INHIBITION OF TUMOR CELL GROWTH BY IMMUNOTOXINS

核糖体抑制肿瘤选择性抗体的偶联 蛋白质(RIP)导致嵌合分子对 靶向肿瘤细胞。由于RIPs使敏感的生物合成失活 细胞内的进程。由它们制成的结合物比 比化疗药物更具细胞毒性。共轭化合物的能力 抑制肿瘤细胞生长依赖于：a)结合，b) 内吞囊泡中RIP的内化和移位 进入细胞质。这一过程被单羧酸离子载体强化。 下降了几个数量级。然而，离子载体并不是有效的。 在体内由于增加的毒性和更快的清除 发行量。因此，开发替代方法非常重要。 提高免疫毒素活性。我们建议做以下几点 实现这一目标的毒素部分的结构变化：a)添加 白喉毒素B片段(CRM-107)易位结构域的研究 它由于点突变而减少了与细胞的结合，b)添加 多赖氨酸，c)添加KDEL易位信号序列，以及 D)并入蜂毒蜂毒素的相应序列 能穿透细胞膜并形成离子通道的多肽。其他 构建包括产生融合蛋白，包含 细菌毒素粘附素和核糖核酸酶A这些新的嵌合毒素 分子应该已经提高了移位的效率。我们有 产生了两种新的单抗(OVX1和OVX2)，它们识别一个 卵巢癌细胞和乳腺癌细胞的独特决定因素。免疫结合物 将在这些抗体和基因工程之间制备 毒素。这些结合物对卵巢肿瘤细胞的抑制作用 将在体外和体内进行研究。该项目的总体目标是 产生针对人类的新型第二代免疫毒素 卵巢上皮性癌细胞。

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.