NEW THERAPEUTIC APPROACHES TO BREAST CANCER

乳腺癌是女性死亡的主要原因之一， 近几十年来稳步增长。 在这个互动的赠款中， 我们提出了两个相互依存的项目， 抗乳腺癌的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Ib期临床试验， 最近开发的抗独特型抗体命名为11 D10。 这 抗体代表高分子量人乳脂肪的内部图像 HMFG主要在人肿瘤中高密度表达， 乳腺癌和其他癌症。 这种抗原在很少的 非肿瘤组织低密度。 该表位由 单克隆抗体MC-10或BrE 1。 以MC-10为免疫原， 抗体或Ab 1，针对其产生抗-ld 11 D10（或Ab 2）。 11 D10用于在小鼠、兔和小鼠中诱导肿瘤特异性抗体， 猴子 此外，与HMFG结合的单克隆抗-抗-Ids或Ab 3 抗原，已经从用11 D10免疫的小鼠获得。 n这 在临床研究中，我们将使用抗ld 11 D 10的明矾沉淀， 在Ib期中主动免疫MC-10阳性乳腺癌患者， 临床试验（1-2年）。 本1期临床试验的目的 1. 确定单个单克隆抗ld抗体的作用 用明矾沉淀对患者产生Ab 3（Ab 1 '）的能力的影响 对自体和/或自体免疫特异性的溶细胞性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Id抗体，和3. 监测临床反应。 在 完成Ib期试验后，我们将在几年内开始II期试验 2-3使用最佳的免疫调节剂量，在第4年，我们将开始 使用选定的抗id 11 D10-细胞因子融合物进行的lb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（RP II）是我们将开发的实验室手臂 构建“第二代”单克隆抗独特型抗体的方法学 将RP 1中使用的11 D10、Ab 2、抗Id抗体连接至 细胞因子IL-2和GM-CSF。 细胞因子已被证明可以增加 蛋白抗原的免疫原性，包括独特型。 我们将创建一个 嵌合抗体11 D10，其将含有两种细胞因子中的任一种 连接至恒定结构域（CH 3）。 H链的可变外显子 对1D 10基因进行克隆和测序。 VH基因将被整合到 转化到质粒中，所述质粒表达与以下基因融合的恒定基因： IL-2或GM-CSF/11 D10杂交瘤细胞重链缺失突变体 将用这些质粒转染细胞系。 函数表达式 将在转染瘤克隆中监测细胞因子和免疫球蛋白， 将选择高产克隆。 这些嵌合抗体将 在小鼠和非人灵长类动物中作为抗独特型肿瘤疫苗进行了测试。 的 用嵌合抗体免疫的动物中的Ab 3滴度将被直接测定。 与用明矾沉淀免疫的动物中的Ab 3滴度相比， Ab 2，如RP 1中所述。 还将在这些组中比较Ab 1 ' 动物。 我们期望观察到显著更高的Ab 3和Ab 1 '滴度 在嵌合Ab 2免疫的动物中。 我们还将比较 两组均对Ab 2产生增殖反应。 如果这些临床前研究 与嵌合的马克隆-Ab 2诱导上级抗肿瘤相关抗原 在动物中的反应，我们将在乳腺癌中启动Ib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.