Isolation and molecular characterization of novel glucarpidases: Enzymes to improve the antibody directed enzyme pro-drug therapy for cancer treatment

Repeated cycles of antibody-directed enzyme pro-drug therapy (ADEPT) and the use of glucarpidase in the detoxification of cytotoxic methotrexate (MTX) are highly desirable during cancer therapy but are hampered by the induced human antibody response to glucarpidase. Novel variants of glucarpidase (formal name: carboxypeptidase G2, CPG2) with epitopes not recognized by the immune system are likely to allow repeated cycles of ADEPT for effective cancer therapy. Towards this aim, over two thousand soil samples were collected and screened for folate hydrolyzing bacteria using folate as the sole carbon source. The work led to the isolation and the characterization of three new glucarpidase producing strains, which were designated as: Pseudomonas lubricans strain SF168, Stenotrophomonas sp SA and Xenophilus azovorans SN213. The CPG2 genes of Xenophilus azovorans SN213 (named Xen CPG2) and Stenotrophomonas sp SA (named Sten CPG2) were cloned and molecularly characterized. Both Xen CPG2 and Sten CPG2 share very close amino acid sequences (99%); we therefore, focused on the study of Xen CPG2. Finally, we demonstrated that a polyclonal antibody raised against our new CPG2, Xen CPG2, does not react with the CPG2 from Pseudomonas sp. strain RS-16 (Ps CPG2) that are currently in clinical use. The two enzymes, therefore could potentially be used consecutively in the ADEPT protocol to minimize the effect of the human antibody response that hampers current treatment with Ps CPG2. The identified novel CPG2 in this study will, therefore, pave the way for safer antibody directed enzyme pro-drug therapy for cancer treatment.

抗体导向酶前药治疗（antibody-directed enzyme pro-drug therapy, ADEPT）的重复循环，以及使用葡萄醛酸酶（glucarpidase）解毒细胞毒性甲氨蝶呤（methotrexate, MTX），在癌症治疗中极具应用价值，但会因人体对葡萄醛酸酶产生的诱导性抗体应答而受阻。可避开免疫系统识别表位的新型葡萄醛酸酶（正式名称：羧肽酶G2，CPG2）变体，有望实现ADEPT的重复循环以开展高效癌症治疗。为达成这一目标，研究团队收集了两千余份土壤样本，以叶酸作为唯一碳源，筛选可水解叶酸的细菌。此项工作成功分离并鉴定出三株新型产葡萄醛酸酶菌株，分别命名为：润滑假单胞菌（Pseudomonas lubricans）SF168、窄食单胞菌属（Stenotrophomonas sp.）SA以及偶氮食酸菌（Xenophilus azovorans）SN213。研究人员对来源于偶氮食酸菌SN213的CPG2基因（命名为Xen CPG2）与窄食单胞菌属SA的CPG2基因（命名为Sten CPG2）进行了克隆与分子表征。Xen CPG2与Sten CPG2的氨基酸序列同源性高达99%，因此研究重点聚焦于Xen CPG2。最终实验证实，针对本研究新型CPG2（Xen CPG2）制备的多克隆抗体，不会与目前临床应用的假单胞菌属菌株RS-16来源的CPG2（Ps CPG2）发生交叉反应。因此，这两种酶可依次应用于ADEPT治疗方案，以减轻当前使用Ps CPG2治疗时受人体抗体应答阻碍的问题。本研究中鉴定出的新型CPG2，将为更安全的癌症抗体导向酶前药治疗铺平道路。