Roth J A, Ames R S, Byers V, Lee H M, Scannon P J
J Immunol. 1986 Mar 15;136(6):2305-10.
Monoclonal antibody 45-2D9 recognizes a 74K Mr glycoprotein determinant on a c-Ha-ras oncogene-transfected cell line (45-342). An immunotoxin was made by conjugating this antibody to the A chain of ricin toxin (RTA). The immunotoxin could mediate essentially complete inhibition of leucine and thymidine incorporation by 45-342 cells prepared as single cell suspensions from tumors grown in vivo. Addition of ammonium chloride to the culture medium potentiated this inhibition, but the magnitude of this effect was dependent on incubation time and cell concentration. The immunotoxin effects were noted at concentrations 100-fold lower than similar effects caused by unconjugated RTA, and the 45-2D9 antibody had no effect in the assay system. Immunotoxins directed against antigens not expressed by 45-342 were not effective, and the 45-2D9 immunotoxin was not specifically toxic to other transfected cells not expressing the gp74 antigen. After a 72-hr incubation, lysis of 80% of the 45-342 cells was demonstrated by trypan blue exclusion. Complete inhibition of 45-342 colony formation was achieved at 10 days with a 10(-9) M concentration of the specific immunotoxin. These results indicate that an immunotoxin with specific reactivity towards an oncogene-transformed cell can be made, and that such cells derived from fresh tumors are susceptible to immunoconjugate-mediated toxicity.
单克隆抗体45-2D9可识别c-Ha-ras癌基因转染细胞系（45-342）上一个74K相对分子质量的糖蛋白决定簇。通过将该抗体与蓖麻毒素A链（RTA）偶联制备了一种免疫毒素。这种免疫毒素能基本完全抑制由体内生长肿瘤制备的单细胞悬液中的45-342细胞掺入亮氨酸和胸苷。向培养基中添加氯化铵可增强这种抑制作用，但这种效应的大小取决于孵育时间和细胞浓度。在比未偶联的RTA产生类似效应低100倍的浓度下就能观察到免疫毒素的作用，且45-2D9抗体在该检测系统中无作用。针对45-342不表达的抗原的免疫毒素无效，且45-2D9免疫毒素对其他不表达gp74抗原的转染细胞无特异性毒性。孵育72小时后，通过台盼蓝排斥试验证明80%的45-342细胞发生裂解。在第10天，10^(-9)M浓度的特异性免疫毒素可完全抑制45-342细胞集落形成。这些结果表明，可以制备一种对癌基因转化细胞具有特异性反应性的免疫毒素，并且来自新鲜肿瘤的此类细胞对免疫偶联物介导的毒性敏感。
