Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody-Drug Conjugates
Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody-Drug Conjugates
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DOI：
10.1021/acs.bioconjchem.5b00224
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发表时间：
2015-11-01
影响因子：
4.7
通讯作者：
van Delft, Floris L.
中科院分区：
文献类型：
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作者：
van Geel, Remon;Wijdeven, Marloes A.;van Delft, Floris L.
提出了一种健壮的、普遍适用的非基因技术，以将单抗转化为稳定和均一的ADC。从天然(非工程的)单抗开始，化学酶方案允许高度控制地将任何给定的有效载荷连接到天冬酰胺-297上的N-糖链上，基于两个阶段：首先，酶重塑(用叠氮修剪和标记)，然后基于无铜点击化学的有效载荷的连接。这项名为GlycoConnect的技术适用于任何类型的免疫球蛋白，而不受糖基化特征的影响。应用于已上市的ADC Kadcyla的两种成分曲妥珠单抗和美坦辛，显示出对GlycoConnect ADC的良好体外和体内疗效。此外，通过体内与一系列基于曲妥珠单抗的糖基化突变体的比较，证明了天然葡聚糖作为连接位点的优越性。对无铜点击探针双环芳酮(BCN)和二苯环芳香辛炔(DBCO)的并列比较表明，BCN的结合效率有惊人的差异，通过在叠氮化物上引入吸电子氟代取代可以进一步提高BCN的结合效率。由此产生的单抗结合物在所有病例中都被发现是高度稳定的，这与所展示的疗效相结合，保证了ADC具有更好的治疗指数。
A robust, generally applicable, nongenetic technology is presented to convert monoclonal antibodies into stable and homogeneous ADCs. Starting from a native (nonengineered) mAb, a chemoenzymatic protocol allows for the highly controlled attachment of any given payload to the N-glycan residing at asparagine-297, based on a two-stage process: first, enzymatic remodeling (trimming and tagging with azide), followed by ligation of the payload based on copper-free click chemistry. The technology, termed GlycoConnect, is applicable to any IgG isotype irrespective of glycosylation profile. Application to trastuzumab and maytansine, both components of the marketed ADC Kadcyla, demonstrate a favorable in vitro and in vivo efficacy for GlycoConnect ADC. Moreover, the superiority of the native glycan as attachment site was demonstrated by in vivo comparison to a range of trastuzumab-based glycosylation mutants. A side-by-side comparison of the copper-free click probes bicyclononyne (BCN) and a dibenzoannulated cydooctyne (DBCO) showed a surprising difference in conjugation efficiency in favor of BCN, which could be even further enhanced by introduction of electron-withdrawing fluoride substitutions onto the azide. The resulting mAb-conjugates were in all cases found to be highly stable, which in combination with the demonstrated efficacy warrants ADCs with a superior therapeutic index.