NEW THERAPEUTIC APPROACHES TO BREAST CANCER

乳腺癌是导致女性死亡的主要原因之一， 近几十年来稳步增长。在这项互动赠与中 我们提出了两个相互依存的发电和发电项目 乳腺癌新型抗独特型抗体疫苗的应用。 在研究项目1(RP 1)中，我们将启动Lb期临床试验 与最近开发的一种抗独特型抗体11D10。这 抗体代表高相对分子质量人乳脂的内在形象 球状蛋白(HMFG)主要由人类肿瘤高密度表达 乳腺癌和选定的其他癌症。这种抗原在极少数细胞中表达。 非肿瘤组织和低密度。该表位由 单抗MC-10或Bre1。以MC-10为免疫源 产生抗LD 11D10(或AB2)的抗体，或AB1。 用11D10诱导小鼠、兔和小鼠的肿瘤特异性抗体。 猴子。此外，与HMFG结合的单抗抗LDS或Ab3s 抗原是从11D10免疫的小鼠中获得的。在这上面 临床研究，我们将使用抗LD 11D10明矾沉淀物 积极免疫MC-10阳性乳腺癌患者1b期 临床试验(1-2年)。这一期临床试验的目标是 目的是：1.确定单抗的效果。 明矾沉淀对患者产生ab3(ab1‘)能力的影响 自体和/或自身特异性细胞溶解T淋巴细胞的应答 同种异体肿瘤细胞；2.确定最佳免疫调节剂量 抗LD抗体；3.监测临床反应。在 Lb阶段试验完成后，我们将在几年内开始第二阶段试验 2-3使用最佳免疫调节剂量，在第4年我们将启动 精选抗id 11D10-细胞因子融合的1b期临床试验 在研究项目II中开发和测试的蛋白质。 研究项目II(RPII)是我们将在其中进行开发的实验室分支 构建“第二代”抗独特型单抗的方法学 将RP 1中使用的11D10、Ab2、抗LD抗体连接到 细胞因子IL-2和GM-CSF。细胞因子已被证明可以增加 蛋白质抗原的免疫原性，包括独特型。我们将创建一个 将包含两种细胞因子之一的嵌合抗体11D10 连接到恒定结构域(CH3)。H链的可变外显子 1D10的基因将被克隆和测序。VH基因将被整合到 转化成表达恒定基因的质粒，融合到两个基因中 IL-2或GM-CSF/11D10杂交瘤细胞重链缺失突变体 将这些质粒导入LINE。泛函的表达 细胞因子和免疫球蛋白将在转染瘤克隆和 将选择高产无性系。这些嵌合抗体将是 在小鼠和非人类灵长类动物中作为抗独特型肿瘤疫苗进行了测试。这个 用该嵌合抗体免疫的动物体内的AB3效价将直接 与沉淀明矾免疫动物的ab3效价比较 AB2，如RP 1中所述。AB1‘也将在这些组中进行比较 关于动物的。我们预计会观察到显著更高的ab3和ab1‘滴度 在嵌合的AB2免疫动物中。我们还将比较 两组对AB2的增殖反应。如果这些临床前研究 嵌合细胞因子-Ab2s诱导高水平抗肿瘤相关抗原 在动物身上的反应，我们将启动乳房的Lb期临床试验 癌症患者。

Breast cancer is among the leading causes of death among women and has steadily increased during recent decades. In this interactive grant proposal we propose two interdependent projects for the generation and application of novel anti-idiotype antibody vaccines against breast cancer. In Research Project 1 (RP 1), we will initiate a Phase lb clinical trial with a recently developed anti-idiotype antibody, designated 11D10. This antibody represents the internal image of the high MW human milk fat globule (HMFG) primarily expressed at high density by tumors of the human breast and selected other cancers. This antigen is expressed in very few non-tumor tissues and at low density. This epitope is identified by the monoclonal antibody MC-10 or BrE1. MC-10 was used as the immunizing antibody, or Ab1, against which anti-ld 11D10 (or Ab2) was generated. 11D10 was used to induce tumor specific antibodies in mice, rabbits and monkeys. Also, monoclonal anti-anti-lds, or Ab3s, which bound to the HMFG antigen, have been obtained from mice immunized with 11D10. n this clinical study, we will use the alum precipitate of anti-ld 11D10 to actively immunize MC-10 positive breast cancer patients in a Phase lb clinical trial (years 1-2). The objectives of this Phase 1 clinical trial are to: 1. Determine the effects of a single monoclonal anti-ld antibody precipitated with alum on patients' ability to generate an Ab3 (Ab1') response to cytolytic T-lymphocytes specific for autologous and/or allogeneic tumor cells; 2. Determine the optimal immunomodulatory dose of the anti-ld antibody, and 3. Monitor clinical responses. At the completion of the Phase lb trial, we will begin a Phase II trial in years 2-3 using the optimum immunomodulatory dose, and in year 4 we will initiate a Phase lb clinical trial with the selected anti-id 11D10-cytokine fusion protein developed and tested in Research Project II. Research Project II (RP II) is the laboratory arm in which we will develop methodologies to construct "second generation" monoclonal anti-idiotype reagents linking the 11D10, Ab2, anti-ld antibody used in RP 1, to the cytokines IL-2 and GM-CSF. Cytokines have been shown to increase the immunogenicity of protein antigens, including idiotypes. We will create a chimeric antibody 11D10 which will contain either of the two cytokines attached to the constant domain (CH3). The variable exon of the H chain gene of 1D10 will be cloned ad sequenced. The VH gene will be incorporated into plasmids which express the constant gene fused to the genes for either IL-2 or GM-CSF/ The heavy chain loss mutant of the 11D10 hybridoma cell line will be transfected with these plasmids. The expression of functional cytokines and immunoglobulin will be monitored in transfectoma clones and high producing clones will be selected. These chimeric antibodies will be tested in mice and non-human primates as anti-idiotype tumor vaccines. The Ab3 titer in animals immunized with the chimeric antibody will be directly compared with the Ab3 titer in animals immunized with the alum precipitated Ab2, as described in RP 1. The Ab1' will also be compared in these groups of animals. We expect to observe a significantly higher Ab3 and Ab1' titer in the chimeric Ab2 immunized animals. We will also compare the proliferative response to Ab2 in both groups. If these preclinical studies with chimeric cytokine-Ab2s induce superior anti-tumor associated antigen responses in animals, we will initiate a Phase lb clinical trial in breast cancer patients.