Zapata G, Ridgway J B, Mordenti J, Osaka G, Wong W L, Bennett G L, Carter P
Department of Process Sciences, Genentech Inc., South San Francisco, CA 94080-4990, USA.
Protein Eng. 1995 Oct;8(10):1057-62. doi: 10.1093/protein/8.10.1057.
We developed a novel bivalent antibody fragment, the linear (L-) F(ab')2, comprising tandem repeats of a heavy chain fragment VH-CH1-VH-CH1 cosecreted with a light chain. Functional humanized L-F(ab')2 directed against p185HER2 was secreted from Escherichia coli at high titer (> or = 100 mg/l) and purified to homogeneity. The L-F(ab')2 binds two equivalents of antigen with an apparent affinity (Kd = 0.46 nM) that is within 3-fold of the corresponding thioether-linked F(ab')2 fragment. The N-terminal site binds antigen with an affinity (Kd = 1.2 nM) that is approximately 4-fold greater than that for the C-terminal site, as shown by the comparison of L-F(ab')2 variants containing a single functional binding site. L-F(ab')2 has greater antiproliferative activity than the thioether-linked F(ab')2 against the p185HER2-overexpressing tumor cell line BT474. Linear and thioether-linked F(ab')2 have very similar pharmacokinetic properties in normal mice, and their serum permanence times are respectively 7- and 8-fold longer than the corresponding Fab fragment. L-F(ab')2 offers a facile route to bivalent antibody fragments that are potentially suitable for clinical applications, and that may have improved biological activity compared with thioether-linked F(ab')2 fragments.
我们研发了一种新型二价抗体片段，即线性（L-）F（ab'）2，它由与轻链共同分泌的重链片段VH-CH1-VH-CH1的串联重复序列组成。针对p185HER2的功能性人源化L-F（ab'）2以高滴度（≥100 mg/l）从大肠杆菌中分泌出来，并纯化至同质。L-F（ab'）2以明显亲和力（Kd = 0.46 nM）结合两个当量的抗原，该亲和力在相应硫醚连接的F（ab'）2片段的3倍以内。如含有单个功能性结合位点的L-F（ab'）2变体的比较所示，N端位点以亲和力（Kd = 1.2 nM）结合抗原，该亲和力比C端位点大约高4倍。L-F（ab'）2对过表达p185HER2的肿瘤细胞系BT474具有比硫醚连接的F（ab'）2更强的抗增殖活性。线性和硫醚连接的F（ab'）2在正常小鼠中具有非常相似的药代动力学特性，它们在血清中的存留时间分别比相应的Fab片段长7倍和8倍。L-F（ab'）2为二价抗体片段提供了一条简便途径，这些片段可能适用于临床应用，并且与硫醚连接的F（ab'）2片段相比可能具有改善的生物学活性。
