Commercial Applications for Enzyme-Mediated Protein Conjugation

Review pubs.acs.org/CR

Commercial Applications for Enzyme-Mediated Protein Conjugation: New Developments in Enzymatic Processes to Deliver Functionalized Proteins on the Commercial Scale Erika M. Milczek* Curie Company, New York, New York 10012, United States ABSTRACT: The field of protein conjugation most commonly refers to the chemical, enzymatic, or chemoenzymatic formation of new covalent bonds between two polypeptides, or between a single polypeptide and a new molecule (polymer, small molecule, nucleic acid, carbohydrate, etc.). Due to the modest selectivity of chemical methods for protein conjugation, there are increased efforts to develop biocatalysts that confer regioselectivity for site-specific modification, thereby complementing the existing toolbox of chemical conjugation strategies. This review summarizes key advances in the use of enzymes to functionalize proteins with commercial relevance. The examples put forth have demonstrated value at the industrial level or show promising industrial potential in the laboratory.

CONTENTS 1. Introduction 2. Overview of Protein Conjugation Reactions 2.1. Oxidoreductases 2.2. Transferases 2.3. Peptidases 2.4. Ligases 3. Nonpharmaceutical Applications of Enzymatic Protein Conjugation 3.1. Food Processing and Edible Packaging 3.1.1. Restructuring Meat with Enzymes 3.1.2. Dairy Production with Enzymes 3.1.3. Baking with Enzymes 3.1.4. Edible Films 3.2. Biomaterials and Biofabrication 3.3. Textiles and Leather Manufacturing 3.4. Promising Applications for Commercialization 3.4.1. The Future of Biomaterials 3.4.2. The Future of Biocatalysts and Fusion Proteins 3.5. Pros and Cons of Enzymatic Cross-Linking Strategies 4. Pharmaceutical Applications of Enzymatic Protein Conjugation 4.1. Antibody−Drug Conjugates and Immunoradiolabeling 4.1.1. Protein−Cytotoxin Conjugates 4.1.2. Protein Carbohydrate Remodeling 4.2. Drug Delivery and Biopharmaceuticals 4.2.1. PEGylation 4.2.2. Hyperglycosylation 4.3. Promising Preclinical Applications 4.3.1. Sortase A 4.3.2. Transglutaminase © 2017 American Chemical Society

4.3.3. Tyrosinase 5. Conclusion Author Information Corresponding Author ORCID Notes Biography Acknowledgments Abbreviations References

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1. INTRODUCTION With an ever-expanding need for proteins in commercial applications,1 tools for protein modification are in high demand. Proteins, peptides, and enzymes have found their way into consumers’ lives through packaged foods, therapeutics, and even household products. A field that was once limited to the food industry has had an explosive impact on driving more environmentally sustainable products to consumers, such as the use of enzymes to replace petroleum-derived chemicals in household products likes laundry detergent. Enzymes often reduce the environmental footprint of these products by reducing volumes for shipping and by replacing synthetic components with biodegradable enzymes.2,3 Since the first reported use of recombinant human insulin ∼30 years ago, the pharmaceutical industry has embraced the use of therapeutic proteins to supplement the small-molecule pharmaceutical market. More than 130 protein therapies are currently on the market, with many more in the clinic awaiting

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Special Issue: Biocatalysis in Industry Received: December 19, 2016 Published: June 19, 2017 119

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Table 1. Applications for Protein Functionalization

approval.4 In an effort to increase shelf life, bioavailability, or pharmacokinetic properties of a biotherapeutic product, posttranslational modifications (PTMs) have emerged as an invaluable tool for bringing biodiversity to market.5,6 Continuing to innovate protein-based products, researchers have turned to functionalizing proteins with both small molecules and macromolecules. A few applications of protein functionalization include (1) cross-linking to conferring greater stability by increasing rigidity,7 (2) delivery of molecular cargo to a specific tissue or receptor (e.g., antibody−drug conjugates, ADCs),8 (3) creation of new materials with unique properties not demonstrated in nature or the lab (i.e., hydrogels),9,10 and (4) armoring proteins with synthetic polymers or biopolymers for robustness (e.g., glycosylation and PEGylation).11 These varied and exciting applications are summarized in Table 1. These technologies have been segregated into two parts in this review. First, section 3 covers protein−protein cross-linking (Table 1, gray shaded entry). The pioneers of this field have largely been food scientists who utilize isolated enzymes to affect the shelf life, taste, and texture of foods. These biocatalysts have since been employed in leather and textile processing, hydrogel development, and sensor production. The second part of this review, section 4, details protein-Xconjugation where X = carbohydrates, polymers, and small organic molecules (Table 1, blue shaded entries). These technologies have primarily been utilized for the development of biotherapeutics. The examples cited in this review have been selected for their potential to be produced in commercially relevant quantities or for having a track record of being carried out on a multikilogram to metric ton scale. A particular focus is paid to enzymes that are commercially available, and the manufacturer will be cited, to aid in the readers’ understanding of why these enzymes are frequently selected by industrial researchers. Finally, the cases described herein are key applications where the use of an enzyme for peptide functionalization provides a clear advantage over genetic manipulation, chemical ligation, or unnatural amino acid incorporation.12−18

For this reason, the bulk of this review will discuss applications in the food industry and therapeutics, where enzymes may circumvent issues of toxicity and/or undesirable heterogeneity in FDA-regulated products. However, there are a few regulatory hurdles in bringing new enzymes to market, particularly for food preparation. Because the product(s) of food “reactions” are consumed by humans, the enzymes must be tested for immunogenicity and toxicity, in order to be generally recognized as safe (GRAS). GRAS status is a certification of the U.S. Food and Drug Administration (FDA) required for enzyme preparations used in the food industry, and premarket approval is required for new enzyme variants. Therefore, it can take considerable effort to bring novel or engineered enzymes to market for food and nutrition. The pharmaceutical industry is primarily focused on homogeneity of the conjugated product, that is, regioselective control. Enzyme-catalyzed protein conjugation chemistry offers regioselective ligations (modifying one subset of a given amino acid: for example, a surface cysteine over an internal cysteine), site-specific ligations (modifying one amino acid residue over others: for example, cysteine over lysine), or even selective ligation of a single residue under mild reaction conditions. Such mild conditions include, but are not limited to, an absence of organic solvents, preservation of functional groups, highly selective reactions, neutral pH, and mild temperature. Furthermore, directed evolution of biocatalysts has shown promise in further enhancing or shifting the regioselectivity of enzymatic transformations while offering high reactivity.19 If one can establish proof of concept for the desired transformation, then the enzyme can often be further optimized through directed evolution or engineering techniques to deliver a high-performing process. However, there are some exceptions. Some enzymes are more difficult to evolve because the model organisms used do not have the appropriate machinery to express the desired protein: for example, highly glycosylated enzymes; pro-sequence cleavage; membranebound proteins, etc. The evolvability of an enzyme will be briefly discussed as well. 120

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Table 2. Post-Translational Modifications by Reaction Class

Scheme 1. Formylglycine-Generating Enzyme-Catalyzed Oxidation of Cysteine to Formylglycine within the CXPXR Taga

a

Condensation of molecular cargo results in the cargo−linker−protein conjugate.

2. OVERVIEW OF PROTEIN CONJUGATION REACTIONS

transferases (section 2.2) to hydrolases (section 2.3) and ligases (section 2.4).

The functional and structural diversity of the proteome in prokaryotic and eukaryotic cells is broadened by the posttranslational modification (PTM) of proteins by specific enzymes.20 A summary of the types of PTMs is presented in Table 2. They have been categorized in the following ways: (1) changes to the polypeptide sequence or connectivity, such as cross-linking proteins, intramolecular bond formation, and proteolytic processing (Table 2, column 1); (2) addition of small molecules like cofactors and methyl, acetyl, sulfate, and phosphate groups (Table 2, column 2); and (3) addition of higher molecular weight molecules or biopolymers, for example, oligosaccharides, nucleotides, and lipids (Table 2, column 3). These enzymatic modifications often occur on functional groups of amino acid side chains at sequence-specific recognition motifs, which can be exploited in the laboratory for targeting specific protein sites. While biocatalytic methods often borrow from chemistries first described in a natural process like PTMs, not all transformations in the following sections are developed from post-translational machinery. In fact, many of the most promising oxidoreductase-catalyzed transformations described here were first observed catalyzing small-molecule oxidations in vivo and were adapted for application to small peptides and proteins in the laboratory. This section will give a high-level summary of the enzymatic strategies adopted in the laboratory and demonstrated at the industrial setting for polypeptide functionalization. These modifications include nearly all classes of enzymes, ranging from oxidoreductases (section 2.1) to

2.1. Oxidoreductases

Oxidoreductases (EC 1) are enzymes that catalyze oxidation or reduction reactions. The most industrially relevant enzymes in this class are glucose oxidase, laccase, catalase, and peroxidase, which are all produced on the industrial scale. Both laccases and peroxidases have proven their utility in functionalizing proteins; however, these enzymes are not technically catalyzing PTMs. There are relatively few oxidative enzymes involved in PTMs that are also commercially used for protein functionalization. These include formylglycine-generating enzyme (FGE), sufhydryl oxidase, and lysyl oxidase. Formylglycine-generating enzyme (FGE, EC 1.8.3 sub-class: the Enzyme Commission is currently reclassifying the enzyme, which will likely move to the 1.8.3 sub-subclass) is responsible for the PTM of human type I sulfatases, which generates their catalytically active form.21 The most studied class of FGEs is the aerobic FGEs isolated from bacterial sources, which recognize the consensus sequence CXPXR for O2-dependent conversion of the cysteine residue to formylglycine (fGly) (Scheme 1). Early reports used this consensus sequence (also called an aldehyde tag) to generate a reactive handle for labeling studies in cell culture.6,22 However, FGE has emerged as a useful tool for developing and scaling biopharmaceuticals because of the exquisite regioselective control the consensus sequence provides to the user. Redwood Bioscience/Catalent has developed a commercial platform, SMARTag, to streamline the use of FGE in generating small libraries of novel therapeutics. 121

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Scheme 2. (A) Laccase- and Peroxidase-Catalyzed Protein Cross-Linking. (B) Tyrosinase Oxidation of Tyrosine Residues To Attach Molecular Cargo or Cross-Link Proteins through Conjugation to the Quinone Intermediate

Scheme 3. Cross-Linking of Proteins through Lysyl Oxidase-Catalyzed Oxidation of Lysine Residues to the Corresponding Aldehydesa

a

Two paths favor dimerization of proteins: aldol condensation and Schiff base formation. Alternatively, molecular cargo can be ligated to the protein by Schiff base formation.

commercial sources for this family of enzymes results in their being frequently tested for many applications beyond food, pulp, and textiles. Multikilogram amounts are generally stock quantities and contribute to short lead times to access laccase. Like laccases, peroxidases (EC 1.11.1.7) generate proteinand small-molecule-based free radicals that can nonenzymatically react to form protein cross-links (Scheme 2A). Again, these reactions typically target tyrosine, cysteine, and lysine residues, resulting in a high degree of heterogeneity in the product composition. One of the major drawbacks of peroxidases is the requirement of H2O2 as an electron acceptor, because excess H2O2 must be removed from food and pharmaceuticals prior to consumption. Additionally, H2O2 can also catalyze background reactions. DSM sells an engineered fungal laccase (MaxiBright) that has GRAS status and was developed to bleach cheese whey for a variety of dairy applications. Perhaps the most promising oxidoreductases for protein cross-linking are tyrosinases (EC 1.14.18.1),24 sulfhydryl oxidases (EC 1.8.3.2),25 and lysyl oxidases (EC 1.4.3.13),26 because of their high fidelity to specific amino acid residues presenting the opportunity for site-selective oxidations. While these oxidative enzymes have shown promise in many

Another useful class of oxidative enzymes is the laccases (EC 1.10.3.2). Laccases are primarily employed in pulp and textile industries for degradation of color-producing aromatic polymers. These multi-copper-binding enzymes catalyze the single-electron oxidation of phenolic compounds, using molecular oxygen as the terminal oxidant. This results in formation of water and free radicals. The free radical organic substrates can further undergo nonenzymatic reactions including disproportionation, polymerization, hydration, and fragmentation; however, the examples in this review will cover radical addition to polypeptides (Scheme 2A). The products of laccase-induced cross-linking are generally heterogeneous, leading to formation of isodityrosine, dityrosine, and disulfide intermolecular bonds between proteins with very little control over location of conjugation. However, because the sole byproduct of catalysis is water, laccases are an attractive option for the food industry.23 Because peptides are poor substrates for laccases, small-molecule mediators are commonly utilized to boost efficiency in cross-linking, which introduces diversity in cross-linking sites. Novozymes (Denmark), Advanced Enzyme Technologies Ltd., and Amano Enzyme USA Co. Ltd. have obtained GRAS status for a laccase enzyme preparation primarily for use in the baking industry. The availability of 122

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Scheme 4. Transglutaminase-Mediated Cross-Linking of Gln and Lys Side Chains To Attach a Payloada to a Protein of Interest

a

Most often the payload is attached through a primary alkylamine.

Scheme 5. Protein Farnesyltransferase-Mediated Conjugation of Cysteine Residue in CAAX Consensus Sequencea

a

Small molecules or functional handles may be installed at the terminal carbon of farnesyl pyrophosphate.

commercial applications, there are few commercial sources for purchasing these enzymes (available in research quantities), which may have limited their adoption in industrial applications. However, it is useful to discuss their chemistry. Tyrosinases are oxygen-dependent copper-containing proteins that catalyze the highly specific four-electron oxidation of tyrosine residues to o-quinones. These quinones readily form cross-links with solvent-exposed lysyl, tyrosyl, and cysteinyl residues (Scheme 2B). Tyrosinase-catalyzed cross-linking processes are highly attractive transformations for the food and pharmaceutical community because the sole byproduct of catalysis is water. Lysyl oxidases are copper-dependent amine oxidases responsible for the oxidative cross-linking of collagen and elastin chains, with concomitant reduction of O2 to hydrogen peroxide. In these reactions, the lysine side chain is oxidized to the corresponding aldehyde, and the two different pathways that can generate cross-links are outlined in Scheme 3. In one scenario, the aldehyde can condense with another aldehyde to form an aldol condensation product. In the second scenario, an amine from a second Lys residue can react with the aldehyde to form a Schiff base. Sulfhydryl oxidases are flavin-dependent oxidases that catalyze the formation of disulfide bonds between cysteine residues of proteins, again using molecular oxygen as the terminal oxidant, with H2O2 as a catalytic byproduct. These interesting biocatalysts can catalyze both intra- and intermolecular disulfide bond formation.

Transglutaminases (TGs, EC 2.3.2.13) are one of the most extensively studied classes of enzymes for protein functionalization because of their ability to polymerize proteins without the constraint of a consensus sequence. TGs are acyltransferases that catalyze transfer of a γ-carboxyamine group of a peptidebound Gln residue to the ε-amino group of lysine, resulting in protein cross-linking. They can also conjugate amine-containing cargo to a protein, with ammonia as the sole byproduct of catalysis (Scheme 4). This promiscuous enzyme tolerates a high degree of diversity in the amine partner provided an alkyl chain, linking the primary amine to the cargo, is of sufficient length to reduce the steric burden. Therefore, molecular cargos fitted with primary amine substrates have been described in numerous laboratory experiments to form the ε-(γ-glutamyl)lysine isopeptide bond.29 TG reactions are, however, limited by the steric environment of the Gln residue because the residue is loaded in the TG active site. Many researchers have exploited this property for regiochemical control when multiple Gln are present in the polypeptide. When amine substrates are not present, TG will catalyze deamination of Gln residues by waterassisted hydrolysis. Early TG studies used calcium-dependent eukaryotic transglutaminases whose native role is to assist in protein polymerization such as blood clotting and keratogenesis. Due to their sluggish reaction kinetics and instability, industry has shifted to using a calcium-independent TG isolated from Streptomyces mobaraensis (often called microbial transglutaminase or mTG).30 Ajinomoto US Inc. is the primary global producer of GRAS enzyme preparations of mTG. Most transferases involved in protein PTMs require a signal sequence, which can prove helpful for researchers targeting a specific region of protein. Two examples of such enzymes are phosphopantetheinyl transferase (PPTase, EC 2.7.8.7) and protein farnesyltransferase (PFTase, EC 2.5.1.58). The lipid transferase, PFTase, ligates farnesyl diphosphate analogues to protein substrates on a cysteine four residues from the Cterminus, with a CAAX recognition sequence signaling PFTase to catalyze the PTM (Scheme 5). Researchers have observed significant promiscuity with farnesyl diphosphate analogues, which turns this reaction into an excellent tool for specific labeling of a single residue at the C-termini of proteins. PPTase can be similarly exploited for its native PTM activity. PPTases post-translationally modify synthases such as fatty acid synthases, polyketide synthases, and nonribosomal peptide

2.2. Transferases

Transferases (EC 2) are enzymes that reversibly catalyze the transfer of a chemical group from one compound to another compound. For example, transpeptidation chemistry has been explored to connect two peptide sequences from different protein partners. Some of the most common industrial enzymes in this class are transglutaminase and fructosyltransferase, which are frequently employed in food processing. Additionally, carbohydrate transferases have been used to decorate proteins with glycans containing bioorthogonal functional handles for further chemical derivatization. A couple of great examples are β-galactoside sialyltransferases (EC 2.4.99.1 and EC 2.4.99.4) and O-GlcNAc transferase (OGTase, EC 2.4.1.255). These glycan remodeling enzymes have been extensively studied for their PTM activity in vivo for the purpose of studying cellular processes8 or PET imaging.27,28 123

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Scheme 6. Phosphopantetheinyl Transferase-Mediated Conjugation of Serine Side Chain in 11-mer Consensus Sequencea

a

Functionalization of the thiol group provides a site for further derivatization.

Scheme 7. Translipidation of Proteinsa Catalyzed by N-Myristoyltransferase

a

Substrate proteins carry a T/SXXXG consensus sequence at the N-terminus.

Scheme 8. Lipoate−Protein Ligase A-Catalyzed Acylation of Lysine Residues with Lipoic Acid within Consensus Sequencea

a

Enzyme is tolerant of substitution at the terminal carbon of lipoic acid analogues.

synthetases, which are carrier proteins fitted with a thiolate domain that tethers growing intermediates on a 4′-phosphopantetheine arm through a reactive thioester linkage (Scheme 6).31 PPTases modify these carrier proteins using coenzyme A for the installation of the 4′-phosphopantetheine arm through formation of a phosphodiester bond with a serine residue on the carrier protein (or protein of interest). ADP is released by 4′-phosphopantetheine transfer to the conserved DSLEFIASKLA motif. Perhaps the most common PPTase is Sfp. Sfp is renowned for its ability to transfer a range of organic molecules bound to the thiol moiety of coenzyme A (purple box in Scheme 6). Another coenzyme A-dependent transformation is lipidation of N-terminal glycine residues in proteins via a T/SXXXG signaling motif. By use of this simple tag, N-myristoyltransferase (NMT, EC 2.3.1.97) can be utilized to add a range of myristic acid analogues to proteins (Scheme 7).32 Lipoate− protein ligase A (LplA, EC 6.3.1.20) catalyzes the acylation of lysine residues embedded in a GFEIDKVWYDLDA tag. Both coumarin and azide acid analogues are well-tolerated by the enzyme, provided the methylene linker is of a suitable length (Scheme 8).

production, and many more. Dozens of hydrolases are produced and stocked on the multikilogram to metric ton scale, providing quick access to industrial researchers without the delay of production. Hydrolases are enzymes that catalyze the hydrolytic cleavage of bonds, like peptide C−N bonds in the case of peptidases. There are many commercially available hydrolytic enzymes; in fact, hydrolases comprise roughly 75% of commercial enzyme sales. These enzymes are more often employed for cleaving bonds rather than forming peptide bonds. However, a few interesting examples have been developed that use the reversibility of hydrolase enzymes to form peptide bonds in manufacturing processes. One such example is a peptidase derived from trypsin (trypsiligase, a quadruple mutant of trypsin).33 Trypsin is produced on the metric ton scale and is readily employed in food and nutrition. This example will be discussed in section 4.1.1.4 as an emerging platform. A widely adopted hydrolase for protein conjugation experiments is sortase, a family of six enzymes (sortases A−F). Sortases are calcium-dependent cysteine transpeptidases that catalyze the cross-linking of proteins bearing a sorting signal, LPXTG.34 One of the more studied enzymes in this family is sortase A (SrtA, EC 3.4.22.70). SrtA is isolated from Staphylococcus aureus and is responsible for the post-translational tethering of surface proteins to the cell wall of bacteria.35 While SrtA is formally classified as a peptidase, it is more aptly described as a transpeptidase (see Scheme 9). The SrtA active-

2.3. Peptidases

Peptidases (EC 3.4) are a subset of the hydrolase family of enzymes that are employed in numerous industrial processes like leather, textiles, food, animal nutrition, fine chemical 124

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Scheme 9. Sortase A-Catalyzed Transpeptidation of Proteins within LPXTG Consensus Sequencea

a

New protein cross-links or small-molecule conjugates are formed at the C- and N-termini.

Scheme 10. Biotin−[acetyl-CoA-carboxylase] Ligase-Catalyzed Acylation of Lysine Residues Embedded in a Consensus Sequence with Biotin and a Biotin Analogue

enzyme-based cross-linking strategies and their potential for different commercial applications are examined.

site cysteine cleaves the Thr−Gly amide bond within the tag sequence LPXTG of the target protein, forming a proteinenzyme-thioester intermediate. This liberates the Gly-containing C-terminal peptide of the tag sequence. An amine nucleophile attacks the covalently bound enzyme-acyl-protein intermediate, resulting in formation of a peptide bond crosslink. In vivo, the terminal amino group of an oligoglycine peptide catalyzes release of the covalently bound protein moiety from the enzyme.36 Researchers often mimic this polyglycine motif when designing substrates for SrtA-catalyzed conjugations.

3.1. Food Processing and Edible Packaging

One of the more established fields that employs enzymes for protein modification is the food industry. Cross-linking of protein molecules into three-dimensional networks is an essential mechanism for engineering food structures with desirable properties. Covalent protein networks can affect a food’s physical properties, shelf life and stability, diffusion/ solubility, sensory properties, and nutritional quality.39 Furthermore, research has demonstrated that modification of proteins in certain foods can alleviate allergic responses to components in the food matrix, such as gluten.40,41 Transglutaminases,29 laccases,23 and peroxidases42 are some of the protein cross-linking enzymes with GRAS status. Food-grade preparations of these enzymes are commercially available from Ajinomoto, Novozymes, and DSM, respectively. The modes of action as well as the commercial applications of these enzymes are summarized in Table 3.

2.4. Ligases

Ligases (EC 6) use the hydrolysis of a diphosphate bond, such as ATP, to catalyze bond formation between two molecules. Because of the requirement for expensive cosubstrates (ATP) or costly cofactor regeneration systems, ligases are less commonly used for commercial or preparative applications. Escherichia coli biotin−[acetyl-CoA-carboxylase] ligase (BirA, EC 6.3.4.15) is a cofactor ligase responsible for the covalent addition of biotin to a Lys residue within an embedded tag, GLNDIEAQKIEWHE.37 BirA is well-studied because of its ability to accept biotin analogues, which facilitates incorporation of bioorthogonal handles for chemical derivatization into the polypeptide backbone of the protein of interest (Scheme 10).

Table 3. Enzymes Frequently Used for Protein CrossLinking in Foods enzyme

3. NONPHARMACEUTICAL APPLICATIONS OF ENZYMATIC PROTEIN CONJUGATION Perhaps the most extensive commercial applications of enzymatic protein modification reside in protein cross-linking.38 Cross-linking allows researchers to modify the physical properties of proteins while often maintaining their integrity and function. Enzymes employed in the food, leather, and biomaterials industries often share the same mechanism for cross-linking. However, these examples illustrate that whether two proteins are fused with a single covalent bond or protein networks are formed through multiple covalent bonds, vastly different properties can be observed with the same enzyme. The most prevalent commercial biocatalysts for cross-linking proteins are transferases, hydrolases, and oxidoreductases. The underlying mechanisms of cross-link formation are illustrated, and the roles of the enzymes in their natural environments are discussed. Additionally, the advantages and drawbacks of the

classification

sulfhydryl oxidase

EC 1.8.3.2

laccase

EC 1.10.3.2

peroxidase

EC 1.11.1.7

tyrosinase

EC 1.14.18.1

transglutaminase

EC 2.3.2.13

mode of action

applications

disulfide bond formation aromatic oxidation (Tyr, Trp) and Cys aromatic oxidation (Tyr) aromatic oxidation (Tyr)

baking (dairy early reports) baking, dairy, allergenicity reduction

isopeptide bond formation (Lys/ GIn)

baking, dairy, allergenicity reduction baking, dairy, allergenicity reduction, meat baking, dairy, meat, cereal, allergenicity reduction

3.1.1. Restructuring Meat with Enzymes. Microbial transglutaminase (mTG), isolated from S. mobaraensis,30 is the most popular cross-linking enzyme in food processing today. mTG is distributed by Ajinomoto US Inc. under the trade name Activa and is safe for use as a general cross-linking agent in food. This enzyme is also called “meat glue” because of its ability to reconstruct scrap meat into larger, more valuable cuts of meat.43 This gluing action can be applied to different types of 125

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meats or fish, influencing the texture of meat gels,44 and altering the mechanical properties of processed meats (i.e., sausage)45 by reacting with myosin and actin, a myofibrillar protein found in muscle filaments.46−48 Because other salts and additives are not needed for mTG to link amino acid side chains, the discovery of mTG opened a new market for the production of “healthy” meat products, making it an invaluable tool for the commercial meat industry.49 mTG is also used commercially in production of dairy and wheat products, as well as in the production of edible films. 3.1.2. Dairy Production with Enzymes. The two primary enzymes found in milk are casein and whey. Both transferases and oxidoreductases can catalyze the cross-linking of casein in milk, which can provide heat-resistant properties and favorable textural changes. Furthermore, cross-linking of milk proteins enhances their water retention properties, providing more stable emulsions.50 Milk gels, like yogurt, are traditionally prepared by acidic fermentation from a lactic starter, which often has the disadvantage of whey/serum separation upon physical impact or change in temperature. Employing enzymatic cross-linking alternatives has circumvented these disadvantages. mTG has been exploited to affect the sensory and water retention properties of yogurt, produce creamier ice cream, and increase the yield of cheese curd. mTG effectively catalyzes the gelation of milk proteins at high temperature to form yogurts51 which alleviates the requirement for dry matter stabilizers that are traditionally used to enhance the strength of acidified casein gels.51−53 This is an important property because casein does not otherwise gel. Kraft Foods has exploited this enhanced mTG stabilization to develop wheyless cream cheese that does not require added stabilizers or emulsifiers.54 Moreover, Kraft Foods, Ajinimoto, and others have demonstrated that cheese curd yield can be significantly enhanced by mTG treatment of dairy preparations, affording a more profitable process.55−59 Oxidative cross-linking of milk proteins with laccase has been explored in the formation of milk gels, albeit with lower efficiency.60 Laccase treatment required higher enzyme loading than mTG for equivalent casein cross-linking; an advantage of these reactions is they can occur at lower temperatures, which may be useful in the production of milk products that are sensitive to heat processing. Early modifications of milk proteins utilized another oxidoreductase, sulfhydryl oxidase. Sulfhydryl oxidase activity was detected in milk in 1975.61 This enzyme was one of the earlier enzymes to be exploited by the food industry, when it was observed that passage through a column containing immobilized sulfhydryl oxidase could eliminate the cooked flavor of milk formed in ultra-hightemperature-treated milk (sterilization).62 3.1.3. Baking with Enzymes. Gliadins and glutenins, the primary proteins in gluten, are readily cross-linked by mTG. In fact, mTG has been exhaustively explored for the production of breads63,64 and pastas65 and has produced desirable changes in texture. mTG has been employed to strengthen noodles, allowing for use of lower-grade flour during production. Furthermore, mTG improved the texture of cooked pasta relative to nontreated pasta by increasing the break strength upon cooking (quantified by SEM analysis).66 Additionally, mTG has been explored for modifying the elasticity and resilience of dough, volume and softness of bread, and textures of cereals. A recent study demonstrated that mTG can be applied to dough to extend its shelf life when frozen. In this study the rheology, microstructure, and baking properties

of mTG-cross-linked and control dough preparations were studied over extended frozen-storage periods. SEM micrographs demonstrated that mTG strengthened the gluten network of both fresh and stored frozen dough, which was further supported by testing the final cooked bread for bread crumb softness.67 Increased occurrence of gluten-related disorders like celiac disease has motivated food scientists to modify gluten during food processing in an effort to avoid an immune response. Transamidation of gluten proteins, with chymotrypsin or mTG, appeared to produce breads with less immunoreactive gluten.40,41,68,69 Oxidative cross-linking of wheat proteins, by use of laccase and tyrosinase, has been studied for improving properties of dough and bread. Laccase addition to dough not only increased its strength and stability but also reduced its stickiness, improving the dough’s machinability.70 However, the mechanisms and final protein matrices of these cross-linked products are less well characterized than those produced with mTG.71 These are not as commonly used as mTG for wheat modifications, likely due to higher enzyme loading and quinone byproducts (which influence the color and aroma).39 Sulfhydryl oxidases, on the other hand, have been shown to efficiently promote disulfide bond formation between cysteine residues in gluten proteins, strengthening the matrix. The concomitant production of hydrogen peroxide, which can further cross-link wheat proteins nonenzymatically, is the likely culprit in the high efficiency of this enzyme.72 Nevertheless, X-zyme in Dusseldorf, Germany, has released the first commercially available recombinant sulfhydryl oxidase, Erv1p from baker’s yeast. The manufacturer markets this protein as a replacement of oxidizing agents, like ascorbic acid, while enhancing gluten strength. 3.1.4. Edible Films. Protective edible protein films have been explored as tools for increasing shelf life and freshness of food. Edible films are intended to help the external packaging in preserving the stability of food, rather than replacing external packaging entirely. A chief limitation in using biopolymer films is their sensitivity to water, which can result in water absorption, swelling, or dissolution of the film. However, when these films are prepared by mTG-catalyzed cross-linking of whey proteins, phaseolin, casein, or whey/chitosan combinations, their resistance to moisture can be greatly improved.73−75 Similarly, tyrosinase and laccase have been employed to cross-link casein films that also demonstrate enhanced water stability.75 These protein-based films provided favorable water and gas barrier efficiency (O2, CO2, and water vapor) as well as being resistant to deformation via increased elasticity and structural stability. Cross-linking also provides greater resistance to solvents, heat, and light.76 Enzymatic crosslinking provides films with promising properties for durable yet biodegradable edible packaging. 3.2. Biomaterials and Biofabrication

Highly absorbent biopolymeric hydrogels have emerged as promising candidates for tissue reconstruction, wound healing, drug release, and protein trafficking. These hydrogels can mimic the natural extracellular matrix, entrapping bioactive molecules and supporting cellular proliferation.26 With several of these hydrogels on the market and in clinical trials, there is a growing demand for efficient and scalable methods for the safe production of stable biomaterials with novel properties.77 The potential for immunogenic and cytotoxic response is of great concern in developing these materials because of their use as 126

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lysyl oxidase found in serum can further rigidify the existing protein−protein framework by continuous cross-linking.89−92 For this reason, these hydrogels are most frequently explored for their applications in replacing cartilage tissue.

injected or implanted materials. Therefore, synthesis of these frameworks by chemical cross-linking may be undesirable if the final products are bioincompatible. The current limitation in the synthesis of biomaterials lies in their potential for toxicity and poor long-term stability. For example, photoinitiatorinduced or chemically induced cross-linking presents risks of cytotoxicity, while physical cross-linking, which relies on noncovalent interactions, may lead to gels that do not have sufficient mechanical strength and stability.78 These limitations can be overcome by instead using enzymes to generate covalently cross-linked hydrogels.26,79 Enzyme-mediated preparation of hydrogels by use of tyrosinases, peroxidases, transferases, and lysyl oxidases has delivered dynamic scaffolds with promising clinical applications. Typically, these enzymatic cross-linking tools are employed to prepare fibrin, elastin, collagen, gelatin, and other peptide-based networks (as well as protein−polysaccharide networks). However, collagen, or its hydrolyzed form gelatin, is the most common polypeptide used in the construction of biomimetic hydrogels and is often partnered with chitosan as a copolymerant.26 Transglutaminase-generated hydrogels are among the most promising materials, due to their facile preparation and high mechanical stability. However, many of the studies demonstrating their utility employ mammalian transglutamases, which are difficult to express recombinantly or obtain at commercial scales.26 mTG is a more promising alternative for commercial preparation of hydrogels. Yung et al.80,81 prepared thermally stable mTG-cross-linked gelatin frameworks, which allowed for the proliferation of encapsulated HEK293 cells and demonstrated promise in trafficking therapeutic proteins. Chen et al.82 have demonstrated that mTG can encapsulate E. coli cells by inducing gelation of a 10% gelatin solution. Proteinase K treatment of the cross-linked gelatin network then proteolyzed the matrix within 1 h, liberating the fully intact E. coli cells. They further illustrated the utility of this methodology by demonstrating the ability to in situ entrap, grow, and release cells under mild conditions, which provides unique opportunities for applications as microfluidic biosensors. Wu et al.83 and Paguirigan et al.84 advanced this field further by utilizing mTG to biofabricate cross-linked protein matrices to attach cell cultures for applications in microfluidic devices. Tyrosinase-cross-linked protein−protein and protein−chitosan hydrogels have also yielded stable hydrogels with unique properties. While the preparation of these gels parallels the observations and properties of the gels described earlier, tyrosinase-prepared gels have not been studied in tissue engineering applications to date. Interestingly, when gelatin and chitosan gels formed upon cross-linking by tyrosinase or transglutaminase are compared, tyrosinase induced faster gelation but produced weaker gels.85 This could have applications in adhesives, wound dressings, and immobilization technologies, where rapid deterioration of the matrix is advantageous. Other useful nontoxic biomaterials derived from the tyrosinase-catalyzed grafting of peptides onto chitosan are silk fibroin86 and silk sericin87 networks. These materials may have interesting biomedical applications due to their unique mechanical properties and adhesiveness; however, the utility of these biomaterials has not yet been demonstrated in the clinic. Lysyl oxidase, which is responsible for stabilizing cross-linked networks in collagen and elastin, is a useful protein-hydrogel tool.88 Many lysyl oxidase-generated matrices share a unique feature of getting mechanically stronger over time because the

3.3. Textiles and Leather Manufacturing

Keratins are the primary protein found in wool. Well-known application of enzymes in the textile industry are the use of hydrolases, cutinases, keratinases, and proteases to hydrolyze keratin fibers, which improves shrink resistance and antifelting behavior of the fabric.93 However, proteolysis of keratin reduces the integrity of the wool by lowering its tensile strength. It has been demonstrated that these fibers can be reinforced by treatment with mTG- or tyrosinase to catalyze cross-linking of the keratin.94,95 These reinforced fibers also become more resilient to damage from wash cycles96 and exposure to oxidants like bleach or hydrogen peroxide.97 Additionally, the mechanical properties of wool have been altered by appending small proteins to the keratin fiber. Cortez et al.98 demonstrate that mTG-grafting of silk proteins onto wool-fibers improves their antifelting properties postwash, increases tensile strength, and reduces area shrinkage. Cui et al. further demonstrated that casein99 and gelatin100 cross-linked-wool fibers have similar positive properties when tested. They further characterized the fibers with SEM to show scale smoothing of the grafted keratin fibers relative to untreated keratin fibers. Another fascinating application is the grafting of antimicrobial peptides onto wool fibers by use of mTG. This strategy has been used to produce clothing with unique odor and stain control properties.101 Both collagen and elastin have been grafted onto wool fibers via tyrosinase-induced cross-linking, to develop antifungal and antibacterial clothing for applications in hospital attire.95,102 Studies at the U.S. Department of Agriculture (USDA) and several academic institutions have demonstrated the value of mTG for grafting gelatin, casein, and whey (as well as other low-cost protein fillers) to fill voids in animal hides in the leather industry.103,104 These studies demonstrate that pretreating leather with a cross-linking agent helps maintain leather integrity throughout the washing steps in leather processing and improves the quality of the final product. 3.4. Promising Applications for Commercialization

3.4.1. The Future of Biomaterials. The use of enzymes for the development of biomaterials is still in its infancy. Many exciting new applications for enzymatic cross-linking have yet to be fully investigated to replace chemical and physical reactions and photoreactions. Other fields where enzymatic biomaterial formation may prove to be useful are micro- and nanocapsule formation for drug delivery,9,105 bioengineered photoelectrochemical106 or electrochemical enzyme biosensors,107 and other novel therapeutic applications.108 As an example of the growth of the hydrogel field, pharmaceutical companies have begun to explore these biomimetic matrices as in vitro human models to find alternatives to animal testing for safety studies.109 Biofabrication of devices can be simply and rapidly produced by mTG-catalyzed cross-linking of durable fibers, like spider silk, fitted with functional molecules for development of biological sensors and probes.83 Moreover, horseradish peroxidase cross-linked multivalent protein polymers are being developed to couple proteins with distinct properties through bifunctional linkers for multifunctional materials without loss of original function.110 3.4.2. The Future of Biocatalysts and Fusion Proteins. An interesting application of this technology would be the 127

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examples presented in this section are primarily from the pharmaceutical industry and benefit from single-isomer composition in the final protein conjugate product. Furthermore, this section highlights examples of highly site-selective enzyme-catalyzed conjugation methods that have been performed on the commercial scale.

formation of cross-linked enzyme aggregates (CLEAs) to produce synthetically valuable enzymes that are more resilient under reaction conditions and offer recyclability of the catalyst. Common formation of CLEAs is through glutaraldehydemediated cross-linking.111 However, Fairhead and ThoenyMeyer112 demonstrated that, by use of phenolic mediators, tyrosinase effectively polymerizes ordered industrially relevant enzymes, like Cal B, allowing for their application in synthetic processes. Fusion proteins are another area of intense interest. The ability to selectively ligate two proteins together in one orientation has long been demonstrated through traditional genetic assembly and intein-based methods.113 However, relatively few techniques allow post-translational fusion in a site-specific manner. Hydrolytic enzymes have been used to semisynthetically assemble peptide fragments, but sortase A is a more promising, fully enzymatic approach for stitching together proteins. The advantage of this approach is that it can generate combinatorial libraries to explore structure−activity relationships (SAR) more rapidly with consistent outcomes. By use of the LPxTG recognition sequence to signal transpeptidation, libraries can be rapidly generated with relatively low molecular biology burden for a range of applications beyond protein− protein fusion, some of which will be described later.114−116 The availability of enzymatic tools to produce biopolymer networks has resulted in an emergence of new and exciting applications for biomaterials that can be easily scaled for commercial applications.

4.1. Antibody−Drug Conjugates and Immunoradiolabeling

Monoclonal antibodies (mAbs) demonstrate high affinity, and therefore specificity, for binding target antigens. In the last two decades, mAbs have given the pharmaceutical community a precise tool for targeted drug delivery in inflammatory diseases and cancers that have antigens that are either uniquely expressed or overexpressed on target cells. Historically, development of chemotherapeutic agents has focused on enhancing drug cytotoxicity; however, the most potent candidates are often too toxic to be of use in the clinic due to off-target effects. Antibody−drug conjugates (ADCs) represent a more targeted and potentially more effective method for delivering the cytotoxin, improving the therapeutic index of chemotherapeutics. ADCs comprise an antibody, a linker, and a payload. The payload is generally a potent cytotoxin that would not be tolerated systemically if circulated in the body. The tumor antigen is selected among those expressed on the cell surface to direct the antibody to the appropriate cells. ADCs are then internalized through receptormediated endocytosis. Inside the cell, the ADC linker is degraded, which liberates the drug, resulting in cell death. Trastuzumab and bevacizumab are mAbs on the market for inhibiting tumor progression in certain forms of cancer, and these are two of the more common mAbs used in ADC studies. These two mAbs will be referred to throughout the following sections. For a more in-depth understanding of ADC design and the history of ADCs, the reader is referred to recent reviews on ADCs for cancer therapy.117,118 Early research on ADCs began nearly 50 years ago,119 but today there are only two FDA-approved ADCs on the market in the United States: brentuximab vedotin, marketed as Adcetris by Seattle Genetics in partnership with Millennium/ Takeda,120 and trastuzumab emtansine, marketed as Kadcyla by Genentech and Immunogen.121 Both drugs feature nonselective conjugation of the drug−linker molecule to the antibody: via acylation of lysine residues with activated esters for Kadcyla121 or via alkylation of cysteine thiols with maleimides for Adcetris.120 In fact, most chemical techniques used to produce antibody−drug conjugates result in a heterogeneous mixture of species with variable drug-to-antibody ratios that can range from 0 to 8. The species exhibit different pharmacokinetics, stability, and safety profiles.117 Studies have revealed that the site of drug attachment not only modulates the pharmacokinetics and stability of the ADCs but also impacts the therapeutic window.122,123 It is largely recognized that sitespecific conjugation techniques are critical for increasing the therapeutic window of these biopharmaceuticals to supply the next generation of ADCs. Several chemical, enzymatic, and chemoenzymatic approaches have been developed to create homogeneous preparations of ADCs. This field has been thoroughly reviewed by pharmaceutical researchers117,124 and academic researchers.118 This review, however, highlights techniques for enzymatic conjugation of payloads to mAbs that have been scaled to produce clinical material or demonstrate the potential for scalability.

3.5. Pros and Cons of Enzymatic Cross-Linking Strategies

Many interesting enzymes are now available for purchase on an industrial scale that can modify the behavior of food proteins, with little or no lead time required. Enzymes used for protein cross-linking are often nontoxic and biodegradable, which makes them advantageous where the product will be injected or consumed, as purification of the product is often unnecessary. The conditions described are functional-group-tolerant and occur at neutral pH and mild temperatures, which are qualities ideal for modification of foods and biomaterials. However, the use of nonhuman enzymes from fungal and microbial sources can come with immunogenicity problems, especially with injectables and food processing. The development of more GRAS enzymes on the commercial scale is necessary for this growing area of research. Most of the examples described above do not require strict control over the site of cross-linking. If homogeneous product compositions are required, new enzymatic methods or directed evolution would be necessary to provide regioselective ligations rather than just site-specific ligations (e.g., modifying cysteines over lysines).

4. PHARMACEUTICAL APPLICATIONS OF ENZYMATIC PROTEIN CONJUGATION By far the most extensive research in this field is in bioorthogonal approaches to labeling proteins in vivo for study of cellular processes, disease states, and tracking biological phenomena. There are many examples of chemical and chemoenzymatic methods of protein modification; however, the selectivity of these methods is often very poor, and they yield heterogeneous mixtures of modified proteins. While moderate chemoselectivity can be achieved through alkalinity or selection of nucleophile/electrophile, the ability to target one amino acid in a polypeptide sequence remains a formidable challenge for chemical cross-linking strategies. The 128

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Scheme 11. Innate Pharma and ETH Strategy for Generating Homogeneous Antibody−Drug Conjugates by Utilizing Deglycosylated Monoclonal Antibodies or Site-Directed Mutagenesisa

a

(A) Enzymatic deglycosylation followed by chemoenzymatic functionalization, using mTG for selective installation of an azide-containing linker. (B) Mutagenic strategies for abolishing glycosylation sites and adding Gln for mTG functionalization.

4.1.1. Protein−Cytotoxin Conjugates. 4.1.1.1. Microbial Transglutaminase. In exploring the reactivity of mTG for the functionalization of tumor-targeting antibodies, researchers in the Schibli lab at the Swiss Federal Institute of Technology (ETH) observed the exclusive acylation of Gln295, which is located at the flexible region of an IgG heavy chain (Fc domain). Human IgG antibodies have a conserved glycosylation site at each Asn297 residue. Point mutation of the Asn297 of the Fc domain deletes the native IgG glycosylation site and further exposes Gln295 to mTG. Exposure of the IgG to mTG in the presence of an amine-bearing linker afforded homogeneous ADC radioimmunotoxins that were tumoruptake-selective in vivo.125 Innate Pharma, in collaboration with the Schibli group at the ETH, investigated two strategies to obtain homogeneous ADCs, generating drug-to-antibody ratios of 2126 or 4.127 With mTG specificity for Gln295, deglycosylation of the IgG yielded ADCs with drug-to-antibody ratio of 2. Unfortunately, direct enzymatic drug−spacer attachment required a molar excess of the drug−spacer derivatives and yielded heterogeneous ADCs with variable drug-to-antibody ratios. However, a chemoenzymatic approach yielded homogeneous ADCs with consistent and reproducible drug-to-antibody ratios. In this approach, the azide-derivatized linker was site-selectively conjugated to the antibody by use of mTG. The azide was then exposed to the drug containing a strained alkyne to deliver the strain-promoted azide−alkyne cycloaddition ADC product. This elegant chemoenzymatic approach can be applied to (1) a variety of antibodies without genetic manipulations to provide ADCs with drug-to-antibody ratios of 2 and (2) N297Q point mutants of IgG, consistently producing drug-to-antibody ratios of 4 (Scheme 11). In a complementary study, the Rinat/Pfizer team engineered an LLQGA mTG recognition sequence (also known as a Qtag) that enabled selective transpeptidation in the presence of all other protein Gln residues.128 They introduced this LLQGA peptide into discrete locations along the IgG1 antibody, and 12 out of the 90 sites were amenable to conjugation with amine coupling partners (Scheme 12). The study interrogated the generality of the recognition sequence by embedding the

Scheme 12. Rinat/Pfizer Strategy for Generating Homogeneous Antibody−Drug Conjugatesa

a

Strategy utilizes a LLQGA tag that mTG recognizes for functionalization of Gln while the native glycosylation pattern is maintained.

sequence into IgG1, IgG2, and IgG4 antibody subtypes as well as an anti-M1S1 antibody (C16) and an anti-Her2 antibody. Comparable conjugation efficiency and selectivity were observed in all cases. To understand the structure−activity relationship at the site of conjugation, heavy-chain and light-chain conjugation sites on the C16 antibody were synthesized and compared in a rat model. This study revealed that the site of conjugation has a significant effect on the pharmacokinetics of the drug. Even though both ADCs had a drug-to-antibody ratio of 2, the lightchain conjugation site demonstrated more favorable pharmacokinetics. Perhaps the most significant finding was that both homogeneous ADCs (heavy- and light-chain modification) had a higher therapeutic window than a conventional heterogeneous cysteine-linked conjugate bearing the same cytotoxic drug. This further illustrates the importance of a toolbox for the scalable production of ADCs, with a particular need for new methods for interrogating different sites on the antibody to fully understand the SAR of the therapeutic agent. An ADC candidate was advanced to phase I clinical trials using this technology at Rinat/Pfizer.129 mTG’s substrate promiscuity in coupling partners (i.e., the cytotoxin) is helpful for conjugation onto antibodies. However, the requirements for deglycosylated mAb, point mutations to access the Gln295 residue, or an engineered tag sequence can limit the diversity of ADC libraries for SAR studies. A complementary approach to the studies described above 129

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Scheme 13. (A) Engineering a Trypsin Quadruple Mutant to Hydrolyze the Arg-Tyr Bond, Followed by Ligation of an Arg Ester Analogue to Load Molecular Cargo on a Protein of Interest. (B) Strategy for Functionalizing the C-terminus of Antibodies by Use of the CTAT Enzyme (Trypsiligase) and CTAT Linker

comes from Dophen Biomedical. Hu et al.130,131 genetically engineered an mTG, rather than the antibody, to enlarge the opening of the active-site pocket to accommodate less-exposed Gln residues in the antibody. This is the first example of direct site-specific conjugation of drug to an unmodified mAb heavy chain at a surface Gln residue. An ADC prepared from this method is anticipated to advance to the clinic in 2017 for phase I clinical trials. 4.1.1.2. Phosphopantetheinyl Transferase. Phosphopantetheinyl transferase (PPTase) has been used by researchers at Novartis to site-specifically ligate cytotoxic payloads onto the heavy and light chains of the α-Her2 antibody trastuzumab.132,133 Introducing 11- and 12-mer PPTase recognition sequences at 110 loop positions directed PPTase selectivity for the site of conjugation. In contrast to mTG conjugation technology that had been shown to conjugate up to 12 sites on an antibody, the Sfp PPTase enzyme could label 63 sites with a cytotoxic payload, which afforded 95 homogeneous ADCs. Similarly to the Rinat/Pfizer study, they found that pharmacokinetic profile, drug clearance, and thermal stability were conjugation-site-dependent in mouse model studies. Interestingly, in vitro cytotoxicity against HER2-positive cell lines was subnanomolar for nearly all ADCs studied and showed no dependence on the conjugation site. This inconsistency between the in vivo and in vitro data further illustrates the challenges in designing efficacious ADCs. Nevertheless, an in vivo xenograft model showed tumor regression and verified that PPTase-mediated conjugation is a viable method for developing efficacious and homogeneous ADCs. 4.1.1.3. Protein Farnesyltransferase. Protein farnesyltransferase (PFTase) has been used exhaustively for the labeling of proteins for imaging and mechanistic studies;8 however, there are relatively few reports of using PFTase as a tool for preparative purposes.134,135 Dozier et al.136 demonstrated that PFTase can be engineered to generate a mutant with a 300-fold rate enhancement. Legochem Biosciences similarly demonstrated the ability to further engineer PFTase and is currently working to commercialize this technology as a platform for generating novel ADCs.137 4.1.1.4. Trypsiligase Peptidase. Another intriguing example for specific conjugation of proteins with cargo is one from

Liebscher et al.33 A highly specific trypsin variant was engineered for the selective modification of N-terminal residues of a diverse collection of proteins with various small-molecule coupling partners.33 A quadruple mutant of trypsin, termed trypsiligase, was capable of cleavage of Tyr-Arg bonds as well as fusion of an ester-bearing ligand onto the newly exposed arginine amine (Scheme 13A). In short, trypsiligase efficiently labeled proteins engineered with an N-terminal HRY tripeptide recognition sequence. The authors speculated that the zymogen-like domain suppresses the hydrolytic activity that is typical for this promiscuous family of peptidases, which favors the desired ligase activity and promotes conjugation. Reasonable stoichiometries (3:1) of ester−small molecule to tagged protein and rapid reaction times, complete within 1 h, make this a very promising technique for future applications in derivatization of therapeutic proteins. Trypsiligase has also been employed for functionalizing the C-terminus of the Fab fragment of antibodies fitted with the YRH tripeptide tag.138 N-terminal coupling partners bearing cargo are well-tolerated, provided they bear an Arg-His linker (Scheme 13B). Eucodis explored the use of trypsiligase, renamed CTAT, to develop anti-Her2 ADCs fitted with a proprietary CTAT linker that directs conjugation to the Cterminus.139 The CTAT ADC performed well in xenograft trials where tumor regression was observed. Eucodis and ProBioGen recently entered into an agreement for commercialization of this technology. 4.1.1.5. Formylglycine-Generating Enzyme. The incorporation of an aldehyde into a protein represents an entirely different chemistry for ligation because aldehyde and ketone side chains are not common in polypeptides. A number of chemical cross-linking strategies have been developed by exposing proteins to aldehydes and ketones, like the wellknown and frequently used glutaraldehyde bifunctional crosslinking agent that promotes Maillard reactions.140 However, glutaraldehyde’s lack of specificity reduces its utility for therapeutic applications. Formylglycine-generating enzyme (FGE) targets small consensus sequences, at a specific cysteine in the CxPxR sequence at N- or C- termini, to generate a formyl glycine at the terminus of the protein. This enzyme can be readily overexpressed in either mammalian or E. coli cells and presents yet another useful strategy for targeting a specific 130

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Scheme 14. Formylglycine-Generating Enzyme Oxidation of Cysteine Thiol To Form Formylglycinea

a

The formylglycine intermediate can react with bioorthogonal reactions with alkoxylamines, hydrazines, or Pictet−Spengler-reacting tryptamines to generated protein−drug conjugates. Redwood Biosciences/Catalent developed a HIPS linker that undergoes a Pictet−Spengler reaction to generated protein−drug conjugates.

Scheme 15. Two Strategies for Overcoming SrtA Transpeptidation Reversibilitya

a

(A) Flow reactor with immobilized SrtA shifts equilibrium toward products by removing the products from the catalyst. (B) Irreversible N-Gly coupling partner provides byproducts that cannot actively participate in catalysis.

4.1.1.6. Sortase A. Sortase is another promising enzyme for antibody functionalization. Sortase A (SrtA) recognizes the Cterminal signaling motif LPXTG and cleaves the Thr-Gly peptide bond.144 The acyl-enzyme intermediate can be attacked by a variety of N-terminal poly-Gly payloads to couple two proteins or peptides of interest. One major disadvantage of this system is that the product of the SrtA reaction also carries the LPXTG sequence. Thus, the product is a substrate for the enzyme, which results in enzymatic consumption (reversibility) of the product over time. SrtA has demonstrated extraordinary value over the years, as many researchers have exploited SrtA for a variety of protein and antibody functionalization purposes. It has been utilized for the development of positron emission tomography (PET) imaging agents,145,146 semisynthesis of proteins,147 and numerous in vivo labeling studies in a variety of cell lines.8 However, the use of SrtA as a preparative technique for isolating functionalized proteins has remained a challenge. The reversibility of the enzyme requires high concentrations of the nucleophile to drive the equilibrium of the reaction, as well as robust purification protocols. To circumvent this limitation, Pentelute and co-workers148 developed a flow-based reactor packed with immobilized SrtA (Scheme 15A). Running the reaction in flow allowed product removal during the reaction, which let the researchers reduce the poly-Gly−nucleophile concentrations. Turnbull and co-

site within a therapeutic protein for post-translational ligation of molecular cargo. When applied to an antibody, FGE enables expression of antibodies with specifically inserted aldehydes suitable for bioorthogonal reactions with alkoxylamines, hydrazines, or Pictet−Spengler-reacting tryptamines.141 Bertozzi and co-workers142 have exploited FGE for in vitro oximination to glycosylate an internal IgG CTPSR site. A key feature of this work is that the recognition sequence was buried in the Fc domain of the antibody, and FGE was able to oxidize the cysteine to generate the aldehyde. This work illustrated that the tag does not need to be located at the N- or C-terminus for FGE to react. Unfortunately, the recombinant Mycobacterium tuberculosis FGE used to generate the fGly residue required stoichiometric dosing, which ultimately limits the utility of the methodology for preparative reactions. Using FGE as a conjugation enzyme, Redwood Bioscience/ Catalent developed the SMARTag platform for aldehyde incorporation to facilitate building ADC libraries using proprietary hydrazino-Pictet−Spengler (HIPS) chemistry and a proprietary 4AP linker (Scheme 14).8 High titer production of the aldehyde-tagged antibodies has been demonstrated on the 100 L scale, which illustrates the viability of FGE for ADC manufacture.143 Redwood Bioscience/Catalent recently entered into a licensing agreement with Triphase Accelerator Corporation to advance their SMARTag generated ADC, CD22−4AP, into the clinic. 131

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Scheme 16. Engineered Sortase A Mutants Deliver a New Strategy for Dual Conjugation Sites

workers149,150 took another approach to overcome reversibility and reduce the requirements for overwhelming concentrations of the nucleophile-coupling partner. They engineered a dipeptide nucleophile that does not release Gly and therefore cannot participate in the reverse reaction. Liu et al.151 at Lilly Research Laboratories took a similar approach and developed irreversible substrates fragment that form a diketopiperazine after incubation with SrtA (Scheme 15B). While these are interesting approaches, they are limited by their substrate scope, as the N-terminus of the protein of interest is the sole site that can be modified. Evolution of the SrtA enzyme by yeast display technology has also proven useful in improving transpeptidase activity.152 Liu and co-workers153 found that the combination of yeast display and fluorescence-activated cell sorting provided an efficient path to identifying a variant with 140-fold enhancement in LPETG-coupling (compared to wild type) after eight rounds of screening. This approach identified two new SrtA variants that recognized the sequences LPXSG and LAXTG (Scheme 16). This discovery opens the door to preparing polyfunctionalized proteins with different cargo. NBE Therapeutics Group has commercialized a platform for sortase-enzyme mediated antibody conjugation (marketed as SMAC-Technology). Beerli et al.154 at NBE demonstrated that SrtA recognition motif LPETG could be added to heavy and light chains of immunoglobulin at their C-termini. These positions were then conjugated with a toxin-ligated (Gly)5 peptide. A chief advantage of this technology is in the employment of cleavable linkers. This could allow (Gly)n to be a universal linker since it would be separated from the released drug. In addition to NBE Therapeutics Group, Bayer Schering Pharma AG has also demonstrated the utility of sortase for antibody conjugation chemistry in a commercial setting. Researchers at Bayer conjugated a (Gly)3-DY647 fluorophore to the C-terminus of an antibody Fab fragment F19 bearing the sequence, LPETGG.155 Unproductive isopeptide bond formation was observed between the LPETGG C-terminus and a Lys side chain in the antibody. This cross-linking phenomenon could be overcome by increasing the concentration of the polyGly−nucleophile. Interestingly, Chilkolti and co-workers156 recently exploited this isopeptidyl background reaction to engineer new coupling partners for SrtA reactions. By exploiting reactive Lys side chains on immunoglobulin, a payload with a LPETG sequence could be loaded onto the antibody. 4.1.1.7. Biotin−[acetyl-CoA-carboxylase] Ligase and Lipoate−Protein Ligase A. Exciting early work by Ting and coworkers37 demonstrated that the E. coli enzyme biotin−[acetylCoA-carboxylase] ligase (BirA), which biotinylates lysines within a recognition sequence (GLNDIEAQKIEWHE), will accept a synthetic keto analog of biotin as a substrate. This substrate promiscuity, although modest, broadens the utility of BirA, similar to FGE or Ambrx technology, inserting a carbonyl

(in this case a ketone) site-specifically into a protein (Scheme 10). Poor reaction kinetics still currently limit its utility; however, this proof of concept does present a new opportunity for protein engineers with an interest in further expanding the toolbox of scalable protein functionalization techniques. In subsequent studies by Ting and co-workers,157,158 the same substrate promiscuity concept was applied to E. coli lipoate− protein ligase A (LplA). The substrate specificity of LplA was altered by a single point mutation, which enabled the introduction of reactive moieties on lipoic acid analogues (R = azide or alkyne, Scheme 8). Like BirA, LplA requires a peptide recognition sequence to direct the enzyme’s activity to the appropriate lysine on the polypeptide (GFEIDKVWYDLDA). In essence, this technology converts a lysine into a functional handle (azide, alkyne, or ketone) that is ready to install molecular cargo that is otherwise biologically inert.159,160 4.1.2. Protein Carbohydrate Remodeling. Glycan remodeling is another area of biocatalyst activity with exciting commercial applications. This is largely because protein glycosylation is one of the most prevalent post-translational modifications (PTMs). Glycosylation can affect protein folding, stability, and intracellular trafficking. Over 40 different types of sugar−amino acid linkages have been identified with combination products ranging from N- and O-glycosylation to Cmannosylation to phosphoglycation to glypiation.161 There are at least 13 different monosaccharides and 8 amino acid residues implicated in protein−carbohydrate chemistry in nature. Roughly 16 glycosylation enzymes have been characterized, and many of these genes have been cloned. N-linked and Olinked are the two major classes of glycoproteins. The site of glycan attachment for N-glycoproteins is the amide side chain of an Asn residue in an NXS/T consensus sequence (where X is anything but proline), while O-glycoproteins bear the sugar on the hydroxyl group of a Ser or Thr residue. There is no known consensus sequence for O-glycoproteins. Because the site for O-linked glycosylation is more difficult to control, researchers have more often targeted sites to produce N-linked glycans. To date, expression of glycoproteins is carried out in eukaryotic cells, primarily mammalian cells, because E. coli lacks the appropriate glycosylation machinery. Production of proteins in mammalian cells is expensive and inefficient (lower titers) in comparison to production of nonglycosylated proteins in E. coli. Currently, much of the research in the space of glycoprotein synthesis is in strain engineering techniques to coexpress the appropriate glycosylation enzymes for whole-cell production of the final product.162,163 Researchers at Glycobia have commercialized platforms based on this technology for expression of human glycopeptide and glycoprotein drugs avoiding mammalian cell culture. For an overview of these strain engineering and synthetic biology advances, the reader is referred to reviews by Wang and Amin164 and Schmaltz et al.165 The following sections are dedicated to biocatalytic advances to deliver glycoproteins via in vitro techniques. 132

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Scheme 17. Chemoenzymatic Synthesis of ADCs by Use of GalactosyltransferaseTo Remodel the Glycan Network for Incorporation of Synthetic Sugars with Bioorthogonal Handles for Chemical Ligation

4.1.2.1. Overview of Techniques for Glycan Modification: Glycosylases and Glycosyltransferases. The classic approach to remodeling N-glycoproteins involves two key manipulations that utilize enzymes in the glycosylase (EC 3.2) and glycosyltransferase (EC 2.4) families. The first step involves enzymatic deglycosylation (or trimming) the N-glycans with an endoglycosidase (EC 3.2.1). Trimming the glycan affords the innermost N-acetylglucosamine (GlcNAc) at the glycosylation site, which can then be elongated with monosaccharides in a stepwise fashion by the appropriate glycosyltransferases (EC 2.4). This stepwise addition of monosaccharides by glycosyltrasferases can be problematic because it requires either (1) strain engineering for in vivo production of the glycoprotein or (2) heterologous expression of each glycosyltransferase for the in vitro semisynthesis of the glycoprotein. Even though there are disadvantages to this technology, this strategy has been the most explored strategy for glycan remodeling of therapeutics. An alternative approach that has emerged in recent years is a convergent chemoenzymatic synthesis of glycoproteins. While this approach has not found its way into larger-scale preparation, it is a promising approach for the discovery and manufacture of future therapeutics. In this approach the Nglycan is preassembled chemically, and an endoglycosidase is employed for the transglycosylation of the N-glycan to the GlcNAc-protein in a single enzymatic step.166 Two primary developments facilitated this approach becoming the state-ofthe-art. First is the identification of competent, synthetic oligosaccharide oxazoline analogues to act as the donor substrates for transglycosylation.167,168 The second advance was the creation of “glycosynthases”, which are single-pointmutation variants of endoglycosidases. The single point mutation of a conserved active-site Asn residue abolished all product hydrolysis activity.169−171 4.1.2.2. Protein Carbohydrate Remodeling for Antibody− Drug Conjugate Synthesis. Interesting glycosyltransferases represent a large, well-characterized family of enzymes that have been explored not only for their potential applications in glycotherapeutics172 but also as a tool for studying glycobiology.173,174 These enzymatic transformations are highly versatile because they transfer sugar phosphates, as glycosyl donors, to a nucleophilic group, usually an alcohol, to form a glycosidic linkage. The product may be an O-, N-, S-, or C-glycoside bearing a monosaccharide, oligosaccharide, or polysaccharide, so it is not a surprise that many researchers have explored these enzymes for ADC development. Synaffix175 and Glykos176 utilized galactosyltransferase, GalT, to introduce synthetic sugars at preexisting glycosylation sites. In the case of Synaffix, the sugars, GlcNAc-S(A)x, bear one or more functional handles (A = azido, keto, or alkynyl group) to facilitate metal-free click chemistry to ligate cytotoxin to antibodies for the synthesis of ADCs (Scheme 17). Limitations

of this technology include (1) the length of synthesis needed to prepare the UDP-functionalized sugars (i.e., UDP-GalNAz) and (2) the poor atom economy of the coupling partner (i.e., UDPleaving group). Nevertheless, both companies have built these platforms for preclinical ADC development. Furthermore, Glykos recently partnered with Sanofi to develop clinically viable ADCs using the GalT technology to combine Glykos’ propriety hydrophilic linkers with Sanofi’s payloads and antibodies. The glycosylation network of antibodies can be an effective handle for site-selective incorporation of a payload. However, this technology would be more powerful if it could be fully executed in a single pot. Because the importance of the glycan framework in ADCs is still unclear, the utility of this methodology has yet to be established in the clinic. In general, interest in ADC therapeutics has resulted in an exponential growth in commercial, off-the-shelf tools for enzymatic protein conjugation. Development of new tools for antibody engineering, enzyme engineering, and expansion of substrate recognition will be valuable tools for driving novel therapeutic proteins to market. With these new platform-based technologies available from commercial sources, it will be interesting to see what effect this will have on the greater protein functionalization field, particularly in the industries described in section 3. 4.2. Drug Delivery and Biopharmaceuticals

In bringing safe and efficacious protein therapeutics to market, scientists face three common challenges: immunogenicity, instability, and short circulating half-lives.177 A number of strategies have been identified to assuage these clinical issues. Perhaps the most common modification is the covalent addition of poly(ethylene glycol) (PEG) to a protein. Other methods for improved clinical profiles include lipid-based delivery vehicles, hyperglycosylation, and polymeric micro/ nanospheres. Biocatalytic transformations have proven useful in providing solutions for two of these types of drug modifications: PEGylation and hyperglycosylation. 4.2.1. PEGylation. Many researchers have reported that poly(ethylene glycol) conjugation (PEGylation) to the surface of proteins can increase solubility and stability in organic solvents, enhance thermostability, and lend favorable pharmacokinetic and pharmacodynamics properties for therapeutic proteins.178 Furthermore, PEGylation has unambiguously been shown to extend the half-life of proteins and reduce immunogenicity.179 There are now 11 FDA-approved, PEGylated-pharmaceuticals currently on the market. Because reproducibility and minimizing batch-to-batch variation is crucial for production of biopharmaceuticals, predictable methods for covalent addition of PEG to a protein are vital. While significant advances in direct enzymatic PEGylation are 133

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Scheme 18. GlycoPEGylation by Use of Sequential in Vitro Site-Directed O-Glycosylation by GalNAc Transferase, Followed by Subsequent Sugar Elongation with a PEGylated Sialylated CMP Derivative Catalyzed by ST6GalNAc-Ia

a

Both stepwise and single-vessel reactions afford a single product without the need for intermediate purification steps.

therapeutic enzyme currently on the market, the yeastproduced α-glucosidase had superior uptake by the lysosome and was more efficiently delivered to the target. Rather than using a strain engineering approach, researchers at Genzyme explored in vitro hyperglycosylation strategies for creating glycoprotein therapies for the treatment of Pompe disease.187 One of these strategies employed galactose oxidase for the direct enzymatic oxidation of the galactosyl protein, generating a reactive aldehyde amenable to further functionalization. This approach was highly selective, generating mannose 6-phosphate-rich α-glucosidase at very specific glycosylation site because it exploits the existing glycosylation pattern. However, enzymatic oxidation was quite limiting in the diversity of glycoproteins that could be generated, due to the high fidelity of galactose oxidase. In fact, chemical oxidation (using periodate) of the sugars resulted in a number of glycoprotein isomers that delivered up to 5-fold enhancement in receptor binding versus the enzyme-treated protein. Again this example demonstrates the importance of having a greater suite of tools for the generation of libraries of biotherapeutics for effective structure−activity relationship studies.

discussed in section 4.3, these examples have not found their way into large-scale syntheses for clinical or commercial deliveries. However, utilizing glycosylation strategies in combination with PEGylation has proven successful for delivering material to the clinic. In fact, researchers have demonstrated the value in targeting glycosylation sites for addition of PEG, a technique often called glycoPEGylation. Zopf and co-workers180 developed a twoenzyme system for effective transfer of the desired PEG-bearing carbohydrate to a protein of interest. First, N-acetylgalactosamine (GalNAc) was ligated to Ser and Thr residues by OGalNAc-transferase. Then exposure of the GalNAc protein with sialyl-PEG substrate (CMP-sialyl-PEG) in the presence of sialyltransferase (ST6GalNAc-I, EC 2.4.99.9) afforded the sialyl-PEG-O-GalNAc glycoprotein (Scheme 18). This is a particularly effective method for producing biotherapeutics because the authors demonstrated that the protein of interest could be expressed in E. coli followed by postexpression enzymatic reaction in a single pot without intermediate purification steps. Notably, Neose Technologies exploited this technique to deliver PEG-recombinant factor IX to the clinic for phase I clinical trials.181,182 GlycoPEGylation afforded a factor IX therapeutic with improved pharmacokinetic properties and good clinical data for enzyme replacement therapy for patients suffering from hemophilia B.183 Novo Nordisk (and Neose Technologies, Inc.) further utilized this strategy to functionalize factor VIIa for improved pharmacokinetic properties and clinical efficacy.184,185 In this study, PEG was covalently attached to either Asn (N-linked) or Ser (O-linked) oligosaccharides in factor VII polypeptides and factor VIIrelated polypeptides for clinical studies. 4.2.2. Hyperglycosylation. Nature’s method for increasing protein stability is to decorate proteins with sugar molecules to increase rigidity and stability by promoting intramolecular Hbonding networks. So it is no surprise that hyperglycosylation of protein therapeutics has been shown to reduce immunogenicity, improve half-life, and increase stability. The biodegradable nature of oligosaccharides provide a clear advantage of hyperglycosylation with respect to PEGylation. In addition, glycosylation is often a preferred approach because of the low interference of added glycans in protein−receptor binding. Researchers at VIB and Oxyrane discovered a bacterial glycosidase that can convert N-glycans modified by yeast-type mannose-Pi-6-mannose to mammalian-type N-glycans with a mannose-6-phosphate substitution.186 This is a significant discovery because enzyme therapies for Pompe disease contain relatively low levels of mannose 6-phosphate, a PTM that aids in lysosomal uptake. Therefore, they engineered a yeast expression system equipped with the glycosidase to circumvent the lower expression yields that plague mammalian cell protein expression systems. By coupling the uncapping glycosidase with α-mannosidase, the researchers were able to produce a Pompe disease enzyme in yeast that bore the appropriate α-glucosidase rich in mannose 6-phosphate. When compared with the

4.3. Promising Preclinical Applications

4.3.1. Sortase A. Exploiting the LPXTG motif to target the C-terminus of a protein of interest, SrtA has been shown to efficiently couple poly-Gly-PEG cargo with molecular masses up to 20 kDa.188 More specifically, this study improved the properties of cytokines by functionalizing granulocyte colonystimulating factor 3 (GCSF-3) and interferon α2 to extend cytokine half-life without losing biological activity. SrtA was utilized to obtain circular polypeptides by covalently joining the N- and C-termini, resulting in an increase in thermal stability. By using two different SrtA variants (engineered to recognize different tagging sequences),153 PEGylation and circularization could be achieved in a single step with two enzymes.188 This Cterminal modification method is particularly intriguing because it provides a complementary approach to classic chemical PEGylation methods, for which functionalization of the Cterminus is not straightforward. 4.3.2. Transglutaminase. A few researchers have demonstrated promising reactivity and regioselectivity of mTGcatalyzed PEGylation of therapeutically interesting proteins. By use of PEGs terminated with a primary amine, mTG can effectively catalyze the site-specific addition of PEG to several proteins at a single or multiple Gln residue(s). 189,190 Interestingly, mTG-mediated PEGylation is often highly selective because it is often the case that only one Gln is accessible to the enzyme. When more than one Gln is accessible to mTG, researchers have used organic solvents to rigidify the protein’s structure and restrict mTG access to Gln residues, thereby reducing the isomers present in the final product mixture. Ethanol has been used to produce a single isomer from mTG-catalyzed-PEGylation of both salmon calcitonin and human growth hormone.191 Ajinomoto has also demonstrated that single-isomer PEGylation can be achieved with mTG. Specifically, recombinant human inter134

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food, pharmaceuticals, materials chemistry, and clothing. Many of these industries favor biocatalytic transformations, particularly when the reaction components are ingested or injected. Other industries, like those producing leather and textiles, require efficient yet mild reaction conditions (neutral pH and low temperatures) to maintain fiber integrity. The biologics sector of the pharmaceutical industry requires regioselective and site-selective transformations to achieve homogeneous biopharmaceutical preparations. This is particularly important because seven of the 10 best selling pharmaceuticals are biologics. Having clear economic motivation is inspiring much of the growth in developing and commercializing platforms for enzyme-mediated protein conjugation. To date, the research and study of new enzymes for protein functionalization is largely guided by what is commercially available. Long lead times for generating newly expressed enzymes that are not commercially available limit the development of these methodologies in many areas that require immediate go/no-go decisions. One way to aid in the discovery of new applications for enzymes would be for those in industry to have ready access to academic constructs without the delays of material transfer agreements (MTA) and confidential disclosure agreements (CDA) for the sale of transfer of material, which contribute to long lead times. Historically, Sigma−Aldrich and Strem have been leaders in bringing promising new chemical catalysts developed in the academic arena to industrial researchers by making them available for purchase in research quantities. To this point, Strem has started to carry biocatalysis kits to streamline bringing new enzyme platforms to market. While the inventory is still small (mostly hydrolytic enzymes), perhaps more enzymes developed in academic laboratories will begin to find their way into the Strem catalog in the future. Of the almost 4000 enzymes that have been identified, roughly 20 are produced on the industrial scale.1 Of these 20 enzymes, roughly 75% are hydrolytic enzymes (primarily carbohydrases, proteases, and lipases), which means there is not much reaction diversity available on the market today. By far the most extensive body of research has been focused on protein−protein cross-linking methods, particularly in the food industry. Ready commercial sources for cross-linking enzymes spurred much of the early PEG and ADC fields. Recent advances in small-molecule−protein conjugates in therapeutics, coupled with growing market interest in bringing new biologics to market, will surely have a positive impact on the quantities and availability of new enzymes on the market. One would anticipate that the growth in biopharmaceuticals will lead to a larger pool of immobilized enzymes on the market. Furthermore, enzyme immobilization allows for catalyst recycling, easy separation of the enzyme from the reaction mixture, and the opportunity to telescope reactions to cut purification costs. Another enzyme-engineering approach that should see growth is directed evolution of protein-modifying enzymes. Given the diversity of reactions catalyzed by mTG and sortase, it is surprising that there are few examples of directed evolution of mTG in the literature and even fewer industrial research and development examples. The lack of competition in the space is likely due to the difficulty associated with expressing enzymes like mTG in high-throughput (HTP) vectors such as E. coli. Numerous other major enzymes in industry have also not been evolved. Hydrolases represent nearly 75% of all industrial enzymes sold globally, yet there are relatively few examples of

leukin 2 is effectively PEGylated by mTG at a single Gln residue, providing a protein−PEG conjugate with a favorable pharmacokinetic profile.192 Researchers at Bio-Ker have explored the use of mTG for direct PEGylation of human GCSF. 193 These studies demonstrated the advantages of mTG for sole specificity of Gln135 conjugation and high PEGylation yields. The sitespecific mono-PEGylated filgrastim was a promising preclinical candidate for treatment of neutropenia in patients undergoing chemotherapy. The conjugate displayed higher in vivo activity than FDA-approved Neulasta that is N-terminally PEGylated. This example, again, underscores the importance of the conjugation site on the efficacy of the therapeutic. At Novo Nordisk, researchers found that mTG could effectively catalyze the PEGylation of human growth hormone (hGH) to delivere Gln40-PEG and Gln141-PEG conjugates of hGH.194,195 Through evolution of the mTG polypeptide, five discrete mutants were identified that exclusively afforded the Gln141-PEG−hGH conjugate. The PEG−hGH conjugates were shown to exhibit better pharmacokinetic profiles than the unmodified hGH.196 While these are interesting proof-ofconcept reactions, the clinical and commercial value of mTG for delivering PEGylated proteins has yet to be demonstrated. Most of the mTG protein conjugation examples discussed in this review focus on the protein of interest bearing the Gln residue to have greater control over site selection. However, the Lys residues on proteins are also available for reaction in mTGcatalyzed transformations. Both Novartis197 and GlaxoSmithKline198 have recently employed mTG to prepare research quantities of Lys-conjugated glycoproteins for vaccine development. In these independent studies, both groups found that the location of carbohydrate conjugation significantly impacts immunogenicity. These studies further illustrate the need for a robust and complete toolbox for selective protein functionalization that can access a variety of amino acid side chains within a protein of interest. 4.3.3. Tyrosinase. An emerging application for enzymecatalyzed protein functionalization is in the synthesis of human serum albumin (HSA) conjugates for drug delivery. USP-grade (pharmaceutical-grade) recombinant HSA is available from Novozymes (marketed as Recombumin) for applications in drug delivery, pharmaceutical formulations, and medical devices. HSA is a blood transport protein that is frequently implicated in the binding of many endogenous and exogenous molecules, including hormones, fatty acids, and pharmaceuticals. Researchers have begun to exploit the binding efficiency and promiscuity of HSA in the development of novel therapeutics for controlled or targeted release.199 There are limited examples of enzymatic conjugation of HSA to therapeutics. One such example is the use of tyrosinase in the preparation of mAb−HSA conjugates. This conjugation strategy employed a phenolic linker molecule (tyrosinase substrate) to enhance conjugation yields of the HSA to the mAb.200 This is the only reported example of enzymatic HSA ligation chemistry to date. It will be interesting to see if some of the protein cross-linking strategies (described in section 3) or ADC ligation methods (section 4.1) find their way into HSA− conjugation research.

5. CONCLUSION Enzyme-catalyzed protein conjugation strategies show significant scientific potential for several reasons. The commercial applications of these transformations span multiple industries: 135

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ACKNOWLEDGMENTS Many thanks to Dale Edmondson (Emory), Valarie Truax (Yale), Cora MacBeth (Emory), Helge Zieler (Primordial Genetics), Rob Garbaccio (Merck), Sean Hu (Dophen), Jeffrey Moore (Merck), and Jesse Dill for helpful discussions in regard to biocatalysis, protein functionalization technologies, clinical landscape, and food industry applications.

hydrolases being evolved in the pharmaceutical sector. This is in part because the natural pool of enzymes is rich but also because of the difficulty in evolving enzymes that may be toxic to the cell because they catalyze proteolysis or cross-linking, which may disrupt cellular processes. Nature avoids this toxicity by adding a prosequence to the enzyme that inhibits its function prior to cleavage. This adds a layer of complication when designing HTP experiments for enzyme evolution. Additionally, enzymes that contain a high degree of posttranslational modification, like glycosylation, can also be a challenge for the current protein expression tools available to researchers. With the growth in commercial applications for protein functionalization, it will be exciting to see if new molecular biology and enzyme engineering tools come to market. HTP expression systems (for evolution) and fermentation expression systems (for industrial production) of biocatalysts with high PTMs are still needed. Moreover, streamlining methods for immobilizing enzymes would aid in bringing new conjugated proteins to market. As demonstrated in the examples in this review, bringing new biologics to market is highly processdependent, unlike small-molecule drugs, whose chemical syntheses do not affect the efficacy of the final therapeutic. For biologics, the process for preparing the drug during the discovery phase is nearly identical to the process used for manufacture of the biologic. The availability of these important enzymatic tools early in the discovery process is crucial for the enzymatic process to be employed in the final manufacturing routes.

ABBREVIATIONS mTG microbial transglutaminase GRAS generally recognized as safe PTM posttranslational modification PPTase phosphopantetheinyl transferase PFTase protein farnesyl transferase ADP adenosine diphosphate ATP adenosine triphosphate UDP uridine diphosphate CMP cytidine monophosphate Sfp surfactin phosphopantetheinyl transferase SrtA sortase A BirA biotin ligase SAR structure−activity relationships mAbs monoclonal antibodies FGE formylglycine generating enzyme ADC antibody−drug conjugate IgG immunoglobulin G GlcNAc N-acetylglucosamine GalT galactosyltransferase PEG poly(ethylene glycol) HTP high-throughput

AUTHOR INFORMATION Corresponding Author

REFERENCES

*E-mail [email protected].

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ORCID

Erika M. Milczek: 0000-0003-3123-2923 Notes

The author declares no competing financial interest. Biography Erika obtained her BS in Chemistry at the University of Tennessee in 2005. She went on to Emory University to start her graduate career in department of chemistry working in the laboratory of Prof. Simon Blakey, where her research focused on developing new methodology for asymmetric C−H amination using Ruthenium-pybox catalysts. After 2 years in the Blakey laboratory, Erika moved to the biochemistry department in the Emory School of Medicine, as an NRSA predoctoral fellow in the laboratory of Prof. Dale Edmondson, studying the structure−function relationship of monoamine oxidase. Completing her doctorate in 2010, Erika joined the laboratory of Prof. John T. Groves at Princeton University as a postdoctoral researcher, where she investigated the use of both biomimetic iron complexes and ironperoxygenases in the oxidation of hydrocarbons to alcohols. In 2012, Erika joined the Biocatalysis Group in the Process Chemistry Department at Merck & Co., where she developed cytochrome P450 oxidation platforms for metabolite ID of active pharmaceutical ingredients as well enzymatic methods for functionalizing therapeutic proteins. In 2016, Erika launched a biotech company that develops and commercializes new consumer products by utilizing engineered enzymes and biocompatible materials. 136

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