Site-Specific Transglutaminase-Mediated Conjugation of Interferon Î±

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Site-Specific Transglutaminase-mediated Conjugation of Interferon Alpha-2b at Glutamine or Lysine Residues Barbara Spolaore, Samanta Raboni, Abhijeet Ajit Satwekar, Antonella Grigoletto, Anna Mero, Isabella Monia Montagner, Antonio Rosato, Gianfranco Pasut, and Angelo Fontana Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00468 • Publication Date (Web): 12 Oct 2016 Downloaded from http://pubs.acs.org on October 20, 2016

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Bioconjugate Chemistry


Site-Specific Transglutaminase-mediated Conjugation of Interferon Alpha-2b at Glutamine or Lysine Residues Barbara Spolaore*,#,§, Samanta Raboni#, Abhijeet Ajit Satwekar§, Antonella Grigoletto#, Anna Mero#, Isabella Monia Montagner†, Antonio Rosato†,∆, Gianfranco Pasut*,#,†, Angelo Fontana#

#

Department of Pharmaceutical and Pharmacological Sciences, University of Padua, via F.

Marzolo 5, 35131 Padua, Italy §

CRIBI Biotechnology Centre, University of Padua, viale G. Colombo 3, 35121 Padua,

Italy †

Veneto Institute of Oncology IOV - IRCCS, Padua, Italy

∆

Department of Surgery, Oncology and Gastroenterology, University of Padua, Padua,

Italy

Running Title: TGase-mediated derivatization of interferon α-2b

*Corresponding Author Dr. Barbara Spolaore CRIBI, University of Padua viale G. Colombo 3, 35121 Padua, Italy Tel: 0039-049-8276155; Fax: 0039-049-8276159; e-mail: [email protected] Prof. Gianfranco Pasut Department of Pharmaceutical and Pharmacological Sciences, University of Padua via F. Marzolo 5, 35131 Padua, Italy Tel: 0039-049-8275694; Fax: 0039-049-8275366; e-mail: [email protected]

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ABSTRACT: Interferon alpha (IFN α) subtypes are important protein drugs that have been used to treat infectious diseases and cancers. Here, we studied the reactivity of IFN α2b to microbial transglutaminase (TGase) with the aim to obtain a site-specific conjugation of this protein drug. Interestingly, TGase allowed the production of two mono-derivatized isomers of IFN with high yields. Characterization by mass spectrometry of the two conjugates indicated that they are exclusively modified at the level of Gln101 if the protein is reacted in the presence of an amino containing ligand (i.e. dansylcadaverine), or at the level of Lys164 if a glutamine containing molecule is used (i.e. carbobenzoxy-Lglutaminyl-glycine, ZQG). We explained the extraordinary specificity of the TGasemediated reaction based on the conformational features of IFN. Indeed, among the 10 Lys and 12 Gln residues of the protein only Gln101 and Lys164 are located in highly flexible protein regions. The TGase-mediated derivatization of IFN was then applied to the production of IFN derivatives conjugated to a 20 kDa polyethylene glycol (PEG), using a PEG-NH2 for Gln101 derivatization and a PEG modified with ZQG for Lys164 derivatization. The two mono-PEGylated isomers of IFN were obtained in good yields, purified and characterized in terms of protein conformation, antiviral activity and pharmacokinetics. Both conjugates maintained a native-like secondary structure, as indicated by far-UV circular dichroism spectra. Importantly, they disclosed a good in vitro antiviral activity retention, about only 1.6- to 1.8-fold lower than that of IFN, and halflives longer, about 5-fold, than that of IFN after intravenous administration to rats. Overall, these results provide evidence that TGase can be used for the development of site-specific derivatives of IFN α-2b possessing interesting antiviral and pharmacokinetic properties.

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INTRODUCTION In recent years many proteins produced in large quantities by recombinant DNA techniques have become important new drugs for treating a variety of diseases. The utility of these proteins can be enhanced by using modified versions of them bearing covalently bound chemical entities such as polymers, fluorescent probes or metal-chelating moieties. For example, the covalent attachment of polyethylene glycol (PEG) chains to proteins is a valuable technique to delay protein clearance and reduce protein immunogenicity while maintaining its biological activity.1,2 In general, a key requirement to obtain a protein conjugate that retains the highest activity is to perform a site-specific derivatization under physiological conditions.3,4 Indeed, commonly used chemical procedures are not specific and several amino acid side chains are involved in the covalent modification causing a decreased bioactivity of the protein derivatives and a time- and labor-consuming effort for the purification and characterization of the heterogeneous mixture of protein conjugates. There is thus a strong interest towards the development of procedures that allow a sitespecific modification of proteins. Microbial transglutaminase (TGase) is a TGase extracted from Streptomyces mobaraensis that catalyzes the reaction between the side-chain of a Gln residue (-CONH2, the acceptor; A) and an amino group (-NH2, the donor; B) of an alkylamine under physiological conditions, according to the reaction A−CONH2 + H2N−B → A−CONH−B + NH3.5 In vivo, the amine donor usually is the epsilon-amino group of lysine residues, thus leading to an intra- or inter-molecular protein crosslinking. However, TGase can catalyze also the covalent attachment of an amino-containing moiety to Gln residues of the target proteins, or the modification of proteins at the level of Lys residues with a ligand containing a Gln residue or a Gln analogue.6–8 Various chemical entities can be incorporated by TGase at the level of protein-bound Gln or Lys residues, allowing numerous biotechnological applications.9,10 For the purposes of protein PEGylation, the derivatization of Gln residues with PEG-NH2 has been exploited to produce mono-conjugated derivatives of different protein drugs.11–14 Recently, Zhou et al. demonstrated that a PEG 5kDa modified with a carbobenzoxy-L-glutaminyl-glycine (ZQG) moiety can be used to derivatize the model protein cytochrome c at the level of Lys residues with high yield.15 This opens the way to exploit also Lys residues for the site-specific PEGylation of proteins. Importantly, the microbial TGase-mediated derivatization is highly site-specific, since folded proteins can be derivatized at the level of a single Gln or Lys residue despite the fact that their polypeptide sequences contain many Gln and Lys residues.16,12,17 Since the 3 ACS Paragon Plus Environment


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sites of TGase derivatization are usually located at the level of flexible regions of a protein, we proposed that the selectivity of this enzymatic reaction is mainly dictated by the conformational features of the substrate. This is also supported by the fact that when the enzymatic reaction is conducted in presence of structuring co-solvents –i.e. methanol or ethanol– the consequent increment of alpha helix percentage in the protein substrate, correlated in turn to structure rigidity, induces a reduction of the number of Gln(s) that are recognized by TGase.11,18 Interferons (IFNs) α-2 are cytokines with antiviral activity as well as antiproliferative and immunomodulatory effects on a variety of cell types.19 Their activity is mediated by the interaction with a cell surface receptor, which is composed of two transmembrane subunits, IFNAR1 and IFNAR2.20 The IFN α subtypes 2a and 2b differ only by residue 23 that is Lys in 2a and Arg in 2b. They show similar activities and have been used clinically in the treatment of viral diseases as hepatitis C and B, and cancer (recombinant IFN α-2a, Roferon-A, Hoffmann-La Roche and recombinant IFN α-2b, intron A, ScheringPlough).19,21,22 Since these IFNs exhibit a relatively short serum half-life in vivo and their bioavailability may be further diminished by neutralizing antibodies, PEGylation has been exploited as an approach to increase their blood residency time and thus the antiviral protection. Two PEG derivatives are already in the market with the commercial name of: i) PegIntron (Schering-Plough), a mixture of 14 positional mono-conjugated isomers of IFN α-2b with a residual antiviral activity of 28%

23

and ii) Pegasys (Hoffmann-La Roche), a

PEGylated form of IFN α-2a mainly (94%) composed of four positional mono-conjugated isomers with a 7% antiviral activity with respect to the unmodified protein.24,25 These conjugates have shown long-serum half-lives and produce a sustained antiviral response. However, the heterogeneous nature of these derivatives as well as their lower activities prompted studies to obtain site-selective IFN mono-conjugates. To this aim, IFN was subjected to protein engineering to insert a free cysteine residue that can react with PEGmaleimide,26,27 to form monoPEGylated dimers,28 to contain an azide-bearing amino acid for PEG conjugation via a click chemistry reaction29 and to be C-terminal derivatized using the enzyme sortase A or exploiting an hydrazine-forming ligation reaction.30–32 Sitespecific PEGylation was also achieved by chemical reactions that allow derivatization at the level of disulfide bonds33 or at the N-terminus of the protein.34,35 In this study, we investigated the application of microbial TGase for the site-specific derivatization of IFN α-2b (hereafter abbreviated as IFN) at the level of Lys or Gln residues. The IFN polypeptide sequence contains 12 Gln and 10 Lys residues (Fig. 1) that 4 ACS Paragon Plus Environment

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could be potentially modified by TGase. We demonstrate that it is possible to produce two mono-derivatized isomers of IFN modified at the level of Gln101 or Lys164 by using TGase. We explained the extraordinary specificity of the TGase-mediated reaction based on the conformational features of the IFN molecule. Then, we produced the two derivatives conjugated to a 20 kDa PEG and we characterized them in terms of protein conformation, antiviral activity and pharmacokinetic. CDLPQTHSLG SRRTLMLLAQ MRRISLFSCL 30 KDRHDFGFPQ EEFGNQFQKA ETIPVLHEMI 60 QQIFNLFSTK DSSAAWDETL LDKFYTELYQ 90 QLNDLEACVI QGVGVTETPL MKEDSILAVR 120 KYFQRITLYL KEKKYSPCAW EVVRAEIMRS 150 FSLSTNLQES LRSKE

165

Figure 1. Amino acid sequence of human IFN α-2b. The Gln and Lys residues potential sites of TGase modification are shown in bold blue (Gln) and red (Lys).

RESULTS AND DISCUSSION TGase-mediated derivatization of IFN at the level of Lys or Gln residues. IFN was modified by TGase at the level of Lys residues using ZQG as Gln containing substrate. RP-HPLC analysis of the reaction mixture of IFN with ZQG after 4 hours of incubation revealed a shift towards a higher retention time of the main chromatographic peak (from 17.0 for native IFN to 17.4 min), and the detection of one new peak at 18.1 min (Fig. 2A). In the RP-HPLC peak at 17.4 min, ESI-MS analysis indicated the formation of a monoZQG-derivative of IFN (IFN1ZQG) and in low percent a di-ZQG-derivative (IFN2ZQG) (Fig. 2D), while the peak at 18.1 min corresponded still to IFN2ZQG and to dimer species of IFN conjugated to one or two ZQG molecules (Table 1). In a separate experiment, the reactivity of Gln residues of IFN towards TGase was investigated using dansylcadaverine (DC) as amino donor. RP-HPLC analysis of the reaction mixture of IFN with DC and TGase after four hours of incubation showed a shift in retention time of the main chromatographic peak (from 17.2 min for native IFN to 17.4 min) and ESI-MS analysis of the eluted protein material confirmed the presence of a mono-derivative of IFN with DC (IFN1DC, Figure 2B and E, Table 1). The RP-HPLC peak at 18.1 min, with low intensity, was also detected in the chromatogram of the reaction mixture after 4 h of incubation 5 ACS Paragon Plus Environment


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(Figure 2B) and ESI-MS analysis demonstrated that it contained a dimer species of IFN conjugated to one molecule of DC (Table 1). A bi-derivative of IFN with two DC molecules (theoretical mass 19902.04 Da) was not detected with a significant signal.

Figure 2. TGase-mediated conjugation of IFN at Lys residues with ZQG and at Gln residues with DC. Left panel. RP-HPLC analyses of the reaction mixtures of IFN with TGase and ZQG (A) or DC (B). A dashed line and a straight line indicate the chromatograms at time 0 h and after 4 h of reaction, respectively. Right panel. Deconvoluted ESI mass spectra of native IFN collected at RT 17.0 min in the RP-HPLC analysis of the reaction mixture of IFN with ZQG and TGase after 0 h of incubation (C), and of the derivatives collected at 17.4 min in the analyses of the reaction mixture with ZQG (D) or DC (E) after 4 h of incubation.


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Table 1. Measured molecular masses of the IFN derivatives produced upon incubation with TGase in the presence of DC or ZQG. Reaction mixture

IFN species

RT

Molecular mass (Da)

(Min)

Founda

Calculatedb

IFN

17.0

19265.12±0.13

19265.16

IFN+TGase+ZQG

IFN

17.4

19265.12±0.53

19265.16

(4h)

IFN

1ZQG

17.4

19585.69±0.13

19585.46

IFN2ZQG

17.4

19906.44±0.41

19905.76

2ZQG

18.1

19906.31±0.35

19905.76

DIMER+1ZQG

18.1

38838.07±0.23

38833.58

DIMER+2ZQG

18.1

39159.48±0.42

39153.88

(incubation time) IFN+TGase+ZQG (0h)

IFN IFN IFN

IFN+TGase+DC (4h)

IFN IFN IFN

17.4

19265.55±0.97

19265.16

1DC

17.4

19583.91±0.13

19583.60

DIMER+1DC

18.1

38833.84±0.49

38831.72

a

Experimental molecular masses determined by ESI-MS. b Calculated average molecular masses.

Dimer species were the main side products of the TGase site-specific modification of IFN. We thus investigated the oligomerization of IFN mediated by TGase in the absence of any ligand. After four hours of incubation of IFN with TGase at the same E/S ratio of the previous derivatization reactions, two new poorly separated peaks were observed by RPHPLC analysis of the reaction mixture (Figure S1A). ESI-MS analysis of the eluting protein material revealed the formation of an IFN dimer in the first eluting product peak and of IFN trimer and tetramer in the second peak (Table S1). The patterns of oligomerization of IFN in different reaction conditions (with DC, with ZQG or with TGase only) were compared by SDS-PAGE analysis (Figure S1B). While in the reaction with DC and ZQG there is the formation of a low amount of dimer due to the competitive presence of the two ligands (Fig. S1B, lane 1 and 2), in the presence of only TGase dimer, trimer, tetramer and even higher molecular weight oligomeric species were formed (Fig. S1B, lane 3). 7 ACS Paragon Plus Environment


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The pattern of the reaction catalyzed by TGase can be summarized as in Scheme 1. IFN has one Lys and one Gln residues as substrate of TGase. In the absence of any ligand, the enzyme catalyzed the formation of the dimer or higher oligomers of the protein, given by the crosslinking between the Lys residue of one protein molecule and the Gln residue of another IFN molecule (Scheme 1A). Working with an excess of a Gln- or primary aminecontaining ligand, the production of the IFN mono-derivatives was favored, while the formation of oligomers decreased significantly (Scheme 1B, C). The dimer and oligomers formed in the presence of a ligand still have a Gln or Lys substrate of TGase (Scheme 1A), thus leading to the conjugation of the ligand to the oligomer as detected in the reaction with ZQG and DC.

Scheme 1. Derivatives produced by the TGase-mediated conjugation of IFN in the presence of the protein only (A), of a Gln-containing ligand (B) or a ligand carrying a primary amine (C). The 3D structure of IFN (PDB code 1ITF) was drawn by using PyMOL (The PyMOL Molecular Graphics System, LLC). The Gln and Lys residues are indicated approximatively at the sites of derivatization identified by MS (residues Gln101 and Lys164).


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Identification of the sites of TGase derivatization of IFN by mass spectrometry. In order to identify the Gln and Lys residues that were specifically modified by TGase, the protein material collected at 17.4 min in the chromatogram of Fig. 2A-B and containing the IFN1ZQG and IFN1DC derivatives, respectively were digested with endoprotease Glu-C and the peptide mixtures were analyzed by LC-MSE (Fig. 3A-C). As reference, also native IFN collected from the RP-HPLC analysis of the reaction mixture at 0 h of incubation was digested and analyzed under the same conditions. The LC-MSE raw data were processed using BiopharmaLynx (Waters) and analyzed using as variable modifications the derivatization of Lys residues with ZQG and of Gln residues with DC (user-created modifiers). ESI-LC-MSE peptide mapping of the digests allowed to achieve a 100% sequence coverage for IFN, IFN1ZQG and IFN1DC with a maximum mass error of 7 ppm and to confirm the identity of most of the peptides by b/y fragment ions with a mass error less than 15 ppm (Table S2, S3 and S4). The mass fingerprinting analysis identified the modification with ZQG on peptide 160–165 of IFN that was conjugated at the level of Lys164 (Table S3). This conjugated peptide eluted in the LC-MSE chromatogram at 11.82 min (Fig. 3B, D). In a separated analysis, this peptide was also subjected to MS/MS analysis confirming the localization of the modification at the level of Lys164 (Figure S2). A secondary site of modification was also detected in peptide 1–42 and localized at the level of Lys31 residue (Fig. 3B, E and Table S3), as confirmed by 5 b/y fragment ions (Figure S3 and Table S5). The intensity of the m/z signals of the modified peptide 1–42 accounts for 4.7% of the total intensity of this peptide (Table S3), indicating a low percentage of derivatization. For the IFN1DC derivative, the modification with DC was localized at the level of peptides 97–107 and 97– 113, that eluted at 18.0 and 17.66 min, respectively in the LC-MSE chromatogram (Fig. 3B, F-G). These peptides resulted modified at the level of Gln101 with a 100% yield, based on m/z signals of the modified and non-modified peptides. The sequence of the modified peptide was confirmed by MSE b/y fragment ions and in the case of peptide 97– 107 by an MS/MS spectrum acquired in a separate analysis (Fig. S4).



11.52

100

13.62

9.76

%

A. IFN

1: TOF MS ES+ BPI 2.12e5

12.62 13.21 12.45

9.26

5.81

15.18 15.71

17.17

19.43

0 6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.45

20.00

13.62

100

B. IFN1ZQG

22.00 1: TOF MS ES+ BPI 6.10e5

11.52 11.82

%

9.76 12.62 9.26

5.81

10.39 10.92

8.66

17.17 15.15

12.45 13.21

15.96 16.83 17.34

18.80

0 6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

11.48

100

22.00 1: TOF MS ES+ BPI 3.92e5

C. IFN1DC

13.62

%

9.76

15.01 12.62 13.21

17.66 18.00

6.00

8.00

10.00

520.2559

12.00 1: TOF MS ES+ 4.39e5

D. IFN1ZQG

18.76

15.91 16.76

10.92

0

100

17.13

9.20

5.64

14.00 100

16.00

18.00

22.00

1: TOF MS ES+ 2.41e4

E. IFN

876.9412

876.4388

520.7595

877.1046

%

%

20.00

876.7717 1ZQG 876.6022

RT 17.1min [M+6H]6+ 1–421ZQG

RT 11.8min [M+2H]2+ 160–1651ZQG

877.2740 876.2694

877.4375

521.2587

877.6010

521.7628

m/z 0 0 516 517 518 519 520 521 522 523 524 525 526 874 697.3465


F. IFN1DC RT 18min [M+2H]2+ 97–1071DC

100

875

876

G. IFN1DC

877

878

698.6911

879

m/z 880


698.3562

RT 17.7min [M+3H]3+ 97–1131DC

697.8486

699.0261

%

100

%

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699.3558

698.3508

699.6909

698.8478

700.0262

699.3503

m/z 0 m/z 0 695 696 697 698 699 700 701 702 703 704 695 696 697 698 699 700 701 702 703 704

Figure 3. LC-MSE analyses of IFN, IFN1DC and IFN1ZQG digested with endoprotease GluC. A-C. BPI chromatograms of the low energy channel generated in the LC-MSE analyses of the digests of IFN (A), IFN1ZQG (B) and IFN1DC (C). Peaks where modified peptides eluted are highlighted in blue. D-G. MS spectra of the modified peptides.


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Molecular basis for the site-specific derivatization of IFN by microbial TGase. IFN has a globular structure characterized by five α-helices and stabilized by two disulfide bridges (Cys1–Cys98 and Cys29–Cys138).36 The sites of TGase derivatization (Gln101 and Lys164) map on stretches with the highest conformational flexibility in the 3D structure of the protein, based on the B-factor analysis of the X-ray structure of a zinc mediated dimer of IFN α-2b (Fig. S5A) and on the NMR structure of IFN α-2a.36,37 Also Lys31 is located in a flexible loop region according to the X-ray structure of the protein.36 In order to probe flexible regions in IFN under the buffer conditions of the TGase reaction, we subjected the protein to a limited proteolysis experiment using proteinase K. In our laboratory, we have demonstrated that the first sites of hydrolysis by proteases invariably occur in regions of the protein substrate characterized by local unfolding and that these regions often correspond with the localization of sites of TGase modification.16,17 Proteinase K is an unspecific protease that is quite useful for detecting disordered regions in proteins. IFN was incubated in PBS buffer with proteinase K and aliquots of the reaction mixture were taken at different times and analysed by SDS-PAGE and RP-HPLC. SDS-PAGE analysis under reducing conditions of the kinetic of the proteolytic reaction of IFN showed the formation of two fragment species of about 11 and 7 kDa that were quite stable to further digestion (Fig. S5B). RP-HPLC analysis of the reaction mixture after 15 minutes of incubation showed the formation of a peak of higher retention time in respect to native IFN (Fig. S5C). ESI-MS analyses of this peak were performed directly on the HPLC fraction and after reduction of the disulfide bridges with tris(2-carboxyethyl)phosphine (TCEP, Table S6). MS spectra indicated the formation of nicked and gapped species of IFN given by fragments covalently linked to each other by the two disulfide bridges of the protein and generated by hydrolysis of the polypeptide chain at the level of Val103–Gly104, Thr106–Glu107 and Leu161–Arg162 peptide bonds. A nicked protein is a two-fragment species given by a fission of a single peptide bond (i.e. 1–106/107–165 and 1–103/104– 165), while a gapped species is a two-fragment species resulting from two peptide bonds cleaved and excision of a protein fragment (i.e. 1–103/107–165, 1–106/107–161 and 1– 103/107–161) (Table S6). The two bands detected in the SDS-PAGE analysis thus corresponded to the formation of fragments 1–103, 1–106 (band at 11 kDa) and of fragments 104–165, 107–165 and 107–161 (band at 7 kDa).



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In agreement with the X-ray and NMR structural analyses of IFN, proteolysis data demonstrate that the polypeptide chain regions encompassing amino acids Val103 and Glu107 and the C-terminal region of the protein (Leu161-Glu165) are the most flexible regions of IFN and in turn that these conformational features determine the site-specificity of the TGase-mediated reaction at the level of Gln101 and Lys164 residues. TGase-mediated PEGylation of IFN with PEG20kDa−NH2 and PEG20kDa−Gln. The conjugation reactions of IFN to ZQG and DC indicated that TGase could be used to prepare, in high yields, IFN derivatives modified with a low molecular mass ligand at the level of Lys164 or Gln101, respectively. We then investigated the feasibility to use the TGase-mediated reaction to conjugate this protein to a 20 kDa PEG functionalized with a primary amine group (PEG20kDa−NH2) or with ZQG (PEG20kDa−Gln) for Gln or Lys residues conjugation, respectively. TGase has been exploited for the PEGylation of diverse proteins at the level of Gln residues, while PEGylation of a pharmaceutical protein at the level of Lys residues using a PEG 20 kDa functionalized with a Gln residue has not been reported previously, to our knowledge. This study thus allowed comparing the two approaches of protein PEGylation via TGase on the same protein. The two sites of derivatization are quite interesting for the production of useful conjugates of this important therapeutic protein. The Gln101 residue is not among the hot spot residues involved in receptor binding, even though an NMR study suggested that this residue is part of a patch composed of residues Val99 ̶ Gly102 and Thr106 involved in the interaction with the receptor subunit IFNAR1.20,38,39 However, previous studies indicated that mutation of the Gln101 residue to a Cys residue in IFN α-2b leads to a mutant with a preserved in vitro biological activity.27 Moreover, the nearby Thr106 is glycosylated in IFNα-2b purified from human leucocytes and the glycosylated protein appears to have a biological activity similar to recombinant IFN, while derivatization of Thr106 with a 20 kDa PEG leads to an IFN α-2b derivative with antiviral activity.40–42 Since to our knowledge no previous PEGylated IFN derivative at the level of Gln101 has been produced and characterized, it would be interesting to test the pharmacokinetic and activity properties of this conjugate. The Lys164 residue is the penultimate residue of the 165 amino acid polypeptide chain of IFN. PEGylated IFN modified at the level of Lys164 is a minor positional isomer in PegIntron and in Pegasys.23,43 In this respect, the TGase-mediated reaction allows to produce an IFN conjugate PEGylated homogenously at the Lys164 residue. The C-terminus of IFN 12 ACS Paragon Plus Environment

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appears not to be important for IFN activity. Indeed, mutants of IFN in which the five Cterminal residues are removed (Leu161-Glu165) were tested for receptor affinity and antiviral and antiproliferative activities.44 The results indicated no significant differences in binding affinity and antiviral and antiproliferative potencies compared to wild-type IFN. This region appears mainly to play a role in differentiating the interaction between different IFNs and the IFNAR2 subunit of the receptor.45 C-terminal PEGylated derivatives of IFN have also been produced using sortase A or an hydrazine-forming ligation reaction.31,32 Both derivatives did not show significant loss in biological activity suggesting that conjugation of the C-terminus of the protein does not significantly affect the interaction with the receptor. In order to maximize the yields of the two PEGylated IFN derivatives, optimization of the reaction conditions

was conducted with PEG20kDa−NH2

testing different IFN

concentrations (0.5, 1.0 and 2.0 mg/ml), IFN/PEG molar ratios (1/10 or 1/20), TGase/IFN weight ratios (1/25 or 1/50) and temperature of incubation (25 °C or 37 °C). Regarding the incubation time, aliquots of the reaction mixtures were analyzed by RP-HPLC after 0, 2, 5 and 8 hours of incubation (data not shown). The reaction conditions that allowed to obtain the highest yield in mono-conjugate were those in which IFN (1.5 mg/ml) was reacted with PEG20kDa−NH2 at 1/20 IFN/PEG molar ratio and in the presence of 1/25 TGase/IFN weight ratio. Incubation was performed for 5 hours under agitation at 37 °C. The TGase-catalyzed reactions of IFN with PEG20kDa−Gln or PEG20kDa−NH2 performed under these optimized conditions were analyzed by RP-HPLC (Fig. 4A), while the eluted species were characterized by SDS-PAGE stained with iodine and Blue Coomassie (Fig. 4B). In the RP-HPLC analysis of the reaction mixture of IFN with PEG20kDa-Gln after 5h of incubation, IFN conjugated to one PEG20kDa (IFN1PEG(K164)) eluted at 17.6 min (Fig. 4A, peak 1). As evidenced by the SDS-PAGE analysis, at the same retention time we observed also the elution of traces of IFN conjugated to two PEG20kDa molecules, the second molecule being likely linked to Lys31 (Fig. 4B). At 18.4 min (peak 2), unreacted IFN coeluted with some PEGylated IFN oligomers. The IFN dimer conjugated to one PEG20kDa eluted at 18.9 min (peak 3), while the IFN dimer and PEGylated IFN oligomers were the main species eluting at 19.4 min (peak 4). The reaction yield in IFN1PEG(K164) derivative, calculated from the areas of the RP-HPLC analysis after 5 h of incubation, is of 54 ± 2% (mean ± SD from three different preparations).



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Figure 4. Derivatization of IFN with PEG20kDa-Gln or PEG20kDa-NH2 mediated by TGase. A. RP-HPLC analyses of the reaction mixtures containing IFN, TGase and PEG20kDa-Gln or PEG20kDa-NH2 after 5 h of incubation. B. SDS-PAGE analysis (4-20%) of the protein material collected from the RP-HPLC analyses reported in A. The same gel was colored with iodine (left) and Coomassie staining (right). In the Coomassie stain, Std are protein standards (SigmaMarker Wide Range), while in the iodine stain, Std are PEG polymers of 10, 20 and 40 kDa. Samples were loaded as follow: 1-4, factions collected in the RPHPLC analysis of the reaction mixture in the presence of PEG20kDa-Gln; 5-9, factions collected in the RP-HPLC analysis of the reaction mixture in the presence of PEG20kDaNH2; 10, IFN. The number of each lane is also reported on the corresponding peak of the RP-HPLC analyses.


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The RP-HPLC chromatogram of the reaction mixture of IFN with PEG20kDa−NH2 after 5 h of incubation displayed a peak at 17.1 min (peak 5) that was shown by SDS-PAGE to contain IFN conjugated to one PEG moiety at the level of Q101 (IFN1PEG(Q101)). At 18.4 min, unreacted IFN eluted with the IFN dimer conjugated to one PEG moiety (peak 6), while at higher retention times the main species detected in the SDS-PAGE analysis were a PEGylated trimer of IFN (RT 19.1 min, peak 7), the IFN dimer and PEGylated IFN oligomers (RT 19.5 min, peak 8) and IFN oligomers (RT 19.7 min, peak 9). Overall, based on the areas of the RP-HPLC peaks the yield in IFN1PEG(Q101) derivative was of 29 ± 2% (mean ± SD from three different preparations). Importantly, the two mono-PEG conjugates of IFN could be both obtained in reasonably good yields. In order to preserve the native and active conformation of IFN, the PEGylated derivatives were purified from the reaction mixtures by strong cation exchange chromatography (Fig. S6), dialyzed against PBS buffer at pH 7.4 and then used for conformational, pharmacokinetic and activity studies. The chemical characterization of the two conjugates of IFN purified by cation exchange chromatography was performed by SDS-PAGE (Fig. S6), MALDI-TOF mass spectrometry and RP-HPLC (Fig. S7), which confirmed the purity of the IFN derivatives. In particular, in the SDS-PAGE analysis the conjugated species displayed a single band at about 60 kDa that was detected by iodine staining, confirming the presence of PEG in such protein band (Fig. S6D). The molecular mass of the two derivatives was measured by MALDI-TOF mass spectrometry that yielded a value of 40453.9 m/z for IFN1PEG(K164) and of 40798.1 m/z for IFN1PEG(Q101), confirming the conjugation of one PEG moiety of 20 kDa to IFN (Fig. S7A). The RP-HPLC chromatograms also showed single peaks at the expected retention times for the PEGylated IFN derivatives (Fig. S7B). Circular dichroism (CD) spectroscopy. IFN has an α-helical secondary structure under physiological conditions. Indeed, the far-UV CD spectrum recorded in PBS buffer, pH 7.4 shows the characteristic two minima at 208 and 222 nm (Fig. 5). Derivatization of this protein with a PEG 20kDa at either Gln101 or Lys164 did not affect the helical structure of the protein as indicated by the far-UV CD spectra of the two IFN derivatives that are superimposable to the spectrum of the native protein (Fig. 5). This result was expected by the fact that the two modified residues are located both at the level of flexible regions of the protein and it is in agreement with the preservation of protein conformation reported for other PEGylated derivatives of IFN.23,46



15 IFN IFN1PEG(Q101) 1PEG(K164) IFN

-3

2 -1 (degcm dmol )

10

[θ] x 10

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5 0 -5 -10 -15 -20 -25 200

210

220

230

240

250

Wavelength (nm) Figure 5. Spectroscopic characterization by circular dichroism of IFN and of the derivatives IFN1PEG(Q101) and IFN1PEG(K164). Far-UV CD spectra were recorded at 20 °C in PBS buffer, pH 7.4. Pharmacokinetic studies. The pharmacokinetics of IFN, IFN1PEG(Q101) and IFN1PEG(K164) after i.v. administration were assessed in rats. The IFN content in serum samples was quantified by human IFN Alpha ELISA kit and the pharmacokinetic data were elaborated using PKSolver software, applying a bi-compartmental model that fitted better the data than a mono-compartmental model. As shown in Figure 6, high plasma levels of IFN were recorded immediately for both the native IFN and its PEGylated forms. The IFN levels for the native protein decreased rapidly within the first 30 min after the injection and fell below the limit of detection after 5 h, whereas the conjugates showed prolonged half-lives with detectable levels of IFN till to 24 h (Fig. 6). PEGylation led to a sustained blood concentration of IFN, as both conjugates showed an increased elimination half-life with respect to free IFN, and reduced clearance and apparent volume of distribution. The key pharmacokinetic parameters are reported in Table 2. The half-lives of IFN1PEG(Q101) and IFN1PEG(K164) were 2.61 and 2.34 h, respectively, corresponding to about 5 fold respect half-life of IFN (0.47 h). Also the bioavailability was greatly improved after PEGylation, 16 ACS Paragon Plus Environment

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since IFN1PEG(Q101) and IFN1PEG(K164) can count of an increase of the area under the curve of about 2.8 to 3.6 fold, respectively, compared to that of IFN. Comparing the two PEGylated derivatives each other, it was demonstrated that the pharmacokinetic behaviours of the conjugates were similar and consequently not affected by site of PEG coupling.

10

‡ 1 Concentration (µg/ml)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


IFNPredicted Predicted IFN

‡

‡

‡

IFN IFN

‡

IFN1PEG(Q101) Predicted IFN1PEG(Q101) Predicted IFN1PEG(Q101) IFN1PEG(Q101)

‡ ‡

0.1

IFN1PEG(K164) Predicted IFN1PEG(K164) Predicted

*

IFN1PEG(K164) IFN1PEG(K164)

0.01

0.001

0.0001 0

5

10

15

20

Time (h)

Figure 6. Pharmacokinetic studies. Pharmacokinetic profiles of IFN, IFN1PEG(Q101) and IFN1PEG(K164) calculated by PkSolver software. Data are presented as mean ± SD (n = 4). * p < 0.05 conjugate vs. IFN; ‡ p < 0.01 conjugate vs IFN (significance was calculated using ANOVA). Table 2. Pharmacokinetic parameters of IFN, IFN1PEG(Q101) and IFN1PEG(K164) after i.v. administration of 0.1 mg/kg IFN (equiv) to Sprague-Dawley rats. AUC(0→∞)

t1/2 α (h)

t1/2 β (h)

IFN

0.01

0.47

1.28

19.26

49.1

IFN1PEG(Q101)

0.03

2.61

3.59

7.10

27.83

IFN1PEG(K164)

0.35

2.34

4.55

5.34

18.49

Compound

Cl (mL/h)

(µg h/mL)

VD (mL)

(t1/2α: distribution half-life, t1/2β: elimination half-life, AUC(0→∞): area under the curve, Cl: clearance, VD: apparent volume of distribution).



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Antiviral assessment. To assess whether the two mono-conjugate isomers of IFN, modified at the level of Gln101 or Lys164, preserved their biological activity, the antiviral potency of IFN-isomers was compared with that of the free cytokine. To this end, Vero cells were pre-incubated with scalar doses of IFN1PEG(K164), IFN1PEG(Q101), and IFN for 24 h to induce the antiviral state, and were subsequently infected with the cytopathic vesicular stomatitis virus (VSV). Finally, cell viability was measured by ATP-based luminescence assay 1 day after. IFN treatment produced a strong resistance to the cytopathic effects of the virus, with a calculated IC50 of 0.272 µg/mL (Fig. 7). However, IFN1PEG(K164) and IFN1PEG(Q101) isomers retained a relevant antiviral activity that was only 1.6- to 1.8-fold (0.45 µg/ml and 0.5 µg/ml, respectively) lower than in IFN. The measured reduction in biological activity is substantially improved in respect to the 14-fold reduction reported for 40 kDa PEG-IFN α-2a (Pegasys) and lower of the approximately 4-fold decrease in antiviral activity measured for the 12 kDa PEG-IFN α-2b (Peg-Intron).24,47

Figure 7. Activity studies. IFN-induced antiviral effects in Vero cell. Cells were incubated with escalating concentrations of IFN, IFN1PEG(K164) and IFN1PEG(Q101), after 24 h were infected with VSV and finally the viability was assessed by ATPlite assay. The values of IC50 reported are the mean ± SE of three viability experiments. The IC50 was calculated from semi-logarithmic dose-response curves by linear interpolation. Values are reported in µg/mL. These data suggest that PEG-conjugation by TGase can be advantageous to obtain two highly active mono-derivatized isomers of IFN. The 1.6- to 1.8-fold reduction in potency with respect to the wild-type protein might be mostly due to PEG steric hindrance on the receptor-interacting area rather than to modification of residues located in regions important for receptor binding (see above).

CONCLUSIONS In this paper, the reactivity of human IFN α-2b towards microbial transglutaminase was studied. We demonstrated that this important therapeutic protein could be selectively 18 ACS Paragon Plus Environment

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modified at the level of Gln101 or Lys164 residues, when an excess of amino donor (i.e. PEG-NH2) or acyl donor substrate (i.e. PEG-Gln) is used, respectively. Consequently, upon the selected enzymatic reaction conditions with TGase it is possible to synthesize two homogenous IFN α-2b mono-conjugates. The high selectivity of the TGase-mediated reaction is determined by the localization of these residues at the level of two highly flexible regions of the protein, as predicted from the structural analyses of IFN α-2b and IFN α-2a and by limited proteolysis experiments.36,37 These results confirmed that the sites of TGase modification in a given protein, at the level of either Gln or Lys residues, are mainly dictated by the conformational features of the protein substrate and that analysis of the 3D structure of a protein can be used to predict residues modified by this enzyme.16,17 Overall, this study further proves that microbial transglutaminase can be an important tool for the site-specific conjugation of proteins. The TGase-mediated reaction can be used for the preparation of two homogenous mono-PEGylated conjugates of IFN in good yields that can be easily purified. Importantly, the two PEGylated IFN derivatives maintained a native conformation and they showed both high specific activity and a desired prolonged pharmacokinetic profile. Moreover, the preparation of the IFN1PEG(K164) derivative demonstrated that PEGylation of a pharmaceutical protein at one Lys residue is achievable with high yield by using TGase, thus establishing that this approach is an efficient method to obtain site-specific polymer conjugation in addition to the TGase-mediated PEGylation of Gln residues.

EXPERIMENTAL PROCEDURES Materials. Non-glycosylated recombinant human interferon α-2b was supplied by ALFA WASSERMANN S.p.A. (Bologna, Italy). V8-protease from Staphylococcus aureus (GluC Sequencing Grade) was from Promega (Madison, WI, USA). Methoxy-PEG-amino 20 kDa (PEG20kDa-NH2) was from NOF Corporation (Tokyo, Japan), while ZQG, DC and all other chemicals where purchased from Sigma-Aldrich (Milwaukee, WI, USA). TGase from Streptomyces mobaraensis was TGase ACTIVA MP from Ajinomoto Co. (Tokyo, Japan). Human IFN Alpha ELISA Kit was provided from Thermo Fisher Scientific (Waltham, MA, USA). Stock solutions of TGase were prepared by dissolving the MP powder in 0.1 M sodium phosphate buffer pH 7.0 in order to obtain a protein concentration of about 1 mg/ml, as determined by UV spectroscopy. Typically, 1 ml of phosphate buffer was added to 200 mg of TGase powder and after vortexing for 5 min and centrifuging for



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10 min at 13200 rpm, protein concentration was measured on the supernatant. This solution was then divided in aliquots and stored at -80 °C until use. The concentration of IFN and TGase solutions was determined on the basis of the absorbance at 280 nm using the

protein’s

extinction

coefficient

generated

by

ProtParam

(http://www.expasy.org/tools/protparam.html; absorbance at 280 nm of a solution 1 g/l is 0.97 for IFN and 1.89 for TGase). TGase-mediated derivatization of IFN with DC or ZQG. IFN was reacted with TGase in PBS buffer, pH 7.2, at a concentration of ~0.67 mg/mL in the presence of DC or ZQG. A reaction was also performed in the presence of only TGase to analyze the products of the TGase-mediated crosslinking of IFN. For the reactions with DC and ZQG, stock solutions of DC (20 mg/mL) and ZQG (34 mg/mL) in dimethyl sulfoxide were added at molar ratios IFN/ligand of 1/30 (DC) or 1/50 (ZQG). TGase was added to the three solutions of IFN (IFN with DC or with ZQG or IFN in the absence of any ligand) at an enzyme/substrate (E/S) weight ratio of 1/25 and the reaction mixtures were incubated at 37 °C. Aliquots were collected after 0 min (before enzyme addition), 15 min, 30 min, 1 h, 2 h and 4 h and reactions were stopped by addition of a solution of iodoacetamide (final concentration 118 µM). Aliquots of the reaction mixtures were analyzed by RP-HPLC on an Agilent series 1100 HPLC with an online UV detection from Agilent Technologies (Waldbroon, Germany). RP-HPLC analyses were performed using a C4 Phenomenex column (Jupiter C4, 300 Å, 5 µm, 150 × 4.60 mm, Phenomenex, Torrance, California, USA) equipped with a SecurityGuard Cartridge Widepore C4 (4 × 3.0 mm ID, Phenomenex) and applying a two steps gradient of acetonitrile (ACN), 0.085 % trifluorocetic acid (TFA) and water, 0.1 % TFA from 5 to 40 % of ACN in 5 min and from 40 to 70% in 20 min. The column was eluted at a flow rate of 0.8 ml/min and the absorbance was read at 226 nm. Fractions collected from RP-HPLC were lyophilized and then analyzed by ESI-MS or subjected to digestion with V8-protease and then analyzed by LC-MSE. About 6 µg of the three reaction mixtures after 4 h of incubation were lyophilized and then analyzed by SDS-PAGE using a gel with 15% acrylamide concentration and stained with Coomassie Brilliant Blue R-250. Proteolytic Digestion of DC- and ZQG-modified IFN. IFN and the IFN1DC and IFN1ZQG derivatives purified by RP-HPLC were lyophilized and dissolved in few µl of an aqueous solution of 0.05% formic acid and then diluted 6 folds with 50 mM NH4HCO3 pH 8.1. Stock solutions of V8-protease were added to obtain a final E/S ratio of approximately 1/50, by weight. Proteolysis was let to proceed at 37 °C overnight in a Thermomixer 20 ACS Paragon Plus Environment

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(Eppendorf), then acidified with a solution of 5mM TCEP in 0.9% formic acid to a final TCEP concentration of 1.2 mM and incubated for 4h at 37 °C in a Thermomixer. The proteolysis mixture was then stored at -20 °C before LC-MSE analysis. Limited proteolysis of IFN with proteinase K. Limited proteolysis of IFN was conducted in PBS buffer pH 7.4, at 25 °C with proteinase K at an E/S of 1/200 by weight. Aliquots from the reaction mixtures were quenched by adding 1% TFA solution and then analysed by Tricine-SDS-PAGE and RP-HPLC. RP-HPLC analysis of the proteolytic mixtures was performed on an Agilent series 1200 HPLC using a C4 Phenomenex column (150 × 4.6 mm), at the flow rate of 0.8 ml/min, with a gradient of ACN, 0.085 % TFA and water, 0.1 % TFA from 5 to 70% of ACN in 40 min. The absorbance was read at 226 nm. Fractions collected from the RP-HPLC analyses were lyophilized and analyzed by MS. For MS analyses, protein samples were dissolved in 0.1% formic acid in ACN: water (1:1). Reduction of the nicked and gapped species was performed upon overnight incubation with 1 mM TCEP at 37 °C, followed by ESI-MS analysis. Synthesis of PEG20kDa-Gln. PEG20kDa-Gln was synthetized starting from PEG-NH2. 38.3 mg (0.2 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (MW 191.7 Da) and 27 mg (0.2 mmol) of 1-hydroxybenzotriazole (MW 135.13 Da) were added to 16.8 mg (49.8 µmol) of ZQG (MW 337.33 Da) previously dissolved in 5 mL of 0.1 M borate buffer/ACN (3:2) mixture pH 8.0. After 1 hour, 250 mg (12.5 µmol) of PEG-NH2 (MW 20000 Da) was added and the pH was adjusted to 8. The reaction was left to stir at room temperature and after 18 hours, 2.5 mg (24.9 µmol) of succinic anhydride (MW 100.08 Da) was added to the mixture. The solution was dialyzed for 24 hours against Milli-Q water and the product PEG20kDa-Gln was then lyophilized (yield: 241 mg, 95%). The absence of free amino groups was verified by 2,4,6-trinitrobenzenesulfonic acid (TNBS) test according to Snyder and Sabocinsky assay.48 TGase-mediated derivatization of IFN with PEG20kDa-Gln and PEG20kDa-NH2. Stock solution of IFN (~ 3 mg/ml, specific activity of 200x106 IU/mg) were dialyzed at 4 °C against PBS, pH 7.4 with two changes of buffer and using a dialysis membrane with a molecular weight cut-off of 6-8000 (Spectra/Por Dialysis Membrane, Spectrum Laboratories Inc., California USA). Reactions were performed at a protein concentration of 1.5 mg/ml. A molar excess of PEG20kDa of 1/20 in respect to IFN was used for both the reaction with PEG20kDa-NH2 and PEG20kDa-Gln. Reaction mixtures were prepared by dissolving the PEG polymer in the volume of PBS buffer needed to dilute the IFN stock



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solution to 1.5 mg/ml. The PEG solution was added under stirring to the IFN solution and an aliquot of the reaction mixture was taken before the addition of TGase as time 0 h. The stock solution of TGase was added in order to obtain an E/S ratio of 1/25, by weight between TGase/IFN. The reactions were incubated in a room at 37 °C under magnetic stirring and were quenched after 5h of incubation upon addition of a stock solution of iodoacetamide at a molar ratio with TGase of 30/1 (iodoacetamide/TGase). An aliquot of the reaction was taken for the RP-HPLC analysis while the remaining reaction mixture was diluted to 1 mg/ml with 10 mM sodium phosphate pH 4.7 and then dialyzed at 4 °C against 10 mM sodium phosphate pH 4.7 with two changes of buffer and using a dialysis membrane with a molecular weight cut-off of 6-8000 (Spectra/Por Dialysis Membrane). Aliquots of the reaction mixtures were analyzed by RP-HPLC on an Agilent series 1100 HPLC with an online UV detection from Agilent Technologies. RP-HPLC analyses were performed using a C18 Phenomenex column (Jupiter C18, 300 Å, 5 µm, 150 × 4.60 mm) equipped with a SecurityGuard Cartridge Widepore C18 (4 × 3.0 mm ID, Phenomenex) applying a two steps gradient of ACN, 0.085 % TFA and water, 0.1% TFA from 5 to 40% of ACN in 5 min and from 40 to 70% in 25 min. The column was eluted at a flow rate of 0.8 ml/min and the absorbance was read at 226 nm. A C18 column was preferred instead of a C4 column because analysis of the reaction mixture with the C4 column resulted in a low recovery of PEG-conjugated IFN. Fractions collected from the RP-HPLC analyses were lyophilized and then analyzed by SDS-PAGE using a Mini-PROTEAN TGX Precast gel 4-20% (BIO-RAD) and stained with iodine and Coomassie Brilliant Blue R-250. Purification of IFN conjugated to PEG20kDa-Gln or PEG20kDa-NH2. Reaction mixtures of IFN in the presence of TGase and PEG20kDa-Gln or PEG20kDa-NH2 after dialysis in 10 mM sodium phosphate pH 4.7 were loaded on a RESOURCE S column (1ml, GE Healthcare Life Sciences). The chromatographic separation was performed using an ÄKTA FPLC (Amersham Biosciences). The cation exchange column was eluted with a gradient of buffer A (10 mM sodium phosphate pH 4.7) and B (0.1 M sodium phosphate, 0.1 M NaCl pH 4.85). After injection of the sample, the column was eluted with 5% B for 10 min and then with a gradient of B from 5% to 35% in 50 min and from 35% to 100% in 30 min. The column was then washed for 10 min with 100% B and for 5 min with 5% B. The flow rate was of 1 ml/min and absorbance was read at 214 nm. IFN derivatives conjugated to PEG20kDa-Gln or PEG20kDa-NH2 collected from the cation exchange column were dialyzed at 4 °C against PBS buffer, pH 7.4 with two changes of 22 ACS Paragon Plus Environment

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buffer and using a dialysis membrane with a molecular weight cut-off of 6-8000 (Spectra/Por Dialysis Membrane) and then concentrated using Amicon Ultra-4 10K (Millipore). About 1 mg of native IFN was also dialyzed at 4 °C against 10 mM sodium phosphate pH 4.7 and then subjected to cation exchange chromatography, dialysis and to the concentration step and used as reference for the wild-type protein. The purified conjugated IFNs and native IFN were analyzed by SDS-PAGE, RP-HPLC, MALDI-TOF mass spectrometry, circular dichroism and used in pharmacokinetic and activity studies. Mass Spectrometry Analyses. Mass spectrometry-based analyses of intact IFN and IFN derivatives (IFN1DC, IFN1ZQG and IFN oligomers) were performed with a Micromass mass spectrometer Q-Tof Micro (Manchester, UK) equipped with an electrospray source. Samples were dissolved in 0.1% formic acid in ACN: water (1:1) and analyzed in MS positive ion mode. Measurements were conducted at a capillary voltage of 3 kV and at cone and extractor voltages of 40 and 1 V, respectively. MS/MS spectra of peptides 97−107 modified with DC and of peptide 160−165 modified with ZQG were acquired on the Q-Tof Micro at variable collision energy values and using argon as collision gas. Instrument control, data acquisition and processing were achieved with Masslynx software (Micromass). MALDI-MS analyses were performed on a REFLEX time-of-flight instrument (4800 Plus MALDI TOF/TOF, AB Sciex, Framingham, Massachusetts, USA) equipped with a SCOUT ion source operating in positive linear mode. Ions generated by a pulsed UV laser beam (nitrogen laser, λ 337 nm) were accelerated to 25 kV. A saturated solution of sinapinic acid in water/ACN (1:1, v/v) was used as a matrix and mixed with the samples dissolved in 0.1% TFA aqueous solution at a v/v ratio 1:1. The peptide mixtures of IFN, IFN1DC and IFN1ZQG digested with endoprotease Glu-C were analyzed using a Xevo G2-S QTof (Waters) equipped with an UPLC1290 Infinity (Agilent Technlogies). The Agilent UPLC was configured with an AdvanceBio Peptide Map Guard (2.1 × 5 mm, 2.7 µm, Agilent technologies) and AdvanceBio Peptide Map column (2.1 × 150 mm, 2.7 µm). Mobile phase A was water and 0.1% formic acid while mobile phase B was 0.1% formic acid in ACN. Peptide separation was performed using a linear gradient from 2% to 65% of B in 25 minutes at a flow rate of 0.2 ml/min, with a column temperature set at 30 °C. The Xevo G2-S QTof was operated in the ESI positive ion, resolution mode and with a detection window between 50-2000 m/z. Source parameters were: capillary (kV) 1.5, sampling cone voltage 30 V, source offset of 80 V. MSE 23 ACS Paragon Plus Environment


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acquisition was performed by alternating two MS data functions: one for acquisition of peptide mass spectra with the collision cell at low energy (6 V), and the second for the collection of peptide fragmentation spectra with the collision cell at elevated energy (linear ramp 20 to 40 V). Analyses were performed with LockSpray™ using a solution of 1 ng/µL LeuEnk in 50:50 ACN/water containing 0.1% formic acid, sampled every 45 sec. MSE data were processed with the BiopharmaLynx 1.3.4 Software (Waters) setting Glu-C as digest reagent and 5 missed cleavages. The following searchable modifications were considered as variable modifications: Met-oxidation, Asn- and Gln-deamidation. In order to detect IFN peptides conjugated to DC or ZQG, the following modified residues were created and included as variable modifiers: Lys residue modified with ZQG (delta mass of 320.1008 Da as side chain modification of Lys) and Gln residue modified with DC (delta mass of 318.1402 Da as side chain modification of Gln). MS ion intensity threshold was set to 250 counts, and the MSE threshold was set at 100 counts. MS mass match tolerance and MSE mass match tolerance were set to 15 ppm. Conjugated peptides were confirmed by at least 4 b/y fragment ions in their correspondent MSE MS/MS fragment spectrum. Circular dichroism. Circular dichroism spectra were recorded at room temperature on a Jasco J-810 spectropolarimeter (Tokyo, Japan) equipped with a Peltier temperature control system set at 20 °C. Protein samples were analyzed at a concentration of 0.14 mg/ml using a cuvette of 1 mm path length. Ethics statement. The study protocol was approved by the Ethics Committee of the University of Padova and the Italian Ministry of Health (CEASA 24/2013), and animals were handled in compliance with national (Italian) Legislative Decree 116/92 guidelines and with the “Guide for the Care and Use of Laboratory Animals” by the National Research Council of the National Academies. Pharmacokinetic study. Pharmacokinetic profiles of free and conjugated IFN were determined in Sprague-Dawley rats (250-350 g). The rats were randomly divided into three groups of 3 animals per group. A dose of 0.1 mg/kg IFN (equiv.) was administered via tail vein to the rats anesthetized with 5% isoflurane gas (mixed with O2 in enclosed cages). At predetermined times blood samples were collected from the tail vein and centrifuged at 1500 × g for 15 min. The IFN content in serum samples was quantified using the Human IFN Alpha ELISA Kit (PBL Assay Science). The pharmacokinetic data elaboration was performed by PkSolver software,49 applying a bi-compartmental model. For each sample a dedicated calibration curve was built using the testing substance to avoid error in the 24 ACS Paragon Plus Environment

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ELISA response owing to PEGylation. Antiviral assay. The African green monkey kidney-derived Vero cell line was grown in Eagle's minimum essential medium (Sigma-Aldrich, Milan, Italy) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Gibco BRL, Paisley, UK), 2 mM L-glutamine (Lonza, Verviers, Belgium), 10 mM HEPES (Lonza), 100 U/mL penicillin/streptomycin (Lonza), hereafter referred as to complete medium. Cell line was maintained at 37 °C in a humidified atmosphere containing 5% CO2. To assess IFN induced antiviral activity, Vero cells were resuspended in completed medium and seeded into 96-well flat-bottomed plates (1 × 105/well) with different concentrations of IFN, IFN1PEG(K164) and IFN1PEG(Q101) isomers for 24 hours. At day after, cells were infected with VSV (MOI 10) and the day 2 the viability was assessed by the ATPlite luminescence adenosine triphosphate detection assay system (PerkinElmer, Zaventem, Belgium), according to the manufacturer's instructions. The lysis solution was added to each well (50 µL), followed by addition of 50µL of substrate solution and finally the luminescence was counted by the TopCount Microplate Counter (PerkinElmer). Within each experiment, determinations were performed in triplicate and experiments were repeated 3 times. The percentage of cell survival was calculated by determining the counts per second (cps) values according to the formula: [(cpstested - cpsblank) / (cpsuntreated control cpsblank)] × 100, with cpsblank referring to the cps of wells that contained only medium and ATPlite solution. The IC50 values were calculated from semi-logarithmic dose-response curves by linear interpolation. Two different preparations of IFN conjugated to PEG20kDaGln or PEG20kDa-NH2 were tested for IFN activity. Samples were filtered using Costar Spin-X centrifuge tube filters 0.22 µm (Corning Incorporated, Corning USA) before use.



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ACKNOWLEDGMENTS We thank Alfa Wassermann S.p.A. (Alanno (PE), Italy) for the generous gift of recombinant human interferon α-2b. We acknowledge also Giacomo Forzato and Alice Menegoli for conducting some experiments.

ASSOCIATED CONTENT Supporting Information TGase-mediated oligomerization of IFN (Fig. S1, Table S1); mass data on peptides of IFN, IFN1ZQG and IFN1DC identified in the digests with endoprotease Glu-C (Table S2-S4); MS/MS mass spectra of peptide 160–165 and 1−42 of IFN modified with ZQG and of peptide 97–107 of IFN modified with DC (Fig. S2-S4, Table S5); limited proteolysis of IFN with proteinase K (Fig. S5, Table S6); purification and chemical characterization of IFN1PEG(K164) and IFN1PEG(Q101) derivatives (Fig. S6, S7). This material is available free of charge via the Internet at http://pubs.acs.org.

ABBREVIATIONS ACN, acetonitrile; cps, counts per second; DC, dansylcadaverine; E/S, enzyme to substrate ratio; IFN, human interferon alpha-2b; PEG, polyethylene glycol; SD, standard deviation; SE, standard error; TFA, trifluoroacetic acid; TGase, transglutaminase; TCEP, tris(2carboxyethyl)phosphine; VSV, vesicular stomatitis virus; ZQG, carbobenzoxy-Lglutaminyl-glycine.

AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected], [email protected].

Present addresses SB: Department of Pharmacy, University of Parma, Parco Area delle Scienza 23/A, 43124 Parma, Italy; AAS: Merck Serono S.p.A., Via Luigi Einaudi, 11, 00012 Guidonia Motecelio, Rome, Italy.

Notes The authors declare no competing financial interest. 26 ACS Paragon Plus Environment

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Funding This work was supported by the Research Projects 2009 (CPDA094592/09) and 2013 (CPDA135898/13) of the University of Padua and by the Italian Association for Cancer Research AIRC (MFAG 15458). A.A.S. and A.G. (PARO112104/11) were supported by grants from the CARIPARO Foundation (Padua).



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Table of Contents (TOC)

Graphical abstract The TGase-mediated conjugation of IFN α-2b can be directed either at Lys164 residue using a Gln-containing ligand or at Gln101 residue using an amine-containing ligand.


Site-Specific Transglutaminase-Mediated Conjugation of Interferon Î±

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