TARGETING ENDOGENOUS ANTIBODIES TO OVARIAN CARCINOMA

描述（由申请人提供）： 人类和旧大陆灵长类动物自然产生大量的 识别特定半乳糖基表位GAL α 1-3GAL的抗体。 我们一直有兴趣测试这些抗Gal抗体是否可以 靶向不需要的癌细胞。具体来说，我们希望探索一个 将这些内源性抗体重定向至卵巢癌的可能性 过表达叶酸受体同种型α（FR-α）的细胞， 叶酸与半乳糖基表位的化学缀合物。最终的结果 应该是靶细胞的细胞溶解为了实现这一目标，目前 本申请涉及叶酸-二半乳糖的全合成， 偶联物和免疫活性卵巢癌模型的建立 小鼠 可介导抗Gal与FR+细胞结合的缀合物的制备 高亲合力是该项目的主要化学目标。 我们的策略是 将多个拷贝，2和4个拷贝的表位引入一个分子， 在它们之间的最佳距离。化学合成将是 在固相载体上进行。结合物将用以下试剂进行检测： FR ~+/Gal ~-人鼻咽癌KB细胞促进肿瘤细胞增殖能力的研究 抗Gal与细胞表面FR结合。相互作用的特异性 将在存在游离叶酸或游离二糖的情况下进行检测 与含有乳糖而不是GAL α 1-3GAL的缀合物一样。的 抗体结合将方便地通过FACS表征 procedure. 该项目的生物学目标是开发一种卵巢癌小鼠模型， 癌症，适用于测试我们的叶酸的抗肿瘤活性 体内缀合物。由于正常小鼠表达半乳糖基表位， 组织，我们将不得不使用α 1，3-半乳糖基转移酶敲除（GT/KO） 小鼠已知GT/KO小鼠产生与人类相同的抗Gal。我们计划 将从这些小鼠培养物中收获的卵巢上皮细胞转化为 肿瘤形成细胞系，我们最近开发的程序。 然后用编码全长FR-α的鼠cDNA转染它们。 最后，将这些GAL-/FR+细胞引入健康小鼠的腹膜中。 GT/KO小鼠。 我们目前的卵巢癌免疫治疗方法是独一无二的， 使用的是天然的内源性抗体免疫调节剂在这 应用的所有小分子都具有MW < 3 kDa，使得药代动力学 最有利于腹膜腔持续活动的特性 减少潜在的副作用。

DESCRIPTION (provided by applicant): Humans and Old World primates naturally produce a significant amount of antibodies which recognize a particular galactosyl epitope, GAL alpha 1-3GAL. We have been interested in testing if these anti-Gal antibodies can be targeted to undesirable cancerous cells. Specifically we wish to explore a possibility of redirecting these endogenous antibodies to ovarian carcinoma cells which overexpress folate receptor isotype alpha (FR-alpha) by means of chemical conjugates of folic acid to the galactosyl epitope. The end result should be the cytolysis of the target cell. Towards this goal, the present application is concerned with the total synthesis of the folate-digalactose conjugates and development of an ovarian cancer model in immune competent mice. Preparation of the conjugate which can mediate anti-Gal binding to FR+ cells with high avidity is the main chemistry goal of the project. Our strategy is to introduce multiple copies, 2 and 4 copies, of the epitopes to one molecule of folic acid at an optimal distance between them. Chemical synthesis will be carried out on a solid-phase support. The conjugates will be tested with FR+/Gal- human nasopharyngeal carcinoma KB cells for their ability of promoting the anti-Gal binding to FR on the cell surface. Specificity of the interaction will be tested in the presence of free folic acid or free disaccharide as well as with a conjugate that contains lactose instead of GAL alpha 1-3GAL. The antibody binding will be conveniently characterized by means of FACS procedure. The biological goal of this project is to develop a mouse model of ovarian cancer that is suitable for testing anti-tumor activity of our folate conjugates in vivo. Since normal mice express the galactosyl epitopes in their tissue, we will have to use alpha 1,3-GALactosyltransferase-knockout (GT/KO) mice. It is known that GT/KO mice produce anti-Gal as in humans. We plan to transform the ovarian epithelial cells harvested from these mice in culture to tumor-forming cell lines following a procedure we have recently developed. They will be then transfected with murine cDNA encoding full length FR-alpha. Finally these GAL-/FR+cells will be introduced into peritoneum of healthy GT/KO mice. Our current approach to immunotherapy of ovarian cancer is unique in that we are using naturally occurring endogenous antibodies. Immune modulators in this application are all small molecules with MW < 3 kDa, rendering pharmacokinetic properties most favorable for sustained activity in peritoneal cavity as well as reduced potential side effects.