INHIBITION OF TUMOR CELL GROWTH BY IMMUNOTOXINS

肿瘤选择性抗体与核糖体抑制性抗体的缀合 蛋白质（RIP）导致嵌合分子，对 靶向肿瘤细胞。 由于RIP对生物合成敏感， 细胞内的过程。由它们制成的共轭物 比化疗药物更有细胞毒性。 缀合物的能力， 抑制肿瘤细胞生长依赖于：a）结合，B） RIP从内吞囊泡的内化和c）易位 到细胞质。 这一过程被一元羧酸离子载体增强 几个数量级。 然而，离子载体不是有效的 由于增加的毒性和更快的清除， 流通 因此，开发替代方法非常重要 以提高免疫毒素活性。 我们建议作出以下规定 为了达到这一目的，毒素部分的结构变化： 白喉毒素B片段（CRM-107）的易位结构域 其与细胞的结合由于点突变而降低，B）添加 c）添加KDEL易位信号序列，和 d）掺入对应于蜂毒肽（一种蜂毒）的序列 能穿透细胞膜并形成离子通道的肽。 其他 构建体包括产生含有 细菌毒素大肠杆菌素和核糖核酸酶A这些新的嵌合毒素 分子应该具有增加的易位效率。 我们有 产生了两种新的单克隆抗体（OVX 1和OVX 2）， 卵巢癌和乳腺癌细胞的独特决定因素。 免疫缀合 将在这些抗体之间制备， 毒素 这些缀合物抑制卵巢肿瘤细胞的功效 将在体外和体内进行研究。 该项目的总体目标是 产生新的第二代免疫毒素， 卵巢上皮癌细胞

Conjugation of tumor selective antibodies to ribosomal inhibitory proteins (RIP) results in chimeric molecules that are highly toxic to target tumor cells. Since RIPs inactivate sensitive biosynthetic processes within the cell. the conjugates made from them are more cytotoxic than chemotherapeutic drugs. The ability of the conjugates to inhibit tumor cell growth is dependent on : a) binding, b) Internalization and c) translocation of RIP from the endocytotic vesicles to the cytoplasm. This process is enhanced by monocarboxylic ionophores by several orders of magnitude. However, ionophores were not efficacious in vivo due to increased toxicity and faster clearance from the circulation. Therefore, it is important to develop alternative methods to improve the immunotoxin activity. We propose to make the following structural changes in the toxin moiety to achieve this goal: a) addition of the translocation domain from Diphtheria toxin B fragment (CRM-107) which has decreased binding to cells due a point mutation, b) addition of poly-lysine , c) addition of the KDEL translocation signal sequence, and d) incorporation of sequences corresponding to melittin, a bee venom peptide which can penetrate cell membranes and form Ion channels. Other constructs include the generation of fusion proteins containing the bacterial toxin colicin and ribonuclease A. These new chimeric toxin molecules should have increased efficiency in translocation. We have generated two new monoclonal antibodies (OVX1 and OVX2) which recognize a unique determinant on ovarian and breast cancer cells. Immunoconjugates will be prepared between these antibodies and genetically engineered toxins. The efficacy of these conjugates to inhibit ovarian tumor cells will be studied in vitro and in vivo. The overall goal of the project is to generate novel, second generation immunotoxins directed to human ovarian epithelial cancer cells.