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发展。尽管我们在癌症生物学方面的知识有了显著的提高，并成功地批准了几种新的靶向治疗方法，但在肿瘤学中，长期疾病控制或治愈仍然是例外，而不是规则。此外，目前使用的抗肿瘤药物具有高度的细胞毒性，治疗往往与严重的副作用有关。特异性识别肿瘤细胞上存在的分子结构的药剂将有利于靶向向肿瘤细胞输送药物。最近，抗体-药物结合物(ADC)领域有了指数级的增长，即单抗(MAb)与高度细胞毒药物相连。ADC生产中的一个主要挑战是获得高度均一的药物-抗体比(DAR)和确定的连接位置。具有相对特异性细胞毒性的肿瘤靶向抗体正在成为治疗某些肿瘤的一线药物，例如过度表达表皮生长因子受体(EGFR)的肿瘤。该联合项目结合了比勒菲尔德大学邻近系的三个小组，他们拥有生物化学(Dierks：甲酰甘氨酸生成酶)、生物技术(Müler：通过细胞展示优化酶、抗体工程)和生物有机/生物偶联化学(Sewald：肽合成、药物偶联物)方面的专业知识，以开发和优化一个强大的酶系统，用于应用两种不同的C(α)-甲酰甘氨酸生成酶对模型多肽和抗体进行双重生物正交衍生物衍生。针对EGFR的抗体将在CHO细胞中与人C(α)-甲酰甘氨酸生成酶FGE共表达。通过合理的设计以及定向进化(使用细胞展示选择技术)将提高酶的活性。在通过定位新生成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是一种针对不同序列标签的原核生物C(α)-甲酰甘氨酸生成酶。第二个甲酰基将用于生物正交引入第二个不同的部分(聚乙二醇残基或其他类型的药物)。这种方法将导致抗体与两种具有不同作用模式或其他功能的药物结合，例如用于摄取和分布研究。获得的ADC将用不同EGFR表达水平的细胞进行细胞毒性测试。

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.