Pegylated endostatin analogs and their applications

Abstract

The present invention provides polyethylene glycol-modified endostatin analogs and applications thereof, wherein the endostatin analogs are coupled with polyethylene glycol at lysine far from the nucleolin binding region or at lysine far from the nucleolin binding region and the amino group at the N-terminus, thereby improving the biological activity of inhibiting tumor cell migration. The polyethylene glycol-modified endostatin analogs of the present invention have higher stability and longer in vivo half-life than unmodified endostatin, and their activity of inhibiting neovascularization is significantly improved.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application number 201780069252.1, application date November 10, 2017, and invention name “PEGylated endostatin analogs and their applications”. The full text of the Chinese invention patent application is hereby incorporated herein by reference. Technical Field

[0002] The present invention relates to a novel recombinant protein drug, and in particular to a pegylated endostatin analog and its application. Background Art

[0003] Neovascularization refers to the formation of new capillaries on existing blood vessels. Tumor growth and migration depend on the formation of neovascularization. Using microvascular endothelial cells in tumors as targets for cancer treatment provides a therapeutic approach for treating tumors (Folkman JN Engl J Med 1971; 285: 1182-1186).

[0004] Endostatin is a 20kDa enzymatic cleavage product of the carboxyl end of collagen XVIII. In 1997, Professor Judah Folkman and others from Harvard University in the United States discovered this protein in the culture medium of hemangioendothelioma cells. It has the activity of inhibiting the proliferation and migration of vascular endothelial cells and angiogenesis in vivo. Further experiments have found that recombinant endostatin can inhibit the growth and migration of various tumors in mice, and can even completely cure tumors without drug resistance. The mechanism of its activity is that it inhibits the growth of vascular endothelial cells and the formation of new blood vessels near the tumor tissue, so that the tumor tissue cannot obtain the large amount of nutrients and oxygen necessary for growth, and finally stops growing or necrotizes (Folkman J. et al. Cell 1997; 88: 277-285; Folkman J. et al. Nature 1997; 390: 404-407). In 2010, Professor Luo Yongzhang and others from Tsinghua University found that endostatin can also significantly inhibit tumor-related lymphangiogenesis (Yongzhang Luo.et al.Journal of Pathology 2010; 222: 249-260; YongzhangLuo.Front.Med.2011,5:336-340.). Recombinant human endostatin prepared by genetic engineering can be used as a tumor treatment drug, and its clinical trials have shown that it can effectively inhibit tumor growth. In China, clinical trials with non-small cell lung cancer as the main indication have shown that it has a relatively outstanding therapeutic effect on tumors.

[0005] For many years, tumor treatment has mainly relied on small molecule compound therapy (chemotherapy) and radiotherapy (radiotherapy). Although both methods are very effective, they also have some toxic and side effects. In contrast, protein drugs have fewer toxic and side effects and do not produce drug resistance, but the use of gastrointestinal administration will produce a serious first pass effect. In order to achieve the highest in vivo activity, improve bioavailability, and reduce drug degradation in the body, protein drugs are usually administered by intravenous or subcutaneous injection. For proteins with small molecular weight, the half-life of the drug in the body after intravenous administration is usually very short. One of the important reasons is that small molecule proteins can be quickly eliminated through renal filtration. Proteins with a hydraulic radius greater than albumin or a molecular weight greater than 66,000 Daltons (66kDa) in the blood can usually be stably retained in the circulatory system, while small molecule protein drugs are quickly eliminated through glomerular filtration. Therefore, in order to maintain an effective therapeutic concentration in the blood, patients need to receive frequent injections or drips. Although this treatment method can achieve the purpose of treatment, it brings serious inconvenience and pain to patients, seriously affects patients' compliance with drug treatment, and increases drug costs. Moreover, long-term and frequent use of certain drugs may also cause some side effects, such as immune reactions.

[0006] Endostatin as a protein drug also has the disadvantages of short half-life and high in vivo clearance rate. At present, the main clinical medication method is frequent administration to maintain effective blood drug concentration, usually daily administration, and long-term medication is required, which makes the patient's mental and economic burden very heavy. The purpose of the present invention is to improve the in vivo metabolic characteristics of the protein, so that it has higher stability and longer in vivo half-life, and even has higher efficacy, thereby achieving the purpose of reducing the frequency of medication, reducing the cost of medication, and alleviating the patient's economic burden.

[0007] Modifying proteins with polymers is a common method for changing and controlling the kinetic properties of drugs, such as half-life, immunological characteristics, and toxicological properties. Among them, polyethylene glycol is the most widely used amphiphilic polymer, which has the characteristics of good solubility, good biocompatibility, non-toxicity and non-immunogenicity. It is a polymer that can be used for drug preparation approved by drug regulatory agencies in many countries and regions, including the US FDA and China SFDA. Coupling proteins with hydrophilic polymers such as polyethylene glycol can reduce proteolysis, increase protein stability, reduce nonspecific adsorption and antigenicity. When the hydrodynamic radius of the conjugate reaches the lower limit of glomerular filtration, the renal clearance efficiency can be greatly reduced. It is an effective method to extend the half-life of protein drugs in vivo (Gianfranco Pasut et.al. Advanced Drug Delivery Reviews 60 (2008) 69-78; FM Veronese et al. Milestones in Drug Therapy 2009, 11-31). With the development of polyethylene glycol modification technology, various groups on protein molecules can be modified by PEG to meet different needs. For example, monomethoxy polyethylene glycol succinimide active ester modifiers (such as mPEG-SC, mPEG-SCM, mPEG-SPA, mPEG-NHS, etc.) are used to randomly modify the N-terminal α-amino group of proteins or polypeptides or the ε-amino group of lysine side chains to achieve multi-site modification of proteins. Monomethoxy polyethylene glycol aldehyde (mPEG-Aldehyde) can specifically react with the N-terminal α-amino group of proteins or polypeptides under weak acidic conditions to form an unstable Schiff base, which is further reduced by sodium cyanoborohydride to generate a stable secondary amine to achieve site-specific modification of the N-terminus. Monomethoxy polyethylene glycol maleimide modifiers (such as mPEG-MAL, mPEG2-MAL, etc.) are used to selectively modify free cysteine ​​in proteins or polypeptides. To date, polyethylene glycol modification technology has been successfully applied to a variety of proteins. The polyethylene glycol-modified protein drugs that have been approved by the FDA include PEG-adenosine deiminase (1990), PEG-asparaginase (1994), PEG-α2b interferon (2001), PEG-granulocyte colony-stimulating factor (2002), PEG-α2a interferon (2002), PEG-α2a interferon and ribavirin mixed preparation (2004), PEG-erythropoietin (2007), PEG-TNFα antibody (2008), PEG-α2b interferon and ribavirin mixed preparation (2008), and PEG-urate oxidase (2010).Representative polyethylene glycol-modified protein drugs that are undergoing clinical research include PEG-arginine deiminase (Polaris Group, Phase III), PEG-growth hormone (Changchun Jinsai Pharmaceutical, Phase III), PEG-β1a interferon (Biogen Idec, Phase III), etc.

[0008] In addition to polyethylene glycol-modified proteins to extend drug half-life, other polymer modifiers can be used, such as dextran, polysucrose, starch, polyalanine, copolymers of oxalic acid and malonic acid, carboxymethyl cellulose, polyvinyl pyrrolidone, poly 1,3-ethylene glycol methyl ether, copolymers of ethylene and maleic hydrazide, polysialic acid, and cyclodextrin. Summary of the invention

[0009] The inventors have found that coupling polyethylene glycol to a lysine far from the nucleolin binding region on endostatin can significantly improve protein activity. Therefore, the present invention provides a polyethylene glycol-modified endostatin analog, wherein the endostatin analog is coupled to polyethylene glycol at a lysine far from the nucleolin binding region, thereby improving its biological activity of inhibiting tumor cell migration. The polyethylene glycol-modified endostatin analog of the present invention has higher stability and longer in vivo half-life than unmodified endostatin, and its activity of inhibiting neovascularization is significantly improved.

[0010] The first aspect of the present invention provides a complex of an endostatin analog and polyethylene glycol; wherein the endostatin analog has a lysine residue at the position corresponding to the 96th position of the natural endostatin amino acid sequence SEQ ID NO.1, and does not have a lysine residue at any other position; and the endostatin analog is coupled to polyethylene glycol only at the lysine residue.

[0011] The second aspect of the present invention provides a complex of an endostatin analog and polyethylene glycol; wherein the endostatin analog has a lysine residue at the position corresponding to the 96th position of the natural endostatin amino acid sequence SEQ ID NO.1, and does not have a lysine residue at any other position; and the endostatin analog is coupled to polyethylene glycol at the N-terminus and the lysine residue.

[0012] In a preferred embodiment, in the complex of the first and second aspects, the endostatin analog is formed by mutation of the lysine residues at positions 76, 107, 118 and 184 of the natural endostatin amino acid sequence SEQ ID NO.1. In a specific embodiment, the sequence of the endostatin analog is shown in SEQ ID NO:3.

[0013] The third aspect of the present invention provides a complex of an endostatin analog and polyethylene glycol; wherein the endostatin analog has a lysine residue at the position corresponding to the 96th position of the natural endostatin amino acid sequence SEQ ID NO.1, and does not have a lysine residue at any other position; and the endostatin analog has an inserted amino acid sequence at its N-terminus; and the endostatin analog is coupled to polyethylene glycol only at the lysine residue.

[0014] The fourth aspect of the present invention provides a complex of an endostatin analog and polyethylene glycol; wherein the endostatin analog has a lysine residue at the position corresponding to the 96th position of the natural endostatin amino acid sequence SEQ ID NO.1, and does not have a lysine residue at any other position, and the endostatin analog has an inserted amino acid sequence at its N-terminus; and the endostatin analog is coupled to polyethylene glycol at the N-terminus and the lysine residue.

[0015] In a preferred embodiment, in the complex of the third aspect and the fourth aspect, the endostatin analog is formed by mutation of the lysine residues at positions 76, 107, 118 and 184 of the natural endostatin amino acid sequence SEQ ID NO.1, and insertion of an amino acid sequence at its N-terminus. In a more preferred embodiment, the inserted amino acid sequence is GGSHHHHH inserted between the methionine M and the histidine H at the N-terminus. In a more preferred embodiment, the sequence of the endostatin analog is shown in SEQ ID NO:8.

[0016] In a preferred embodiment, in the endostatin analog in the coupled complex of the present invention, the lysine residues at positions 76, 107, 118 and 184 of the natural endostatin amino acid sequence SEQ ID NO.1 are mutated to X1, X3, X4, X5, respectively, wherein X1, X3, X4 or X5 is independently any natural amino acid except lysine. X1, X3, X4 or X5 is independently preferably a water-soluble amino acid, further preferably any one of the charged amino acids arginine, histidine, glutamic acid and aspartic acid, further preferably any one of the positively charged amino acids arginine and histidine, and most preferably arginine.

[0017] In a preferred embodiment, in the complex of the present invention, the amino acid sequence inserted at the N-terminus of the endostatin analog is MGGSHHHHH.

[0018] In a preferred embodiment, in the complex of the present invention, the endostatin analog is covalently coupled to polyethylene glycol.

[0019] In a preferred embodiment, the polyethylene glycol has an average molecular weight of 5,000 to 40,000 Daltons.

[0020] In a more preferred embodiment, the polyethylene glycol has an average molecular weight of 20,000 to 40,000 Daltons.

[0021] In a preferred embodiment, the polyethylene glycol is monomethoxy polyethylene glycol, monoglucose polyethylene glycol or monogalactose polyethylene glycol.

[0022] In a preferred embodiment, the polyethylene glycol is linear or branched.

[0023] In a preferred embodiment, the polyethylene glycol is monomethoxy polyethylene glycol.

[0024] In some embodiments, polyethylene glycol is coupled to the amino group of the endostatin analog.

[0025] In some embodiments, polyethylene glycol is coupled to the endostatin analog lysine side chain epsilon-amino group.

[0026] In some embodiments, polyethylene glycol is coupled to the N-terminal α-amino group and the ε-amino group of a lysine residue of the endostatin analog.

[0027] In a more preferred embodiment, the coupling of polyethylene glycol to the amino group of the endostatin analog can be carried out using monomethoxypolyethylene glycol propionaldehyde (mPEG-ALD), monomethoxypolyethylene glycol butyraldehyde (mPEG-ButyrALD), monomethoxypolyethylene glycol succinimidyl carbonate (mPEG-SC), monomethoxypolyethylene glycol succinimidyl acetate (mPEG-SCM), monomethoxypolyethylene glycol succinimidyl propionate (mPEG-SPA), monomethoxypolyethylene glycol succinimidyl butyrate (mPEG-SBA), monomethoxypolyethylene glycol succinimidyl α-methylbutyrate (mPEG-SMA), and monomethoxypolyethylene glycol NHS ester (mPEG-NHS) for coupling reaction.

[0028] The present invention also provides a pharmaceutical composition, which comprises the above-mentioned complex of the endostatin analog of the present invention and polyethylene glycol and a pharmaceutically acceptable carrier.

[0029] The present invention also provides a method for treating diseases caused by neovascularization or neolymphaticization, comprising administering the above-mentioned complex or pharmaceutical composition of the endostatin analog and polyethylene glycol to a patient.

[0030] The present invention also provides use of the endostatin analog and polyethylene glycol coupling complex or pharmaceutical composition of the present invention in treating diseases caused by neovascularization or neolymphatic formation.

[0031] In a preferred embodiment, the disease caused by neovascularization or neolymphaticization is a tumor.

[0032] The present invention also provides use of the coupling complex of the present invention in preparing a drug for inhibiting the formation of new blood vessels or new lymphatic vessels in vivo or in vitro.

[0033] When the complex of the present invention is used to inhibit the formation of new blood vessels or new lymphatic vessels in vivo, its half-life is significantly prolonged compared with that of endostatin, and its inhibition rate is significantly increased compared with that of endostatin under the same administration conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1: SDS-PAGE electrophoresis analysis results of the modified samples of 20kDa mPEG-AL N-terminal single-modified endostatin and the subsequent chromatography purified samples. Lane 1 is a low molecular weight standard, from top to bottom, 116, 66, 45, 30, 25, 18.4, 14.4 kDa; Lane 2 is a sample of endostatin modified with 20kDa mPEG-ALD; Lanes 3 to 7 are fractions 1, 2, 3, 4, and 5 obtained by elution with 100, 150, 200, 350, and 500 mM NaCl during chromatography purification, respectively, of which the 200 mM NaCl elution fraction (fraction 3) in Lane 5 is the N-terminal single-modified product fraction.

[0035] Figure 2 : SDS-PAGE electrophoresis analysis results of the modified samples of 20kDa mPEG-ALD-terminal single-modified endostatin K1 analogs and subsequent chromatography-purified samples. Lane 1 is a low molecular weight standard, which is 116, 66, 45, 30, 25, 18.4, and 14.4 kDa from top to bottom; Lane 2 is a sample of endostatin K1 analogs modified with 20kD mPEG-ALD; Lanes 3 to 7 are fractions 1, 2, 3, 4, and 5 obtained by elution with 100, 150, 150, 350, and 500 mM NaCl during chromatography-purification, respectively, and the 150 mM NaCl elution fractions (fractions 2 and 3) in lanes 4 and 5 are both 20kDa polyethylene glycol N-terminal single-modified endostatin K1 analogs.

[0036] Figure 3 : Results of electrophoresis analysis of 20kDa mPEG-SPA double-modified endostatin K1 analog lysine side chain amino group and N-terminal amino group double-modified and purified samples. Lane 1 is a low molecular weight standard, from top to bottom, 116, 66, 45, 30, 25, 18.4, 14.4 kDa; Lane 2 is a sample of endostatin modified with 20kDa mPEG-SPA; Lanes 3 to 8 are 25, 50, 75, 100, 200 and 500mM NaCl elution fractions 1, 2, 3, 4, 5, 6 during chromatography purification, respectively, of which the 100mM NaCl elution fraction (fraction 4) in lane 6 is the K1 analog double-modified product fraction.

[0037] Figure 4:The SDS-PAGE electrophoresis results of the modified samples and the subsequent purified samples of the 20kDa endostatin ESC analog without PEG-MAL modified thiol groups. Lane 1 is the sample of endostatin ESC analog modified with 20kDamPEG-MAL; Lanes 2 to 5 are the 100, 150, 200 and 500mM NaCl elution fractions during chromatography purification, and Lane 6 is the low molecular weight standard, which are 116, 66, 45, 30, 25, 18.4, and 14.4kDa from top to bottom; Among them, the 200mMNaCl elution fraction (fraction 3) in Lane 4 is the ESC analog single thiol modification product fraction.

[0038] Figure 5 :The results of SDS-PAGE electrophoresis analysis of the modified samples of endostatin K1 analogs with lysine side amino groups modified by 20kDamPEG-ALD and the subsequent purified samples. Lane 1 is a low molecular weight standard, which is 116, 66, 45, 30, 25, 18.4, and 14.4 kDa from top to bottom; Lane 2 is a sample of endostatin modified with 20kDamPEG-ALD; Lanes 3 to 7 are 50, 100, 125, 200, and 500 mM NaCl elution fractions during chromatography purification, respectively, of which lanes 4 and 5 are both 20kDa polyethylene glycol-modified endostatin analogs. The modification site analysis of the two samples confirmed that the sample in lane 4 is a polyethylene glycol N-terminal single modification product and a small amount of lysine side amino-side single modification product, and the sample in lane 5 is a endostatin K1 analog lysine side amino-side single modification product.

[0039] Figure 6 : The tertiary structure diagram of human endostatin indicates the N-terminus, C-terminus, and the positions of lysine residue K1 at position 76, lysine residue K2 at position 96, lysine residue K3 at position 107, lysine residue K4 at position 118, and the region where the protein binds to the receptor Nucleolin.

[0040] Figure 7 : The tertiary structure diagram of human endostatin indicates the N-terminus, C-terminus, and the positions of the 76th lysine residue K1, the 96th lysine residue K2, the 107th lysine residue K3, the 118th lysine residue K4, and the 127th asparagine residue of the human endostatin protein.

[0041] Figure 8 : The sequence of the endostatin analogs involved in the present invention. DETAILED DESCRIPTION

[0042] The term "endostatin (ES)" used herein may refer to natural endostatin, preferably human endostatin, for example, having the sequence of SEQ ID NO: 1, but is not limited thereto, for example, it may also be natural endostatin from other mammals, such as mice, rats, pigs, dogs, rabbits, sheep, goats, cats, etc. "Endostatin" may also refer to a functional variant of endostatin, such as an engineered functional variant, which has one or more amino acid substitutions, deletions or additions compared to natural endostatin, and has substantially the same biological function, such as the activity of inhibiting endothelial cell proliferation, migration and angiogenesis in vivo. "Endostatin" may also refer to a derivative or modified product of natural endostatin or its functional variant, such as a polyethylene glycol-modified product.

[0043] The term "functional variant" as used herein includes a mutant of endostatin containing one or more (e.g., 1-5, 1-10 or 1-15, specifically, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or even more) amino acid substitutions, deletions or additions in the amino acid sequence, and the mutant has a biological activity similar to that of endostatin in inhibiting vascular endothelial cell proliferation, migration and angiogenesis in vivo. The biological activity of the "functional variant" of endostatin can be, for example, 30% or more, 50% or more, 60% or more, 70% or more, 80% or more or 90% or more of that of natural endostatin, such as natural human endostatin. The "functional variant" can be a naturally occurring mutant or an artificial mutant, such as a mutant obtained by site-directed mutagenesis, or a mutant produced by a genetic recombination method.

[0044] The biological activity of the "functional variant" can be detected by a method well known in the art for detecting the activity of endostatin. For example, HMEC cells can be selected, and the inhibition rate of the functional variant on HMEC cell migration can be analyzed by the Migration (Tranwell Assay) method, and the number of cells can be counted to reflect the protein activity (see Luo Yongzhang et al., Endostatin inhibits tumourlymphangiogenesis and lymphatic metastasis via cell surface nucleolin on lymphangiogenic endothelial cells (J Pathol 2010; 222: 249-260).

[0045] In the present invention, "endostatin analogs" and "endostatin functional variants" can be used interchangeably. In some embodiments, the "endostatin analogs" of the present invention have a lysine residue at the position corresponding to the 96th position of natural human endostatin (e.g., SEQ ID NO: 1), and do not have a lysine residue at any other position, so that polyethylene glycol is only coupled to the lysine residue of the endostatin analog or is coupled to the amino terminal and the lysine residue at the same time, but not to any other amino acid residue. The coupled complex obtained in this way has higher stability and longer in vivo half-life than natural human endostatin or the amino terminal monopolyethylene glycol coupled complex of natural human endostatin, and its angiogenesis inhibition activity is significantly improved.

[0046] The endostatin analogs of the present invention can be obtained by modifying natural endostatin (e.g., natural human endostatin or natural mammalian endostatin), specifically, mutating the lysine residues at positions 76, 107, 118, and 184 in natural human endostatin (e.g., SEQ ID NO: 1) to retain only the lysine residue at position 96 to obtain an endostatin analog, or mutating the lysine residues at positions 76, 107, 118, and 184 in natural endostatin of other mammals corresponding to those in natural human endostatin (e.g., SEQ ID NO: 1) to retain only the lysine residue at position 96 to obtain an endostatin analog. The endostatin analogs of the present invention can also be obtained by modifying known naturally occurring or artificially synthesized functional variants of endostatin. Specifically, if the endostatin functional variant has a lysine residue at the position corresponding to the 96th position of natural endostatin, the lysine residue is kept unchanged, and all other lysine residues of the endostatin functional variant are mutated; if the endostatin functional variant has a non-lysine residue at the position corresponding to the 96th position of natural endostatin, the amino acid at the position is mutated to a lysine residue, and all other lysine residues of the endostatin functional variant are mutated.

[0047] The functional variants of endostatin that can be modified to obtain the endostatin analogs of the present invention include ES variants whose first amino acid M is randomly deleted when human ES is recombinantly expressed in Escherichia coli. The functional variants of endostatin that can be modified to obtain the endostatin analogs of the present invention also include ES variants with four amino acids missing from the N-terminus due to random cleavage of the N-terminus when ES is recombinantly expressed in yeast. Functional variants of endostatin that can be modified to obtain the endostatin analogs of the present invention include YH-16, which is an ES variant obtained by adding 9 additional amino acids (MGGSHHHHH) to the N-terminus of ES to increase soluble expression and facilitate purification (Fu Y et al. IUBMB Life 2009; 61: 613-626; Wang J et al. Zhongguofei ai za zhi 2005; 8: 283-290; Han B et al. J Thorac Oncol 2011; 6 (6): 1104-1109, all of which are incorporated herein by reference). The functional variants of endostatin that can be modified to obtain the endostatin analogs of the present invention also include those endostatin mutants disclosed in PCT International Application PCT / CN2012 / 081210, such as ES006, ES008, ES011, S02, S09, Z006, Z008, ZN1, etc. (the entire contents of which are incorporated herein by reference). The functional variants of endostatin that can be modified to obtain the endostatin analogs of the present invention also include those endostatin mutants disclosed in PCT International Publication No. WO2016 / 070798, such as 003, 007, Z101, 009, S03, 36, 249, 381, 57, 114, 124, 125, 160, 163, 119 (the entire contents of which are incorporated herein by reference).

[0048] The "position corresponding to the natural endostatin amino acid sequence SEQ ID NO: 1" in the present invention refers to the position on the endostatin functional variant corresponding to the corresponding amino acid residue on SEQ ID NO: 1 after comparing the endostatin functional variant with the natural endostatin amino acid sequence SEQ ID NO: 1 using software or algorithms well known in the art. The software or algorithms include but are not limited to BLAST and FASTA.

[0049] "Polyethylene glycol (PEG)" of the present invention can be monomethoxy polyethylene glycol, monoglucose polyethylene glycol or monogalactose polyethylene glycol. The polyethylene glycol used can be linear or branched, and can have a molecular weight of, for example, about 5kD to about 50kD, about 20kD to about 40kD or, for example, about 20kD.

[0050] The term "polyethylene glycol modification" or "polyethylene glycol coupling" used herein may refer to chemically coupling a polyethylene glycol modifier molecule to a protein molecule. The group of the polyethylene glycol modifier involved in the coupling reaction is the activation group introduced during the activation process. The group of the protein is mainly a free amino group, a sulfhydryl group, etc., preferably an amino group. Coupling with polyethylene glycol (PEG) can prolong the half-life of the coupled complex in vivo, avoid protease degradation, or increase solubility. Methods for modifying proteins with polyethylene glycol are well known to those skilled in the art.

[0051] The term "polyethylene glycol modifier" includes but is not limited to monomethoxy polyethylene glycol modifier, which is an activated polyethylene glycol obtained by blocking a hydroxyl group at one end of a polyethylene glycol molecule with a methoxy group and activating a hydroxyl group at the other end with a suitable activation method. Since the reactivity of the hydroxyl group itself is very low, the reactivity of the polyethylene glycol molecule after activation is greatly improved, and it is called a "polyethylene glycol modifier". The selection of the activating group, the mechanism of activation, and the modification reaction mechanism of the polyethylene glycol modifier obtained by activation are well known in the art and have been reported in many documents. Polyethylene glycol modifiers can be obtained commercially. The polyethylene glycol modifiers that can be used include, but are not limited to, polyethylene glycol monomethoxypolyethylene glycol propionaldehyde (mPEG-ALD), monomethoxypolyethylene glycol butyraldehyde (mPEG-ButyrALD), monomethoxypolyethylene glycol succinimidyl carbonate (mPEG-SC), monomethoxypolyethylene glycol succinimidyl acetate (mPEG-SCM), monomethoxypolyethylene glycol succinimidyl propionate (mPEG-SPA), monomethoxypolyethylene glycol succinimidyl butyrate (mPEG-SBA), monomethoxypolyethylene glycol succinimidyl α-methylbutyrate (mPEG-SMA), and monomethoxypolyethylene glycol NHS ester (mPEG-NHS).

[0052] In the present invention, polyethylene glycol is coupled to the amino group of the endostatin analog, for example, to the ε-amino group of the lysine side chain of the endostatin analog, or to the α-amino group at the N-terminus of the endostatin analog and the ε-amino group of the lysine residue.

[0053] The endostatin K1 analogue described in the present invention (e.g., the embodiment) refers to an endostatin analogue with an amino acid sequence of SEQ ID NO.2, wherein the lysine residues at positions 96, 107, 118, and 184 of the endostatin amino acid sequence SEQ ID NO.1 from the N-terminus are mutated to X2, X3, X4, and X5, respectively. Wherein X2, X3, X4, and X5 are any natural amino acids other than lysine, preferably water-soluble amino acids, more preferably one of the charged amino acids arginine, histidine, glutamic acid, and aspartic acid, more preferably one of the positively charged amino acids arginine and histidine, and most preferably arginine.

[0054] The endostatin K2 analogue described in the present invention (e.g., the embodiment) refers to an endostatin analogue with an amino acid sequence of SEQ ID NO.3, wherein the lysine residues at positions 76, 107, 118, and 184 of the endostatin amino acid sequence SEQ ID NO.1 from the N-terminus are mutated to X1, X3, X4, and X5, respectively. Wherein X1, X3, X4, and X5 are any natural amino acids other than lysine, preferably water-soluble amino acids, more preferably one of the charged amino acids arginine, histidine, glutamic acid, and aspartic acid, more preferably one of the positively charged amino acids arginine and histidine, and most preferably arginine.

[0055] The endostatin K3 analogue described in the present invention (e.g., the embodiment) refers to an endostatin analogue with an amino acid sequence of SEQ ID NO.4, wherein the lysine residues at positions 76, 96, 118, and 184 of the endostatin amino acid sequence SEQ ID NO.1 from the N-terminus are mutated to X1, X2, X4, and X5, respectively. Wherein X1, X2, X4, and X5 are any natural amino acids other than lysine, preferably water-soluble amino acids, more preferably one of the charged amino acids arginine, histidine, glutamic acid, and aspartic acid, more preferably one of the positively charged amino acids arginine and histidine, and most preferably arginine.

[0056] The endostatin K4 analogue described in the present invention (e.g., the embodiment) refers to an endostatin analogue with an amino acid sequence of SEQ ID NO.5, wherein the lysine residues at positions 76, 96, 107, and 184 of the endostatin amino acid sequence SEQ ID NO.1 from the N-terminus are mutated to X1, X2, X3, and X5, respectively. Wherein X1, X2, X3, and X5 are any natural amino acids other than lysine, preferably water-soluble amino acids, more preferably one of the charged amino acids arginine, histidine, glutamic acid, and aspartic acid, more preferably one of the positively charged amino acids arginine and histidine, and most preferably arginine.

[0057] The endostatin ESC analogue described in the present invention (eg, the examples) refers to an endostatin analogue having an amino acid sequence of SEQ ID NO.6, wherein a cysteine ​​residue C is connected to the C-terminus of the endostatin amino acid sequence of SEQ ID NO.1.

[0058] The endostatin NK1, NK2, NK3, NK4, and NESC analogs described in the present invention (e.g., the embodiments) refer to endostatin analogs having amino acid sequences of SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, and SEQ ID NO. 11, respectively. The endostatin NK1, NK2, NK3, NK4, and NESC analogs are obtained by inserting the amino acid sequence GGSHHHHH between the methionine M and the histidine H at the N-terminus of the above-mentioned endostatin K1, K2, K3, K4, and ESC analogs, respectively.

[0059] The endostatin ESK analogue described in the present invention (e.g., the embodiment) refers to an endostatin analogue with an amino acid sequence of SEQ ID NO.12, wherein the lysine residues at positions 76, 96, 107, 118, and 184 of the endostatin amino acid sequence SEQ ID NO.1 from the N-terminus are mutated to X1, X2, X3, X4, and X5, respectively, and the 127th residue asparagine is mutated to lysine. Wherein X1, X2, X3, X4, and X5 are any natural amino acids other than lysine, preferably water-soluble amino acids, more preferably one of the charged amino acids arginine, histidine, glutamic acid, and aspartic acid, more preferably one of the positively charged amino acids arginine and histidine, and most preferably arginine.

[0060] The endostatin NESK analogue described in the present invention (eg, the examples) refers to an endostatin analogue with an amino acid sequence of SEQ ID NO. 13, wherein the amino acid sequence GGSHHHHH is inserted between the methionine M and the histidine H at the N-terminus of the above endostatin ESK analogue.

[0061] The term "single modification" as used herein refers to a product obtained by modifying one endostatin or endostatin analog with one PEG molecule.

[0062] The term "double modification" as used herein refers to a product obtained by modifying one endostatin or endostatin analog with two PEG molecules.

[0063] The present invention also provides a pharmaceutical composition, which comprises the endostatin analog of the present invention and the polyethylene glycol coupling complex as described above, for treating diseases caused by neovascularization or neolymphatics. In some embodiments, the disease caused by neovascularization or neolymphatics is a tumor, including but not limited to lung cancer, breast cancer, etc. Suitably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0064] The term "pharmaceutically acceptable carrier" as used herein refers to solid or liquid diluents, fillers, antioxidants, stabilizers and other substances that can be safely administered. Depending on the route of administration, various carriers well known in the art can be used, including, but not limited to, sugars, starch, cellulose and its derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffer, emulsifiers, isotonic saline, and / or pyrogen-free water, etc.

[0065] The pharmaceutical composition may also be a sustained-release preparation in a form selected from microcapsules, hydrogels, microspheres, micro-osmotic pumps or liposomes.

[0066] The present invention also provides a kit comprising the above-mentioned complex of the endostatin analog of the present invention and polyethylene glycol and instructions for use.

[0067] The present invention also provides a method for treating a disease caused by neovascularization or neolymphatic formation, comprising administering to a subject a therapeutically effective amount of the endostatin analog and polyethylene glycol coupling complex of the present invention as described above. In some embodiments, the disease caused by neovascularization or neolymphatic formation is a tumor, including but not limited to lung cancer, breast cancer, colon cancer, etc.

[0068] The term "therapeutically effective amount" as used herein refers to the amount of active compound sufficient to induce the biological or medical response desired by the clinician in the subject. The "therapeutically effective amount" of the endostatin analog and polyethylene glycol coupling complex can be determined by those skilled in the art based on factors such as the route of administration, the subject's weight, age, and condition. For example, a typical daily dose range can be 0.01 mg to 100 mg of active ingredient per kg body weight.

[0069] The drugs provided by the present invention can be prepared into clinically acceptable dosage forms such as powders and injections. The pharmaceutical composition of the present invention can be administered to a subject by any appropriate route, for example, by oral administration, intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneal administration, rectal administration, sublingual administration, or by inhalation, transdermal administration, and the like.

[0070] Unless otherwise indicated, the scientific and technical terms used in this specification should have the meanings commonly understood by those of ordinary skill in the art. Generally, the nomenclature and techniques related to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry used in this specification are well known and commonly used in the art.

[0071] Unless otherwise specified, the methods and techniques used in this specification are generally performed according to well-known and conventional methods in the art and in the manner described in various references described or cited in this specification.

[0072] In the following examples, N-terminal amino-polyethylene glycol mono-modified products, N-terminal amino- and lysine amino-polyethylene glycol double-modified products, lysine amino-polyethylene glycol mono-modified products and C-terminal thiol-polyethylene glycol mono-modified products of endostatin and endostatin K1, K2, K3, K4, ESC, NK1, NK2, NK3, NK4, NESC, ESK, NESK analogs were prepared and purified, and their inhibition rates on HMEC cell migration were detected. In the endostatin K1, K2, K3, K4, NK1, NK2, NK3, NK4, ESK, NESK analogs used in the following examples, the mutated lysine was mutated from lysine to arginine.

[0073] Example 1: Coupling of 20 kDa polyethylene glycol to the N-terminal amino group of natural human endostatin

[0074] The natural human endostatin was dialyzed into a 30mM sodium acetate solution at pH 5.0±1.0, the protein concentration was determined, and the protein concentration was adjusted to between 5 and 15mg / ml. The amount of 20kDa monomethoxypolyethylene glycol propionaldehyde (mPEG-ALD) to be added was calculated according to the molar ratio of the target protein to polyethylene glycol of 1:3, and the amount of reducing agent sodium cyanoborohydride was calculated according to the final solution volume, and its concentration was 20mM. The required monomethoxypolyethylene glycol propionaldehyde (mPEG-ALD) and sodium cyanoborohydride were weighed and added to the target protein, stirred evenly, and then allowed to stand at room temperature for 6 to 8 hours. The electrophoresis diagram of the modification results is attached. Figure 1 The content of each band of the electrophoresis gel was analyzed using Bio-Rad's 1D gel quantification software Quantity One and compared with the molecular weight standard. The results showed that the proportion of endostatin being single-modified (product molecular weight 40kDa) was more than 60%, that is, one endostatin was modified with a monomethoxy polyethylene glycol with a molecular weight of 20kDa.

[0075] Example 2: Purification of the product of coupling 20 kDa polyethylene glycol with the N-terminal amino group of natural human endostatin

[0076] The product solution of the coupling reaction of monomethoxypolyethylene glycol propionaldehyde (mPEG-ALD) and natural human endostatin in Example 1 was purified using a cationic chromatography column. Specifically, GE Healthcare's SPFF medium was selected, and the pH value of the reaction solution was adjusted to 5.0-7.0. The cationic column was balanced with 20mM NaH2PO4 (pH value to 5.0-7.0) and loaded. Then, gradient elution was performed using 20mM NaH2PO4, 500mM NaCl pH5.0-7.0), and different components were collected according to 280nm ultraviolet absorption. SDS-PAGE analysis was performed, and the analysis results are shown in Figure 1 .Depend on Figure 1 It can be seen that component 3 is the product of 20kDa polyethylene glycol mono-modified endostatin (molecular weight 40kDa). Since the pKa values ​​of the N-terminal amino group and the lysine side chain amino group are 8.0 and 10.5 respectively, the reaction activity of the N-terminal amino group when modified under pH 5.0±1.0 is 10^(10.5-8.0)=316 times that of the lysine side chain amino group, it can be considered that the mono-modified product is an N-terminal mono-modified product.

[0077] Example 3: Coupling of 20 kDa polyethylene glycol to the N-terminal amino group of endostatin K1 analog

[0078] The endostatin K1 analog was dialyzed into a 30mM sodium acetate solution at pH 5.0±1.0, the protein concentration was determined, and the protein concentration was adjusted to between 5 and 15mg / ml. The amount of 20kDa monomethoxypolyethylene glycol propionaldehyde (mPEG-ALD) to be added was calculated according to the molar ratio of the target protein to polyethylene glycol of 1:3, and the amount of reducing agent sodium cyanoborohydride was calculated according to the final solution volume, and its concentration was 20mM. Weigh the required monomethoxypolyethylene glycol propionaldehyde (mPEG-ALD) and sodium cyanoborohydride, add them to the target protein, stir evenly and let stand at room temperature for 6 to 8 hours. The electrophoresis diagram of the modification results is shown in Figure 2 The content of each band of the electrophoresis gel was analyzed using Bio-Rad's 1D gel quantification software Quantity One and compared with the molecular weight standard. The results showed that the proportion of endostatin K1 analogs that were modified by a single modification was more than 60%, that is, one endostatin K1 analog was modified by a monomethoxy polyethylene glycol with a molecular weight of 20 kDa.

[0079] Example 4: Purification of the product of coupling of 20 kDa polyethylene glycol with the N-terminal amino group of endostatin K1 analog

[0080] The product solution of the coupling reaction of monomethoxypolyethylene glycol propionaldehyde (mPEG-ALD) and endostatin K1 analog in Example 3 was purified using a cationic chromatography column. GE Healthcare's SPFF medium was selected, and the pH value of the reaction solution was adjusted to 5.0-7.0. The cationic column was equilibrated with 20mM NaH2PO4 (pH value to 5.0-7.0) and loaded. Gradient elution was then performed using 20mM NaH2PO4, 500mM NaCl pH5.0-7.0), and different components were collected based on 280nm ultraviolet absorption. SDS-PAGE analysis was performed, and the analysis results are shown in Figure 2 .Depend on Figure 2 It can be seen that components 2 and 3 are both products of 20 kDa polyethylene glycol-monomodified endostatin K1 analogs. Based on the same modification principle analysis as in Example 2, it can be considered that the monomodified products are N-terminal monomodified products.

[0081] Example 5: Double coupling of 20 kDa polyethylene glycol with lysine side chain amino group and N-terminal amino group of endostatin K1 analog

[0082] The endostatin K1 analog was dialyzed into a 20mM NaH2PO4 solution at pH 8.5, the protein concentration was determined, and the protein concentration was adjusted to between 5 and 15 mg / ml. The amount of 20kDa monomethoxypolyethylene glycol succinimidyl propionate to be added was calculated according to the molar ratio of the target protein to polyethylene glycol of 1:10, and added to the target protein. After stirring evenly, it was allowed to stand at room temperature for 60 to 120 minutes. The electrophoresis diagram of the modification results is shown in Figure 3 The content of each band of the electrophoresis gel was analyzed using Bio-Rad's 1D gel quantification software Quantity One and compared with the molecular weight standard. At this time, the proportion of endostatin K1 analogs that were double-modified (molecular weight 60kDa) was more than 50%, that is, one endostatin K1 analog was modified with two monomethoxy polyethylene glycols with a molecular weight of 20kDa.

[0083] Example 6: Purification of the double coupling product of 20 kDa polyethylene glycol and endostatin K1 analog lysine side chain amino group and N-terminal amino group

[0084] The product solution of the coupling reaction of monomethoxypolyethylene glycol succinimidyl propionate and endostatin K1 analog in Example 5 was purified by a cationic chromatography column. GE Healthcare SPFF medium was selected, and the pH value of the reaction solution was adjusted to 5.0-7.0. The cationic column was balanced with 20mM NaH2PO4 (pH value to 5.0-7.0) and loaded. Then, 20mM NaH2PO4, 500mM NaCl pH5.0-7.0) was used for gradient elution, and different components were collected according to the ultraviolet absorption at 280nm. SDS-PAGE analysis was performed, and the analysis results are shown in Figure 3 .Depend on Figure 3 It can be seen that component 4 is the product of 20kDa polyethylene glycol double-modified endostatin K1 analog.

[0085] Example 7: Coupling of 20 kDa polyethylene glycol to the N-terminal amino group of endostatin K2 analogs and purification

[0086] The method of Example 3 was used for N-terminal coupling, and the method of Example 4 was used for purification of the modified product, and similar experimental results were obtained.

[0087] Example 8: Coupling of 20 kDa polyethylene glycol with lysine side chain amino groups and N-terminal amino groups of endostatin K2 analogs and purification

[0088] The coupling was performed using the method of Example 5, and the modified product was purified using the method of Example 6, and similar experimental results were obtained.

[0089] Example 9: Coupling of 20 kDa polyethylene glycol to the N-terminal amino group of endostatin K3 analogs and purification

[0090] The method of Example 3 was used for N-terminal coupling, and the method of Example 4 was used for purification of the modified product, and similar experimental results were obtained.

[0091] Example 10: Coupling of 20 kDa polyethylene glycol with lysine side chain amino groups and N-terminal amino groups of endostatin K3 analogs and purification

[0092] The coupling was performed using the method of Example 5, and the modified product was purified using the method of Example 6, and similar experimental results were obtained.

[0093] Example 11: Coupling of 20 kDa polyethylene glycol to the N-terminal amino group of endostatin K4 analogs and purification

[0094] The method of Example 3 was used for N-terminal coupling, and the method of Example 4 was used for purification of the modified product, and similar experimental results were obtained.

[0095] Example 12: Coupling of 20 kDa polyethylene glycol with lysine side chain amino groups and N-terminal amino groups of endostatin K4 analogs and purification

[0096] The method of Example 5 was used for N-terminal coupling, and the method of Example 6 was used for purification of the modified product to obtain similar experimental results.

[0097] Example 13: 20 kDa polyethylene glycol conjugated to endostatin ESC analogs

[0098] The endostatin ESC analog was dialyzed into a 20mM NaH2PO4 solution at pH 7.5, the protein concentration was determined, and the protein concentration was adjusted to between 5 and 15 mg / ml. The amount of 20kDa monomethoxy polyethylene glycol maleimide (mPEG-MAL) to be added was calculated according to the molar ratio of the target protein to polyethylene glycol of 1:5, and added to the target protein. After stirring evenly, it was allowed to stand at room temperature for 6 to 8 hours. The electrophoresis diagram of the modification results is shown in Figure 4 . Bio-Rad's 1D gel quantification software QuantityOne was used to analyze the content of each band of the electrophoresis gel and compare it with the molecular weight standard. At this time, the proportion of endostatin ESC analogs being single-modified (molecular weight 40kDa) was more than 80%, that is, one endostatin ESC analog molecule was modified by one monomethoxy polyethylene glycol maleimide molecule with a molecular weight of 20kDa. Since monomethoxy polyethylene glycol maleimide can only react with free thiol groups, it can be considered that the polyethylene glycol modification site of this product is the free thiol group of endostatin ESC analogs.

[0099] Example 14: Purification of the conjugated product of 20 kDa polyethylene glycol and endostatin ESC analogs

[0100] The product solution of the coupling reaction of monomethoxypolyethylene glycol maleimide (mPEG-MAL) and endostatin ESC analog in Example 13 was purified using a cationic chromatography column. GE Healthcare's SPFF medium was selected, and the pH value of the reaction solution was adjusted to 5.0-7.0. The cationic column was balanced with 20mM NaH2PO4 (pH value to 5.0-7.0) and loaded. Gradient elution was then performed using 20mM NaH2PO4 and 500mM NaCl (pH5.0-7.0), and different components were collected based on 280nm ultraviolet absorption. SDS-PAGE analysis was performed, and the analysis results are shown in Figure 4 .Depend on Figure 4 It can be seen that component 3 is the product of 20kDa polyethylene glycol-modified endostatin ESC analog.

[0101] Example 15: Coupling of 20 kDa polyethylene glycol to the N-terminal amino group of endostatin NK1, NK2, NK3, NK4, ESK, NESK analogs and purification

[0102] The method of Example 3 was used for N-terminal coupling, and the method of Example 4 was used for purification of the modified product to obtain similar experimental results.

[0103] Example 16: Coupling of 20 kDa polyethylene glycol with lysine side chain amino groups and N-terminal amino groups of endostatin NK1, NK2, NK3, NK4, ESK, NESK analogs and purification

[0104] The coupling was performed using the method of Example 5, and the modified product was purified using the method of Example 6, and similar experimental results were obtained.

[0105] Example 17: Purification of the product of coupling of 20 kDa polyethylene glycol with endostatin K1 analog lysine side chain amino group

[0106] The endostatin K1 analog was dialyzed into a 20mM sodium dihydrogen phosphate solution at pH 8.5±0.5, the protein concentration was determined, and the protein concentration was adjusted to between 10 and 20 mg / ml. The amount of 20kDa monomethoxy polyethylene glycol propionaldehyde (mPEG-ALD) to be added was calculated according to the molar ratio of the target protein to polyethylene glycol of 1:1, and the amount of reducing agent sodium cyanoborohydride was calculated according to the final solution volume, the concentration of which was 10mM, the required monomethoxy polyethylene glycol propionaldehyde (mPEG-ALD) and sodium cyanoborohydride were weighed, added to the target protein, stirred evenly, and allowed to stand at room temperature for 6 to 8 hours. The reaction product solution was purified by a cationic chromatography column, specifically GE Healthcare Macrocap SP medium, and the pH of the reaction solution was adjusted to 4.5±0.5. The cationic column was balanced with 30mM NaAc (pH to 4.0-5.0) and loaded. Then 30mMNaAc, 500mMNaCl (pH4.0-5.0) were used for gradient elution, and different components were collected according to the 280nm UV absorption. The modified sample and each component collected after subsequent purification were analyzed by SDS-PAGE electrophoresis. The analysis results are shown in Figure 5 The two single modified components collected in lanes 4 and 5 were analyzed for modification sites by trypsin digestion followed by mass spectrometry analysis, and it was confirmed that the sample component in lane 5 was a single modification product of the amino group of the lysine side chain.

[0107] Example 18: Coupling of 20 kDa polyethylene glycol with lysine side chain amino groups of endostatin K2, K3, NK1, NK2, NK3 analogs and purification

[0108] The method of Example 17 was used to carry out coupling and purification of the modified product, and similar experimental results were obtained.

[0109] Example 19: Activity determination of modified products

[0110] All target purified products obtained from Examples 1 to 18 were subjected to cell activity assays to screen the optimal modification scheme. HMEC cells were selected, and the Migration (Tranwell Assay) method was used to count the number of cells to reflect protein activity (see Luo Yongzhang et al., Endostatin inhibits tumour lymphangiogenesis and lymphatic metastasis via cell surface nucleolin on lymphangiogenic endothelial cells (J Pathol 2010; 222: 249-260), and the inhibition rate of each modified product on HMEC cell migration was analyzed. The results are shown in Table 1:

[0111] Table 1: Determination results of the inhibition rate of each modified product on HMEC cell migration

[0112]

[0113]

[0114] The above activity results show that the inhibition rate of HMEC cell migration by the single N-terminal modification products of all analogs is lower than that by the single N-terminal modification product of endostatin, indicating that the mutation has a certain effect on the protein activity; the results of single modification of the lysine side chain amino group show that the product with single modification of the second lysine side chain amino group (i.e., endostatin K2 analog) has the highest inhibition rate on HMEC cell migration, which is higher than the single N-terminal modification product of endostatin; among the products with double modification of the N-terminal amino group and the lysine side chain amino group, the product with double modification of the second lysine side chain amino group and the N-terminal amino group (i.e., endostatin K2 analog) has the highest inhibition rate on HMEC cell migration among all products.

[0115] Binding to the tertiary structure of the endostatin molecule ( Figure 6) were comprehensively analyzed, and there are 6 amino groups that can be modified with polyethylene glycol in the endostatin molecule, namely the N-terminal amino group and 5 lysine side chain amino groups. The 5th lysine residue is located at the C-terminus, close to the N-terminus. Due to steric hindrance, it is difficult to achieve a single modification of the 5th lysine side chain amino group. The difficulty of preparation is reduced by adding a cysteine ​​residue behind it for thiol modification. Among the above 6 modified sites, the second lysine residue is farthest from the nucleolin binding region of the protein, followed by the N-terminus. The polyethylene glycol modification of the ethylene glycol long chain has the least effect on the binding region. Therefore, the N-terminal amino group and the second lysine side chain amino group are modified singly or double, and the product has a better inhibition rate on HMEC cell migration than other site modification schemes, which fully shows that choosing to modify away from the nucleolin binding region is the best choice for endostatin.

[0116] To verify the above conclusion, we introduced a lysine residue at position 127 (i.e., endostatin ESK analog) based on the tertiary structural characteristics of the endostatin molecule, which was also far away from the nucleolin binding region and the second lysine residue for modification. The results showed that the HMEC cell migration inhibition rate of the double-modified product of the analog was lower than that of the endostatin K2 analog, indicating that double modification of the endostatin K2 analog is the best choice for endostatin or its analogs.

[0117] See also Figure 6 and Figure 7, the effects of polyethylene glycol modification on protein activity were different in different regions of the ES molecule surface. The N-terminal single modification and K2 single modification products significantly increased the inhibition rate of HMEC cell migration inhibition activity. The double modification products of these two sites increased the inhibition rate of HMEC cell migration inhibition activity most significantly. K2 was farthest from the nucleolin binding region, and single modification increased the protein's HMEC cell migration inhibition activity the most. K1, K3, K4 and C-terminal modification products increased the protein's HMEC cell migration inhibition activity less, or decreased, and the double modification products also obtained similar results, indicating that K2, which is far away from the nucleolin binding region, is the best modification choice. In order to further increase the half-life of the product, double modification of N-terminal and K2 is the best choice. In order to confirm whether there is an insertable modification site similar to K2 in the K2 region far from K3 and K4, point mutations were made on N127 of the natural structure of ES to form a new modification site, and related experiments were carried out. The results confirmed that the increase in HMEC cell migration activity of the modified products in this region (whether single modification or double modification with N-terminal) was less than the effect of the corresponding modification products of K2. This fully demonstrates that double modification of K2 and N-terminus is the best choice for increasing protein activity and extending half-life.

Claims

1. A complex of an endostatin analogue coupled to polyethylene glycol; wherein the sequence of the endostatin analogue is as shown in SEQ ID NO.3, wherein the amino acids at positions 76, 107, 118, and 184 are arginine; and the endostatin analogue is coupled to polyethylene glycol only at the lysine residue at position 96, or the endostatin analogue is coupled to polyethylene glycol at the N-terminus and the lysine residue at position 96; wherein the molecular weight of the polyethylene glycol is about 5,000 to about 50,000 Daltons.

2. The coupled complex according to claim 1, wherein the endostatin analog is formed by mutation of the lysine residues at positions 76, 107, 118 and 184 of the natural endostatin amino acid sequence SEQ ID NO.

1.

3. A complex of an endostatin analogue coupled to polyethylene glycol; wherein the sequence of the endostatin analogue is as shown in SEQ ID NO.8, wherein the amino acids at positions 84, 115, 126, and 192 are arginine; and the endostatin analogue is coupled to polyethylene glycol only at a lysine residue, or the endostatin analogue is coupled to polyethylene glycol at the N-terminus and a lysine residue; wherein the molecular weight of the polyethylene glycol is about 5,000 to about 50,000 Daltons.

4. The coupled complex of claim 3, wherein the endostatin analog is formed by mutation of the lysine residues at positions 76, 107, 118 and 184 of the natural endostatin amino acid sequence SEQ ID NO.1, and insertion of GGSHHHHH between the methionine M and the histidine H at its N-terminus.

5. The coupled complex according to any one of claims 1 to 4, wherein the endostatin analog is coupled to polyethylene glycol via a covalent bond.

6. The coupled complex of any one of claims 1 to 4, wherein the molecular weight of the polyethylene glycol is about 20,000 Daltons.

7. The coupled complex according to any one of claims 1 to 4, wherein the polyethylene glycol is monomethoxy polyethylene glycol, monoglucose polyethylene glycol or monogalactose polyethylene glycol. The coupled complex according to claim 7 , wherein the polyethylene glycol is monomethoxypolyethylene glycol.

9. The coupled complex according to any one of claims 1 to 4, wherein the polyethylene glycol is linear or branched.

10. The coupled complex of any one of claims 1 to 4, wherein the polyethylene glycol is coupled to the amino group of the endostatin analog using monomethoxypolyethylene glycol propionaldehyde (mPEG-ALD), monomethoxypolyethylene glycol butyraldehyde (mPEG-ButyrALD), monomethoxypolyethylene glycol succinimidyl carbonate (mPEG-SC), monomethoxypolyethylene glycol succinimidyl acetate (mPEG-SCM), monomethoxypolyethylene glycol succinimidyl propionate (mPEG-SPA), monomethoxypolyethylene glycol succinimidyl butyrate (mPEG-SBA), monomethoxypolyethylene glycol succinimidyl α-methylbutyrate (mPEG-SMA), or monomethoxypolyethylene glycol NHS ester (mPEG-NHS).

11. A pharmaceutical composition comprising the coupled complex according to any one of claims 1 to 10 and optionally a pharmaceutically acceptable carrier.

12. Use of the coupled complex according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 11 in the preparation of a medicament for treating a tumor caused by neovascularization or neolymphatic formation, wherein the tumor is lung cancer, breast cancer or colon cancer.

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