双特异性 FpFs:临床前抗体开发的多功能工具。

IF 4.2 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY RSC Chemical Biology Pub Date : 2024-09-27 DOI:10.1039/D4CB00130C
Matthew Collins, Nkiru Ibeanu, Wiktoria Roksana Grabowska, Sahar Awwad, Peng T. Khaw, Steve Brocchini and Hanieh Khalili
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摘要

我们以前曾将 FpFs 1̲(Fab-PEG-Fab)描述为 IgG 的结合模拟物。FpFs 是用二(双砜)共轭试剂 3̲制备的,这种试剂会与每个 Fab 的可触及二硫化物发生二硫重桥共轭(方案 1)。我们现在制备了双特异性 FpFs 2̲(bsFpF 和 Fab1-PEG-Fab2),作为潜在的双特异性抗体模拟物,目的是将 bsFpFs 用于临床前抗体开发,因为在临床前研究中寻找双特异性抗体可能具有挑战性。二(双砜)试剂 3̲首先用于制备 bsFpF 2̲,方法是先将第一个 Fab 然后将第二个 Fab 顺序连接到另一个靶点(方案 2)。为了改进 bsFpF 的合成,我们研究了不对称共轭试剂双砜双硫醚 1̲6̲,其每个末端的硫醇共轭反应活性不同(方案 4)。为了探索在制备 bsFpF 家族时使用普通中间体的优势,我们研究了用蛋白质共轭-连接方法合成 bsFpF(方案 5)。我们研究了在 PEG 的一个末端带有用于共轭的双砜基,而在另一个末端带有连接基的试剂。双砜基 PEG 反环辛烯(TCO)2̲8̲ 和双砜基 PEG 四嗪(Tz)3̲0̲ 被用来制备针对各种治疗靶点(TNF-α、IL6R、IL17 和 VEGF)和组织亲和性靶点(透明质酸和胶原蛋白 II)的多种 bsFpF。表面等离子共振(SPR)结合研究表明,未修饰的 Fab、单共轭 PEG-Fab 和 bsFpF 中相应 Fab 的解离速率常数(k d)差别不大。bsFpF中的Fab结合速率(k a)比PEG-Fab慢,这可能是因为质量差异影响了SPR结果。这些观察结果表明,每种 Fab 都会独立于其他 Fab 与靶标结合,因此可以使用相应的 PEG-Fab 共轭物来估计 bsFpF 的结合曲线。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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Bispecific FpFs: a versatile tool for preclinical antibody development†

We previously described FpFs (Fab–PEG–Fab) as binding mimetics of IgGs. FpFs are prepared with di(bis-sulfone) conjugation reagents that undergo disulfide rebridging conjugation with the accessible disulfide of each Fab (Scheme 1). We have now prepared bispecific FpFs (bsFpF and Fab1–PEG–Fab2) as potential bispecific antibody mimetics with the intent that bsFpFs could be used in preclinical antibody development since sourcing bispecific antibodies may be challenging during preclinical research. The di(bis-sulfone) reagent was first used to prepare a bsFpF by the sequential conjugation of a first Fab and then a second Fab to another target (Scheme 2). Seeking to improve bsFpF synthesis, the asymmetric conjugation reagent, bis-sulfone bis-sulfide , with different thiol conjugation reactivities at each terminus (Scheme 4) was examined and the bsFpFs appeared to be formed at similar conversion to the di(bis-sulfone) reagent . To explore the advantages of using common intermediates in the preparation of bsFpF families, we investigated bsFpF synthesis with a protein conjugation–ligation approach (Scheme 5). Reagents with a bis-sulfone moiety for conjugation on one PEG terminus and a ligation moiety on the other terminus were examined. Bis-sulfone PEG trans-cyclooctene (TCO) and bis-sulfone PEG tetrazine (Tz) were used to prepare several bsFpFs targeting various therapeutic targets (TNF-α, IL6R, IL17, and VEGF) and tissue affinity targets (hyaluronic acid and collagen II). Surface plasmon resonance (SPR) binding studies indicated that there was little difference between the dissociation rate constant (kd) for the unmodified Fab, mono-conjugated PEG–Fab and the corresponding Fab in a bsFpF. The Fab association rate (ka) in the bsFpF was slower than for PEG–Fab, which may be because of mass differences that influence SPR results. These observations suggest that each Fab will bind to its target independently of the other Fab and that bsFpF binding profiles can be estimated using the corresponding PEG–Fab conjugates.

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发文量
128
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