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

Abstract

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.

Production of “biobetter” glucarpidase variants to improve drug detoxification and antibody directed enzyme prodrug therapy for cancer treatment

“…However, it was important to verify that the conjugated forms of CPG2 retained enzyme activityit remained possible that attachment of large additional molecules might sterically hinder access to the active site of CPG2. Surprisingly, enzyme activity studies indicate that HSA-CPG2 has slightly increased catalytic activity relative to free CPG2the V max of the former was 48.72 ±4.389 µM/min whereas unconjugated CPG2 has a V max of 24.35 ±1.91 µM/min 13 . In the case of PEGylated CPG2, the catalytic activity found to be slightly lower than that of unconjugated CPG2 (Vmax value of PEG-CPG2: 20.69 ±1.428 µM/min).…”

“…In our previous work 13 we isolated a novel glucarpidase whose raised antibodies did not cross-react with the one in clinical use. In principle, therefore, it would be possible to delay the production of antibodies in a patient by alternating the use of the two versions of CPG2.…”

“…paradoxus strain RS-16 13 . We also demonstrated that antibodies raised against the newly isolated glucarpidase do not react with the one from V. paradoxus strain RS-16.…”

In vitrostudies on CNGRC-CPG2 fusion proteins for ligand-directed enzyme prodrug therapy for targeted cancer therapy

“…Cloning of the gene encoding a His-tagged version of the CPG2 enzyme from Xenophilus azovorans SN213 has been previously described [32]. The following PCR primers were used to prepare DNA fragments encoding the single (CNGRC-CPG2) and double (CNGRC-CPG2-CNGRC) protein constructs:…”

Coupling of a Novel TIMP3 Peptide to Carboxypeptidase G2 for Pro-Drug Activation at the Tumour Site

“…A Pseudomonas aeruginosa strain RS-16-derived enzyme, carboxypeptidase G2 (CPG2 or glucarpidase), has been used in this staged therapy [ 6 ]. This is partly because its activity is not found in humans, reducing the chance of toxicity to healthy tissue, as the prodrug will be activated only by the localized exogenous enzyme.…”