Maria L Jaramillo, Traian Sulea, Yves Durocher, Mauro Acchione, Melissa J Schur, Anna Robotham, John F Kelly, Marie-France Goneau, Alma Robert, Yuneivy Cepero-Donates, Michel Gilbert
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引用次数: 1
摘要
有效的合成抗体-药物偶联物(adc)的工艺需要:1)有效载荷的位点特异性结合,以避免与目标表位的结合受到干扰;2)最佳的药物/抗体比例,以获得足够的效力,同时避免聚集或溶解度问题;3)均质产品,以方便监管机构的批准。在传统adc中,药物分子随机附着在抗体表面残基(通常为赖氨酸或胱氨酸)上,这可能干扰表位结合和靶向,导致整体产物异质性、长期胶体不稳定和不利的药代动力学。在这里,我们提出了一种更可控的生成adc的过程,其中药物通过功能化唾液酸在一个狭窄的比例范围内特异性地偶联到Fab n -链聚糖。利用细菌唾液转移酶,我们将n -叠氮多乙酰神经氨酸(Neu5NAz)掺入西妥昔单抗的Fab聚糖中。由于只有约20%的人IgG1具有Fab聚糖,我们通过使用分子模型在其他治疗性单克隆抗体的Fab恒定区引入n -糖基化位点,扩展了该方法的应用。我们使用曲妥珠单抗作为模型,将Neu5NAz纳入我们设计的新型Fab聚糖中。adc是通过点击掺入的Neu5NAz生成的,该Neu5NAz与单甲基aurisatin E (MMAE)连接在以二苯并环辛(DBCO)为末端的自溶连接体上。通过这一过程,我们获得了西妥昔单抗- mmae和曲妥珠单抗- mmae,药抗比在1.3 - 2.5之间。我们证实这些adc仍然有效地结合它们的靶标,并且在细胞毒性试验中与通过标准偶联方案获得的对照adc一样有效。与Fab聚糖的定点偶联具有避免对依赖于Fc聚糖结构的效应函数的潜在干扰的额外好处。
A glyco-engineering approach for site-specific conjugation to Fab glycans.
Effective processes for synthesizing antibody-drug conjugates (ADCs) require: 1) site-specific incorporation of the payload to avoid interference with binding to the target epitope, 2) optimal drug/antibody ratio to achieve sufficient potency while avoiding aggregation or solubility problems, and 3) a homogeneous product to facilitate approval by regulatory agencies. In conventional ADCs, the drug molecules are chemically attached randomly to antibody surface residues (typically Lys or Cys), which can interfere with epitope binding and targeting, and lead to overall product heterogeneity, long-term colloidal instability and unfavorable pharmacokinetics. Here, we present a more controlled process for generating ADCs where drug is specifically conjugated to only Fab N-linked glycans in a narrow ratio range through functionalized sialic acids. Using a bacterial sialytransferase, we incorporated N-azidoacetylneuraminic acid (Neu5NAz) into the Fab glycan of cetuximab. Since only about 20% of human IgG1 have a Fab glycan, we extended the application of this approach by using molecular modeling to introduce N-glycosylation sites in the Fab constant region of other therapeutic monoclonal antibodies. We used trastuzumab as a model for the incorporation of Neu5NAz in the novel Fab glycans that we designed. ADCs were generated by clicking the incorporated Neu5NAz with monomethyl auristatin E (MMAE) attached to a self-immolative linker terminated with dibenzocyclooctyne (DBCO). Through this process, we obtained cetuximab-MMAE and trastuzumab-MMAE with drug/antibody ratios in the range of 1.3 to 2.5. We confirmed that these ADCs still bind their targets efficiently and are as potent in cytotoxicity assays as control ADCs obtained by standard conjugation protocols. The site-directed conjugation to Fab glycans has the additional benefit of avoiding potential interference with effector functions that depend on Fc glycan structure.
期刊介绍:
mAbs is a multi-disciplinary journal dedicated to the art and science of antibody research and development. The journal has a strong scientific and medical focus, but also strives to serve a broader readership. The articles are thus of interest to scientists, clinical researchers, and physicians, as well as the wider mAb community, including our readers involved in technology transfer, legal issues, investment, strategic planning and the regulation of therapeutics.