Chemoenzymatic Site-Selective Attachment to Antibodies: Using Glycosylation Sites to Post-Translationally Position Cargo and to Program Function

化学酶位点选择性附着抗体：使用糖基化位点翻译后定位货物并编程功能

• 批准号: 1812345
• 金额: --
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1812345
• 关键词: Chemoenzymatic; Site; Selective; Attachment; Antibodies

Targeted delivery of molecules through conjugation of probes and/or cytotoxics to antibodies represents a promising approach to therapy and diagnosis for many diseases, including but not limited to cancer. More than 30 IgG-type antibodies have been approved for therapy yet the use of conjugates has lagged behind. Current methods for generating Ab conjugates typically rely on non-specific chemistries, which create heterogeneous mixtures of modified products with variable profiles. These can also alter Ab function detrimentally, due to direct modification of functional amino acids. Site-selective strategies based on new linking chemistries to study function are needed in order to generate homogenous conjugates.The Davis Group have recently discovered and demonstrated an enzymatic transformation using the newly-discovered synthetic activity of the enzyme EndoS that is highly efficient, site-selective and does not target the amino acid side chains as with other antibody conjugate but instead allows attachment to the N-linked glycosylation site that is found in immunoglobulins. This biocatalytic activity now allows processing of previously refractory antibodies including eukaryotic proteins and antibodies from human sources. This novel chemistry will convert the glycan side chain to a site for attachment of payloads, such as labels, imaging motifs, functional peptides or toxins and allow the modulation of Ab function via glycans. Advantageously, this chemistry will also increase the purity of the mixtures of carbohydrates found at this site creating purer therapeutic product. Finally, it will create a linkage that is highly stable in blood due to the high chemical stability of glycosidic linkages but will be readily degraded in the lysosome of cells by endogenous glycosidases, which are present for normal degradation and carbohydrate recycling. Aims and objectives:- Generate carbohydrate reagents that will allow the site-selective alteration of glycans in Abs- Test EndoS-catalyzed attachment of these reagents to representative Abs- Use these reagents in 2 Ab alteration strategies: a) direct attachment of payload to Ab glycosylation site (direct enzymatic)b) creation of an unnatural reactive functional group in the carbohydrate that can be used as a 'tag' for subsequent attachment (two-step chemoenzymatic conjugation)- Site-selectively attach payloads (e.g. model toxins or peptide antigens) using both strategies to representative antibodies (isolated & recombinant sources)- Study & explore release modes through degradation of carbohydrate linkages by lysosomal glycosidases- Selection of an Ab for a validated biomarker target & application of methods to that therapeutically-relevant Ab- The study of the use of this construct in cellulo and evaluation of efficacy in model e.g., xenograft, in vivo systems

通过将探针和/或细胞毒素缀合至抗体来靶向递送分子代表了治疗和诊断许多疾病（包括但不限于癌症）的有前景的方法。超过30种IgG型抗体已被批准用于治疗，但缀合物的使用已经落后。目前用于产生Ab缀合物的方法通常依赖于非特异性化学，其产生具有可变特征的修饰产物的异质混合物。由于功能性氨基酸的直接修饰，这些也可以间接地改变Ab功能。需要基于新的连接化学的位点选择性策略来研究功能，以产生同质缀合物。Davis Group最近发现并证明了使用新发现的酶EndoS的合成活性的酶转化，位点选择性的，并且不像其他抗体缀合物那样靶向氨基酸侧链，而是允许连接到N-在免疫球蛋白中发现的连接的糖基化位点。这种生物催化活性现在允许加工以前难处理的抗体，包括真核蛋白和来自人源的抗体。这种新的化学反应将聚糖侧链转化为用于连接有效载荷（例如标记、成像基序、功能肽或毒素）的位点，并允许通过聚糖调节Ab功能。有利地，该化学过程还将增加在该位点发现的碳水化合物混合物的纯度，从而产生更纯的治疗产品。最后，由于糖苷键的高化学稳定性，它将产生在血液中高度稳定的键，但在细胞的溶酶体中容易被内源性糖苷酶降解，内源性糖苷酶存在用于正常降解和碳水化合物再循环。目的和目标：-生成允许Ab中聚糖位点选择性改变的碳水化合物试剂-检测这些试剂与代表性Ab的EndoS催化连接-在2种Ab改变策略中使用这些试剂：a）将有效载荷直接连接至Ab糖基化位点（直接酶促）B）在碳水化合物中产生非天然反应性官能团，其可用作随后连接的“标签”（两步化学酶结合）-位点选择性连接有效载荷（例如模型毒素或肽抗原）使用这两种策略对代表性抗体（分离和重组来源）-研究和探索通过溶酶体糖苷酶降解碳水化合物键的释放模式-为经验证的生物标志物靶标选择Ab &方法在治疗相关Ab上的应用-在细胞中使用该构建体的研究和在模型中的功效评价，异种移植，体内系统