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

通过探针和/或细胞毒素与抗体结合的分子靶向递送代表了许多疾病的治疗和诊断的有希望的方法，包括但不限于癌症。超过30种igg型抗体已被批准用于治疗，但结合物的使用却滞后。目前产生Ab偶联物的方法通常依赖于非特异性化学反应，这种化学反应产生具有可变轮廓的修饰产物的异质混合物。由于功能性氨基酸的直接修饰，这些也会有害地改变Ab的功能。为了生成齐次共轭物，需要基于新的连接化学来研究功能的位点选择策略。Davis小组最近发现并展示了一种酶转化，利用新发现的酶的合成活性EndoS，它是高效的，位点选择性的，不像其他抗体偶联物那样靶向氨基酸侧链，而是允许附着在免疫球蛋白中发现的n -链糖基化位点上。这种生物催化活性现在允许处理以前难处理的抗体，包括真核蛋白和人源抗体。这种新的化学反应将把聚糖侧链转化为有效载荷（如标签、成像基元、功能肽或毒素）的附着位点，并允许通过聚糖调节Ab功能。有利的是，这种化学反应也将增加在该位点发现的碳水化合物混合物的纯度，从而产生更纯净的治疗产品。最后，由于糖苷键的高化学稳定性，它将在血液中产生高度稳定的键，但在细胞的溶酶体中很容易被内源性糖苷酶降解，内源性糖苷酶存在于正常降解和碳水化合物循环中。目的和目标：-生成碳水化合物试剂，允许对Abs中的聚糖进行位点选择性改变-测试这些试剂在endos催化下与代表性Abs的附着-在2种Ab改变策略中使用这些试剂：a)将有效载荷直接附着在Ab糖基化位点（直接酶促）b)在碳水化合物中产生非自然反应性官能团，可作为后续附着的“标签”（两步化学酶偶联）-使用两种策略将有效载荷（例如模型毒素或肽抗原）选择性地附着在代表性抗体（分离和重组源）上-研究和探索通过溶酶体降解碳水化合物键的释放模式糖苷酶-选择一种Ab作为有效的生物标志物靶点，并应用治疗相关Ab的方法-研究这种结构在细胞中的使用，并评估其在模型（如异种移植、体内系统）中的功效

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