C-terminal site-specific PEGylation of a truncated thrombomodulin mutant with retention of full bioactivity

Addition of poly(ethylene glycol) to bioactive proteins (PEGylation) improves their plasma half-life, enhances stability against proteolytic cleavage, and may also decrease protein immunogenicity. Characteristically, PEGylation usually involves a reaction to available lysine amino groups, some of which may be within or near a bioactive site. Thus, most protocols are nonspecific and result in a loss of protein activity. We report herein a strategy for site-specific PEGylation of a thrombomodulin (TM) derivative at the C terminus. A truncated TM mutant consisting of epidermal growth factor (EGF)-like domains 4-6 was expressed in Escherichia coli with a C-terminal azido-methionine. The TM mutant was site-specifically conjugated to a methyl-PEG-triarylphosphine compound via the Staudinger reaction. Enzymatic activity of the TM construct before and after PEGylation was unchanged, which confirms the utility of this site-specific PEGylation scheme.     

重组溶葡萄球菌酶的PEG定点修饰

为获得高抗菌活性聚乙二醇(PEG)定点修饰的溶葡萄球菌酶(Lysn),根据该酶的高级结构,在它的催化域和结合域上优选8个位点(Q9、N13、N40、T172、N174、G197、V240和T244),将其分别突变成半胱氨酸,纯化后的溶葡萄球菌酶突变体经DTT处理后与20 kDa的单甲氧基聚乙二醇马来酰亚胺(m PEG-MAL)进行定点修饰反应,RP-HPLC分析显示PEG修饰率大于70%,修饰产物经MacroCap SP阳离子交换层析纯化后,PEG化溶葡萄球菌酶的纯度大于95%。比浊法和最小抑菌浓度(MIC)实验表明20K-PEG-Lysn V240C和20K-PEG-Lysn T244C的抗菌活性维持在原来的50%左右。研究结果为溶葡萄球菌酶全身给药治疗金黄色葡萄球菌感染奠定基础。     

Site-specific PEGylation at histidine tags

The efficacy of protein-based medicines can be compromised by their rapid clearance from the blood circulatory system. Achieving optimal pharmacokinetics is a key requirement for the successful development of safe protein-based medicines. Protein PEGylation is a clinically proven strategy to increase the circulation half-life of protein-based medicines. One limitation of PEGylation is that there are few strategies that achieve site-specific conjugation of PEG to the protein. Here, we describe the covalent conjugation of PEG site-specifically to a polyhistidine tag (His-tag) on a protein. His-tag site-specific PEGylation was achieved with a domain antibody (dAb) that had a 6-histidine His-tag on the C-terminus (dAb-His(6)) and interferon α-2a (IFN) that had an 8-histidine His-tag on the N-terminus (His(8)-IFN). The site of PEGylation at the His-tag for both dAb-His(6)-PEG and PEG-His(8)-IFN was confirmed by digestion, chromatographic, and mass-spectral studies. A methionine was also inserted directly after the N-terminal His-tag in IFN to give His(8)Met-IFN. Cyanogen bromide digestion studies of PEG-His(8)Met-IFN were also consistent with PEGylation at the His-tag. By using increased stoichiometries of the PEGylation reagent, it was possible to conjugate two separate PEG molecules to the His-tag of both the dAb and IFN proteins. Stability studies followed by in vitro evaluation confirmed that these PEGylated proteins retained their biological activity. In vivo PK studies showed that all of the His-tag PEGylated samples displayed extended circulation half-lives. Together, our results indicate that site-specific, covalent PEG conjugation at a His-tag can be achieved and biological activity maintained with therapeutically relevant proteins.     

Creation of a lysine-deficient LIGHT mutant with the capacity for site-specific PEGylation and low affinity for a decoy receptor

The cytokine LIGHT is a promising candidate for cancer therapy. However, the therapeutic effect of LIGHT as a systemic anticancer agent is currently insufficient because of its instability and its binding to nonfunctional soluble decoy receptor 3 (DcR3), which is overexpressed in various tumors. Modification of proteins with polyethylene glycol (PEGylation) can improve their in vivo stability, but PEGylation may occur randomly at all lysine residues and the NH₂-terminus; therefore, PEGylated proteins are generally heterogeneous and have decreased bioactivity. In this study, we attempted to create a lysine-deficient LIGHT mutant that could be PEGylated site-specifically and would have lower affinity for DcR3. We prepared phage libraries expressing LIGHT mutants in which all the lysine residues were replaced with other amino acids. A lysine-deficient LIGHT mutant [mLIGHT-Lys(−)] was isolated by panning against lymphotoxin β receptor (LTβR). mLIGHT-Lys(−) could be site-specifically PEGylated at its NH₂-terminus, yielding molecular uniformity and in vitro bioactivity equal to that of non-PEGylated, wild-type LIGHT. Furthermore, mLIGHT-Lys(−) was not trapped by the nonfunctional DcR3, despite binding to its functional receptors. These results suggest that mLIGHT-Lys(−) might be a useful candidate for cancer therapy.     

Site-specific PEGylation of protein disulfide bonds using a three-carbon bridge

The covalent conjugation of a functionalized poly(ethylene glycol) (PEG) to multiple nucleophilic amine residues results in a heterogeneous mixture of PEG positional isomers. Their physicochemical, biological, and pharmaceutical properties vary with the site of conjugation of PEG. Yields are low because of inefficient conjugation chemistry and production costs high because of complex purification procedures. Our solution to these fundamental problems in PEGylating proteins has been to exploit the latent conjugation selectivity of the two sulfur atoms that are derived from the ubiquitous disulfide bonds of proteins. This approach to PEGylation involves two steps: (1) disulfide reduction to release the two cysteine thiols and (2) re-forming the disulfide by bis-alkylation via a three-carbon bridge to which PEG was covalently attached. During this process, irreversible denaturation of the protein did not occur. Mechanistically, the conjugation is conducted by a sequential, interactive bis-alkylation using alpha,beta-unsaturated beta'-monosulfone functionalized PEG reagents. The combination of (a) maintaining the protein's tertiary structure after disulfide reduction, (b) the mechanism for bis-thiol selectivity of the PEG reagent, and (c) the steric shielding of PEG ensure that only one PEG molecule is conjugated at each disulfide bond. PEG was site-specifically conjugated via a three-carbon bridge to 2 equiv of the tripeptide glutathione, the cyclic peptide hormone somatostatin, the tetrameric protein l-asparaginase, and to the disulfides in interferon alpha-2b (IFN). SDS-PAGE, mass spectral, and NMR analyses were used to confirm conjugation, thiol selectivity, and connectivity. The biological activity of the l-asparaginase did not change after the attachment of four PEG molecules. In the case of IFN, a small reduction in biological activity was seen with the single-bridged IFN (without PEG attached). A significantly larger reduction in biological activity was seen with the three-carbon disulfide single-bridged PEG-IFNs and with the double-bridged IFN (without PEG attached). The reduction of the PEG-IFN's in vitro biological activity was a consequence of the steric shielding caused by PEG, and it was comparable to that seen with all other forms of PEG-IFNs reported. However, when a three-carbon bridge was used to attach PEG, our PEG-IFN's biological activity was found to be independent of the length of the PEG. This property has not previously been described for PEG-IFNs. Our studies therefore suggest that peptides, proteins, enzymes, and antibody fragments can be site-specifically PEGylated across a native disulfide bond using three-carbon bridges without destroying their tertiary structure or abolishing their biological activity. The stoichiometric efficiency of this approach also enables recycling of any unreacted protein. It therefore offers the potential to make PEGylated biopharmaceuticals as cost-effective medicines for global use.     

Site-specific PEGylation of proteins containing unnatural amino acids

Here, we report a generally applicable PEGylation methodology based on the site-specific incorporation of para-azidophenylalanine into proteins in yeast. The azido group was used in a mild [3+2] cycloaddition reaction with an alkyne derivatized PEG reagent to afford selectively PEGylated protein. This strategy should be useful for the generation of selectively PEGylated proteins for therapeutic applications.     

Site-specific PEGylation of bone morphogenetic protein-2 cysteine analogues

Three cysteine analogues of bone morphogenetic protein (BMP)-2, BMP2A2C, BMP2N56C, and BMP2E96C, were generated in order to enable the attachment of SH-reactive poly(ethylene glycol) (PEG) at specific sites. Three different approaches (Ap) were used for SH-specific PEGylation: (Ap1) reaction of glutathione activated proteins with thiol PEG; (Ap2) reaction of DTT reduced proteins with orthopyridyl disulfide PEG; (Ap3) reaction of DTT reduced proteins with maleimide PEG. Non-, mono-, and di-PEGylated BMP-2 analogues could be separated by RP-HPLC. Trypsin digestion of PEGylated proteins and Trypsin and GluC double-digestion of N-ethylmaleimide-labeled proteins confirmed that the modifications were site-specific. Surface plasmon resonance analysis of type I and type II receptor binding of the PEGylated BMP-2 analogues revealed that all three PEGylation approaches were equivalent. PEGylation at positions 2 and 96 caused a similar decrease in receptor affinity. PEGylation at position 56 resulted in a larger decrease in affinity for both types of receptors. Mono-PEGylated BMP-2 analogues exhibited intermediate affinities in comparison with unmodified and di-PEGylated proteins. However, the biological activity of the PEGylated BMP-2 analogues as measured in alkaline phosphatase assay was higher than BMP-2 wild-type for the PEGylated BMP2A2C, slightly reduced for the BMP2N56C, and strongly reduced for the BMP2E96C. These results taken together indicate that specific attachment of PEG at engineered sites of BMP-2 is possible and that the attachment site is critical for biological activity. Furthermore, the biological activity of PEGylated BMP-2 analogues in cell culture seems to be determined not only by receptor affinity, but also by other factors such as protein solubility and stability. It is also discussed that the attached PEG interferes with the binding of BMP-2 to modulator proteins, co-receptors, or heparinic sites of proteoglycans in the extracellular matrix.     

Site-specific PEGylation of native disulfide bonds in therapeutic proteins

Native disulfide bonds in therapeutic proteins are crucial for tertiary structure and biological activity and are therefore considered unsuitable for chemical modification. We show that native disulfides in human interferon alpha-2b and in a fragment of an antibody to CD4(+) can be modified by site-specific bisalkylation of the two cysteine sulfur atoms to form a three-carbon PEGylated bridge. The yield of PEGylated protein is high, and tertiary structure and biological activity are retained.     

Towards minimized gonadotropins with full bioactivity.

Gonadotropins are highly complex glycoprotein hormones consisting of two noncovalently associated subunits, which are heavily glycosylated. Using the X-ray structure of human choriogonadotropin and structure/activity relationships we aimed to design 'minimized' gonadotropins of reduced complexity. Our results show that it is possible to reduce the size of natural human choriogonadotropin by one-third of its molecular weight while retaining its wild-type biopotency. To our knowledge, such 'mini'-human choriogonadotropins represent the smallest gonadotropins described so far with an lutropin/choriogonadotropin receptor affinity and in vitro biological activity comparable with that of natural human choriogonadotropin. It provides an important step towards the structure/function-based design of small molecule drugs to the human gonadotropin receptors.     

蛋白药物的聚乙二醇定点修饰策略与最佳位点

聚乙二醇修饰是一种改善蛋白质药物临床药效行之有效的方法。聚乙二醇修饰具有延长蛋白质药物在体内的半衰期、降低免疫原性和延缓蛋白酶降解、提高稳定性和溶解性等优点。而聚乙二醇的定点修饰由于能够获得均一性和高活性保留率的产物,并能提高产率,已经引起了广泛关注。综述了近年来聚乙二醇定点修饰蛋白质药物方面的研究进展,着重介绍了聚乙二醇定点修饰的策略及最佳修饰位点,并对聚乙二醇定点修饰技术的发展趋势进行了展望。     

人白细胞介素11的定点聚乙二醇修饰

利用人白细胞介素11（hIL-11）无半胱氨酸（Cys）残基这一特点,通过定点突变将一个Cys残基引入hIL-11的N末端。然后,利用与Cys 巯基特异性反应的mPEG-马来酰亚胺将mPEG偶联到预先选定的位点,经层析纯化得到hIL-11的定点PEG修饰物。利用依赖型细胞株7TD1测定其生物学活性,结果表明,其体外生物学活性保持原有hIL-11活性的30%左右。定点聚乙二醇修饰方法为定向改造hIL-11,提高其药效的应用研究打下基础。     

Optimal site-specific PEGylation of mutant TNF-alpha improves its antitumor potency

Recently, we created a lysine-deficient mutant tumor necrosis factor-alpha [mTNF-alpha-Lys(-)] with full bioactivity in vitro compared with wild-type TNF-alpha (wTNF-alpha), and site-specific PEGylation of mTNF-alpha-Lys(-) was found to selectively enhance its in vivo antitumor activity. In this study, we attempted to optimize this PEGylation of mTNF-alpha-Lys(-) to further improve its therapeutic potency. mTNF-alpha-Lys(-) was site-specifically modified at its N-terminus with linear polyethylene glycol (LPEG) or branched PEG (BPEG). While randomly mono-PEGylated wTNF-alpha (ran-LPEG5K-wTNF-alpha) with 5 kDa of LPEG (LPEG5K) had about only 4% in vitro bioactivity of wTNF-alpha, mono-PEGylated mTNF-alpha-Lys(-) [sp-PEG-mTNF-alpha-Lys(-)] with LPEG5K, LPEG20K, BPEG10K, and BPEG40K had 82%, 58%, 93%, and 65% bioactivities of mTNF-alpha-Lys(-), respectively. sp-LPEG-mTNF-alpha-Lys(-) and sp-BPEG10K-mTNF-alpha-Lys(-) had much superior antitumor activity to those of both unmodified TNF-alphas and ran-LPEG5K-wTNF-alpha, though sp-BPEG40K-mTNF-alpha-Lys(-) did not show in vivo antitumor activity. Thus, the molecular shape and weight of PEG may strongly influence the in vivo antitumor activity of sp-PEG-mTNF-alpha-Lys(-).     

Site-specific PEGylation of engineered cysteine analogues of recombinant human granulocyte-macrophage colony-stimulating factor

Granulocyte macrophage colony-stimulating factor (GM-CSF) stimulates proliferation of hematopoietic cells of the macrophage and granulocyte lineages and is used clinically to treat neutropenia and other myeloid disorders. Because of its short circulating half-life, GM-CSF is administered to patients by daily injection. We describe here the engineering of highly potent, long-acting human GM-CSF proteins through site-specific modification of GM-CSF cysteine analogues with a cysteine-reactive poly(ethylene glycol) (PEG) reagent. Thirteen cysteine analogues of GM-CSF were constructed, primarily in nonhelical regions of the protein believed to lie away from the major receptor binding sites. The GM-CSF cysteine analogues were properly processed but insoluble following secretion into the Escherichia coli periplasm. The proteins were refolded and purified by column chromatography. Ten of the cysteine analogues could be modified with a 5-kDa maleimide PEG, and seven of the mono-PEGylated proteins were purified by ion-exchange column chromatography. Biological activities of the 13 cysteine analogues and 7 PEGylated cysteine analogues were comparable to that of wild-type GM-CSF in an in vitro cell proliferation assay using human TF-1 cells. One cysteine analogue was modified with larger 10-, 20-, and 40-kDa PEGs, with only minimal loss of in vitro bioactivity. Pharmacokinetic experiments in rats demonstrated that the PEGylated proteins had up to 47-fold longer circulating half-lives than wild-type GM-CSF. These data demonstrate the utility of site-specific PEGylation for creating highly potent, long-acting GM-CSF analogues and provide further evidence that the nonhelical regions of human GM-CSF examined are largely nonessential for biological activity of the protein.     

Effect of oriented or random PEGylation on bioactivity of a factor VIII inhibitor blocking peptide

Peptides as therapeutic substances are efficient agents in the treatment of several diseases. However, they often have to be chemically modified in order to be suited as therapeutic agent. Conjugation to large carrier molecules is often required. A critical step is the identification of available sites for chemical reaction, without influencing bioactivity. Peptide 238/S1 with the sequence NH(2)-PYWKWQYKYD-COOH previously selected from a combinatorial decapeptide library, has the ability to block inhibitory antibodies against blood clotting factor VIII (FVIII) and therefore, it constitutes a lead for developing a drug to treat patients suffering from development of such antibodies. The aims of this study were (i) to identify sites of the peptide, which are suited for modification without losing bioactivity and (ii) to find out the influence of molecular size of polyethylene glycol (PEG) for bioactivity of the peptide. The contribution of each amino acid residue to biological functionality was investigated by mutational analysis. This method confirmed that the N-terminus is crucial for activity, whereas both lysine residues could be exchanged by other L-amino acids. Using mutational analysis it was possible to identify peptides with higher reactivity compared to the wild type 238/S1. PEGylation experiments demonstrated that conjugation of the peptide to PEG 20,000 resulted in a loss of reactivity, while PEG 5,000 could maintain the bioactivity when conjugated in a site directed manner. The peptide lost its neutralization properties when PEG was coupled to the N-terminus, again indicating that this part of the peptide is important for functionality.     

集成干扰素突变体IIFN72C的构建及其聚乙二醇定点修饰

目的：设计构建集成干扰素突变体IIFN72C并进行聚乙二醇定点修饰,以获得高活性的长效干扰素分子。方法：利用蛋白质分子同源模建,选择在集成干扰素分子IIFN的第72位引入半胱氨酸残基构成集成干扰素突变体IIFN72C。诱导表达后经包涵体变复性和层析纯化,与单甲氧基聚乙二醇（mPEGMAL）定点偶联。修饰产物经纯化后,以SDSPAGE考察其纯度,用WISHVSV系统进行生物活性测定。结果：IIFN72C以包涵体形式表达,表达量占菌体总蛋白的30%以上,比活性与突变前相当;修饰产物大多数为单修饰体,纯化后纯度大于98%,比活性保留约为修饰前的8%。结论：成功设计并表达IIFN72C用于PEG定点修饰,修饰产物活性保留得以提高。     

Studies on the Site-specific PEGylation Induced Interferences Instigated in Uricase Quantification Using the Bradford Method

Uricase from Bacillus fastidiosus was site-specifically PEGylated using methoxypolyethyleneglycol-maleimide (mPEG-mal) of different molecular weights (750 Da, 5 kDa, 10 kDa) via Thiol PEGylation strategy. The obtained monoPEGylated uricase conjugates were characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the molecular weight of single subunit of the conjugate was found to be 42.6, 48.1 and 56.3 kDa with respect to different molecular weights of m-PEG-mal. The influence of PEGylation on the quantification of uricase using protein quantification techniques like Bradford assay, RP-HPLC detection and Dumbroff method has been evaluated. A gradual decline in the absorbance value was observed with the advancement of the PEGylation reaction, indicating an interferences in the protein quantification due to PEGylation. The extent of interference highly dependence on mPEG-mal concentration and its chain length. The present study indicates that the quantification of PEGylation induced interferences caused in protein measured ought to be prudently measured at every discrete step of the PEGylation process.     

Overexpression, effective renaturation, and bioactivity of novel single-chain antibodies against TNF-alpha

Neutralization of tumor necrosis factor-alpha (TNF-alpha) has become an effective therapeutic strategy for TNF-related autoimmune diseases. Due to the limitations of the large molecular inhibitors in the therapy, development of novel TNF-alpha inhibitors is very attractive and useful. In this study, based on the previously designed domain antibody, two novel human anti-TNF single-chain antibodies were constructed using modular consensus frameworks of human antibody as scaffold to display the antagonistic peptides. A variety of expression plasmids were used to determine the optimal expression system. The single-chain antibodies were always overexpressed in E.coli BL21(DE3) host as inclusion bodies. Under the optimized refolding conditions, the inclusion bodies were renatured successfully and the refolded single-chain antibodies could bind with TNF-alpha and block TNF-induced cytotoxicity on L929 cells. The bioactivity of the single-chain antibodies was significantly increased over the domain antibody.     

Solid-phase PEGylation of recombinant interferon alpha-2a for site-specific modification: process performance, characterization, and in vitro bioactivity

'Solid-phase' PEGylation, in which a conjugation reaction attaches proteins to a solid matrix, has distinct advantages over the conventional, solution-phase process. We report a case study in which recombinant interferon (rhIFN) alpha-2a was adsorbed to a cation-exchange resin and PEGylated at the N-terminus by 5, 10, and 20 kDa mPEG aldehydes through reductive alkylation. After PEGylation, a salt gradient elution efficiently purified the mono-PEGylate of unwanted species such as unmodified IFN and unreacted PEG. Mono-PEGylation and purification were integrated into a single, chromatographic step. Depending on the molecular weight of the mPEG aldehyde, the mono-PEGylation yield ranged from 50 to 65%. Major problems associated with the solution-phase process such as random or uncontrollable multi-PEGylation and post-PEGylation purification difficulties were overcome. N-terminus sequencing and MALDI-TOF mass spectrophometry confirmed that the PEG molecule was conjugated only to the N-terminus. A cell proliferation study indicated reduced antiviral activity of the mono-PEGylate compared to that of the unmodified IFN. As higher molecular weight PEG was conjugated, in vitro bioactivity and antibody binding activity, as measured by a surface plasmon resonance biosensor, decreased. Nevertheless, trypsin resistance and thermal stability were considerably improved .