Benhar I, Pastan I
Laboratory of Molecular Biology, Division of Cancer Biology Diagnosis and Centers, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255, USA.
Clin Cancer Res. 1995 Sep;1(9):1023-9.
The mAb B1 (mouse IgG1 kappa) recognizes a carbohydrate epitope on human carcinoma cells (I. Pastan et al., Cancer Res., 51: 3781-3787, 1991). We have generated plasmids encoding immunotoxins in which the Fv domain of B1, either as a single-chain Fv or as a disulfide-stabilized Fv (dsFv), was fused to PE38, a truncated form of Pseudomonas exotoxin A. To compare the activities of the two types of recombinant immunotoxins, the proteins were prepared from cytoplasmic inclusion bodies produced in Escherichia coli. The immunotoxins were evaluated for stability, antigen binding, specific cytotoxicity, pharmacokinetics, and antitumor activity in a nude mouse model. Although the single-chain immunotoxin is relatively stable when incubated at 37 degreesC (t(1/2) approximately 4 h), the dsFv immunotoxin is much more stable, with no loss of activity after 8 h at 37 degreesC. The single-chain immunotoxin has a 2-fold better binding affinity and cytotoxicity toward antigen-positive cultured cells than the dsFv immunotoxin. The half-lives in the blood of mice of B1(Fv)PE38 (single-chain) and B1(dsFv)PE38 (disulfide-stabilized) are 23 and 27 min, respectively. Their therapeutic potential was evaluated in athymic nude mice bearing human epidermoid carcinoma xenografts. Both immunotoxins caused complete regressions of the s.c. (30-40 mm3) tumors when given i.v. in three doses of 0.025 mg/kg every other day. This is one-twentieth of the mouse LD50. Recombinant immunotoxins containing the B1(Fv) are 2-3-fold more potent antitumor agents than previously described immunotoxins containing the B3(Fv) (Brinkmann et al., Proc. Natl. Acad. Sci. USA, 88: 8616-8620, 1991), which also target LeY and related carbohydrates in human tumors, but have a similar toxicity in mice. Thus, their therapeutic window is 2-3-fold larger. In addition, B1(dsFv)PE38 has only a 50% decrease in the apparent binding affinity of B1(Fv)PE38, whereas B3(dsFv)PE38 has a much greater loss in antigen binding.
单克隆抗体B1（小鼠IgG1 κ）可识别人类癌细胞上的一种碳水化合物表位（I. 帕斯坦等人，《癌症研究》，51: 3781 - 3787, 1991）。我们构建了编码免疫毒素的质粒，其中B1的Fv结构域，以单链Fv或二硫键稳定化Fv（dsFv）的形式，与PE38融合，PE38是铜绿假单胞菌外毒素A的截短形式。为比较这两种重组免疫毒素的活性，从大肠杆菌产生的细胞质包涵体中制备了这些蛋白质。对免疫毒素在裸鼠模型中的稳定性、抗原结合能力、特异性细胞毒性、药代动力学和抗肿瘤活性进行了评估。虽然单链免疫毒素在37℃孵育时相对稳定（半衰期约4小时），但dsFv免疫毒素更稳定，在37℃孵育8小时后活性无损失。单链免疫毒素对抗原阳性培养细胞的结合亲和力和细胞毒性比dsFv免疫毒素高2倍。B1(Fv)PE38（单链）和B1(dsFv)PE38（二硫键稳定化）在小鼠血液中的半衰期分别为23分钟和27分钟。在携带人表皮样癌异种移植物的无胸腺裸鼠中评估了它们的治疗潜力。当每隔一天静脉注射三次，剂量为0.025 mg/kg时，两种免疫毒素都使皮下（30 - 40 mm3）肿瘤完全消退。这是小鼠半数致死量的二十分之一。含有B1(Fv)的重组免疫毒素作为抗肿瘤药物的效力比先前描述的含有B3(Fv)的免疫毒素高2 - 3倍（布林克曼等人，《美国国家科学院院刊》，88: 8616 - 8620, 1991），后者也靶向人类肿瘤中的LeY及相关碳水化合物，但在小鼠中具有相似的毒性。因此，它们的治疗窗口大2 - 3倍。此外，B1(dsFv)PE38的表观结合亲和力仅比B1(Fv)PE38降低50%，而B3(dsFv)PE38的抗原结合损失则大得多。
