B7 GENE TRANSFER FOR BREAST CANCER IMMUNOTHERAPY

越来越多的证据表明，许多人类肿瘤，包括乳腺癌， 表达肿瘤特异性抗原，但这些抗原的表达 这些恶性细胞表面的“外来”蛋白质不能 诱导足够的免疫反应。 尽管如此，在体外和体内， 使用分离和刺激的肿瘤浸润淋巴细胞的研究， 已经表明，免疫增强策略可以导致 有效的抗肿瘤免疫。 同样，转染的肿瘤细胞系 与淋巴因子基因如IL-2或IL-4的结合已经被高度呈现， 免疫原性。 因此，淋巴细胞不能排斥内源性 肿瘤可能与恶性细胞不能激活 辅助T细胞产生细胞因子，导致扩增， 细胞毒性T细胞和其他免疫效应物群体的活化。 T细胞的生产性刺激需要两个信号。 第一 是抗原特异性的，由T细胞受体的接合产生。 该第二信号可以通过T细胞附属物的结合来递送， 分子CD 28与其在活化的细胞表面上表达的配体B7结合， 抗原呈递细胞 其他研究表明， 传递第二信号（即，单独刺激T细胞受体） 会导致T细胞无反应性 事实上，阻断B7介导的T细胞 刺激可以导致长期器官和组织移植 生存 因此，非免疫细胞上B7表达的缺乏可能 代表了对自身抗原免疫耐受的正常机制， 当细胞发生肿瘤时， 转化并表达肿瘤抗原。 本提案的目标 检测B7基因转移在体外诱导 和对乳腺癌的体内免疫应答。 初步实验 使用低严格性筛选大鼠脾细胞cDNA文库， 鼠B7探针产生了几个阳性克隆。 具体目标 1，我们将完成这些初步的实验，并克隆一个全长 大鼠B7 cDNA。 在具体目标2中，我们将大鼠B7转染到先前的 建立大鼠原代乳腺癌细胞系，并检测其对乳腺癌细胞的杀伤能力。 当这些细胞被移植到肿瘤细胞中时， 移植到其他未经治疗的同基因受体中。 If B7基因 转移是有效的，这些研究也将确定是否B7+ 乳腺癌细胞可以诱导针对未转染细胞的反应 当后者在同一时间或在稍后的时间点移植时。 平行的体外研究将测试B7+细胞诱导 增殖性或细胞毒性T细胞反应。 具体目标3： 将人B7（先前克隆的）转染入人乳腺癌细胞 我们以前推导出的线，并确定这是否是 能够在自体淋巴细胞中诱导体外免疫应答。 在最后一个具体目标中，我们将寻求开发可靠的方法， 从人类乳腺癌中获得细胞系，因为如果B7基因转移 证明是有效的，那么最终应用于人类 治疗工具将需要从以下来源获得细胞系的能力： 乳腺癌患者。

Accumulating evidence indicates that many human tumors, including breast cancer, express tumor-specific antigens, but that the expression of these "foreign" proteins on the surface of these malignant cells fails to induce an adequate immune response. Nonetheless, in vitro and in vivo studies using isolated and stimulated tumor infiltrating lymphocytes, have indicated that immunological enhancing strategies can lead to effective anti-tumor immunity. Similarly, tumor cell lines transfected with lymphokine genes such as IL-2 or IL-4 have been rendered highly immunogenic. Thus, the failure of lymphocytes to reject endogenous tumors may be related to the inability of the malignant cells to activate helper T cells to produce cytokines leading to the expansion and activation of cytotoxic T cells and other immune effector populations. The productive stimulation of T cells requires two signals. The first is antigen-specific and results from engagement of the T cell receptor. This second signal can be delivered by binding of the T cell accessory molecule CD28 to its ligand B7 expressed on the surface of activated antigen presenting cells. Additional studies have shown that failure to deliver a second signal (i.e., stimulation of the T cell receptor alone) can lead to a T cell anergy. Indeed, blockade of B7-mediated T cell stimulation can lead to long-lasting organ and tissue transplant survival. Thus the lack of expression of B7 on non-immune cells may represent a normal mechanism of immunologic tolerance to self-antigens, a mechanism which may be undesirable when cells undergo neoplastic transformation and express tumor antigens. The goals of this proposal are to examine the efficacy of B7 gene transfer in inducing an in vitro and in vivo immune response to breast cancer. Preliminary experiments using low stringency screening of a rat splenocyte cDNA library with a murine B7 probe have yielded several positive clones. In specific aim 1, we will complete these initial experiments, and clone a full length rat B7 cDNA. In specific aim 2, we will transfect rat B7 into previously established rat primary breast carcinoma cell lines, and test the ability of this maneuver to induce anti-tumor immunity when these cells are transplanted into otherwise untreated syngeneic recipients. If B7 gene transfer is effective, these studies will also determine whether B7+ breast cancer cells can induce a response against non-transfected cells when the latter are transplanted at the same time or at a later point. Parallel in vitro studies will test the ability of B7+ cells to induce a proliferative or cytotoxic T cell response. In specific aim 3, we will transfect human B7 (previously cloned) into human mammary carcinoma cell lines which we have previously derived, and determine whether this is able to induce an in vitro immune response in autologous lymphocytes. In the last specific aim, we will seek to develop reliable methods to derive cell lines from human breast cancers, since if B7 gene transfer proves effective, then ultimate application of this as a human therapeutic tool will require the capability of deriving cell lines from breast cancer patients.