Glycan Directed Grafting-from Polymerization of Immunoglobulin G

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Glycan Directed Grafting-from Polymerization of Immunoglobulin G: Site-selectively Modified IgGpolymer Conjugates with Preserved Biological Activity Chih-Hung Chou, and Po-Chiao Lin Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00669 • Publication Date (Web): 11 Jun 2018 Downloaded from http://pubs.acs.org on June 12, 2018

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Glycan Directed Grafting-from Polymerization of Immunoglobulin G: Site-selectively Modified IgG-polymer

Conjugates

with

Preserved

Biological Activity Chih-Hung Chou, and Po-Chiao Lin* Department of Chemistry, National Sun Yat-sen University 70, Lienhai Road, Kaohsiung 80424, Taiwan E mail: [email protected]

KEYWORDS: Protein-polymer, ATRP, Antibody-PNIPAAm conjugates

ACS Paragon Plus Environment

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ABSTRACT

Antibody and related antibody drugs for the treatment of malignancies have led to the progress of targeted cancer therapy. Preparation of diverse antibody conjugates is critical for preclinical and clinical applications. However, precise control in tagging molecules at specific locations on antibodies is essential to preserve their native function. In this study, a synthetic boronic acid (BA)-tosyl initiator was used to trigger a glycan-directed modification of IgGs and the obtained IgG macroinitiators allowed a growth of the poly N-isopropylacrylamide (PNIPAAm) chains specifically at Fc-domains. . Therefore, the PNIPAAm chains are located away from the critical antigen-binding domains (Fab) which could reasonably prevent the loss of biological activity after the attachment of polymer chains. According to the proposed strategy, a site-selectively modified anti-concanavalin A (Con A) antibody-PNIPAAm conjugates showed six times higher efficiency in the binding of targeted Con A antigen to a randomly conjugated anti-Con A antibody-PNIPAAm conjugates. In this study, we developed the first chemical strategy for the site-specific preparation of IgG-polymer conjugates with conserved biological activity as well as intact glycan structures.

INTRODUCTION In the post-genome era, understanding the function, modification, and regulation of proteins remains an important but daunting task.1 Numerous proteins, such as enzymes, effectors, and antibody (Ab), are known to regulate important biomolecular interactions and have potential as powerful treatments in many diseases. In recent decades, protein-polymer conjugates have been successfully applied in diverse biomedical applications, specifically for improving


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pharmacokinetic properties, affinity separation, as microfluidic devices, and as switches in enzymatic reactions.2-6 For instance, the PEGylation of therapeutic proteins, especially Abs, has been demonstrated to significantly improve both physical and thermal stability as well as reduce immunogenicity. For these reasons, PEGylated proteins have been approved by the U.S. Food and Drug Administration (FDA) for therapeutic use in diabetes, cancer, and hepatitis C.7-8 However, non-selective polymer conjugation results in multiple polymer attachments at random grafting sites. The heterogeneous mixtures of polymerized positional isomers could considerably reduce their native bioactivity.9 Recent advances in site-selective protein-polymer ligation have rapidly grown to attain homogeneity of protein-polymer conjugates.10-12 Two methods, grafting-to and grafting-from, depending on polymerization occurring before or after the protein conjugation are major synthetic routes for the preparation of homogenous protein-polymer conjugates. In the concept of grafting-to, the pendant polymers attach to the proteins of interest via specific chemical reactions. With the high reactivity of the sulfhydryl group and relatively sparse occurrence of cysteine residues in proteins, cysteine modification provides a useful method in chemoselective protein modification. An incorporation of a cysteine residue to maleimide or divinyl sulfone-conjugated polymers via Michael addition showed a convenient way in the preparation of protein-polymer conjugates.13-16 Using a pyridine disulfideconjugated polymerization initiator, the Maynard group has successfully developed a cysteinespecific strategy in the preparation of BSA and T4 lysozyme-polymer conjugates with conserved bioactivity.17 Moreover, an activity-directed grafting-to synthesis of protein-polymer conjugates was developed using biotin-ended poly(N-isopropylacrylamide) to non-covalently associate with streptavidin.18 The grafting-to method have also contributed in the homodimeric19 and


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heterodimeric20 protein-polymer conjugations. The prefabricated polymers can be synthesized in harsh or bioincompatible conditions, and are easier to be characterized. However, the steric hinderance and aqueous solubility of polymers can lead to low conjugation efficiency. Subsequently, the difficulty for separating protein-polymer conjugates from unreacted synthetic polymers can considerably limit its practical uses. In contrast, grafting-from method directly elongates polymer chains from proteins can prevent above-mentioned drawbacks, but a well characterization of attached polymers could be very challenging. To a similar concept, using a pyridine disulfide functionalized initiator reacted with targeted T4 lysozyme and the formed macroinitiator can then trigger the growth of polymer chains. The obtained T4 lysozymepolymer conjugates showed no statistical differences in bioactivity.21 Similarly, Davis and coworkers used a pyridine disulfide-conjugated chain-transfer agent (CTA) to prepare BSApolymer conjugates via reversible addition fragmentation transfer polymerization.22 Moreover, the sulfhydryl group of cysteine can proceed via Michael addition to prepare the macroinitiator and macro-CTA, leading to direct polymer growth from the surface of the protein.23-24 A sitespecific (C-terminal) preparation of green fluorescence protein (GFP)-PEG conjugates by inteinmediated protein ligation provides a general methodology for improving pharmacokinetic profiles.25 Besides those chemoreactivity directed conjugation methods, affinity-directed protein macroinitiator preparation provided an alternative to achive site-selective conjugation. Biotinylated initiator was demonstrated to specifically bind streptavidin, forming the streptavidin initiator, and subsequently triggering polymer chain growth to finally produce streptavidinpolymer conjugates.26 Although the active sites were blocked and could be at risk of dissociation, no need of genetic engineering techniques allows this strategy to target endogenous proteins and forms homogeneous protein-polymer conjugates.


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In this research, we aimed to propose an affinity-directed strategy in the preparation of Abpolymer conjugates which can conserve the Ab’s native structure as well as bioactivity. Immunoglobulin G (IgG) is the most abundant class of antibodies, constituting about 80 % of the total serum immunoglobulin, and dominating the recognition, neutralization, and elimination of foreign antigens and pathogens. IgG-based drugs are believed to be the most rapidly expanding class of pharmaceuticals for treating a variety of human diseases, especially cancers.27-28 The IgG molecule consists of two identical heavy chains and two light chains which is endowed with a variable region, comprising the antigen recognition site (Fab domain, pink part of IgG in Figure 1) and a constant region (Fc domain, cyan part of IgG in Figure 1) which bind to effector molecules. The Fc domain, composed of two paired Ig domains, mediates the interaction of Ab molecules and Fcγ receptors, and thereby facilitates the ingestion and destruction of potentially harmful antigens recognized by IgG. Two oligosaccharide chains attached at Asn297 of the Fc domain were found to be closely related to antibody’s function. Difference in glycosylation might considerably affect antibody function in the studies of the remission of rheumatoid arthritis,29 and inflammatory response.30-31 The integrity of Fab domains and of Fc domain with glycan chains is crucial for the complete activity of IgG Ab. Therefore, a good control of polymer conjugation sites would allow for the production of homogeneous, modified, but completely functional Ab conjugates. Conventionally, the preparation of Ab-polymer conjugates is conducted by random conjugation at reactive amino acid residues, including amino (lysine and histidine), thiol (cysteine), or hydroxyl (serine, threonine, and tyrosine) groups. The formation of subsequent peptide bonds, disulfides, and carboxylic esters is highly efficient; however, conjugation at multiple and random sites is known to considerably reduce the affinity of binding to target proteins. When conjugation


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sites are located very close to antigen-binding domains, the biological function of the Ab is reduced due to steric hindrance. Further, the conventional uncontrolled conjugation strategy may have considerable batch-to-batch variation in the preparation of Ab-polymer conjugates, thereby resulting in inconsistent and heterogeneous products. To reduce the loss of Ab function, siteselective modifications of Abs are usually targeted at the Fc domain, which is distant from the antigen binding sites. Methods involving the specific reduction of interchain disulfides, followed by alkylation, are popularly used for site-specific Ab modification.32-35 However, native disulfide bonds of a protein are usually crucial to its structure and function, and cannot, therefore, be easily modified.36-37 To avoid altering the native structure of Ab, recombinant techniques provide an ideal tool for the production of Abs fused with a protein, such as green fluorescent protein.38 Schultz and co-workers genetically incorporated unnatural amino acids (UAAs) into Abs, placing ketone functional groups on their residues. The ketone groups can then selectively react with aminooxy-derived ligands to achieve site-specific Ab modification.39 Moreover, enzymatic reactions have also been used to introduce molecules of interest at a specific position in Abs. For instance, an acyl-transfer reaction between the γ-carboxamide group of glutamine and a primary ε-amino group, mediated by transglutaminase, has been successfully used in site-specific and stoichiometric modification of Abs.40 Francis and co-workers developed a pyridoxal 5’phosphate-catalyzed biomimetic transamination reaction that introduces a ketone group on the N-termini of monoclonal anti-FLAG IgG.41 Alternatively, carbohydrate residues of Ab Fc domains offer a unique chemical reactivity for Ab site-specific modification. The diol group-rich carbohydrates allow specific oxidative cleavage by periodate; resultant aldehyde groups can then be used as sites for attachment of amino or hydrazine derivatives to the Ab.42-48 Recently, Lin and co-workers proposed a glycan-


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mediated approach combined with photo-crosslinking to conduct a site-specific Ab immobilization.49 All above mentioned methods can specifically introduce tag molecules at the Fc domains of Abs of interest. However, the necessity of genetic engineering limited their use in native Abpolymer conjugate synthesis. Although glycan-directed modification can prevent the loss of antigen binding ability, the glycan chain function could be considerably affected. To overcome this limitation, a Fc-specific strategy

conserving its glycan structures is critical to the

preparation of diverse Ab-polymer conjugates.50 To explore the general concept of activitydirected synthesis of protein-polymer conjugates, in this research, ligand-directed tosyl chemistry51 was applied in the study of Ab-polymer conjugates. Very recently, we successfully applied this traceless labeling strategy to glycoproteins of interest with synthetic boronic acid (BA)-tosyl chemical probes.52 According to the well established synthetic method,53 a new synthetic probes is proposed to selectively introduce the initiators at the Fc domain of IgG as the macroinitiator and subsequently used in the preparation of Ab-polymer conjugates. Probe molecules were composed of three general elements: an affinity ligand as a targeting head, a reactive benzenesulfonyl group located behind the head part as an ideal leaving group, and a terminal initiator for the growth of polymer chains. The general principle of this strategy is illustrated in Figure 1. The affinity ligand, BA, specifically binds to glycosylated IgG, triggers specific binding to the glycan chain, and thereby draws the probe molecule very close to the molecular surface of the Fc domain in IgG (Figure 1, step a). The formation of the boronate ring induces a proximity effect, facilitating an SN2 substitution reaction with a nucleophilic residue on the labeled IgG, and subsequently releasing the benzenesulfonyl part (Figure 1, step b). Concurrently, the initiator is covalently transferred onto the surface of the target protein to be the


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desired protein macroinitiator for further polymer chain growth. Notably, the boronate-blocked glycans can be treated with polyol molecules, such as glycerol or sorbitol, to render native glycans with the release of BA-benzenesulfonate.

Figure 1. General principal of BA-tosyl initiator directed Fc-selective polymerization of antibodies. (a) IgG with treatment of BA-tosyl initiator, (b) covalent transfer of initiator, (c) the release of BA-benzenesulfonate and subsequent grafting from polymerization.

In this research, we proposed an intrinsic nature-directed traceless synthesis of protein-polymer conjugates in a site-selective manner. A model using biotin and streptavidin was carefully studied to validate the labeling process with conserved biological activity. Then, human IgG were used to prepare corresponding Ab-polymer conjugates followed by careful characterization of the conjugates. Very importantly, a comparison of site-specific anti-Con A antibody-polymers


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with randomly conjugated anti-Con A antibody-polymers in the Con A-binding assay is discussed.

EXPERIMENTAL SECTION General methods and materials All chemicals were purchased from Sigma-Aldrich and Acros. Anti-Con A antibody was purchased from Vector laboratories (CA, USA). The monomeric acrylamide/bisacrylamide solution (40%, 29:1) was purchased from Bio-Rad. The BCA protein assay reagent kit was obtained from Pierce. Sodium dodecyl sulfate (SDS) was purchased from GE Healthcare. IgG from human serum (reagent grade, >95%) was purchased from Sigma-Aldrich. Monoclonal antibiotin antibody (peroxidase conjugated) for western blotting was purchased from Sigma-Aldrich. Water was obtained using a Barnstead™ Easypure™ RoDi Water Purification System (Thermo Scientific, US).

Biotin-tosyl initiator directed synthesis of streptavidin synthesis. Streptavidin was dissolved in 10% dimethyl sulfoxide (DMSO)(666 µL, 10-5 M) aqueous solution. Then, 13 µL of a biotin-tosyl initiator stock solution (5.0 mg of biotin-tosyl initiator in 680 µL of dimethyl sulfoxide, 10-3 M) was added to the streptavidin solution. After incubating at 37°C for 24 hours, excess biotin-tosyl initiators were removed by column chromatography (Sephadex® G-25). The collected streptavidin macroinitiators were then concentrated by centrifugal filters (Amicon® Ultra Centrifugal Filters，Merck Millipore, Germany) to obtain the purified streptavidin macroinitiator.


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Atom transfer radical polymerization from the streptavidin macroinitiator. The obtained streptavidin macroinitiator (50 µg, 9.4 mmol) and NIPAAm (5.7 mg, 50 µmol) were dissolved in 48 µL of degassed ddH2O in an argon atmosphere. An oxygen-free catalyst solution (11 µL, obtained by dissolving 1.0 mg of CuBr and 4.18 mg of 2,2’-bipyridine in 78 µL degassed water) was added to the reaction mixture and initiated the polymerization. The reaction was monitored by reverse phase HPLC with the consumption of monomers.(Figure S5). After 1.5 h, the monomer showed non-distinguishable change and the reaction was then stopped by deactivating Cu (I) catalyst in open air. The formed streptavidin-PNIPAAm conjugates were heated to 45 oC, it will be precipitated and separated from reaction mixtures by centrifugation. Then, the precipitate was redissolved in chilled ddH2O followed by a heating precipitation process to remove the reactants and catalysts. The washing step was repeated three times to purify streptavidin-PNIPAAm conjugates.

Competition of streptavidin-PNIPAAm conjugates by amino biotin. Streptavidin macroinitiator (300 µg, 5.66 mmol) was prepared by treating streptavidin with biotin-tosyl initiator 1 and succinidimyl biotin were respectively dissolved in 20 µL deionized distilled water. Then, 50 µL of an amino biotin stock solution (100 mM in ddH2O) was added to the two streptavidin macroinitiator solutions. After incubating at room temperature for 2 hours, excess amino biotins were separated by column chromatography (Sephadex® G-25). The collected streptavidin macroinitiators were then concentrated by centrifugal filters to obtain the purified macroinitiators. These two obtained streptavidin macroinitiators were then subjected to


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polymerization conditions (90 mM CuBr, 180 mM 2,2-bipyridine and 50 µmole NIPAAm) The streptavidin-PPNIPAAm conjugates were then analyzed by SDS-PAGE.

BA-tosyl initiator directed labeling of anti-Con A antibody. Anti-Con A antibody was dissolved in PBS (pH 8.0, 0.1M) buffer containing 10% dimethyl sulfoxide (DMSO)(400 µL, 5x10-6 M). Then, 20 µL of a BA-tosyl initiator stock solution (1.0 mg of BA-tosyl initiator in 8.3 mL of DMSO, 2x10-4 M) was added to anti-Con A Ab solution. After incubating at room temperature for 24 hours, the excess BA-tosyl initiator was removed by column chromatography (Sephadex® G-25). The collected anti-Con A antibody macroinitiators were then concentrated by centrifugal filters to obtain the purified macroinitiator.

Atom transfer radical polymerization in anti-Con A antibody. The obtained anti-Con A macroinitiator (50 µg, 3.33 mmol) and NIPAAm (17 mg, 150 µmol) were dissolved in 63 µL of degassed water in an argon atmosphere. An oxygen-free catalyst solution (20 µL obtained by dissolving 1.0 mg of CuBr and 4.2 mg of 2,2’-bipyridine in 78 µL degassed water) was added to start the polymerization. The reaction was stopped after 1.5 hours by exposure to air. The reverse phase HPLC was used to monitor the progress of polymerization as shown in Figure S5. The formed antibody-PNIPAAm conjugates were heated to 45 oC, it will be precipitated and separated from reaction mixtures by centrifugation. The precipitates were then dissolved in chilled ddH2O followed by a heating precipitation process to remove the reactants and catalysts. The washing step was repeated three times to obtain purified antibodyPNIPAAm conjugates. The protein conversion yield is determined by bicinchoninic acid (BCA) assay as 87%.


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Enrichment of biotinylated Con A by anti-Con A Ab-PNIPAAm conjugates. The obtained anti-Con A Ab-polymer conjugates were then incubated with biotinylated Con A solution (17 µg in 15 µL aqueous solution). After incubating at room temperature for 2 hours, excess biotinylated Con A was then separated by centrifugation at 45°C to collect the precipitates as the biotinylated Con A-anti ConA Ab-PNIPAAm complex. Then, 15 µL stripping buffer (glycine-HCl, pH 2.5, 0.1M) was used to dissolve the precipitates and release biotinylated Con A from anti-Con A Ab-PNIPAAm conjugates. The eluted biotinylated Con A was neutralized by Tris buffer (pH 10.0, 1M) and directly analyzed by western blotting with anti-biotin monoclonal Ab.

RESULTS and DISCUSSION

Figure 2. Chemical structures of the biotin-tosyl initiator (1), biotin initiator (2) and BA-tosyl initiator (3).

Synthesis of Ligand-Tosyl Initiators. To demonstrate this concept, two polymer initiator transfer molecules (biotin-tosyl initiator and BA-tosyl initiator) and control molecule (biotin initiator) were designed (Figure 2); the details of the syntheses of biotin-tosyl initiator and BA-


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tosyl initiator are described in Scheme 1. To model the biotin-streptavidin interaction, biotintosyl initiator was synthesized from the highly reactive 3-(chlorosulfonyl)benzoyl chloride (CSBC). A one-pot two-step procedure was engaged to sequentially conjugate CSBC with spacer 5 and ATRP initiator 6.53 The formed compound 7 was then treated with trifluoroacetic acid (TFA) to expose the reactive amine group. This compound was further reacted with activated succimidinyl biotin 9 to obtain the biotin-tosyl initiator. Simultaneously, BA-tosyl initiator 3 was designed and synthesized as a initiator transfer for Abs of interest. 3-Aminophenyl boronic acid (BA) was first protected with pinacol to facilitate purification. Because of the strong binding affinity to silica gel, the BA-containing molecules are difficult to purify by silica gel column chromatography. The protected amino BApin 10 and initiator 6 were then sequentially added to the starting material CSBC in the presence of diisopropylethyl amine (DIEA) and N,Ndimethylaminopyridine (DMAP). The obtained pinacol-protected BApin-tosyl initiator was then deprotected with NaIO4 to furnish the BA-tosyl initiator 3 for advanced study in site-selective IgG modification. To demonstrate the importance of the key components (the targeting head and the reactive tosyl group) in BA-tosyl initiator 3, control molecule (the biotin-initiator) was synthesized through conventional amide formation. The details of these corresponding syntheses are included in the supporting information.


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Scheme 1. Syntheses of (A) biotin-tosyl initiator and (B) BA-tosyl initiator

Reaction Conditions for the Preparation of Thermoresponsive Protein-Polymer Conjugates. To grow uniform lengths of polymer chains, we used controlled radical polymerizations (CRPs);

10,54

CRPs have emerged as powerful methods in the preparation of

well-defined protein-polymer and peptide-polymer conjugates. Atom transfer radical polymerization (ATRP)55-56 is an ideal CRP method for the preparation of bioreactive polymeric scaffolds due to its user-friendly operation and potential to work under physiological conditions. In a low concentration of Cu(I), the obtained protein macroinitiator can directly serve to extend the polymer chain on the molecular surface of proteins. Here, N-isopropylacrylamide (NIPAAm) was used as the monomer in ATRP to assemble the thermo-responsive smart polymer (PNIPAAm)57 growing from the protein of interest. The PNIPAAm chain allows for instant precipitation when the temperature rises above the low critical solution temperature (LCST). The


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unique property makes the PNIPAAm-conjugates be easily separated from the unreacted mixtures and resuspends in buffer while raising temperature up to LCST. In this research, reaction conditions for the PNIPAAm grafting from the protein of interest by ATRP was optimized with a model protein, bovine serum albumin (BSA). BSA was first conjugated with the ATRP initiator by random amide bond formation which experimental procedures are shown in supporting information. To optimize the grafting-from ATRP in biomacromolecules, different Cu catalysts and ligands have been carefully studied. In 2005, Maynard and co-workers were used streptavidin-biotin initiator to grafting-from PNIPAAm through ATRP on streptavidin26. And they also tried to utilize sacrificial initiator to improve polymerization process. The resultant BSA macroinitiator was then used in the optimization of the ATRP reaction under physiological condition. Different Cu(I) catalysts and ligands such as Tris(2-dimethylaminoethyl)amine, 1,1,4,7,10,10-Hexamethyltriethylenetetramine, and 2,2’-bipyridine have been examined by screening reactant equivalents and reaction time. A sacrificial initiator was also used and evaluated while screening conditions. The conditions to degas the protein of interest was defined as 15 µM protein in PBS buffer at pH 8.0, three times, and then treated with 2,2’-bipyridine (180 mM) and CuBr (90 mM) at 25°C for 12 hours. This condition would be generally used in the following studies of protein-polymer preparation.


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Biotin-tosyl

initiator-directed

synthesis

of

Streptavidin-PNIPAAm

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Conjugates.

Scheme 2. Illustration of biotin-tosyl initiator used in the preparation of Streptavidin-PNIPAAm conjugates with conserved biotin binding affinity. The lanes i-vii of the insert figure represent the streptavidin conjugates i-vii in SDS-PAGE. (a) 2, 10% DMSO, 37oC, 24h; (b) amino biotin (25), rt, 2h; (c) CuBr, 2,2’-bipyridine, N-isopropylacrylamide; (d) 1, 10% DMSO, 37oC, 24h; (e) amino biotin (25), rt, 2h; (f) CuBr, 2,2’-bipyridine, N-isopropylacrylamide. To demonstrate this site-selective strategy in the preparation of protein-polymer conjugates, a binding pair of biotin and streptavidin was selected as a model to evaluate this affinity-directed chemical strategy in the preparation of protein-polymer conjugates with conserved protein function. A SDS-PAGE inserted in the Scheme 2 clearly indicates the formation of all intermediates during this affinity-directed protein modification. As illustrated in Scheme 2, the biotin-tosyl initiator and its control molecule, biotin-initiator, were treated with streptavidin individually. The biotin-tosyl initiator can covalently transfers the initiator group to streptavidin and leave biotin tosylate which non-covalently associated with streptavidin as the intermediate


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(ii). The negatively charged biotin tosylate may make the protein band be shifted to a lower position in SDS-PAGE. In contrast, a non-covalently associated streptavidin/biotin initiator intermediate (v) can be found by treating streptavidin with the control molecule biotin-initiator. Without the tosyl group as the initiator transferring agent, although intermediate (v) can still grow polymer chains by terminal initiator, the non-covalent association is in a risk of dissociation. More importantly, the resultant streptavidin-PNIPAAm conjugates would certainly lose the biotin-binding activity if trying to keep the protein-polymer conjugate’s integrity. . To demonstrate the importance of the developed ligand-tosyl protein-polymer synthesis, intermediates (ii) and (v) were subjected to the dissociation of streptavidin-biotin complexes. Trying to remove the bound biotin from streptavidin, a high concentration of biotin solution is necessary due to the high affinity (KD = 10-15 M). However, no observed dissociation can be observed while incubated the mixture with high concentrations of free biotin (1M in DMSO) which cannot be prepared in neutral aqueous solution neither at 25°C nor heating at 70 °C. An acidic condition (pH 1.5) has also been adopted to release the bound biotins, but still worked poorly. Then, a water soluble amino biotin was synthesized and successfully used to remove biotin form the complexes. The biotin benzenesulfonate was released by competition (intermediate iii) and regained the biotin binding site. The initiator attached on the surface of the streptavidin initiated ATRP by treatment with CuBr, 2,2’-bipyridine and NIPAAm to grow PNIPAAm chains. The resultant thermo-responsive streptavidin-PNIPAAm conjugate (iv) considerably increased in molecular weight as well as be endowed the thermo-responsive property. After heating and denaturing, no distinguished band can be observed by SDS-PAGE, which strongly supports the success in the formation of this streptavidin-PNIPAAm conjugates (lane iv, Scheme 2). In contrast, amino biotins successfully displaced the biotin from the biotin


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initiator-treated streptavidin (v) and therefore release the biotin-initiator from streptavidin. A clear streptavidin band of product (vii) strongly supports no or very few streptavidin-PNIPAAm conjugates formed in the same polymerization condition (lane vii, Scheme 2). The results of this model study fully support the importance of this initiator-transferring chemistry in the preparation of protein-polymer conjugates. The simple activity-based synthesis of a protein macroinitiator, such as biotin-streptavidin, may suffer ligand dissociation. The biotin-tosyl initiator directed specific recognition with streptavidin by non-covalent interaction, then the proximity effect triggered the second covalent conjugation. The resultant stable streptavidin macroinitiator was successfully used in the preparation of streptavidin-PNIPAAm conjugates, which highly conserved affinity to biotin.

Preparation and Characterization of Human IgG-PNIPAAm Conjugates with Boronic Acid (BA)-Tosyl Initiator. With the success of streptavidin-PNIPAAm conjugates, this activity-directed synthesis of protein-polymer conjugates was extended to the application of the preparation of IgG-PNIPAAm conjugates. BA-tosyl initiator 3 was used to selectively recognize glycan chains of IgG antibody specifically on the Fc domain (Figure 1). The resultant mixture was then purified by Sephadex® G-25 column chromatography to remove excess BA-tosyl initiator 3, generating the Fc-specific IgG macroinitiator. To optimize conditions for ATRP, different amounts of NIPAAm were treated with hIgG macroinitiator (15 µM, human immunoglobulin G from serum, SigmaAldrich) in the presence of CuBr (90 mM) and 2,2’-bipyridine (180 mM). To the treatment of NIPAAm monomers 50 µmole, the obtained IgG-PNIPAAm conjugates were analyzed by SDSPAGE electrophoresis. Because all samples were denatured in 95 °C, those polymer-attached


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fragments would be precipitated and therefore cannot be observed in SDS-PAGE. In Figure 3A, a gradual reduction of heavy chain bands in SDS-PAGE with nearly consistent bands of light chains strongly supports the selective growth of PNIPAAm chains on heavy chains of IgG. The conjugated heavy chains and unreacted light chains could be dissociated by treatment the reducing agent, β-mercaptomethanol, and the thermal sensitive IgG heavy chain-PNIPAAm conjugates would be precipitated in heating condition which led to the reduction of heavy chain bands in SDS-PAGE. This result reveals that this BA-tosyl initiator strategy can be efficiently used in Fc domain-specific IgG-polymer conjugation. To compare with conventional random protein-polymer conjugates, human IgG was added with initiator succinimidyl ester to prepare randomly-conjugated IgG-initiators conjugates. The multiple and randomly located initiators on IgG initiated the growth of PNIPAAm chains at both light chains and heavy chains. As shown in Figure 3B (lane 4), only very weak bands of both light chains and heavy chains can be observed after random polymer growth, indicating uncontrollable grafting on the whole IgG molecule.


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Figure 3. (A) lane 1: BA-tosyl initiator; lane 2: lane 1 after polymerization. (B) lane 1: BA-tosyl initiator; lane 2: lane 1 after polymerization; lane 3: OSu-initiator; lane 4: lane 3 after polymerization.

The obtained IgG conjugates exhibited a temperature responsive phase change and the corresponding values of LCST were determined by UV illumination at 280 nm. In the screening range of 20-65°C, the LCST was at 38°C. (Figure 4A). To further characterize properties of these obtained IgG-PNIPAAm conjugates, the obtained IgG-polymer conjugates have been further analyzed by fast protein liquid chromatography (FPLC) (Figure 4B). While the IgGinitiator molecule gives the retention time at 60 mins, the formed IgG-PNIPAAm conjugate’s retention time was shifted to 50 minutes. Subsequently, dynamic light scattering (DLS) was used to determine the corresponding hydrodynamic diameters of IgG-PNIPAAm conjugates prepared by this method. The diameter of IgG was determined to be 4.9 ± 0.4 nm. Then, polymerization of IgG macroinitiator with NIPAAm monomers formed IgG-PNIPAAm conjugates with diameter increasing to 59.2 ± 9.7 nm (Figure S4B).


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Figure 4. (A) Determination of LCST of IgG-PNIPAAm conjugates by UV-Vis spectrometer. (B) FPLC diagram of IgG-initiator, IgG-PNIPAAm conjugates.

Figure 5. Polyacrylamide gel electrophoresis (SDSPAGE, left) and Western blotting (right) of hIgG after treatment with BA-m-tosyl-alkyne followed by biotinylation. Coomassie blue staining of the SDS-PAGE gel (left) shows that heavy chain and light chain cleavage by β-ME. The signal was revealed by Western blotting with an anti-biotin monoclonal antibody. To further demonstrate the site-specifically modification on Fc domain, a BA-tosyl-alkyne was synthesized and treated with IgG to transfer a terminal acetylene group on Fc domain. Subsequently, in the presence of Cu (I) catalyst and azido biotin, the IgG molecule would be further tagged a biotin. Monoclonal anti-biotin antibody was then used in Western Blot experiment to indicate the existing of biotins. As shown in Figure 5, only a band located at heavy chains (~55 kDa) can be observed which strongly supports the specific modification site at heavy chains by BA-tosyl chemistry. Furthermore, an ester bond, BA-tosyl-ester-initiator 29, was introduced to displace the amide bond in the initiator linkage which allows the dissociation of polymer chains from conjugated IgG by basic hydrolysis. As shown in Figure S6, the intensity of IgG’s heavy chain on SDS-PAGE electrophoresis was considerably reduced after polymerization as usual. By contrast, light chain showed no distinguishable change. Under the basic condition, the ester bond could be cleaved and released the NIPAAm chains from IgG.58 As the result, the intensity of IgG’s heavy chain was recovered which strongly supports the specific polymerization site on heavy chains. (Figure S6) Site-Specific Conjugated anti-Con A antibody-PNIPAAm Conjugates in the Enrichment of Con A.


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Through the characterization of IgG-PNIPAAm conjugates, the BA-tosyl strategy was demonstrated to specifically attach the PNIPAAm chains of interest on IgG at the Fc domain. Accordingly, the antigen binding (Fab) domains of modified IgG should be completely exposed to environment and preserve its native function. To further evaluate this, an anti-Con A (Concanavalin A) antibody was treated with BA-tosyl initiator followed by ATRP with NIPAAm, 2,2-bipyridine and CuBr; meanwhile, a control experiment was carried out by treating the antiCon A Ab with succinimidyl initiator (OSu initiator) to generate a randomly conjugated anti-Con A Ab-PNIPAAm conjugate. The site-selective anti Con-A Ab-PNIPAAm and random anti-Con A Ab-PNIPAAm conjugates (50 µg) were mixed with biotinylated Con A (17 µg, 0.64 nmol) respectively. After Incubation for 30 mins, the reaction mixtures were then heated to 40 °C to precipitate the anti Con-A Ab -PNIPAAm-biotinylated Con A complexes. Then, the bound biotinylated Con A precipitates were separated from unbound ones by centrifuging. The biotinylated Con A were dissociated from anti-Con A Ab in acidic conditions (stripping buffer, glycine-HCl, pH 2.5) and collected for subsequent analysis by western blotting with anti-biotin monoclonal Ab. In Figure 6, a strong band (lane 1) represented the extraction of Con A by site-selective anti-Con A Ab but a much weaker biotinylated Con A band (lane 2) can be observed using random anti-Con A Ab. The quantitative analysis of this western blotting result showed about six times higher intensity of Con A being biotinylated by BA-tosyl initiator rather than the use of random conjugated antiCon A antibody-PNIPAAm conjugates. These experiments were repeated three times and gave the consistent results. Using the same amount of anti-Con A Ab, the randomly conjugated Ab considerably lost its native function in binding its substrate Con A, which can be attributed to the block of Fab domain by randomly located PNIPAAm chains. Therefore, site-selective grafting in


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the preparation of IgG-polymer conjugates is certainly an important issue for biomedical applications.

Figure 6. Enrichment of biotinylated Con A by (A) BA-tosyl initiator mediated anti-Con A AbPNIPAAm conjugates and (B) OSu-initiator mediated anti-Con A Ab-PNIPAAm conjugates (lane 1, site selective) (lane 2, random). (C) The results of western blotting and its quantitative analysis of eluted biotinylated Con A. CONCLUSION Considering the importance of protein-polymer drugs in modern therapeutics, a need of new methods in the preparation of protein-polymer conjugates is emerging to provide well-defined architectures of protein-polymer conjugates instead of heterogeneous structures. In this research, a new chemical strategy was proposed to prepare the protein macroinitiator in a site-selective manner for use in the preparation of antibody-polymer conjugates. This activity-based strategy can effectively preserve the native function of modified proteins. To the best of our knowledge, we have conducted the first study in the preparation of Fc-directed IgG-polymer conjugates with native glycans, preserving the inherent structure of IgG molecules. A comparison in the


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enrichment of biotinylated Con A by anti-Con A Ab-pNIPAAm conjugates strongly supported the importance of site-selectivity when attaching polymers to antibodies of interest. ASSOCIATED CONTENT Supplementary information (Figure S1 to S6) include all, 1H NMR, 12C NMR, Mass and UVVis spectra, as well as reverse phase HPLC. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author Prof. Po-Chiao Lin Email : [email protected] ACKNOWLEDGMENT This work is supported by grants from Ministry of Science and Technology (MOST 103-2113M-110-010- and MOST 103-2627-M-007-004-) and National Sun Yat-sen University (01C030703 and 01A06802). We also thank the support from National Sun Yat-sen UniversityKaohsiung Medical University Joint Research Center, Kaohsiung, Taiwan. REFERENCES 1. Goffeau, A.; Barrell, B. G.; Bussey, H.; Davis, R. W.; Dujon, B.; Feldmann, H.; Galibert, F.; Hoheisel, J. D.; Jacq, C.; Johnston, M.; Louis, E. J.; Mewes, H. W.; Murakami, Y.; Philippsen, P.; Tettelin, H.; Oliver, S. G. Life with 6000 genes. Science 1996, 274, 546-567.


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Wavelike

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Graphic Abstract


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Glycan Directed Grafting-from Polymerization of Immunoglobulin G

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