2匹配的肿瘤 HLA-A2阴性的肿瘤。 这表明， 共享肿瘤抗原。 其中一个最近被我们确认为 一种衍生自Her 2/neu的九个氨基酸的肽，Her 2/neu是一种原癌基因， 在20-30%的卵巢癌和乳腺癌中过度表达。 使用 现在常用的方法是用酸洗脱HLA结合的肽， 肿瘤细胞和HPLC分级，我们有初步证据表明， 在卵巢和乳腺中存在多种共有的肿瘤抗原 癌 因此，早期应用于黑色素瘤的方法 对其他恶性肿瘤也有效。 所以我们 选择表达这些抗原但不表达这些抗原的肿瘤细胞系， 过表达Her 2/neu，以鉴定这些共同抗原的性质。 我们希望通过质谱分析，我们能够做到这一点。 我们 目前正在研究来自卵巢和乳腺的肽组分， 癌细胞系