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

摘要

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.