IDENTIFICATION OF RESIDUES THAT STABILIZE THE SINGLE-CHAIN FV OF MONOCLONAL-ANTIBODIES B3
IDENTIFICATION OF RESIDUES THAT STABILIZE THE SINGLE-CHAIN FV OF MONOCLONAL-ANTIBODIES B3
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DOI：
10.1074/jbc.270.40.23373
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发表时间：
1995-10-06
影响因子：
4.8
通讯作者：
PASTAN, I
中科院分区：
文献类型：
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作者：
BENHAR, I;PASTAN, I
B3（Fv）-PE 38是一种重组单链免疫毒素，其中单链形式的B3抗体的Fv部分（其用作靶向部分）与PE 38融合，PE 38是假单胞菌外毒素A的截短形式，其用作细胞毒性部分。B3（Fv）-PE 38对许多人癌细胞系具有特异性细胞毒性，目前正在临床试验中进行评估。单克隆抗体B3（IgG 1 k）和B5（IgMk）识别人癌细胞上的相关碳水化合物表位。这些抗体的Fv区先前已被克隆并分别表达为单链Fv-免疫毒素B3（Fv）PE 38和B5（Fv）-PE 38。B3（Fv）-PE 38免疫毒素以比B5（Fv）-PE 38更高的亲和力结合抗原阳性癌细胞，并且是比B5（Fv）-PE 38更有效的细胞毒性剂。然而，它不太稳定，在37 ℃下孵育后迅速聚集。两个Fv的V-L结构域非常相似，仅三个残基不同，即第四和第七个Fr 1残基以及第五个CDR 1残基。两种Fv的V-H结构域差异很大。为了研究不同的V-L残基是否会影响B3（Fv）的稳定性，我们构建了含有B3 V（H）和B5 V（L）的嵌合免疫毒素。与B3（Fv）-PE 38相比，该嵌合体具有改善的稳定性和更高的表观抗原结合亲和力和细胞毒活性。位点特异性诱变用于显示V-L M4 L突变在稳定B3（Fv）中具有重要作用，尽管残基V-L Ser-7和V-L Ile-28也在增加的稳定性中起作用。当在体内模型系统中测试时，含有B3 V（H）和B5 V（L）的嵌合体在人异种移植小鼠模型中具有改善的抗肿瘤活性。这些研究表明，通常使用简并（“家族特异性”）引物克隆Fv片段可能会引入不稳定突变。
B3(Fv)-PE38 is a recombinant single-chain immunotoxin in which the Fv portion of the B3 antibody in a single-chain form, which serves as the targeting moiety, is fused to PE38, a truncated form of Pseudomonas exotoxin A, which serves as the cytotoxic moiety, B3(FV)-PE38 is specifically cytotoxic to many human cancer cell lines and is currently evaluated in a clinical trial. Monoclonal antibodies B3 (IgG1k) and B5 (IgMk) recognize related carbohydrate epitopes on human carcinoma cells. The Fv regions of these antibodies were previously cloned and expressed as the single chain Fv-immunotoxins B3(Fv) PE38 and B5(Fv)-PE38, respectively. The B3(Fv)-PE38 immunotoxin binds to antigen positive cancer cells with a higher affinity than B5(Fv)-PE38 and is a more potent cytotoxic agent than B5(Fv)-PE38. However, it is less stable and rapidly aggregates upon incubation at 37 degrees C. The V-L domains of the two Fvs are very similar, differing by only three residues, the fourth and seventh Fr1 residues and the fifth CDR1 residue. The V-H domains of the two Fvs vary considerably. To investigate whether any of the different V-L residues may influence the stability of the B3(Fv), we constructed a chimeric immunotoxin containing the B3V(H) and the B5V(L). This chimera had an improved stability and a higher apparent antigen binding affinity and cytotoxic activity when compared with B3(Fv)-PE38. Site-specific mutagenesis was used to show that the V-L M4L mutation has an important role in stabilizing B3(Fv), although residues V-L Ser-7 and V-L Ile-28 also play a role in the increased stability. When tested in an in vivo model system, the chimera containing the B3V(H) and the B5V(L) had an improved antitumor activity in a human xenograft mouse model. These studies indicate that the common use of degenerate (''family-specific'') primers to clone Fv fragments may introduce destabilizing mutations.