Protein therapeutics versus small molecule drugs

Small molecule drugs have several advantages, including oral bioavailability, ability to reach intracellular targets, ease of manufacturing, and generally a long shelf life. These characteristics make them favorable over protein drugs in the pharmaceutical industry (see the section titled "Challenges in Pharmaceutical Translation of New Therapeutic Proteins," in this chapter). However, small molecule drugs, which typically have a molecular weight less than 1000 Daltons, have limited surface area available to contact a target protein. Furthermore, forming a favorable interaction requires the presence of a deep hydrophobic pocket in the target protein, limiting the number of potential druggable targets (Hopkins and Groom 2002; Hopkins and Groom 2003). In contrast, protein drugs are usually large in size and do not have this limitation, making them indispensable therapeutic tools for human disease treatment. By using protein therapeutics, some debilitating diseases that were previously untreatable, such as chronic renal failure, dwarfism, and infertility, are now successfully managed (Johnson-Leger et al. 2006).

Protein therapeutics have higher binding selectivity and specificity compared to small molecule drugs; therefore they can target specific steps in disease pathology. For example, before the advent of protein therapeutics, drugs used to suppress the immune system in chronic inflammatory disorders were limited to small molecule drugs, such as corticosteroids and cyclosporine A. These drugs act broadly and inhibit both protective and harmful immune responses indiscriminately, thus having serious side effects. In contrast, mAbs such as Infliximab (Remicade®, Centocor Inc.) are considered immune-modulating. Infliximab targets tumor necrosis factor-a, a key proinflammatory cytokine in the pathogenesis of chronic immune disorders, and leaves the protective immune response intact (Rutgeerts et al. 2006). In the past decades, the development of new protein therapeutics has revised the treatment paradigm of certain diseases (Flamant and Bourreille 2007; Gergely and Fekete 2007), and they are gradually replacing or supplementing small molecule drug therapies (Johnson-Leger et al. 2006; Eng 2007; Flamant and Bourreille 2007; Gergely and Fekete 2007).

Sources of Protein Therapeutics

The human body has evolved an elegant immune system that helps combat and control diseases. Insufficient, deficient, or improper action of any molecular component of the immune system results in disorders to various extents. Therefore, extrinsic modulation of the immune system using natural human immune regulators represents an appealing strategy to cure diseases, and the human genome, completely sequenced in 2003, provides a huge source for drug target mining. In fact, before the introduction of recombinant DNA technology, therapeutic proteins such as growth hormone and follicle stimulating hormone were isolated directly from the human body. With the advance of recombinant DNA technology, therapeutic proteins in the market now include recombinant antibodies, hormones, cytokines, interferons, and enzymes of human origin produced industrially in bacterial, yeast, or mammalian expression systems (Johnson-Leger et al. 2006).

In addition to human immunoregulatory proteins, many viruses are also "experts" at manipulating the human immune system to facilitate their propagation in the host. Viruses evade or subvert host immune detection and destruction by encoding and expressing a diverse array of immunomodulatory proteins, which target pathways of antibody response, cytokine-mediated signaling, and major histocompatibility complex (MHC)-restricted antigen presentation (Tortorella et al. 2000; Alcami 2003). After eons of coevolution, these virus-engineered immunomodulatory proteins have exquisite potency and specificity unrivalled by commercial pharmaceuticals, providing a powerful platform of protein therapeutics development (Lucas and McFadden 2004). Therefore, similar to the concept of human genome mining described previously, human "virome" mining has been proposed to uncover more drug candidates (Anderson et al. 2003; DeFilippis et al. 2003).

Targets of Protein Therapeutics and Modes of Action

Any molecule that has implications in the pathogenesis of a disease is a potential target for protein therapeutics. In contrast to small molecule drugs that are able to diffuse across cell membranes, protein therapeutics typically cannot traverse this cellular barrier due to their large size. Therefore, they almost exclusively target cell surface receptors or extracellular molecules. In recent years, researchers have also explored the possibility of directing protein therapeutics to intracellular targets (Stocks 2004; Bernal et al. 2007).

Broadly speaking, protein therapeutics have three different modes of action based on the pathology of a disease. First, if the disease is caused by unwanted extracellular molecules such as cell metabolites or cell lysate, enzyme therapeutics can degrade these targets. Second, if the disease is caused by a deficiency in certain proteins, such as enzymes, protein therapeutics can be used to replace them and restore an individual's health. Third, if the disease involves improper immune responses or dysregulated signaling pathways, such as chronic inflammatory diseases, autoimmune diseases, infectious diseases, and cancers, protein therapeutics act as inhibitors or activators of cell surface receptors. Among these three categories, the last one has attracted the most attention of researchers and represents an active area of research (see the sections titled "Examples of Protein Therapeutics," and "New Classes of Therapeutic Proteins under Development" in this chapter).


Protein therapeutics clearly have indisputable importance among modern pharmaceuticals. However, they remain at an early stage of development and application, and substantial improvement must be made in almost all aspects, including drug target identification, protein engineering and design, protein expression and purification, drug delivery, and marketing. Here we will focus on recent advances in protein engineering and design technologies.

Challenges in Pharmaceutical Translation of New Therapeutic Proteins

Although the human genome represents a rich source of candidate proteins for therapeutics, these proteins were not evolved for therapeutic purposes, and thus do not have optimal affinity, specificity, activity, and/or stability for disease treatment. Protein instability and immunogenicity are among the key challenges affecting the success of protein therapeutics. Proteins often have limited physical and chemical stability, which has several implications: (1) They have short half-life in the body, which in turn leads to limited efficacy and frequent dosage; (2) they are difficult to produce and have short shelf life, both of which are responsible for high cost of pharmaceutical commercialization; (3) they need to be administered through injection because they are quickly digested in the intestines if taken orally, which affects patient compliance and therapeutic outcomes (Kefalides 1998). Therefore, development of protein therapeutics with improved stability, efficacy, pharmacokinetics, pharmacodynamics, and expression productivity is required.

Safety is the priority criterion of all drugs. Immunogenicity is a unique issue associated with protein therapeutics. The human immune system responds to pathogens through recognition of their proteins or processed protein products. Similarly, patients who receive protein therapeutics can potentially develop immune responses against the protein drug, producing antidrug antibodies (Barbosa and Celis 2007; De Groot and Scott 2007). Such immune responses can reduce the efficacy of protein drugs, and in rare cases, they can lead to life-threatening situations (Barbosa and Celis 2007; De Groot and Scott 2007). Therefore, an immunogenicity assessment is essential to protein therapeutics development.

Strategies for Designing Effective Protein Therapeutics

To address the issues in protein therapeutics development described previously, various protein engineering and design strategies have been developed, including protein post-translational modification, protein fusions, and genetic engineering, as illustrated in Figure 7.1.

strategies for improving Pharmacokinetics

The efficacy of a therapeutic protein in the human body can be improved by a number of strategies, including fusions, glycosylation, and chemical modification. Recombinant DNA technology enables the fusion of a protein of interest to an endogenous human protein (for example, human serum albumin, an antibody Fc fragment, and transferrin), thereby increasing the effective size of the protein and reducing clearance in the kidneys, which occurs for proteins below a molecular weight of approximately 70,000 Daltons (Caliceti and Veronese 2003; Beals and Shanafelt 2006). Fusing a protein to an antibody Fc fragment affords a second benefit: This takes advantage of the interaction between Fc and the FcRn receptor, which protects IgG antibodies in the body by allowing them to be released into the plasma rather than degraded in the lysosome, a natural "recycling" system (Lobo et al. 2004).

Glycosylation, the term for decoration of a protein's surface with carbohydrates, also increases protein size, thus reducing renal clearance. In addition, glycosylation can further help enhance protein solubility, stabilize against damage from heat and free radicals, and shield a protein from proteolysis and immune surveillance, ultimately resulting in enhanced serum half-life (Sinclair and Elliott 2005). Targeted introduction of glycosylation motifs into the sequence of a therapeutic protein is termed glycoengineering. Production of recombinant protein by mammalian cell

Genetic engineering: #

• Rational design

• Semi-rational design

• Directed evolution • Random mutagenesis

• Family shuffling

• Family shuffling

Protein fusions:

• Transferrin

Post-translational modifications:

Glycosylation PEGylation

FIGuRE 7.1 (see color insert following page 178) Strategies for designing effective protein therapeutics. StEP = staggered extension process, ITCHY = incremental truncation for the creation of hybrid enzymes, SHIPREC = sequence homology-independent protein recombination (see Chapter 4 for a detailed description of each method).

Protein fusions:

• Transferrin

Post-translational modifications:

Glycosylation PEGylation

FIGuRE 7.1 (see color insert following page 178) Strategies for designing effective protein therapeutics. StEP = staggered extension process, ITCHY = incremental truncation for the creation of hybrid enzymes, SHIPREC = sequence homology-independent protein recombination (see Chapter 4 for a detailed description of each method).

culture, particularly Chinese hamster ovary (CHO) cells, is used industrially to synthesize glycosylated therapeutic proteins. The use of CHO cell culture for protein production does have drawbacks, namely significantly higher cost versus culture of bacteria or yeast. It is also possible that CHO cell cultures can harbor viral or prion contamination, and the glycosylation by CHO cells can be heterogeneous, leading to therapeutic proteins with a range of efficacies (Sethuraman and Stadheim 2006).

Finally, chemical modification by PEGylation, the conjugation of polyethylene glycol (PEG), is a common strategy to enhance the serum half-life of protein therapeutics (Beals and Shanafelt 2006). The increased size of the protein-PEG conjugate reduces clearance from the kidneys, and the bulky PEG molecule also protects therapeutic proteins from degradation by proteases via steric hindrance (Veronese and Pasut 2005). However, PEGylation also suffers from several limitations. Low molecular weight proteins are especially susceptible to partial or complete inactiva-tion after conjugation of a PEG molecule (Shechter et al. 2008). Further, interference of protein-protein binding is beneficial vis-à-vis proteases, but reduces the effectiveness of antibody-based protein therapeutics or those acting through a receptor (Kubetzko et al. 2005; Shechter et al. 2008). This has led to the development of "reversible PEGylation," where the conjugated PEG molecule can be considered a prodrug, and undergoes spontaneous hydrolysis under physiological conditions to release the active therapeutic protein (Shechter et al. 2008).

Strategies for Reducing immunogenicity

In general, reduction of immunogenicity involves altering protein therapeutics such that they can avoid immune surveillance. This includes avoidance of antibodies, binding to antigen presenting cell (APC) surface receptors leading to receptor-mediated endocytosis, subsequent proteolysis to peptide fragments that bind to MHC class II molecules, and, finally, avoidance of binding by B and T cell receptors (Chirino et al. 2004). Many strategies are available to reduce immunogenicity and are similar to techniques used to improve pharmacokinetics. PEGylation, which possesses other useful properties as discussed previously, is nontoxic and reduces immunogenicity and antigenicity (Caliceti and Veronese 2003). Specifically, the PEG molecule shields immunoreactive sites on recombinant proteins from recognition by antibodies or surface receptors (Caliceti and Veronese 2003). PEG also enhances solubility, which prevents the accumulation of highly immunogenic protein aggregates (Chirino et al. 2004). Conjugated PEG also deters proteolysis, which may help PEGylated therapeutics to avoid cleavage into peptides capable of display on MHC class II molecules.

Glycosylation, which helps to enhance the serum half-life of therapeutic proteins, is also thought to interfere with antibody binding (De Groot and Scott 2007). Ideally, glycosylation should be of a human pattern for greatest effectiveness (Brooks 2006). This has led to considerable interest in humanizing the glycosylation pathways of the organisms currently used to express recombinant proteins, and fully humanized, sialylated glycoproteins can be produced from the yeast Pichia pastoris (Hamilton et al. 2003). Changes to the primary sequence of a therapeutic protein can also help reduce immunogenicity. The humanization of murine and mouse-human chimeric antibodies (i.e., the removal of as much nonhuman content from the constant and variable regions as possible) helps to reduce the formation of human antimouse antibodies (De Groot and Scott 2007). In addition, identifying and eliminating antibody and T-cell epitopes and class II MHC agretopes are strategies currently employed to reduce the immunogenicity of the next generation of protein therapeutics (Chirino et al. 2004).

Genetic Engineering

Genetic engineering strategies, consisting of three broad categories—rational design, directed evolution, and semirational design—have long been valuable tools in engineering proteins with altered physical and chemical properties and/or creating novel functions. In the context of protein therapeutics, these well-established methods are facing new challenges due to consideration of additional engineering parameters including pharmacokinetics, pharmacodynamics, and immunogenicity as described in the section in this chapter titled "Challenges in Pharmaceutical Translation of New Therapeutic Proteins." Numerous rational and semirational design strategies have been developed (detailed in other chapters in this volume) and applied to the engineering of protein therapeutics. Rational or computation design methods have been applied to improve stability and solubility, or to predict and to reduce immu-nogenicity of protein therapeutics (reviewed in Marshall et al. 2003; Rosenberg and Goldblum 2006; De Groot and Moise 2007). The primary drawback of rational design is the requirement for knowledge of protein structure, mechanisms, and protein structure-function relationships to a certain extent. In contrast, directed evolution does not have this limitation because it creates molecular diversity at the DNA level in a stochastic manner (see Chapter 4). This also leads to the key challenge of directed evolution: how to find the variant with desired property in a library of up to a billion variants. Therefore, high-throughput selection or screening methods are highly desirable for directed evolution. A variety of library selection and screening methods have been developed for different applications (reviewed in Arnold and Georgiou 2003). For each directed evolution experiment, the selection or screening method must be prudently chosen or developed, because the first principle of directed evolution is "you get what you select (screen) for."

Among the library selection and screening approaches, display technologies have been increasingly used in therapeutic protein engineering, and have proved to be especially powerful for engineering protein drugs for improved affinity and specificity. The shared principle of different display technologies is to create a physical linkage between the genotype and the protein displayed on the platform, so that a library of target protein variants is directly accessible to binding analysis and thus selectable and recoverable for further engineering. By using surface display, in vitro affinity maturation of an antibody yielded variants with the highest affinity reported (femtomolar range, which is orders of magnitude beyond natural antibodies) (Boder et al. 2000), and new classes of therapeutic proteins are being developed (see the section titled "New Classes of Therapeutic Proteins under Development," in this chapter). Over the past decades, a number of display platforms have been developed, including phage display, cell surface display, and cell-free display (see Chapters 1-3). These different platforms have advantages and disadvantages that make them more conducive to certain protein engineering applications.

Phage display was the earliest developed platform and has since been utilized most often for protein engineering (Sergeeva et al. 2006). Recent advances have enabled selection of phage libraries in more complex biological systems, such as cultured cells and in vivo (Sergeeva et al. 2006). Although phage display has been successfully used for engineering of peptides, antibodies, and for epitope mapping, it has achieved limited success with more complex human membrane proteins such as MHC and T cell receptors (TCRs) (see the section titled "New Classes of Therapeutic Proteins under Development," in this chapter). This is because the bacterial host required for phage propagation has limited ability in terms of protein folding and post-transla-tional modifications that are important for mammalian protein functions.

A cell surface display library is usually generated by transforming cells with DNA variants and screening for mutants with a desired phenotype by fluorescence activated cell sorting (FACS). FACS enables high-throughput enrichment of positive clones in a quantitative manner, but is not applicable for phage display libraries due to the small size of phage particles (Georgiou et al. 1997; Boder and Wittrup 1998). Several different cell types have been explored for their ability to display protein libraries, including bacteria, yeast, insect, and mammalian cells. Among these platforms, yeast display has attracted the most attention. Yeast display has the advantage of possessing post-translational processing pathways, which enable folding and gly-cosylation of complex human proteins (Kondo and Ueda 2004). Starting with the same library, yeast display was shown to sample the immune antibody repertoire considerably more fully than phage display, selecting twice as many novel antibodies as phage display (Bowley et al. 2007). Studies have also shown that the surface display level of a protein on yeast cell surface is strongly correlated with both thermal stability and soluble expression level (Shusta et al. 1999).

Cell-free display (also known as in vitro display) represents an emerging technology that has proven useful for discovery and engineering of therapeutic proteins with high affinity (FitzGerald 2000; Rothe et al. 2006). For example, ribosome (polysome) display, which was the first cell-free system developed for complete in vitro protein engineering, has been shown to generate antibodies with higher affinities (picomolar range) than those obtained from a phage display library (nanomolar range) (Groves et al. 2006). The biggest advantage of cell-free display methods is that the transcription and/or translation steps completely take place in vitro, abolishing the need of introducing DNA into host cells, which often limits the library size accessible to other display approaches. Library sizes created by in vitro display platforms are usually several orders of magnitude higher than that obtained with other display methods (up to 1014-1015) (FitzGerald 2000). In addition, the cell-free feature of in vitro display methods might make them more amenable to automation, potentially allowing ultra high-throughput identification of new drug targets on a genomic level (FitzGerald 2000).

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