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Dually Reactive Long Recombinant Linkers for Bioconjugations as an Alternative to PEG

Covalent cross-linking of biomolecules can be useful in pursuit of tissue targeting or dual targeting of two receptors on cell surfaces for avidity effects. Long linkers (>10 kDa) can be advantageous for such purposes, and poly(ethylene glycol) (PEG) linkers are most commonly used due to the high aqueous solubility of PEG and its relative inertness toward biological targets. However, PEG is non-biodegradable, and available PEG linkers longer than 5 kDa are heterogeneous (polydisperse), which means that conjugates based on such materials will be mixtures. We describe here recombinant linkers of distinct lengths, which can be expressed in yeast, which are polar, and which carry orthogonal reactivity at each end of the linker, thus allowing chemoselective cross-linking of proteins. A conjugate between insulin and either of the two trypsin inhibitor peptides/proteins exemplifies the technology, using a GQAP-based linker of molecular weight of 17 848, having one amine at the N-terminal, and one Cys, at the C-terminal. Notably, yeast-based expression systems typically give products with mixed disulfides when expressing proteins that are equipped with one unpaired Cys, namely, mixed disulfides with glutathione, free Cys amino acid, and/or a protein homodimer. To obtain a homogeneous linker, we worked out conditions for transforming the linker with mixed disulfides into a linker with a homogeneous disulfide, using excess 4-mercaptophenylacetic acid. Subsequently, the N-terminal amine of the linker was transformed into an azide, and the C-terminal Cys disulfide was reduced to a free thiol and reacted with halo-acetyl insulin. The N-terminal azide was finally conjugated to either of the two types of alkyne-containing trypsin inhibitor peptides/proteins. This reaction sequence allowed the cross-linked proteins to carry internal disulfides, as no reduction step was needed after protein conjugations. The insulin–trypsin inhibitor conjugates were shown to be stabilized toward enzymatic digestions and to have partially retained binding to the insulin receptor.


■ INTRODUCTION
Linkers based on poly(ethylene glycol) (PEG) are the most common types for chemical cross-linking of biomolecules. 1 Long PEG chains (20−40 kDa) are also commonly used for modification of biomolecules in pursuit of prolonged half-life, due to slower absorption of large PEG derivatives from the relevant injection site, hindrance of kidney-mediated clearance, and/or partial protection from enzymatic breakdown. 2, 3 Advantages of PEG are its high solubility in water and most organic solvents, its large hydrodynamic volume, and its relative inertness to interactions with biomolecules. PEG is however nondegradable in vivo, so accumulation of PEG in various tissues upon chronic administration of PEGylated drugs has been reported in some cases, 4 whereas excretion via bile has been described in other cases. 5 PEG can be purchased with various functional groups at one or both ends, for making either PEGylated proteins or cross-linked constructs. Notably, long PEG chains are prepared by polymerization of ethylene oxide and fractioned for size, so long PEG chains (>10 kDa) are always heterogeneous, i.e., they consist of polydisperse mixtures of chain lengths with a polydispersity index of nearly 1.05 in best cases.
Polar protein sequences with PEG-like properties, sometimes called "recombinant PEG", have in recent years been described by Alvarez,6 Amumix (mixed sequences of GEDSTAP residues, termed "XTEN"), 7 XL-protein (PAS repeats), 8 Novo Nordisk (GQAP-like repeats), 9,10 SOBI, 11,12 and others. The advantages of these materials are their biodegradable nature and distinct size, i.e., one precise molecular weight. Furthermore, recombinant PEG can often be introduced by simple extension of the expressed protein (fusion protein), and the hydrodynamic volumes of the polar extensions are similar to what PEG provides. Immunogenicity of the non-native protein sequences can be a problem, but the risk seems to be low due to the polar nature of the sequences (no hydrophobic binding motifs) and disordered structures. XTEN fusions of other human growth hormones are in latestage human clinical trials without serious immunogenicity problems. 13 Both XTEN and PAS have high content of serine residues, and this can be a problem for expressions using yeast-based systems, since yeast typically gives mixtures of O-mannosylated products when there are many serine residues in the sequences. 10,11,14 XTEN and PAS fusions are typically expressed in Escherichia coli-based systems where O-mannosylation is not a problem, but expression in E. coli typically gives protein products in inclusion bodies, which must be refolded and disulfide-paired, and this is often difficult. Expressions from yeast on the other hand often provide folded, disulfidepaired protein excreted into the medium. 15 Another drawback of XTEN can be that the high content of charged amino acids, such as glutamate, can lead to an increase in viscosity of protein solutions/formulations when high protein concentrations are needed in pharmaceutical formulations, presumably due to the more extended character of the multicharged sequences vs uncharged sequences. For these reasons, we prefer sequences such as GQAP as recombinant PEG. The repeat sequences contain Gln/Q residues, which might be seen as a problem with regard to long-term stability, due to potential side-chain deamidations, but we have not encountered this problem during handling of the materials, and the half-life for Gln deamidations of peptides in aqueous solution at 37°C is known to be in the range of 2−50 years. 16 This is in sharp contrast to Asn/N, which often deamidates readily. 16 XTEN, PAS, GQAP, etc. have mainly been used for making fusion proteins, but the sequences could also be used for making protein conjugations, i.e., used as "recombinant PEG linkers" for cross-linking biomolecules, via introduction of reactive residues along the sequence. 17 We exemplify here the design and expression of GQAP-based linkers equipped with orthogonal reactivity at either end and use of such a linker for cross-linking prefolded cysteine-rich proteins, namely insulin and two trypsin inhibitor peptides/proteins. The insulin conjugates are not meant to be new drug candidates but were designed as model compounds for demonstrating the applicability of the homogeneous linkers in cross-linking complex biomolecules.

■ RESULTS AND DISCUSSION
(GQAP)49-GQEP-Cys 1 (Scheme 1, MW 17 848) was expressed in yeast using a single-chain insulin precursor as an expression anchor, having GGGGK-(GQAP)49-GQEP-Cys as extension from A21, in 142 mg/L yield. 15 The free linker 1 was isolated after cleavage at the lysine by Achromobactor lyticus protease (ALP) and purified by ion-exchange Scheme 1. (a) MPAA (10 equiv), pH 9, 18 h, 64−69% Yield; (b) 50 equiv of Im-SO 2 -Azide, 10 mM Phosphate Buffer, pH 7.5, 22 h, 74−85% Yield; (c) 1.25 equiv of DTT, pH 7.5, 2 h, 60−86% Yield; (d) 1.7 equiv of B29Nε-Iodoacetyl Insulin 5, pH 7.5, 2 h, 44−52% Yield; and (e) 1.9 equiv of 7 or 8, CuI, THPTA, N 2 Purge, pH 7.5, 2−5 h, 47−70% Yield 1 1 Products 9 and 10 are drawn showing the CH 2 -triazole side chain from R2, which leaves Gly from Prg in the inhibitor peptides/proteins. chromatography. The reason for expressing the recombinant linker with insulin-GGGGK as the expression anchor is that this setup improves expression yields and gives the product that is secreted into the yeast medium. To be able to perform orthogonal conjugations with the linker, we did not require more than one amino group (the N-terminal on the linker). To allow the linker to be used for conjugations of prefolded, disulfide-paired proteins using robust conjugation reactions, we decided to transform the N-terminal amine into an azide, for performing triazole/click chemistry, CuAAC. 18,19 But first we had to handle the Cys in the C-terminal of the linker. As expected for yeast-based systems, the expressed linker 1 was found to contain mixed disulfides at the unpaired Cys, namely, disulfides with both glutathions, free cysteine amino acid, and a Cys−Cys-linked homodimer. 20 To simplify the planned protein conjugations and purification, we transformed the linker with mixed disulfides into a linker with a homogeneous disulfide via treatment with excess 4-mercaptophenylacetic acid (MPAA) overnight at pH 9. MPAA has been promoted for use in native chemical ligation reactions and is an odor-free thiol. 21 The homogeneous linker (GQAP)49-GQEP-Cys(MPAA) 2 was isolated in 64−69% yield by reversed-phase highperformance liquid chromatography (RP-HPLC) (see Scheme 1). Next, the N-terminal amine was transformed into an azide by treatment with excess imidazolyl-sulfonyl-azide hydrogen sulfate 22,23 overnight in 10 mM phosphate buffer, pH 7.5, and the azido product 3 was isolated by RP-HPLC in 74−85% yield. The disulfide was next reduced to free thiol by treatment with a slight excess of dithiothreitol (DTT) for 2 h at pH 7.5. This reaction was performed gently and carefully followed by liquid chromatography mass spectrometry (LCMS), as DTT can reduce not just disulfides but also azides under rougher conditions. The azide-linker thiol 4 was isolated by RP-HPLC in 60−86% yield. B29Nε-iodoacetyl desB30 human insulin 5 was prepared by the reaction of desB30 human insulin with succinimidyl iodoacetate at pH 10.5. 24 The amine in the lysine side chain is more basic than the two N-terminal amines in insulin (A1 and B1, pK a ∼ 7), and the lysine amine (pK a ∼ 10) is therefore the most reactive at pH above its pK a , so B29Nε products are always the main product from insulin acylation above pH 10. 25 The thiol on the linker was reacted with B29Nε-iodoacetyl insulin 5 for 2 h at pH 7.5, and conjugate 6 was isolated by RP-HPLC in 44−52% yield. Finally, the azido group on the linker−insulin conjugate 6 was reacted under CuI catalysis (CuAAC, "click coupling") with either the sunflower trypsin inhibitor (SFTI) 26 or Bowman−Birk trypsin inhibitor (BBI) 27 containing propargylglycine (Prg) in positions 14 and 27, respectively (7 and 8). The required alkyne-containing SFTI peptide 7 was prepared by solid-phase synthesis of CFP-Prg-GRCTKSIPPI-hydrazide, which was backbone-cyclized using the nitrite method, 28 which transformed the acylhydrazide via acyl-azide into peptide thioester, which in turn spontaneously cyclized by internal native chemical ligation. The alkyne-containing BBI protein 8 was similarly prepared via hydrazides, by segmental native chemical ligations of DDESSKPCCDQCACTKSNPPQCR-hydrazide 8a with CSD-Prg-RLNSCHSACKSCICALSYPAQCF-hydrazide 8b and CVDITDFCYEPCKPSEDDKEN 8c, as previously described for normal BBI. 27 Upon CuAAC reaction between insulin-linker-azide 6 and either of the two alkyne enzyme inhibitors 7 and 8, using CuI and THPTA under N 2 , the two final bivalent protein conjugates insulin-linker-SFTI 9 and insulin-linker-BBI 10 were isolated by RP-HPLC in 48−70 and 47−50% yield, respectively.
The human insulin receptor (HIR) affinities of the conjugates were measured as an assessment of preservation of insulin bioactivity: insulin-SFTI 9 and insulin-BBI 10 showed HIR affinities relative to human insulin of 8.5 and 7.4%, respectively. HIR affinities are classically reported relative to human insulin, 29 and affinities in the given range will usually preserve full insulin bioactivity in vivo. 30 Protection of the insulin conjugates against enzymatic breakdown was tested by treatments with chymotrypsin and trypsin, monitored using an HPLC method. First, it was examined whether conjugating SFTI and BBI to insulin had preserved the inhibitory activity against the enzymes. Indeed, insulin-SFTI 9 and insulin-BBI 10 were both found to be protected against tryptic degradation (see Figure 1 and Table   1). As expected, insulin-BBI 10 was also protected toward chymotryptic degradation, whereas insulin-SFTI 9 was only partially protected against chymotrypsin. Human insulin (HI) itself was rapidly degraded by both trypsin and chymotrypsin, whereas addition of equimolar concentrations of free BBI 8 to insulin considerably slowed down HI degradation by both enzymes, while addition of equimolar concentrations of free SFTI 7 only slowed down insulin degradation by chymotrypsin, not trypsin, as expected ( Figure 1 and Table 1).  Next, it was examined whether there was an advantage of covalent attachment of SFTI to insulin over a simple mixture of the two compounds. In this experiment, the amount of trypsin was increased up to 10-fold. Compared to HI + free Prg-SFTI 7, the degradation half-life of insulin-SFTI 9 was twice as long as that using an enzyme/substrate ratio of 1:4 and 3 times as long as that using enzyme/substrate ratio of 1:2, thereby demonstrating an advantage of the covalent attachment of insulin and inhibitor under such harsh conditions (Figure 2 and Table 2). As a perspective, it could be imagined that the insulin-inhibitor conjugates would be better protected against enzymatic breakdown upon dramatic dilution, for example, upon oral dosing, since the conjugate would retain high local concentration of the inhibitor even after high dilution, whereas the free inhibitor would be diluted away from insulin when dosing the mixture of insulin and inhibitor to a large volume (the gut). We do not intend to pursue these compounds with oral dosing, but they illustrate the principle.

■ CONCLUSIONS
Long linkers of repeating GQAP units can be expressed in yeast, providing compounds with PEG-like properties. The linkers have a distinct molecular weight, contrary to PEG, and are expected to be biodegradable in vivo like XTEN and other recombinant PEG materials. Equipping the linkers with orthogonal functional groups at either terminal made them useful for cross-linking of folded proteins, as exemplified using N-terminal amine and C-terminal thiol (Cys). To ensure robust conjugation chemistry, we transformed the N-terminal amine into an azide for the triazol/click reaction and used thiol alkylation chemistry at the C-terminus, via transformation of Cys mixed disulfides from yeast expression into a homogeneous MPAA disulfide. The azide/thiol-functionalized linker was used for making conjugates of insulin with peptide/ protein-based enzyme inhibitors. It was shown that the conjugates were better protected toward enzymatic breakdown than simple mixtures of insulin and inhibitors, and that the conjugates bind to the insulin receptor.
It is possible to make functionalized linkers with amine on one end and a single carboxylate on the other end, so it should be possible to perform simple amide-based conjugations, although amide couplings are sometimes harder to achieve at high dilutions that are often necessary when handling big molecules, due to competing hydrolysis of active esters.

■ EXPERIMENTAL PROCEDURES
Solid-phase peptide synthesis (SPPS) was performed on a SymphonyX synthesizer from Gyros Protein Technologies (Tucson, AZ 85714) on a 450 μmol scale using a 5-fold excess of Fmoc-amino acids (300 mM in dimethylformamide (DMF) with 300 mM Oxyma Pure) relative to resin loading (typical loading of the hydrazine resin was 0.6 mmol/g). Fmocdeprotection was performed using 20% piperidine in DMF with 0.1 M Oxyma Pure. Coupling was performed using amino acid/Oxyma Pure/DIC/collidine (3:3:3:4) in DMF. DMF and dichloromethane (DCM) washes (7 mL, 0.5 min, 2 × 2 each) were performed between deprotection and coupling steps. Coupling times were generally 120 min. After synthesis, the resin was washed with dichloromethane, and the peptide was cleaved from the resin by a 3 h treatment with trifluoroacetic acid/triisopropylsilane/dithiothreitol/water (92.5:2.5:2.55:2.5) followed by precipitation with diethyl ether. The peptide was dissolved in 0−20% acetonitrile in water and purified by standard RP-HPLC on a C18 column using acetonitrile/ water/trifluoroacetic acid (TFA).
Expression of (GQAP)49-GQEP-Cys(mixed disulfide) 1. An insulin precursor extended from A21 with GGGGGK-(GQAP)49-GQEP-Cys was expressed in yeast (Saccharomyces cerevisiae), 15 142 mg/L, and cleaved with ALP to release the free linker (GQAP)49-GQEP-Cys(mixed disulfide) 1, which was isolated by ion-exchange chromatography as previously described. 31 As expected from yeast-based expression, Cys in the linker was found to be mixed disulfides with glutathione, free Cys amino acid, and a homodimer.