Center for Biologics Evaluation and Research, U. S. Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, MD 20993, USA.
Int J Mol Sci. 2022 Jul 23;23(15):8134. doi: 10.3390/ijms23158134.
Single-chain variable fragments (scFv) are antigen-recognizing variable fragments of antibodies (FV) where both subunits (V and V) are connected via an artificial linker. One particular scFv, iKM33, directed against blood coagulation factor VIII (FVIII) was shown to inhibit major FVIII functions and is useful in FVIII research. We aimed to investigate the properties of iKM33 enabled with protease-dependent disintegration. Three variants of iKM33 bearing thrombin cleavage sites within the linker were expressed using a baculovirus system and purified by two-step chromatography. All proteins retained strong binding to FVIII by surface plasmon resonance, and upon thrombin cleavage, dissociated into V and V as shown by size-exclusion chromatography. However, in FVIII activity and low-density lipoprotein receptor-related protein 1 binding assays, the thrombin-cleaved iKM33 variants were still inhibitory. In a pull-down assay using an FVIII-affinity sorbent, the isolated V, a mixture of V and V, and intact iKM33 were carried over via FVIII analyzed by electrophoresis. We concluded that the isolated V and V assembled into scFv-like heterodimer on FVIII, and the isolated V alone also bound FVIII. We discuss the potential use of both protease-cleavable scFvs and isolated Fv subunits retaining high affinity to the antigens in various practical applications such as therapeutics, diagnostics, and research.
单链可变片段 (scFv) 是抗体的抗原识别可变片段，其中两个亚基 (V 和 V) 通过人工接头连接。一种针对凝血因子 VIII (FVIII) 的特定 scFv，即 iKM33，被证明可以抑制 FVIII 的主要功能，在 FVIII 研究中很有用。我们旨在研究具有蛋白酶依赖性解体功能的 iKM33 的特性。使用杆状病毒系统表达了三种在接头内带有凝血酶切割位点的 iKM33 变体，并通过两步色谱法进行纯化。所有蛋白质通过表面等离子体共振保留了对 FVIII 的强结合，并且在凝血酶切割后，如通过尺寸排阻色谱法所示，解离为 V 和 V。然而，在 FVIII 活性和低密度脂蛋白受体相关蛋白 1 结合测定中，凝血酶切割的 iKM33 变体仍然具有抑制作用。在使用 FVIII 亲和吸附剂的下拉测定中，通过电泳分析，分离的 V、V 和 V 的混合物以及完整的 iKM33 通过 FVIII 被携带。我们得出结论，分离的 V 和 V 在 FVIII 上组装成 scFv 样异源二聚体，并且分离的 V 本身也结合 FVIII。我们讨论了在治疗、诊断和研究等各种实际应用中使用既具有蛋白酶可切割性又具有高亲和力的 scFv 和分离的 Fv 亚基的潜在用途。
