On June 23, 2022, Huang Wei and Tang Feng, Associate Researchers at the Shanghai Institutes for Drug Research, Chinese Academy of Sciences, published online in the journal Angewandte Chemie International Edition a research paper entitled “A Traceless Site-Specific Conjugation on A Traceless Site-Specific Conjugation on Native Antibodies Enables Efficient One-Step Payload Assembly”.
The team developed a novel ligand-directed acylation reagent based on a thioester structure that automatically releases the redundant ligand structure while modifying the site-specific lysine of the antibody, enabling the first “one-step”, “no-trace” strategy for the chemical preparation of an Antibody-Drug Conjugate (ADC). This is the first “one-step” and “no-trace” strategy for the preparation of ADCs by chemical means.
Huang Wei and Tang Feng are the corresponding authors of the paper, and Zeng Yue, a PhD student at the Shanghai Institute of Pharmaceutical Sciences, is the first author.
Antibody-Drug Conjugate (ADC) is a complex formed by covalently coupling a small molecule cytotoxic drug to an antibody to achieve targeted delivery of the small molecule drug using the specificity of the antibody to achieve selective tumour killing. In recent years, it has been found that quantitative coupling of small molecule drugs to specific sites of antibodies to form targeted ADC drugs has a better therapeutic index and is gradually becoming the focus of research and development in the field of ADC drugs. However, due to the complex amino acid composition of antibodies, targeted coupling of small molecule drugs by chemical means has been challenging. One potential approach is ligand-directed targeted coupling, but the difficulty of redundant ligand release or the complexity of the release process limits the application of this technique in targeted ADC drug development.
图1：基于配体导向技术的定点ADC制备方法
Recently, a novel ligand-directed acyl transfer reagent was developed by the team of researcher Wei Huang and associate researcher Feng Tang at the Shanghai Institute of Pharmaceutical Sciences, Chinese Academy of Sciences, enabling the one-step preparation of targeted ADC drugs. The reagent uses a thioester structure as the “acyl” donor, and under the guidance of the ligand, the lysine at the specific site of the Fc domain attacks the thioester structure and undergoes an “S-to-N” acyl transfer reaction, forming an amide bond and releasing the ligand structure with sulfhydryl groups. This is a “one-step” approach to the targeted coupling and ligand release of small molecule drugs.
The team first screened several common active ester forms and found that a flexible thioester fragment could be ligand-directed to achieve quantitative azide modifications of natural antibodies, specifically adding two azide groups. However, the team found that the transfer efficiency was significantly reduced after replacing the azide moiety with a larger biotin fragment, requiring a longer incubation time to achieve the coupling of the 2 biotin. The team then identified from the crystal structure of the ligand-antibody Fc structural domain that the position where the amino acid at position 10 of the Fc-binding peptide is located has a larger spatial structure to accommodate a larger compound fragment, and therefore deduced that attaching the thioester structure at this site would facilitate the reaction. The mass spectrometry results showed that the ligand-thioester-biotin coupling based on this site had a significantly higher reactivity, coupling two biotin molecules to the antibody within 2 hours.
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图2：Fc配体-硫酯-生物素复合物结构优化实现生物素的高效偶联
随后，该研究团队利用该技术快速地实现了天然抗体曲妥珠单抗的定点叠氮化修饰、生物素化修饰以及多种小分子药物修饰，均特异性地偶联2分子修饰，具有极高的位点选择性。同时，该技术也同样适用于利妥昔单抗、帕妥珠单抗、贝伐单抗等IgG1抗体，帕尼单抗等IgG2抗体，纳武利尤单抗等IgG4抗体，具有很好的底物普适性。
图3：新技术实现天然抗体的多样化细胞毒分子修饰
The team then used the technique to rapidly achieve targeted azide modifications of the natural antibody trastuzumab, biotinylated modifications and a variety of small molecule drug modifications, all specifically coupled to 2 molecule modifications, with a very high site selectivity. The technique is also applicable to IgG1 antibodies such as rituximab, patuximab and bevacizumab, IgG2 antibodies such as panitumumab and IgG4 antibodies such as nabulizumab, with excellent substrate generality.
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图4：基于该技术的定点ADC药物体内外活性
In summary, the team has achieved a “one-step”, “no-trace” quantitative modification of natural antibody-specific lysine by designing novel acyl transfer reagents and optimising the ligand structure. This technique is the first to achieve efficient and homogeneous preparation of site-specific ADC compounds by chemical means, without the need to engineer antibodies, without relying on bio-orthogonal reactions, and compatible with a wide range of substrate structures and antibody types, providing an important technical approach for the development of site-specific ADC drugs.
Related paper info. https://doi.org/10.1002/anie.202204132
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