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  • One of the therapeutic proteins used in cancer


    One of the therapeutic proteins used in cancer therapy is glucarpidase, also known as Carboxypeptidase G2, CPG2, which originates from the bacterium Variovorax paradoxus (old name, Pseudomonas sp. strain RS-16). It has no mammalian equivalent (Thompson et al., 1994; Kamlage, 1994) and is a zinc-dependent dimeric protein with two subunits of 41 kDa.(Minton et al., 1983; Kalghatgi and Bertino, 1981) Glucarpidase has a relatively restricted specificity and hydrolyzes the C-terminal glutamic (S)-(+)-Dimethindene maleate receptor residue of folic acid and folate analogues such as methotrexate.(Sherwood et al., 1985) The mechanism of action of glucarpidase is, therefore, to lower systemic methotrexate levels by rapidly causing methotrexate to be converted to glutamate and 4‑deoxy‑4‑amino‑N 10-methylpteroic acid (DAMPA), both of which undergo hepatic metabolism. The enzyme can also be used in a targeted cancer therapy technique known as Antibody Directed Enzyme Prodrug Therapy (ADEPT), which has already been implemented for cancer treatment.(Bagshawe et al., 1988) ADEPT consists of two steps (Fig. 1), which result in the production of a powerful cytotoxic drug only in the vicinity of the tumor. In the first step, a tumor-selective antibody is chemically linked to an enzyme and then administered intravenously to the patient. The second step includes the injection of a non-toxic drug precursor (Prodrug). The enzyme, which accumulates at the tumor site via the tumor-specific antibody, converts the prodrug into an active drug. This therapy, therefore, produces a powerful cytotoxic drug in the vicinity of the tumor with little toxicity elsewhere in the patient body. One of the enzymes that has been used in the ADEPT is the glucarpidase from V. paradoxus strain RS-16, which when applied has been shown to result in antitumor activity in different types of cancer.(Sharma et al., 2005; Martin et al., 1997; Bagsgawe et al., 1995; Rappold and Bacher, 1974) We recently isolated a new glucarpidase that shares 94% amino acid identity with the one produced by V. paradoxus strain RS-16.(Rashidi et al., 2018) We also demonstrated that antibodies raised against the newly isolated glucarpidase do not react with the one from V. paradoxus strain RS-16. In this work, we report the production of long-acting variants of our glucarpidase to overcome the complications related to the multiple cycles of ADEPT. A number of strategies have been developed to address the issue of immunogenicity and to improve the pharmacokinetics of protein and peptide therapeutics. These include PEGylation, i.e. the attachment of polyethylene glycol (PEG) polymer chains, fusion with human serum albumin (HSA), fusion with non-structured polypeptides, and fusion with the constant fragment (Fc) domain of a human immunoglobulin (Ig)G.(Schellenberger et al., 2009) In this study, we focused our work on the production of ‘biobetter’ glucarpidases by using PEGylation (PEG) and fusion with the human serum albumin (HSA). PEGylation technology has already been used successfully to produce long-acting proteins. For example, PEGylated forms of interferon α2b and interferon α2a, which are known commercially as Pegintron and Pegasys respectively, have been used for the treatment of patients with melanoma and hepatitis B. Similarly, a PEGylated version of granulocyte colony-stimulating factor has been used for the treatment of chemotherapy-induced neutropenia.(Herndon et al., 2012; Barnard, 2001; Maullu et al., 2009) Human serum albumin, which has a circulation half-life of nineteen days (Peters Jr, 1985), has also been used to extend the half-life of biopharmaceuticals and to maintain their bioactivity.(Ru et al., 2016; Kim et al., 2013) Protein therapeutics that have been improved using this strategy include vascular endothelial growth factor,(Muller et al., 2007) interferon,(Tian et al., 2013; Zhao et al., 2009) and interleukin‑2.(Melder et al., 2005)