IMMUNOTOXIN AND ONCOTOXIN THERAPY OF CANCER CELLS

假单胞菌外毒素(PE)或转基因形式的PE已被 与单抗或生长因子相结合以产生 细胞特异性细胞毒剂。突变的PE分子在哪个结构域I 已经被生长因子、CD4或单链抗体所取代 通过基因融合创造了结合区域，嵌合蛋白 在大肠杆菌中生产，并提纯到接近均一的水平。我们有 构建并研究了以下嵌合毒素的活性： 转化生长因子α-PE40、IL-2-PE40、IL-4-PE40、IL-6-PE40、IGF1-PE40、酸性成纤维细胞生长因子-PE40、 CD4-PE40、抗转铁蛋白(Fv)-PE40和抗转铁蛋白(Fv)-PE40。转化生长因子α-PE40 通过EGF受体杀死细胞的药物现在已被证明具有 腹腔注射抗肿瘤药物对小鼠的抗肿瘤作用 抗肿瘤和抗皮下肿瘤。IL-2-PE40对 用IL2受体杀伤小鼠和大鼠细胞，但对 灵长类和人类细胞。为了克服这一缺陷，单一的链条 构建了免疫毒素抗Tac(Fv)-PE40。 对含有IL2受体的人和灵长类细胞的细胞毒作用，包括 直接从成人T细胞白血病患者体内分离的细胞。 IL6-PE40及其变异体IL6-PE664GIu对几种骨髓瘤有细胞毒作用 细胞系和肝癌细胞系；最少有400个受体的细胞 每个细胞都可以被杀死。因为肿瘤依赖于新的血液 补充，我们构建了具有细胞毒性的酸性成纤维细胞生长因子-PE40和PE664Glu 对携带成纤维细胞生长因子受体的细胞。已经联合检测了CD4-PE40 与AZT联合使用，并显示出协同作用以阻止艾滋病毒的传播 培养中的感染。一种单抗，B3，与许多 结肠癌、乳腺癌、肺癌和卵巢肿瘤已被分离，这些基因 克隆了可变区的编码，并制成了单链免疫毒素。 B3(Fv)-PE40具有较强的抗肿瘤作用 裸鼠，目前正在进行临床前开发。 具有更高活性的假单胞菌外毒素突变体是由 将羧基末端从REDLK改为KDEL。这些分子是 对靶细胞的细胞毒性要高出两到十倍。延长笔记时间 用免疫毒素治疗，这种毒素具有很强的免疫原性， 免疫抑制剂15脱氧精瓜灵已被研究，该药物 能抑制小鼠PE的一次抗体形成。已修改 插入外源多肽的PE克隆已被构建 进入PE的移位结构域，从而将这些插入引入 靶细胞的胞浆。

Pseudomonas exotoxin (PE) or genetically modified forms of PE have been attached to monoclonal antibodies (mAbs) or growth factors to create cell-specific cytotoxic agents. Mutant PE molecules in which domain I has been replaced by growth factors, CD4 or single chain antibody combining regions have been created by gene fusion, the chimeric proteins produced in E. coli, and purified to near homogeneity. We have constructed and studied the activity of the following chimeric toxins: TGFalpha-PE40, IL2-PE40; IL4-PE40, IL6-PE40, IGF1-PE40, acidic FGF-PE40, CD4-PE40, antiTac(Fv)-PE40 and antitransferrin(Fv)-PE40. TGFalpha-PE40 which kills cells with EGF receptors has now been shown to have an antitumor effect in mice when injected I.P. against intraperitoneal tumors and against subcutaneous tumors. IL2-PE40 is very effective in killing mouse and rat cells with IL2 receptors but is less active against primate and human cells. To overcome this deficiency, a single chain immunotoxin anti-Tac(Fv)-PE40 was constructed which is extremely cytotoxic to human and primate cells containing IL2 receptors including cells directly isolated from patients with adult T cell leukemia. IL6-PE40 and a variant, IL6-PE664GIu, is cytotoxic to several myeloma cells lines and hepatoma cell lines; cells with as few as 400 receptors per cell can be killed. Because tumors are dependent on a new blood supply, we constructed acidic FGF-PE40 and PE664Glu which are cytotoxic to FGF receptor bearing cells. CD4-PE40 has been tested in combination with AZT and shown to act synergistically to arrest the spread of HIV infection in culture. A monoclonal antibody, B3, reactive with many colon, breast, lung and ovary tumors has been isolated, the genes encoding the variable regions cloned and a single chain immunotoxin made. B3(Fv)-PE40 has a strong antitumor effect against human tumors growing in nude mice and is currently undergoing preclinical development. Pseudomonas exotoxin mutants with increased activity have been created by changing the carboxy terminus from REDLK to KDEL. These molecules are two to ten-fold more cytotoxic to target cells. To pen-nit prolonged therapy with immunotoxins which are very immunogenic, the effect of an immunosuppressive agent, 15deoxyspergualin, has been studied and the drug shown to suppress primary antibody formation to PE in mice. Modified clones of PE have been constructed with foreign polypeptides inserted into the translocating domain of PE which introduces these inserts into the cytosol of target cells.