Institut National de la Santé et de la Recherche Médicale, UMR_S 1176, Université Paris-Saclay, Le Kremlin-Bicêtre, France.
Institut National de la Santé et de la Recherche Médicale, UMR_S 1148, Université de Paris, Paris, France.
J Thromb Haemost. 2020 Sep;18(9):2155-2168. doi: 10.1111/jth.14940.
Protease nexin-1 (PN-1) is a member of the serine protease inhibitor (Serpin)-family, with thrombin as its main target. Current polyclonal and monoclonal antibodies against PN-1 frequently cross-react with plasminogen activator inhibitor-1 (PAI-1), a structurally and functionally homologous Serpin.
Here, we aimed to develop inhibitory single-domain antibodies (VHHs) that show specific binding to both human (hPN-1) and murine (mPN-1) PN-1.
PN-1-binding VHHs were isolated via phage-display using llama-derived or synthetic VHH-libraries. Following bacterial expression, purified VHHs were analyzed in binding and activity assays.
By using a llama-derived library, 2 PN-1 specific VHHs were obtained (KB-PN1-01 and KB-PN1-02). Despite their specificity, none displayed inhibitory activity toward hPN-1 or mPN-1. From the synthetic library, 4 VHHs (H12, B11, F06, A08) could be isolated that combined efficient binding to both hPN-1 and mPN-1 with negligible binding to PAI-1. Of these, B11, F06, and A08 were able to fully restore thrombin activity by blocking PN-1. As monovalent VHH, half-maximal inhibitory concentration values for hPN-1 were 50 ± 10, 290 ± 30, and 960 ± 390 nmol/L, for B11, F06, and A08, respectively, and 1580 ± 240, 560 ± 130, and 2880 ± 770 nmol/L for mPN-1. The inhibitory potential was improved 4- to 7-fold when bivalent VHHs were engineered. Importantly, all VHHs could block PN-1 activity in plasma as well as PN-1 released from activated platelets, one of the main sources of PN-1 during hemostasis. In conclusion, we report the generation of inhibitory anti-PN-1 antibodies using a specific approach to avoid cross-reactivity with the homologous Serpin PAI-1.
蛋白酶抑制剂-1（PN-1）是丝氨酸蛋白酶抑制剂（Serpin）家族的一员，其主要靶标为凝血酶。目前针对 PN-1 的多克隆和单克隆抗体经常与结构和功能上同源的纤溶酶原激活物抑制剂-1（PAI-1）发生交叉反应。
本研究旨在开发对人（hPN-1）和鼠（mPN-1）PN-1 均具有特异性结合的抑制性单域抗体（VHH）。
采用噬菌体展示技术，使用来自骆驼科的或合成的 VHH 文库分离与 PN-1 结合的 VHH。经细菌表达后，对纯化的 VHH 进行结合和活性分析。
通过使用骆驼科来源的文库，获得了 2 种特异性的 PN-1 VHH（KB-PN1-01 和 KB-PN1-02）。尽管具有特异性，但它们均未显示对 hPN-1 或 mPN-1 的抑制活性。从合成文库中分离出的 4 种 VHH（H12、B11、F06、A08）可与 hPN-1 和 mPN-1 高效结合，与 PAI-1 结合微弱。其中，B11、F06 和 A08 可通过阻断 PN-1 完全恢复凝血酶活性。作为单价 VHH，对 hPN-1 的半最大抑制浓度值分别为 50±10、290±30 和 960±390nmol/L，对 B11、F06 和 A08 分别为 1580±240、560±130 和 2880±770nmol/L，对 mPN-1 分别为 1580±240、560±130 和 2880±770nmol/L。当构建二价 VHH 时，抑制潜力提高了 4 至 7 倍。重要的是，所有 VHH 均可阻断血浆中的 PN-1 活性以及在止血过程中作为 PN-1 主要来源之一的活化血小板中释放的 PN-1 活性。总之，我们使用一种特定的方法生成了针对 PN-1 的抑制性抗体，从而避免与同源 Serpin PAI-1 发生交叉反应。
