Development of a high‐affinity antibody‐binding peptide for site‐specific modification.
Development of a high‐affinity antibody‐binding peptide for site‐specific modification.
复制标题
开发用于位点特异性修饰的高亲和力抗体结合肽。
作者：
Muguruma;K.;Osawa;R.;Fukuda;A.;Ishikawa;N.;Fujita;K.;Taguchi;A.;Takayama;K.;Taniguchi;A.;Ito;Y.;Hayashi;Y.
免疫球蛋白G（IgG）结合肽（如15-IgBP）是抗体位点特异性修饰和制备均质抗体-药物偶联物的方便工具。肽如15-IgBP可以通过Lys 8的β-氨基以亲和力依赖性方式选择性地与人IgG的片段可结晶区交联。之前，我们发现肽15-Lys 8Leu具有高亲和力（Kd=8.19 nM），这是由于Leu 8中存在γ-二甲基基团。 然而，与抗体交联所需的伯氨基已经丢失。在这里，我们报告了一种新的非天然氨基酸，4-（2-氨基乙基氨基甲酰基）亮氨酸（Aecl）的设计和合成，它具有γ-二甲基片段和一个伯氨基。合成了含有Aecl 8的肽（15-Lys 8Aecl），并且显示出比15-IgBP（Kd =267 nM）高10倍的结合亲和力（Kd =24.3 nM）。 在Aecl 8的侧链处具有N-羟基琥珀酰亚胺酯的异硫氰酸黄绿素（FITC）标记的15-Lys 8Aecl（FITC-15-Lys 8Aecl（OSu））成功地用荧光团标记了抗体（曲妥珠单抗，Herceptin®）。这种肽支架具有很强的结合亲和力和交联能力，并且可以是用感兴趣的分子（例如药物）选择性化学修饰抗体的有用工具。
Immunoglobulin G (IgG)‐binding peptides such as 15‐IgBP are convenient tools for the site‐specific modification of antibodies and the preparation of homogeneous antibody–drug conjugates. A peptide such as 15‐IgBP can be selectively crosslinked to the fragment crystallizable region of human IgG in an affinity‐dependent manner via the ϵ‐amino group of Lys8. Previously, we found that the peptide 15‐Lys8Leu has a high affinity (Kd=8.19 nM) due to the presence of the γ‐dimethyl group in Leu8. The primary amino group required for the crosslinking to the antibodies has, however, been lost. Here, we report the design and synthesis of a novel unnatural amino acid, 4‐(2‐aminoethylcarbamoyl)leucine (Aecl), which possesses both the γ‐dimethyl fragment and a primary amino group. A peptide containing Aecl8 (15‐Lys8Aecl) was synthesized and showed a binding affinity ten times higher (Kd=24.3 nM) than that of 15‐IgBP (Kd=267 nM). Fluorescein isothiocyanate (FITC)‐labeled 15‐Lys8Aecl with anN‐hydroxy succinimide ester at the side chain of Aecl8 (FITC‐15‐Lys8Aecl(OSu)) successfully labeled an antibody (trastuzumab, Herceptin®) with the fluorophore. This peptide scaffold has both strong binding affinity and crosslinking capability, and could be a useful tool for the selective chemical modification of antibodies with molecules of interest such as drugs.