Benhar I, Pastan I
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Protein Eng. 1994 Dec;7(12):1509-15. doi: 10.1093/protein/7.12.1509.
The mAbs B1 (IgG1 kappa) and B5 (IgM kappa) recognize carbohydrate epitopes on human carcinoma cells. The Fv regions of these antibodies were separately cloned from hybridoma RNA using reverse transcription and PCR with oligonucleotide primers designed according to the amino acid sequences of the N-termini. The Fv regions also provide sequences for translation initiation in Escherichia coli (Fr1 oligos) and sequences of the constant region of the heavy and light domains (CH1 or C-kappa oligos). Following the determination of the DNA sequence of the Fvs, primers were designed according to the 3' ends of the VH and VL domains. These also provided for a peptide linker at the C-terminus of the VH and a short connector at the C-terminus of the VL (Fr4 oligos). The VH and VL were then each PCR-amplified using their corresponding Fr1 and phosphorylated Fr4 oligos. The resulting PCR products were annealed as 'mutagenic primers' to a uracil-containing single-stranded template obtained from an expression plasmid encoding a single-chain immunotoxin in which the B3 single-chain Fv is fused to a truncated form of Pseudomonas exotoxin (PE). Thus, the B1 and B5 variable domains replaced their corresponding B3 domains in the expression plasmid by 'variable domain shuffling' without subcloning. The resulting B1(Fv)-PE38 and B5(Fv)-PE38 were expressed in E. coli and purified to near homogeneity. Both show specific cytotoxicities to human carcinoma cell lines, but B1(Fv)-PE38 is much more active, reflecting its higher affinity to the target cells.
单克隆抗体B1（IgG1 κ）和B5（IgM κ）可识别人类癌细胞上的碳水化合物表位。利用逆转录技术从杂交瘤RNA中分别克隆这些抗体的Fv区域，并使用根据N端氨基酸序列设计的寡核苷酸引物进行PCR扩增。Fv区域还提供了大肠杆菌中的翻译起始序列（Fr1寡核苷酸）以及重链和轻链恒定区的序列（CH1或C-κ寡核苷酸）。在确定Fv的DNA序列后，根据VH和VL结构域的3'端设计引物。这些引物还在VH的C端提供了一个肽接头，在VL的C端提供了一个短连接子（Fr4寡核苷酸）。然后分别使用它们相应的Fr1和磷酸化的Fr4寡核苷酸对VH和VL进行PCR扩增。将所得的PCR产物作为“诱变引物”与从编码单链免疫毒素的表达质粒获得的含尿嘧啶的单链模板退火，其中B3单链Fv与铜绿假单胞菌外毒素（PE）的截短形式融合。因此，通过“可变结构域改组”，B1和B5可变结构域在表达质粒中取代了它们相应的B3结构域，而无需亚克隆。所得的B1（Fv）-PE38和B5（Fv）-PE38在大肠杆菌中表达并纯化至接近均一。两者对人癌细胞系均显示出特异性细胞毒性，但B1（Fv）-PE38活性更高，这反映了其对靶细胞的亲和力更高。
