Strategies for the production of longacting therapeutics and efficient drug delivery for cancer treatment

Protein therapeutics play a significant role in treating many diseases. They, however, suffer from patient's proteases degradation and antibody neutralization which lead to short plasma half-lives. One of the ways to overcome these pitfalls is the frequent injection of the drug albeit at the cost of patient compliance which affects the quality of life of patients. There are several techniques available to extend the half-life of therapeutics. Two of the most common protocols are PEGylation and fusion with human serum albumin. These two techniques improve stability, reduce immunogenicity, and increase drug resistance to proteases. These factors lead to the reduction of injection frequency which increases patient compliance and improve quality of life. Both techniques have already been used in many FDA approved drugs. This review describes many technologies to produce long-acting drugs with the attention of PEGylation and the genetic fusion with human serum albumin. The report also discusses the latest modified therapeutics in the field and their application in cancer therapy. We compare the modification methods and discuss the pitfalls of these modified drugs.


Abstract
Protein therapeutics play a significant role in treating many diseases. They, however, suffer from patient's proteases degradation and antibody neutralization which lead to short plasma half-lives. One of the ways to overcome these pitfalls is the frequent injection of the drug albeit at the cost of patient compliance which affects the quality of life of patients.
There are several techniques available to extend the half-life of therapeutics. Two of the most common protocols are PEGylation and fusion with human serum albumin. These two techniques improve stability, reduce immunogenicity, and increase drug resistance to proteases. These factors lead to the reduction of injection frequency which increases patient compliance and improve quality of life.
Both techniques have already been used in many FDA approved drugs.
This review describes many technologies to produce long-acting drugs with the attention of PEGylation and the genetic fusion with human serum albumin. The report also discusses the latest modified therapeutics in the field and their application in cancer therapy. We compare the modification methods and discuss the pitfalls of these modified drugs.

Introduction
Proteins therapeutic can be defined as proteins that are either naturally produced in the body or created in the laboratory and introduced into the patient with the aim of improving or curing a pathological condition. They are usually acquired from either microbial cells or by genetically modifying an animal or plant, and their uses range from oncology to inflammation to infectious diseases.1 Proteins therapeutic also have the advantage that they function naturally as either pharmacokinetic or pharmacodynamic drugs, as they usually serve to replace an absent protein, and the body responds as if the protein is naturally occurring.2 Proteins often have multiple highly specific and complex functions that cannot be mimicked by simple chemical compounds. However, in common with small-molecule drugs, there are three major parameters influence their therapeutic efficacy: time (t1/2 or half-life), toxicity and targeted binding.3 The body produces many diverse proteins that are used as therapeutics. In the case of diseases caused by the mutation or deletion of a protein-coding gene, the protein therapeutic generally replaces the abnormal or missing protein in question without the need to go through gene therapy. Protein therapeutics have multiple advantages over small-molecule drugs. In particular, the clinical development and approval time of protein therapeutics by national drug approval agencies such as the Food and Drug Administration (FDA) is generally faster than that of small-molecule drugs.1 Protein therapeutics are categorized as having either an enzymatic or regulatory activity.
They can have specifications based on their pharmacological activity, in which they replace a protein that is deficient or abnormal. Alternatively, they can augment an existing pathway, provide a novel function or activity; interfere with a molecule or organism; or deliver other compounds (including other proteins), such as a radionuclide, cytotoxic drug, or effector protein. 4 The first promoted recombinant therapeutic protein was human insulin (Humulin R) which was first produced in 1982 and has become one of the best-selling biologics worldwide after FDA approval. 5 There are now multiple approved protein therapeutics, and many of these proteins have molecular mass below 50 kDa and a short terminal halflife in the range of minutes to hours.6 These limitations have led to the development and implementation of half-life extension approaches to lengthen the time that these recombinant proteins remain in the blood and to improve their pharmacokinetic properties as well.7 To achieve therapeutically effective concentration over a prolonged period of time, the drug is typically applied at a local region or subcutaneously so that it is only slowly absorbed into the bloodstream. Thus, factors such as the clearance rate, volume of circulation and the bioavailability of the therapeutic drug all influence its effective half-life.7 This review discusses some key strategies to extend the half-lives of therapeutic proteins and their applications. In particular, it focuses on two approaches, the attachment of polyethylene glycol moieties to proteins (protein PEGylation) and fusion with human serum albumin, as these are most often used and have proved especially useful.

Advantages of modified proteins over unmodified ones
In contrast to small-molecule drugs, proteins are readily amenable to site-specific alterations through genetic engineering. In principle, therefore, it is possible to build in features that allow them to remain active for longer in the body and or to improve their tolerance. These features include: resistance to proteolysis; delayed clearance; reduced capacity to cause local irritation; increased half-life; lower toxicity; increased stability and solubility, and decreased immunogenicity. 12,13 Many of protein therapeutic drugs have now been developed and approved. Many exhibit short half-lives in plasma and hence strategies to improve their pharmacokinetic properties, which influence distribution and excretion,13 are becoming increasingly important. Increasing the size and hydrodynamic radius of the protein, or peptide aims to decrease kidney filtration and to increase the net negative charge of the target protein or peptide has a similar effect, as the net charge of the protein contributes to renal filtration. It has been suggested that the proteoglycans of the endothelial cells and the glomerular basement membrane contribute to an anionic barrier, which partially prevents the passage of negatively-charged plasma macromolecules.14 Another approach is to increase the degree to which the therapeutic peptide or protein interacts with serum components, e.g. albumin or immunoglobulins, which tends to increase the half-life of the circulating targeted protein. 15

Strategies for producing long-acting protein therapeutics
Significant effort has been expended to discover different approaches to extend the halflives of protein drugs, not least by evading or interfering with their common clearance pathway. Modifications to protein drugs that prolong their half-lives include conjugation or fusion to specific moieties and the discovery of variants of the therapeutic protein drugs. 4  Table 1). 22 On the other hand, changes in structure or sequence of protein molecules (e.g. through glycosylation or PEGylation) may cause changes in the pharmacokinetic properties of these compounds. The size of a therapeutic protein may hinder its passage across a biological membrane. Other factors that affect its half-life include its immunogenicity, the level of the corresponding endogenous protein, the period of drug administration, and the rate and site of drug delivery.13 Gene modification can be used to create therapeutic proteins with altered isoelectric points and protein dynamics.23 Such mutations can also modulate both enzyme selectivity and the intrinsic activity of the enzyme. In one example, both the activity and the specificity of Neprilysin, a protease that degrades amyloid beta and hence might be of use in the treatment of Alzheimer's disease, were altered through site-specific mutagenesis.
The engineered Neprilysin double mutant G399V/G714K showed a ~20-fold increase in activity on amyloid beta 1-40 but a ~3,200-fold reduction in activity on other peptides.
Further, this therapeutic drug is therefore, a promising candidate for the in vivo treatment of Alzheimer's disease. 24 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. 10 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.7 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, nonionic and biocompatible, it is widely employed in the field of polymer-based drug delivery. PEG moieties are made from multiple units of ethylene oxide that create long chains of amphiphilic inert molecules. 44 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.45 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.46 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. 47 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 (Figure 2).  Nhydroxysuccinimidyl-activated esters, which produce an amide linkage between PEGepoxide, and PEG-aldehyde, PEG-tresylate and PEG-carbonyl imidazole, which will provide a urethane linkage. The activated PEG compound will react with one or all exposed free amino groups contained within the protein groups, with regards to steric hindrance. By regulating the concentration of the reagents whether through the protein, or reaction conditions, in reference to the standard methods of amine condensation, one can control the degree of PEGylation of the free amino groups exposed on the folded protein.
Another option is to use the thiol groups of cysteine residues, which can be modified by use of PEG-maleimide and vinyl sulfone. However, changes in PEGylation interactions or reaction conditions can result in changes in the functional properties of the therapeutic proteins. [50][51][52] A study was conducted to optimize site-specific PEGylation of Exendin-4 (Ex4-Cys), an analogue of glucagon-like peptide-1 (GLP-1) with anti-diabetic properties, using a highmolecular-weight trimeric PEG. PEGylation of the C-terminus (C40-tPEG-Ex4-Cys) was carried out using Ex4-Cys and activated trimeric PEG. The resulting C40-tPEG50K-Ex4Cys derivative had a better t1/2 in circulating blood (7.53-fold increase) and its AUCinf (a measure of total exposure to the drug) relative to Ex4-Cys was increased over 45-fold.
Further, its hypoglycemic duration, a measure of its pharmacologic activity, was increased 8-fold relative to that of native Ex4-Cys, with a dose of 25 nM/kg.52

Fusion to Human Serum Albumin
Human serum albumin (HSA) is one of the best-characterized proteins in the pharmaceutical field. It is responsible for transporting endogenous and exogenous compounds and has a long average half-life (around 19 days). In part, this is due to its size -it is around 66 kDa, which is almost at the boundary of the kidney's filtration capacity stimulating factor (G-CSF) was prolonged by genetic fusion to domain III of I to its Nterminus. 64

Diseases that have been treated with PEGylated proteins
Several PEGylated molecules have been approved for clinical use. For example, PEGylated interferon for such infections, PEG-interferon alfa-2b, was approved by the FDA in August 2001.55 Table 2 lists some PEGylated products that have received FDA approval. 8

PEGylation to improve drug delivery and targeting of cancer cells
The number of therapeutics involving drug delivery has increased markedly, especially for cancer treatment ( Table 3). 65 While most of the PEGylated products to date are nonprotein-based, the use of peptide-and protein-based PEGylated products is now being investigated. In principle, PEGylation of proteins, due to its enhanced permeability and retention (EPR) effect, is an excellent way to achieve a longer circulation time and for drug delivery to a tumor site.66 For example, a succinimide-activated PEG derivative has been used to PEGylate the εamino groups of lysine residues of xanthine oxidase, which mediates anticancer activity because of its ability to generate cytotoxic reactive oxygen species. In animal studies, this derivative exhibited 2.8-fold higher tumor accumulation at solid tumors when compared to the native enzyme in a 24 hr injection period. 65 Bispecific antibodies have been studied as a method in cancer immunotherapy, and the use of PEGylation is an effective method to improve their antitumor efficiency.
Sitespecific PEGylation has been used to modify a bispecific single-domain antibody- PEGylation can be combined with other strategies to improve drug delivery. For example, it has been used in conjunction with niosomes, i.e. non-ionic surfactant-based vesicles that can carry various drugs within them, to improve cell targeting. Niosomes are first rendered magnetic with Fe3O4@SiO2 nanoparticles prior to modifying their surface by PEGylation. In this case, the role of PEGylation increases the bioavailability of niosomes, and magnetization makes them capable of targeting specific tissues. In one application, carboplatin, an antitumor drug, was loaded into PEGylated magnetic niosomes, leading to an increased drug release rate (Figure 3). Moreover, using an external magnetic field significantly increases their toxicity towards cancerous cells.69 In addition to the use of drug encapsulation using a vesicular carrier, drugs can be delivered to a tumor site by attaching them to a drug delivery module via acid-cleavable linkers, which can be hydrolyzed in the acidic environment of the tumor. Alternatively, some other type of specialized linkage can be used that permits the drug to be released in situ within the tumor microenvironment. Thus, both pro-drug and active targeting strategies can be used.12 To minimize the loss of activity reversible PEGylation has been developed and a large number of cleavable linkages, mediated in vivo by specific enzymes, hydrolytic cleavage or reduction, have been identified. 8,70,71 The use of pH sensitive cleavable PEG has proved to be an effective approach in which cleavage of a PEG-lipid moiety is triggered in the vicinity of the tumor. In order to achieve a tumor-specific cleavable PEG system, the enzymes specifically expressed in the tumor have also been exploited for cleavage, e.g. matrix metalloproteinases (MMP).66 Another comparable example in facilitating drug delivery to tumor cells is the peptide-loaded pHsensitive PEGylation to liposomes (PEG-PpHL) which are characterized and delivered to cis-platinum resistant ovarian cancer C13 cells. The carrier entraps the drug and exhibits a pH-dependent release in the tumor site. Moreover, the PEGylated PpHL behaved differently against macrophage cells due to its ability to protect liposomes from the cells of the reticuloendothelial system.72

Diseases which have been treated with proteins linked to HAS
A number of therapeutic products conjugated to HSA have now been approved for clinical use ( Table 2) The side effects of GH therapy were reported to be rare and it was shown to have a favorable overall safety profile.76

HSA fusion to improve drug delivery and targeting of cancer cells
Fusion of therapeutic proteins to HSA has proved to be a viable and effective way to increase the solubility and/or delivery of molecules for cancer therapy.77 The physicochemical properties of HSA, which facilitate coupling to drugs, and its preferential uptake in tumor tissue make it an almost ideal carrier for drug delivery.78 In pancreatic cancer chemotherapy the gemcitabine (GEM) nanocarriers have received extensive attention in recent years. Linking HSA to GEM/IR780 resulted in a complex that had elevated levels in blood and a long-term circulation in tumor tissues when compared to the free IR780. 79 Fusion with HSA can also be used to target and inhibit essential intracellular pathways.
It has been recently reported that a fusion protein consisting of HSA linked to PEGylation is considered as one of the best approaches for passive targeting of anticancer therapeutics, based upon the concentration gradient between the intracellular and extracellular space that is created due to the high concentration of the drug in the tumor area. 8 The clearance of the PEG chain depends on its seizes. The molecule less than 400 Da would be degraded by alcohol dehydrogenases and lead to the formation of toxic metabolites. The elimination mechanism of longer PEG chains, depends on their molecular mass. PEG below 20 kDa, are eliminated by renal filtration. The PEGylated proteins conjugated with PEG molecule larger than 20kDa are cleared by different pathways such as liver uptake and degradation of the protein part by proteases. It is also the same mechanism for clearing of large protein molecules with molecular masses above anti-PEG antibodies appeared after the first dose, but 5 of the 10 responders had preexisting antibodies, even though they had not previously been exposed to PEGloticase.
In phase 3 trials, high levels of antibodies to PEGloticase was the main reason for the loss of efficacy. 91 As indicated above, the enzyme-linked immunosorbent assays (ELISA) technique is an efficient method to analyze anti-PEG antibody responses. Direct and competitive ELISAs can be used in combination to determine the PEG-specificity of Ab responses induced after treatment with a PEGylated protein (PEG-Pr), as well as pre-existing anti-PEG Abs.
Both anti-PEG IgM and IgG can bind to polymers composed of repeated subunits.89 PEG-modified recombinant mammalian urate oxidase (PEG-uricase), a treatment for patients with chronic gout, was investigated for the presence of anti-PEG antibodies. In 5 of 13 patients, low-titer IgM and IgG antibodies against PEG-uricase were detected.
This correlated with the plasma uricase activity being not detectable beyond ten days after injection. As in the other study 106, the elicited antibodies were directed against the PEG moiety rather than the uricase protein. Conversely, the relatively low titer antibodies did not inhibit uricase catalytic activity. Instead, the uricase activity was decreased due to rapid clearance of the circulating uricase. This is due to the binding of the antibody against the unabsorbed PEG-uricase at the injection site after dosing.

Effects of the HSA moiety on protein immunogenicity and stability
HSA is the only therapeutic protein that is stable as a liquid at room temperature. This stability is primarily due to the presence of 17 disulfide linkages present in the molecule.
The stability of albumin makes its storage and handling easier than typical proteins, thus making itself well suited as an excipient. The high stability of the protein also allows it to be heated at a temperature of 60°C for 10 hours, without significant denaturation, which facilitates virus inactivation during manufacturing. HSA is used as a stabilizer for proteins due to its amphiphilic properties, which makes it appropriate as an additive to prevent adsorption of the active protein, via the competitive adsorption mechanisms. HSA may also stabilize the native conformation of the active molecule, thereby helping it to maintain its bioactivity throughout the product shelf life.94 While HSA is largely non-immunogenic, the same cannot be said of proteins that bind to HSA. For example, the albumin binding domain of streptococcal protein G is of concern due to its bacterial origin. Accordingly, it has been engineered to reduce its immunogenicity by removing the T-cell epitopes. Based on the existing literature and use of in silico programs for predicting T-cell epitopes, several derivatives have been produced and one (ABD094) is currently being clinically evaluated. 95 Besides, its long serum half-life, HSA has been found to accumulate in many tumors as a result of their enhanced vascular permeability and the increased retention of albumin in tumor interstitium.96, 97 These findings have been validated by radiolabeled or dyeconjugated albumins, which have been shown to have high uptake into tumors.98 Based on this property, HSA is considered to be a suitable system for drug delivery to tumor tissue.99, 100 By implication, it is assumed that fusion proteins bearing albumin binding domains will also accumulate inside tumors following their association with HSA.
In the case of constructs such as ABD-rIL-2, this induces the recruitment of cytotoxic T cells to tumor sites. 75 Studies Currently, there is no way of predicting which fusion structure will be most effective -it is necessary to use trial and error to test different constructs and determine which is best. This last property is particularly useful in the case of PEGylated liposomes and niosomes, greatly increasing their utility for drug delivery. 47 The FDA approval of several PEGylated therapeutic proteins highlights their advantages. Some of the most important advantages are their prolonged body-residence time, which allows a drug to be administered less frequently, which arises from their increased resistance to degradative agents such as proteases or nucleases, and decreases in immunogenicity. Given these advantages, it is perhaps unsurprisingly that PEGylation has allowed the creation of blockbuster products such as Pegasys (peginterferon alfa-2a) and PEG-Intron (PEG-Intron (Peginterferon alfa-2b).16 The unusually long circulation time of HSA (19 days) has similarly encouraged researchers to use it to prolong the serum half-lives of other proteins either through genetic fusion or by chemical conjugation. 102. It has been known for some time that HSA's longevity in serum is due in large part to its electrostatic repulsion in kidneys and to FcRnmediated recycling in the endothelium. [14][15][16]103 However, it was initially unclear if fusion with HSA would increase the longevity of other proteins attached to it or if this would simply result in a decrease in HSA longevity. Fortunately, subsequent investigations proved these concerns to be largely unfounded.
In contrast to PEGylated proteins which tend to have reduced absorption in the body relative to their native counterparts, proteins conjugated to albumin tend to accumulate in certain locales in vivo. This means that albumin-based drug carrier systems have particular applications in the field of chemotherapy as they can improve the passive tumor targeting properties of anti-cancer drugs.
Proliferating tumor cells utilize albumin and other plasma proteins for their nutrition and take up albumin by fluid phase endocytosis at a greater rate than normal tissues. After lysosomal digestion, the derived amino acids serve as a source for nitrogen and energy in the tumor cells. These favorable properties make albumin an attractive choice as a drug carrier where the conjugates enjoy the same favorable tumor targeting properties as albumin itself, e.g. high tumor uptake rates, low liver uptake rates and a very long biologic half-life.
Both approaches have the ability to conjugate to proteins without comprising the critical property of the target protein. In mice, the serum half-life using the HSA and the PEG was typically around 9-and 7-fold greater, respectively, than that of the sfGFP-WT. Although the binding affinity of HSA to a mouse is much greater than that of a human, it is still much greater than that of a PEGconjugated protein in human. A disadvantage in both techniques is that the handling and chemical modification of HSA during modification can lead to slight denaturation which may generate a significant immune response.21 The hydrophobic moieties present on the polymers can bind to proteins through hydrophobic interactions (e.g., PEG with aromatic groups). Additionally, these polymers can also destabilize the native protein conformation by stabilizing unfolded protein conformations. Protein excipients, for example human serum albumin (HSA), stabilize biopharmaceuticals by competitively adsorbing to surfaces and interfaces and preventing interface induced aggregation of the drug product.55 The production of long-acting protein therapeutics using techniques such as PEGylation, and others to overcome the patient's immunogenicity has been established and covered extensively in the literature. We successfully produced two forms of long-acting glucarpidase using PEGYlation and HSA fusion with glucarpidase. The two forms produced are more resistant to proteases than the free enzyme ( Figure 5). They also less immunogenic than the free glucarpidase (  In our recent work, we established a different and new strategy to overcome the immunogenicity problem. We showed that isolation of a novel form of the protein used with different epitops could also minimize the patient immune response. We showed that the antibody of one form of the enzyme does not neutralize the other form (Figure 7). 10 We therefore proposed that the two forms of the proteins or the enzymes could be used consecutively instead of using one form of the enzyme.

Nanonization and Drug development
We discussed different strategies to overcome the immunogenicity of drugs and many problems related to drug development and their clinical applications Another major obstacle to drug development is poor water solubility of many therapeutics. This poor water solubility affects the drug bioavailability which in turn reduce its efficiency. This presents another major pitfall to their clinical applications.
Many conventional formulations to improve solubility may lead to poor pharmacokinetics and low or loss of bioavailability. Drug PEGylation, in addition to many advantages, may increase solubility. Nanonization techniques, however, offers an efficient way to overcome problems related to drug development.
Different methods of drug nanonization have been established. [104][105][106][107][108] The use of nanonization to improved drug development and drug delivery and to overcome the solubility problem have been successful in several drugs. 105,[109][110][111][112][113][114][115][116][117] In summary, the use of techniques such as drug PEGylation and fusion with human serum albumin followed by drug nanonization could solve many problems related to drug development such as immunogenicity, proteases degradation and drug bioavailability.

Conclusions
Modification of therapeutic molecules by chemical conjugation with Human serum albumin (HSA), polyethylene glycol (PEG) or other known molecules has been established to enhance the drug pharmaceutical properties. This has been shown in the successful approval of more than 12 modified drugs by the FDA. Despite the fact that the added moiety improves the pharmacologic and pharmaceutical properties of the drug, most of the adverse effects of the modified drugs are due to the active part of the medicine and not to the added moiety. The use of external moieties, such as PEG and HSA, as delivery of the drug to the cancer cell is an added advantage of the process and will pave the way for more research in this area.