Department of Early Discovery Biochemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
Protein Eng Des Sel. 2012 Mar;25(3):127-33. doi: 10.1093/protein/gzr067. Epub 2012 Jan 18.
The serine protease hepsin is highly upregulated in prostate cancer and is implicated in tumor progression. Therefore, specific inhibition of hepsin enzymatic activity by an antibody constitutes an attractive therapeutic approach. Here, we report the identification of the anti-hepsin antibody Fab25 by screening of a Fab phage display library with a restricted chemical diversity at the complementary determining regions. Hepsin with its S1 pocket occupied by 3,4-dichloro-isocoumarin was used as the 'bait' for library screening. Fab25 was highly specific and it potently inhibited hepsin activity toward a panel of synthetic and macromolecular substrates. Biochemical and enzymatic studies with synthetic substrates of variable length suggested that Fab25 acts as an allosteric inhibitor based on non-competitive inhibition kinetics. Isothermal titration calorimetric experiments showed that the high-affinity (K(D) 6.1 nM) binding of Fab25 with hepsin is enthalpically driven. Despite an unusually long CDR-H3 loop with several potential hepsin cleavage sites (Lys, Arg residues), Fab25 was not processed by hepsin. Antibody-25 should be valuable for investigating hepsin's role in cancer progression and for potential therapeutic applications. Furthermore, the herein presented phage display strategy using an active site-modified protease should be widely applicable for identifying potential allosteric anti-protease antibodies.
丝氨酸蛋白酶组织蛋白酶 H 在前列腺癌中高度上调，并与肿瘤进展有关。因此，通过抗体特异性抑制组织蛋白酶 H 的酶活性构成了一种有吸引力的治疗方法。在这里，我们通过用限定化学多样性的互补决定区筛选 Fab 噬菌体展示文库来报告抗组织蛋白酶 H 的 Fab25 抗体的鉴定。用其 S1 口袋被 3,4-二氯异香豆素占据的组织蛋白酶 H 作为文库筛选的“诱饵”。Fab25 具有高度特异性，它能够强烈抑制针对一系列合成和大分子底物的组织蛋白酶 H 活性。用不同长度的合成底物进行生化和酶学研究表明，Fab25 基于非竞争性抑制动力学作为别构抑制剂起作用。等温滴定量热实验表明，Fab25 与组织蛋白酶 H 的高亲和力（K(D) 6.1 nM）结合是由焓驱动的。尽管 CDR-H3 环异常长，有几个潜在的组织蛋白酶切割位点（Lys、Arg 残基），但 Fab25 没有被组织蛋白酶 H 切割。抗体 25 应该对研究组织蛋白酶 H 在癌症进展中的作用以及潜在的治疗应用具有重要价值。此外，本文中提出的使用活性位点修饰蛋白酶的噬菌体展示策略应该广泛适用于鉴定潜在的别构抗蛋白酶抗体。
