One strategy which is different from the above is to
One strategy which is different from the above is to isolate a similar enzyme to the one under study which will not be recognized by the antibody of the original protein. This approach will lead to prolonging an enzyme’s activity. For example, a novel variant of Carboxypeptidase G2 (CPG2), which has been used in drug detoxification and ADEPT is used in targeted therapy for cancer, especially in the ADEPT strategy mentioned above .
A leading approach to improving the half-life of a protein therapeutic is to reduce its renal clearance rate, e.g. by increasing its size above the renal cut-off of 40–50 kDa. Several ways can achieve this, including chemical and post-translational modification as well as by genetic engineering . Table 1 lists different modifications that can create favorable new features in therapeutic proteins. Two of the wildly used approaches to extend the half-life of therapeutics and improve drug delivery, are PEGylation and albumination, this review will focus on the use of the two techniques and discuss their application in cancer therapy. This part will include our recent work on the glucarpidase PEGylation and albumination.
Protein PEGylation using polyethylene glycol (PEG) Polyethylene glycol (PEG) is a neutral polyether polymer. Because it is water soluble, non-ionic and biocompatible, it is widely employed in the field of polymer-based drug delivery. PEG moieties are made from multiple units of Zoledronic Acid australia oxide that create long chains of amphiphilic inert molecules . In 1990 the FDA approved the first PEGylated product, and ever since it has been extensively used in post-production modification methodology to improve the physicochemical properties, and hence the biomedical efficacy, of therapeutic proteins. PEGylated pharmaceuticals have proven their applicability and safety over many years. Thus, PEGylation plays an essential role in prolonging the residence time in the circulation of the relatively small therapeutic drugs such as peptides, proteins, nanobodies and scaffolds, which is achieved by increasing their molecular size to above that needed for half-life extension . As indicated above, a key advantage of using PEGylated proteins is that patients require fewer doses to maintain the necessary therapeutic levels in the circulation. More recently, releasable PEG moieties have been developed that can be removed from a therapeutic protein under controlled conditions. This strategy allows administration of the protein in a pro-drug format prior to reconstitution of the native protein under appropriate conditions . A wide range of biologically important molecules have been conjugated to PEG to take advantage of its advantages (Table 1). Moreover, site-specific PEGylation offers opportunities to create novel proteins and peptides and peptides of medicinal interest . It is essential to add a functional group to the PEG at one or both termini which will enable its conjugation to a protein. By choosing the functional group judiciously, it is possible to attach PEG moieties to specific amino acid side chains or to the N-terminus of a protein (Fig. 2). PEGylation of proteins can be performed by chemically reacting a specific chemical functionalities within a protein (e.g. the side chains of lysine, histidine, arginine, cysteine, aspartic acid, glutamic acid, threonine, tyrosine, and serine as well as the N-terminal amino and the C-terminal carboxylic acid groups) with a suitable PEGylation reagent . As the degree of modification increases, the likelihood of antigenicity generally decreases whereas the circulatory half-life of the therapeutic protein is extended. Due to reactions with different nucleophilic groups on the protein, even mono-PEGylation leads to positional isomers that can differ significantly in their biological and biomedical properties mainly in body residence time and immunogenicity. However, it should be noted that conjugation might sometimes lead to the formation of new epitopes as a consequence of, e.g., partial protein denaturation after conjugation or the use of an inappropriate spacer between protein and PEG chain .