It is generally considered that a protein's native disulfide bonds cannot be modified because they are crucial to its structure and function^{1, 2}. Covalent conjugation of poly(ethylene glycol) (PEG) to therapeutic proteins increases their in vivo stability by protecting the protein from degradation, masking its immunogenic sites and reducing clearance³. Typically, PEGylation uses nonspecific reactions with nucleophilic residues and produces mixtures of PEGylated positional isomers⁴. To solve this problem, we exploited the reactivity of the two sulfur atoms of a native disulfide for selective conjugation of PEG using a thiol-specific, cross-functionalized PEG monosulfone (Fig. 1a). Mechanistically, the conjugated double bond in the PEG monosulfone is necessary to initiate a sequence of addition-elimination reactions^{5, 6}. After addition of thiol, elimination of sulfinic acid generates another conjugated double bond for the second thiol (Supplementary Scheme 1 and Supplementary Methods online). This leads to the formation of a three-carbon bridge between two sulfur atoms. Disulfide-scrambling reactions are inhibited because of thiol propinquity in the nondenatured protein and by having the bisalkylation functionality at the end of PEG.

Figure 1: Structural characterization.

(a) PEG monosulfone. (b) Silver-stained gel of the non-PEGylated three-carbon (190 Da) disulfide-bridged IFN. Lanes: (1) M_w markers (kDa); (2) IFN; (3) reduced IFN; (4) 1 equiv. bissulfone showing IFN (upper), single-bridged (middle) and double-bridged (lower) IFN; (5 and 6) 2 and 4 equiv., respectively, showing single-bridged (upper) and double-bridged (lower) IFN; (7) 6 equiv. showing double-bridged IFN. (c) MALDI-TOF–MS of IFN (left), Cys-CCC-Cys IFN (middle) and double-bridged IFN (right). (d) Gels stained with colloidal blue (protein) and barium iodide (PEG, right). Lanes: (1) 20 kDa PEG; (2) IFN; (3) IFN with reduced disulfide; (4) PEGylation reaction mixture; (5) IFN with both disulfides reduced.

Full figure and legend (52K) Figures, schemes & tables index

We used interferon -2b (IFN) because it is representative of four-helical-bundle proteins with accessible disulfide bonds. Theoretically, the effect of introducing a three-carbon bridge is determined using stochastic dynamics simulations. The bridged IFN isomers Cys1-CCC-Cys98 and Cys29-CCC-Cys138 are within the conformational flexibility of the crystal and NMR-based structures of interferon -2a, indicating that IFN's tertiary structure is preserved⁷ (Supplementary Results 1 online).

We found that a three-carbon disulfide-bridged PEG-IFN can be prepared when one protein equivalent (equiv.) of PEG monosulfone is used after reducing both disulfides. Conjugation is conducted at pH 7.8 and 4 °C for 2 h after removal of excess dithiothreitol. If two equivalents of PEG monosulfone are used, both disulfides undergo conjugation. As a control, we conjugated a non-PEG precursor to IFN. SDS-PAGE gels showed IFN's conjugation to precursor and PEG monosulfone, with MALDI-TOF–MS confirming the M_w of the isomers Cys1-CCC-Cys98 and Cys29-CCC-Cys138 (Fig. 1b–d) and of their trypsin-digested fragments (Supplementary Results 2 online). The three-carbon–bridged PEG-IFNs were purified by cation-exchange chromatography followed by size-exclusion chromatography (SEC)-HPLC with confirmation by western immunoblotting. The SEC-HPLC chromatogram showed a three-carbon disulfide single-bridged PEG-IFN (that is, Cys1-CC[PEG]C-Cys98 or Cys29-CC[PEG]C-Cys138, yield 65%), a three-carbon disulfide double-bridged PEG-IFN (Cys1-CC[PEG]C-Cys98 and Cys29-CC[PEG]C-Cys138, yield 23.5%), IFN (yield 4.9%) and aggregated IFN (yield 6.6%) (Supplementary Results 3 online).

The reaction can be simplified by in situ conversion of the PEG bissulfone to the PEG monosulfone at pH 7.8 during protein conjugation. Competitive reactions of the PEG monosulfone with other nucleophilic residues are not seen (Supplementary Results 4 online). MALDI-TOF–MS confirmed the M_w of the two-bridged PEG-IFN isomers, and CD confirmed the preservation of IFN's -helical structure (Supplementary Results 2).

Interferon -2b has distinct effects in vitro: it blocks infection of human A549 (lung epithelial) cells by encephalomyocarditis (EMC) virus, it induces 2',5'-oligoadenylate synthetase (2',5'-OAS) mRNA synthesis, and it upregulates major histocompatibility (MHC) class I expression on immunoregulatory cells (Supplementary Methods). Using SEC-HPLC, we found that the unreacted IFN and the non-PEGylated three-carbon disulfide single-bridged IFN both showed a small reduction in antiviral activity compared to IFN (Fig. 2a,b). Our results also showed that insertion of a three-carbon disulfide bridge contributed 11%, and addition of PEG contributed 89% to the reduction in the PEG-IFN's biological activity. Because PEG reduces protein immunogenicity, the PEG-IFNs have a lower affinity for MHC class I molecules than IFN (Supplementary Results 5 online). Uniquely, the PEG's length does not affect its biological activities. The PEG-IFN's biological activities (8% of IFN) are similar to those of the PEG-IFN in clinical use (7%)^{8, 9, 10}; the enhanced in vivo therapeutic efficacy compensating for the reduced in vitro activity¹⁰. Our PEG-IFNs are stable in aqueous solution for 3 months at 4 °C; and in human serum for 30 h at 37 °C. After subcutaneous administration in mice, the 20 kDa PEG-IFN's half-life is 12 h compared to 1 h for IFN.

Figure 2: Biological activities.

(a) Antiviral activity in A549 cells infected with EMC virus (n = 6). (b) 2',5'-OAS mRNA synthesis in Molt-4 cells (n = 3). IFN (gray); unreacted IFN recovered after SEC-HPLC (red); non-PEGylated three-carbon disulfide-bridged IFN (green); three-carbon disulfide single-bridged 10 kDa PEG-IFN (orange); three-carbon disulfide single-bridged 20 kDa PEG-IFN (blue). (c) Immunoblot with an antibody to Fab. M_w markers (left) are in kDa. Lanes: (1) Fab; (2) reduced Fab; (3) three-carbon disulfide single-bridged 20 kDa PEG-Fab. (d) Inhibition of HIV-1 entry into human C8166 (T-lymphoblastoid) cells as determined by real-time PCR for RU5, the first DNA transcript of HIV-1 to be synthesized after viral entry (n = 3). Data presented as mean s.e.m.

Full figure and legend (56K) Figures, schemes & tables index

We applied this approach to a human CD4 receptor–blocking antibody fragment (Fab). Entry of HIV-1 into cells requires viral gp120 to bind the D1 domain of human CD4. The IgG₁ monoclonal antibody Q4120/ADP318 (which binds the D1 domain of CD4; ref. 11) was digested to make Fab and PEGylated after reduction of its interchain disulfide (Fig. 2c). At a saturating dose, the PEG-Fab was as effective as Fab at blocking HIV-1 entry into CD4⁺ T-lymphocyte cells (Fig. 2d).

Our studies also include the PEGylation of L-asparaginase without loss of enzyme activity or immunogenicity¹². The accessible native disulfide bonds of proteins can therefore be modified by the site-specific insertion of a three-carbon PEGylated bridge. Our approach differs fundamentally from conjugation of PEG to amine residues^{8, 9, 10}, where the biological activity of the PEGylated positional isomers depends upon conjugation conditions and the size of PEG⁴. It also makes engineering free cysteines into proteins for thiol-selective PEGylation unnecessary. As the biological activities of our PEGylated proteins are independent of PEG size, only their in vivo pharmacokinetics need optimizing before clinical trials.

Note: Supplementary information is available on the Nature Chemical Biology website.

Acknowledgments

The authors thank the National Institute for Biological Standards and Control (UK) for Q4120/ADP318 antibody. This work was supported by the Wellcome Trust (068309) and BBSRC-UK (BB/D003636/1).

Ancillary details

Received 21 February 2006; Accepted 28 March 2006; Published online 23 April 2006.

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1. Faculty of Medicine, Imperial College London, Hammersmith Hospital, Ducane Road, London W12 0NN, UK.
2. Department of Pharmaceutics, The School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, UK.
3. Department of Pharmaceutical and Biological Chemistry, The School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, UK.

Correspondence to: Sunil Shaunak¹ Email: s.shaunak@imperial.ac.uk

Correspondence to: Steve Brocchini² Email: steve.brocchini@pharmacy.ac.uk