ISOLATION & CHARACTERIZATION OF PEPTIDE VESICLES FROM HUMAN OPTIC NERVE

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• 批准号: 6345213
• 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: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6345213
• 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.

我们开发了一种分离、扩展和选择的方法 来自实体卵巢癌或卵巢癌的肿瘤反应性T淋巴细胞 恶性腹水。通常，新鲜分离的T细胞被放置在 48个抗CD3单抗包被的组织培养瓶 几个小时。随后，T细胞每7-10天重新激活一次 用三次照射的自体肿瘤细胞进行筛选 肿瘤反应性T细胞。在最终重新激活后，T细胞 对自体和同种异体肿瘤细胞进行识别测试。 这些研究表明，肿瘤反应性T细胞确实可以 从几乎所有的标本扩展而来。他们还表明， 肿瘤细胞的识别是HLAI类限制性T细胞 受体介导的。进一步研究表明，尤其是人类白细胞抗原-A2 是呈递肿瘤抗原的主要分子。由于人类白细胞抗原A2是 大约50%的人表达了，我们决定把重点放在 人类白细胞抗原A2限制性肿瘤抗原。最近的一项发现是 BLA-A2限制性T细胞能更好地识别HLA-A2相合的肿瘤 细胞数高于人类白细胞抗原A2阴性肿瘤。这表明， 共同的肿瘤抗原。其中之一最近被我们确认为 来自Her2/neu的九个氨基酸多肽，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 目前常用的酸洗脱法对人白细胞抗原结合多肽进行洗脱。 肿瘤CER和高效液相分离，我们有初步证据表明 卵巢和乳腺中都存在多种共有的肿瘤抗原 癌症。因此，早期应用于黑色素瘤的方法 似乎对其他恶性肿瘤也有效。因此，我们有 表达这些抗原但不表达这些抗原的选定肿瘤细胞系 过表达Her2/neu，以鉴定这些常见抗原的性质。 我们希望通过质谱学，我们将能够做到这一点。我们是 目前正在研究从卵巢和乳房中提取的多肽 癌细胞系。