Twofold bio-orthogonal derivatization of antibody fragments by two different C(alpha)-formylglycine generating enzymes for generation of antibody-drug conjugates

肿瘤治疗是一个重要的研究目标，并继续推动新型生物药物的开发。尽管我们在癌症生物学方面的知识有了显着的提高，并且成功批准了几种新型靶向疗法，但长期疾病控制或治愈仍然是肿瘤学中的例外而不是规则。此外，目前使用的抗肿瘤药物是高度细胞毒性的，并且治疗通常与严重的副作用相关。特异性识别肿瘤细胞上存在的分子结构的药剂将有利于将药物靶向递送至肿瘤细胞。最近，抗体-药物缀合物（ADC）领域出现了指数增长，其中单克隆抗体（mAb）与高细胞毒性药物连接。ADC生产中的主要挑战是达到具有均匀药物-抗体比（DAR）和确定的缀合位点位置的高均一性。具有相对特异性细胞毒性的肿瘤靶向抗体正在成为一些肿瘤（例如表皮生长因子受体（EGFR）过表达肿瘤）的一线治疗。该联合项目将比勒费尔德大学邻近部门的三个小组与生物化学专业结合起来（Dierks：甲酰甘氨酸生成酶），生物技术（Müller：通过细胞展示、抗体工程进行酶优化）和生物有机/生物缀合物化学（Sewald：肽合成，药物偶联物），以便开发和优化用于双重生物-使用两种不同的C（α）-甲酰甘氨酸生成酶对模型肽和抗体进行正交衍生。针对EGFR的抗体将与人C（α）-甲酰甘氨酸生成酶FGE在CHO细胞中共表达。酶活性将通过合理设计以及定向进化（使用细胞展示选择技术）来改善。在通过寻址新产生的甲酰基连接第一部分（小分子药物或荧光团）后，抗体将在体外被AtsB进一步转化，AtsB是一种靶向不同序列标签的原核C（α）-甲酰基甘氨酸生成酶。第二个甲酰基将用于生物正交引入第二个不同的部分（PEG残基或另一种类型的药物）。这种方法将导致具有两种药物的抗体缀合物，所述两种药物具有不同的作用模式或其他功能，例如用于摄取和分布研究。将使用具有不同EGFR表达水平的细胞在细胞毒性测定中测试获得的ADC。

Tumor therapy is an important research objective and continues to drive the development of novel biopharmaceuticals. Despite a remarkable improvement of our knowledge in cancer biology and the successful approval of several novel targeted therapies, long term disease control or cure remain the exception rather than the rule in oncology. Moreover, currently used anti-tumor drugs are highly cytotoxic and therapies often are associated with severe side effects. Pharmaceutical agents that specifically recognize molecular structures present on tumor cells would be advantageous to target the delivery of a drug to a tumor cell. Recently, there has been an exponential growth in the field of antibody-drug conjugates (ADCs), where a monoclonal antibody (mAb) is linked to a highly cytotoxic drug. A major challenge in ADC production is to reach a high homogeneity with a uniform drug-antibody ratio (DAR) and a defined location of conjugation site. Tumor-targeting antibodies with relative specific cell toxicity are emerging as first line treatments for some tumors, for example Epidermal Growth Factor Receptor (EGFR) overexpressing tumors. The joint project combines three groups from neighboring departments at Bielefeld University with expertise in biochemistry (Dierks: formylglycine-generating enzymes), biotechnology (Müller: enzyme optimization by cell display, antibody engineering), and bioorganic/bioconjugate chemistry (Sewald: peptide synthesis, drug conjugates) in order to develop and optimize a robust enzymatic system for the twofold bio-orthogonal derivatization of model peptides and antibodies applying two different C(alpha)-formylglycine generating enzymes. Antibodies directed against EGFR will be co-expressed with the human C(alpha)-formylglycine generating enzyme FGE in CHO cells. Enzyme activity will be improved by rational design as well as by directed evolution (using cell display selection technology). After ligation of a first moiety (small-molecule drug or fluorophore) by addressing the newly generated formyl group, the antibody will be further converted in vitro by AtsB, a prokaryotic C(alpha)-formylglycine generating enzyme targeting a different sequence tag. The second formyl group will be used for bio-orthogonal introduction of a second, different moiety (a PEG residue or another type of drug). This approach will lead to antibody conjugates with two drugs having different modes of action or other functionalities, e.g. for uptake and distribution studies. Obtained ADCs will be tested in cytotoxicity assays using cells with different EGFR expression levels.