Evaluation of Two Chemoenzymatic Glycan Remodeling Approaches to Generate Site-Specific Antibody-Drug Conjugates.
Evaluation of Two Chemoenzymatic Glycan Remodeling Approaches to Generate Site-Specific Antibody-Drug Conjugates.
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DOI：
10.3390/antib12040071
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发表时间：
2023-11-03
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Fc-糖基特异性抗体-药物缀合代表了制备位点特异性抗体-药物缀合物（ADC）的有希望的方向。在本研究中，我们进行了系统的评价，两个内切糖苷酶催化的化学酶糖工程技术，以制备糖位点特异性ADC。在前两步方法中，将抗体去糖基化，然后用修饰的完整N-聚糖恶唑啉再糖基化。在第二种一锅法中，将抗体去糖基化并同时用官能化二糖恶唑啉糖基化。为了综合评价，我们首先优化并放大了叠氮聚糖恶唑啉的制备。之后，我们证明了一锅法聚糖重塑方法对所有IgG亚类都有效。随后，我们使用两种技术路线，用两种不同的接头-有效载荷组合组装各自的ADCS，并进行全身性体外和体内评价。所有制备的ADC都实现了高均一性，并在具有最小聚集体的缓冲液中表现出优异的稳定性，以及在大鼠血清中的优异稳定性。所有ADC均显示出对BT-474乳腺癌细胞的有效杀伤。转到小鼠研究，由两种技术途径制备的ADC在BT-474异种移植模型中显示出有效且相似的功效，其与由随机缀合产生的FDA批准的ADC相当。这些ADC还表现出优异的安全性，并且在测试剂量下不会引起体重减轻。
Fc-glycosite-specific antibody–drug conjugation represents a promising direction for the preparation of site-specific antibody–drug conjugates (ADCs). In the present research, we conducted a systemic evaluation of two endoglycosidase-catalyzed chemoenzymatic glycoengineering technologies to prepare glycosite-specific ADCs. In the first two-step approach, the antibody was deglycosylated and then reglycosylated with a modified intact N-glycan oxazoline. In the second one-pot approach, antibodies were deglycosylated and simultaneously glycosylated with a functionalized disaccharide oxazoline. For the comprehensive evaluation, we first optimized and scaled-up the preparation of azido glycan oxazolines. Afterwards, we proved that the one-pot glycan-remodeling approach was efficient for all IgG subclasses. Subsequently, we assembled respective ADCS using two technology routes, with two different linker-payloads combinations, and performed systemic in vitro and in vivo evaluations. All the prepared ADCs achieved high homogeneity and illustrated excellent stability in buffers with minimum aggregates, and exceptional stability in rat serum. All ADCs displayed a potent killing of BT-474 breast cancer cells. Moving to the mouse study, the ADCs prepared from two technology routes displayed potent and similar efficacy in a BT-474 xenograft model, which was comparable to an FDA-approved ADC generated from random conjugation. These ADCs also demonstrated excellent safety and did not cause body weight loss at the tested dosages.