CN115703828A

CN115703828A - Anti-angiogenesis medicine

Info

Publication number: CN115703828A
Application number: CN202110910557.9A
Authority: CN
Inventors: 刘鹏
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-08-09
Filing date: 2021-08-09
Publication date: 2023-02-17
Also published as: WO2023016410A1

Abstract

The invention relates to a reduced human vascular endothelial inhibin or an analogue thereof, a modified reduced human vascular endothelial inhibin or an analogue thereof, wherein the reduced human vascular endothelial inhibin or the analogue thereof and the modified reduced human vascular endothelial inhibin or the analogue thereof have better anti-angiogenesis and anti-tumor activity compared with the existing oxidized human vascular endothelial inhibin.

Description

Anti-angiogenesis medicine

Technical Field

The invention relates to the technical field of biological medicines, in particular to reduced human vascular endothelial inhibin or an analogue thereof and modified reduced human vascular endothelial inhibin or an analogue thereof. The invention also relates to a modified protein or polypeptide, which is modified to make the protein or polypeptide which is not soluble in water under physiological pH value become soluble in water, thereby improving the drug property of the protein or polypeptide.

Background

The Endostatin (Endostatin) is a section of enzyme digestion product with the molecular weight of 20kDa at the carboxyl end of the collagen XVIII. This protein was found in cultures of vascular endothelial cells by professor Judah Folkman, harvard university, USA 1997, and has activity in inhibiting vascular endothelial Cell proliferation, migration, and angiogenesis in vivo (O' Reilly MS, boehm T, shing Y, fukai N, vasios G, lane WS, flynn E, birkhead JR, olsen BR, folkman J: endostatin: an endogenesis inhibitor of angiogenisis and tumor growth. Cell 277-85,1997. Further studies by professor Judah Folkman et al found: the recombinant vascular endothelial inhibin can inhibit the growth and metastasis of various tumors in a mouse body, even can completely cure the tumors, and does not generate drug resistance; the mechanism of its activity is that it inhibits the formation of new blood vessels in the vicinity of tumor tissue by inhibiting the growth of vascular endothelial cells, rendering the tumor tissue deprived of the macronutrients and oxygen necessary for growth, and eventually stopping growth or necrosis (Boehm T, folkman J, browder T, O' Reilly MS: anatomical thermal of experimental cancer patients not requiring cultured drug resistance. Nature 390: 404-7, 1997).

The recombinant human vascular endothelial inhibin prepared by genetic engineering can be used as a tumor treatment drug. The only commercially available recombinant human endostatin drug is currently marketed under the trade name "Endotar". Initial studies found that Endostatin has very high anti-angiogenic and anti-Tumor activities, but subsequent basic and clinical studies showed that the activities were not ideal (B. Kim Lee Sim, nicholes J. MacDonald and Edward R. Gubish: angiostatin and Endostatin: endogenesis Inhibitors of Tumor growth. Cancer and Metastasis Reviews 19 181-190,2000). Therefore, in practical applications, vascular endostatin with better antitumor activity is needed.

Disclosure of Invention

In view of the above, the present invention provides a reduced human endostatin or an analog thereof and a modified reduced human endostatin or an analog thereof, wherein the reduced human endostatin or the analog thereof and the modified reduced human endostatin or the analog thereof have better anti-angiogenic and anti-tumor activities compared to the existing oxidized human endostatin.

In view of the above objects, a first aspect of the present invention provides a reduced form of human endostatin or an analog thereof, the reduced form of human endostatin comprising at most one pair of disulfide bonds formed from thiol groups on two cysteine residues of a human endostatin molecule.

In a preferred embodiment of the present invention, the reduced form of the human endostatin analogue includes at least one of the following or a combination thereof:

-deletion or substitution of one or more cysteines on the basis of the amino acid sequence of native angiostatin;

-a partial peptide stretch of native vascular endostatin; or

-altering the amino acid sequence of native endostatin;

wherein the reduced human endostatin analogue comprises at most one pair of disulfide bonds.

In a preferred embodiment of the invention, the amino acid sequence of the reduced human endostatin is shown in SEQ ID NO 1, 2, 3 or 4.

In a preferred embodiment of the present invention, the amino acid sequence of the reduced human endostatin is shown in SEQ ID NO. 1, wherein the disulfide bond formation condition comprises:

-the thiol group of Cys33 and Cys173 residues in the reduced human endostatin molecule do not form a disulfide bond;

-the thiol group of Cys135 and Cys165 residues in the reduced human endostatin molecule do not form a disulfide bond; or alternatively

None of the above two disulfide bonds are formed.

In a second aspect, the invention also provides a modified reduced form of human endostatin or an analogue thereof, which comprises at most one pair of disulfide bonds formed from thiol groups on two cysteine residues of a human endostatin molecule.

In a preferred embodiment of the present invention, the reduced human endostatin analog comprises at least one of the following or a combination thereof:

-a partial peptide of natural endostatin; or

-altering the amino acid sequence of natural endostatin;

In a preferred embodiment of the invention, the amino acid sequence of the reduced human vascular endostatin is shown in SEQ ID NO 1, 2, 3 or 4.

In a preferred embodiment of the invention, the amino acid sequence of the reduced human vascular endostatin is shown in SEQ ID NO 2 or 4.

-the thiol groups on Cys135 and Cys165 residues in the reduced human endostatin molecule do not form a disulfide bond; or alternatively

None of the above two disulfide bonds are formed.

In a preferred embodiment of the present invention, the modifier is selected from any one of a high molecular polymer, a protein molecule or a fragment thereof, a small molecular substance, or a combination thereof.

In a preferred embodiment of the invention, the protein molecule or fragment thereof comprises albumin, an immunoglobulin, a cytokine or a fragment thereof and the like, preferably an immunoglobulin Fc fragment. The reduced human vascular endothelial inhibin or the analogue thereof modified by the immunoglobulin Fc fragment can be obtained by chemical modification or fusion expression. When the reduced human vascular endothelial inhibin modified by the immunoglobulin Fc fragment is expressed by fusion, the reduced human vascular endothelial inhibin modified by the immunoglobulin Fc fragment is the fusion protein formed by the immunoglobulin Fc fragment and the reduced human vascular endothelial inhibin.

In a preferred embodiment of the present invention, the high molecular polymer is polyethylene glycol.

In a preferred embodiment of the invention, the amino acid sequence of the reduced human endostatin is shown in SEQ ID NO. 2 or 4.

In a preferred embodiment of the invention, a molecule of reduced human endostatin or an analog thereof is coupled to one or more polyethylene glycol molecules.

In a preferred embodiment of the present invention, the coupling site of the reduced human endostatin or the analog thereof and the polyethylene glycol is one of the N-terminal alpha-amino group of the reduced human endostatin or the analog thereof, the epsilon-amino group of the lysine residue side chain, the sulfhydryl group of the cysteine residue side chain, the carboxyl group of the aspartic acid residue side chain, the carboxyl group of the glutamic acid residue side chain, or the combination thereof.

In a preferred embodiment of the present invention, the site of coupling of the reduced human endostatin or the analog thereof to the polyethylene glycol is an alpha-amino group at the N-terminal of the reduced human endostatin or the analog thereof.

In a preferred embodiment of the invention, the polyethylene glycol molecule has an average molecular weight of between 1,000 and 100,000 daltons.

In a preferred embodiment of the invention, the polyethylene glycol molecule has an average molecular weight of between 5,000 and 40,000 daltons.

In a third aspect, the present invention provides a pharmaceutical composition, which comprises the above reduced human endostatin or its analog or the above modified reduced human endostatin or its analog, and a pharmaceutically acceptable carrier.

In a preferred embodiment of the invention, the pharmaceutical composition is a sustained release formulation.

In a fourth aspect, the invention provides a modified protein or polypeptide which is not soluble in water at physiological pH, which is soluble in water after modification at physiological pH; the modifier is selected from any one of high molecular polymer, protein molecule or fragment thereof, small molecular substance or combination thereof.

From the above, it can be seen that the reduced human endostatin or the analog thereof and the modified reduced human endostatin or the analog thereof provided by the invention have better anti-angiogenesis and anti-tumor activities compared with the existing oxidized human endostatin.

Drawings

FIG. 1 is an electrophoresis diagram of SDS-PAGE electrophoresis detection of reduced recombinant human endostatin and polyethylene glycol (PEG) modified reduced recombinant human endostatin; wherein, L1 and L2 are polyethylene glycol (PEG) modified reduction state recombinant human vascular endothelial inhibin, and L3 and L4 are reduction state recombinant human vascular endothelial inhibin.

FIG. 2 is a comparison graph of the activity of PEG-modified reduced recombinant human endostatin, endotar (Endostar) and PEG-modified Endotar (Endotar) for inhibiting migration of HMEC cells (human microvascular endothelial cells); wherein, B is blank, L1 is activity of inhibiting HMEC cell migration by using the degree of administration with concentration of 10 mug/ml, L2 is activity of inhibiting HMEC cell migration by using polyethylene glycol modified degree (PEG-Endostar) with administration concentration of 10 mug/ml, L3 is activity of inhibiting HMEC cell migration by using polyethylene glycol modified reduced state recombinant human vascular endothelial inhibin (nES) with administration concentration of 1 mug/ml, L4 is activity of inhibiting HMEC cell migration by using polyethylene glycol modified reduced state recombinant human vascular endothelial inhibin (nES) with administration concentration of 2 mug/ml, and L5 is activity of inhibiting HMEC cell migration by using polyethylene glycol modified reduced state recombinant human vascular endothelial inhibin (nES) with administration concentration of 5 mug/ml.

FIG. 3 is a graph showing the comparison of the components, the degree of inhibition of HMEC cell migration by polyethylene glycol-modified degree of inhibition, obtained by purifying polyethylene glycol-modified reduced recombinant human vascular endostatin by cation exchange chromatography (medium is sulfopropyl dextran gel (SP for short)); wherein B is blank, L1 is activity of inhibiting migration of HMEC cells at an administration concentration of 10. Mu.g/ml, L2 is activity of inhibiting migration of HMEC cells at an administration concentration of 10. Mu.g/ml for polyethylene glycol-modified emnity, L3 is activity of inhibiting migration of HMEC cells at nES (FT (effluent)), L4 is activity of inhibiting migration of HMEC cells at nES (25 mS/cm) at an administration concentration of 2ug/ml, L5 is activity of inhibiting migration of HMEC cells at nES (30 mS/cm) at an administration concentration of 2. Mu.g/ml, and L6 is activity of inhibiting migration of HMEC cells at nES (40 mS/cm) at an administration concentration of 2. Mu.g/ml.

FIG. 4 is a graph comparing the activity of a nES (25 mS/cm) fraction, an enricher and a polyethylene glycol modified enricher to inhibit HMEC cell migration at various dosing concentrations; wherein B is blank, L1 is activity of inhibiting migration of HMEC cells by an saturation administered at a concentration of 10. Mu.g/ml, L2 is activity of inhibiting migration of HMEC cells by a polyethylene glycol-modified saturation administered at a concentration of 10. Mu.g/ml, L3 is activity of inhibiting migration of HMEC cells by a nES (25 mS/cm) fraction administered at a concentration of 0.1ug/ml, L4 is activity of inhibiting migration of HMEC cells by a nES (25 mS/cm) fraction administered at a concentration of 0.2. Mu.g/ml, L5 is activity of inhibiting migration of HMEC cells by a nES (25 mS/cm) fraction administered at a concentration of 0.5. Mu.g/ml, and L6 is activity of inhibiting migration of HMEC cells by nES (25 mS/cm) fraction administered at a concentration of 1.0. Mu.g/ml.

FIG. 5 is a graph comparing the activity of placebo or PEG-modified recombinant human endostatin in inhibiting HMEC cell proliferation; wherein, the a picture is the activity of blank control for inhibiting HMEC cell proliferation, and the b picture is the activity of polyethylene glycol modified reduced recombinant human vascular endothelial inhibin for inhibiting HMEC cell proliferation.

FIG. 6 is a schematic diagram of the spatial structure of a prior art human endostatin in an oxidized state;

FIG. 7 is a schematic diagram showing the disulfide bond pairing of endostatin in the prior art oxidized form.

Detailed Description

It is to be noted that, unless otherwise defined, technical terms used in the present specification are the ordinary meanings as understood by those skilled in the art.

The experimental procedures in the following examples are conventional unless otherwise specified. The pharmaceutical agent materials and the like used in the following examples are all commercially available products unless otherwise specified.

Definition of

Herein, neovascularization refers to the formation of new capillaries over the existing blood vessels. Various diseases are known to be associated with neovascularization, such as tumors and macular degeneration. Taking tumors as an example, the growth and migration of tumors is dependent on the production of new blood vessels. The microvascular endothelial cells in the tumor are used as the target point of cancer treatment, so that a treatment mode is provided for treating the tumor.

Herein, HMEC refers to Human Microvascular Endothelial cells (Human Microvascular Endothelial Cell lines); HUVEC refers to Human Umbilical Vein Endothelial Cells (Human Umbilical vessel Endothelial Cells).

Protein renaturation in this context means that the protein can be denatured under suitable conditions to restore its native conformation and biological activity, a phenomenon known as renaturation (renaturation) of the protein. In general, renaturation causes proteins inactivated by a change in steric structure to recover the original steric structure and regain activity, but a protein does not necessarily have only one steric structure. In contrast, as for endostatin, the inventors found that endostatin which is insoluble at physiological pH becomes soluble by so-called "renaturation", but also loses its activity because the highly active state of endostatin is rather an insoluble state.

In this context, idodine is an antitumor drug developed with human endostatin as an active ingredient.

The recombinant human endostatin injection is a 1.1 class anti-tumor vascular targeted drug which is researched and developed by scientists in China by adding 9 amino acids on the basis of natural human endostatin.

It is known to those skilled in the art that the human endostatin molecule contains four cysteine residues, which may form disulfide bonds. Herein, "reduced human endostatin" refers to human endostatin that contains at most one (1 or 0) pair of disulfide bonds. Taking fig. 7 as an example, in "reduced human endostatin", no disulfide bond is formed between the thiol groups of Cys33 and Cys173 residues, or between Cys135 and Cys165 residues, or between both of these two disulfide bonds, in the human endostatin molecule. "oxidized form human endostatin" refers to human endostatin that contains two pairs of disulfide bonds. Also in FIG. 7, for example, in "oxidized form of human endostatin", cys33 of the human endostatin molecule forms a disulfide bond with the thiol group of Cys173 residue, and Cys135 also forms a disulfide bond with the thiol group of Cys165 residue.

As used herein, a "reduced human endostatin analog" includes a cysteine mutation in native endostatin, a partial peptide of native endostatin, a change in the amino acid sequence of native endostatin, or a combination of the three, provided that the condition "comprising at most one pair of disulfide bonds" is satisfied, while still retaining the anti-angiogenic and anti-tumor activities of reduced human endostatin. The cysteine mutation of the natural vascular endothelial inhibin refers to the deletion or substitution of one or more cysteine(s) on the basis of the amino acid sequence of the natural vascular endothelial inhibin; the alteration of the amino acid sequence of natural endostatin means that a part of the amino acid sequence is inserted, deleted or substituted on the basis of the amino acid sequence of natural endostatin. The reduced human endostatin analogue contains at most one pair of disulfide bonds.

As used herein, "insoluble at physiological pH" means that the reduced form of human endostatin is insoluble in water at about pH7.4, e.g., between pH7.35 and 7.45. The reduced form of human endostatin of the present invention is not soluble in water at physiological pH, but is soluble in water at other pH, e.g., at a pH less than 5.5.

"conductivity" herein refers to the ability of a solution to conduct an electrical current. The conductivity of the solution may be altered by changing the salt concentration in the solution, e.g. the conductivity of the elution buffer may be increased by increasing the salt concentration in the elution buffer. Salts that may be used to increase conductivity include, but are not limited to, potassium chloride (KCl), sodium chloride (NaCl), potassium carbonate, sodium acetate, potassium sulfate, sodium sulfate, citrate, phosphate, or mixtures of these salts.

1.The reduced human vascular endothelial inhibin or the analogue thereof of the invention

It is generally recognized by those skilled in the art that native and active endostatin is two disulfide bonds (as shown in FIG. 6) and soluble at physiological pH (e.g., pH 7.4). The native folded structure is necessary for the function of endostatin. The sulfhydryl groups of the four cysteine residues on the endostatin molecule form a unique intramolecular nested disulfide bond. These two pairs of intramolecular disulfide bonds are important in stabilizing the secondary and tertiary structures of the protein, respectively. Prior art documents (Zhou H et al, constraints of discrete bonds in a structured to the structure, stability, and Biological functions of endostatin, journal of Biological Chemistry,2005,280: endostatin is a globular protein with two nested disulfide bonds, cys33-Cys173 and Cys135-Cys165 (FIG. 7); the inhibitory activity of endostatin on endothelial cell migration and proliferation may be closely related to the structure of disulfide bond retention, especially the disulfide bond Cys135-Cys165.

The inventors of the present invention have surprisingly found that: the highly active structure of endostatin is human endostatin in a reduced state, which contains at most one pair of disulfide bonds and is insoluble at physiological pH. The invention also proves that the activity of the vascular endothelial inhibin is related to the state of cysteine and the protein space structure of the vascular endothelial inhibin, but the invention discovers that the high-activity structure of the vascular endothelial inhibin is reduced state human vascular endothelial inhibin instead of oxidized state human vascular endothelial inhibin which is generally considered in the prior art. Therefore, the reduced human vascular endothelial inhibin of the invention is structurally different from the oxidized human vascular endothelial inhibin, and has higher biological activity. Such a highly active structure is not limited to reduced human endostatin containing a cysteine residue in its molecule, but includes reduced human endostatin analogs as long as the condition of "containing at most one pair of disulfide bonds" is satisfied. Preferably, the amino acid sequence of the reduced human vascular endothelial inhibin is shown in SEQ ID NO 1, 2, 3 or 4.

The reduced human endostatin analogue of the invention comprises at least one or the combination of the following:

-deletion or substitution of one or more (meaning two or more, up to four) cysteines on the basis of the amino acid sequence of native endostatin;

-a partial peptide stretch of native vascular endostatin; or

-altering the amino acid sequence of native endostatin;

The reduced human endostatin analogue molecule of the present invention may contain four cysteine residues, three cysteine residues, two cysteine residues, one cysteine residue or 0 cysteine residue, but no matter there are several cysteine residues, at most one pair of disulfide bonds is formed.

The invention does not limit the position of cysteine which does not form disulfide bond in the reduced human vascular endothelial inhibin, and the position of the cysteine can be changed according to the amino acid sequence of the reduced human vascular endothelial inhibin. Preferably, the amino acid sequence of the reduced human vascular endostatin is shown in SEQ ID NO 1, wherein the disulfide bond formation condition comprises:

-the thiol group of Cys135 and Cys165 residues in the reduced human endostatin molecule do not form a disulfide bond; or

None of the above two disulfide bonds are formed.

2.The invention relates to a preparation method of reduced human endostatin or analogues thereof

The reduced human vascular endothelial inhibin of the invention is directly purified from inclusion bodies expressed by escherichia coli.Those skilled in the art know that: inclusion bodies are high-density, insoluble protein particles coated by membranes formed when exogenous genes are efficiently expressed in prokaryotic cells, particularly escherichia coli. Biologically active proteins in cells often exist as soluble proteins or molecular complexes, and functional proteins always fold into specific three-dimensional structures. The proteins in inclusion bodies are aggregates in unfolded state and have no biological activity. For vascular endostatin, it is usually obtained by genetic engineering (e.g., using an E.coli expression system). The recombinant human vascular endothelial inhibin produced by the escherichia coli expression system has no natural folding structure, is poor in water solubility and is easy to form precipitates. In order to obtain recombinant human endostatin which has two pairs of disulfide bonds and is soluble in water at physiological pH, the precipitated form of recombinant human endostatin is typically refolded (renatured). Chinese patent 00107569.1 (title of the invention: method for producing endostatin) discloses: the nucleotide coding sequence of the human vascular endothelial inhibin is modified to produce the recombinant human vascular endothelial inhibin (rhEndostatin, named as Endostar, chinese name: endu) with an additional amino acid sequence (MGGSHHHHH) at the N terminal, and the processing process of the inclusion body is specifically disclosed: the inclusion body expressed by the Escherichia coli is dissolved in Tris buffer solution containing 7M guanidine hydrochloride and 50mM mercaptoethanol, placed for 1 hour, and centrifuged to obtain supernatant. Using Ni to the supernatant ²⁺ Column purification from Ni ²⁺ The eluted endostatin-containing protein on the column was rapidly diluted at a 1. During renaturation, free thiol groups on cysteines are disulfide bonded by addition of, for example, oxidized glutathione. The recombinant human endostatin after renaturation has two pairs of disulfide bonds and is water-soluble under physiological pH conditions (such as pH 7.4).

The prior art generally holds that endostatin in inclusion bodies has no native folded structure and is inactive. The reduced human vascular endothelial inhibin is directly purified from inclusion bodies expressed by escherichia coli, and the activity of the reduced human vascular endothelial inhibin is far higher than that of oxidized human vascular endothelial inhibin. Therefore, the invention provides a method for preparing the reduced human vascular endothelial inhibin, which comprises the steps of transforming a vector containing a nucleotide sequence for coding the human vascular endothelial inhibin into a prokaryotic expression system (such as escherichia coli), obtaining an inclusion body through fermentation expression, dissolving and purifying the inclusion body, and obtaining the reduced human vascular endothelial inhibin.

The Escherichia coli expression protein can automatically add methionine at the N end, so that the nucleotide sequence for coding human vascular endostatin is the nucleotide sequence with the coding amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 3. SEQ ID NO 1 differs from SEQ ID NO 2 in that: SEQ ID NO 2 has one more methionine at the N-terminus compared to SEQ ID NO 1, and SEQ ID NO 3 differs from SEQ ID NO 4 in that: SEQ ID NO. 4 has one more methionine at the N-terminus compared to SEQ ID NO. 3. And SEQ ID NO 2 differs from SEQ ID NO 4 in that: the N-terminal of SEQ ID NO. 4 has an additional amino acid sequence (MGGSHHHHH), and SEQ ID NO. 4 is the amino acid sequence of the existing product. The difference between the reduced recombinant human vascular endothelial inhibin of the invention and Endu is that the reduced recombinant human vascular endothelial inhibin of the invention is not renatured, and the inclusion body expressed by escherichia coli fermentation is dissolved and directly purified from the inclusion body dissolved solution.

Amino acid sequence 1 (SEQ ID NO: 1):

HSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGL AGTFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSWEALFSG SEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSDPNGRRLTESY CETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENSFMT ASK

amino acid sequence 2 (SEQ ID NO: 2):

MHSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVG LAGTFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSWEALFS GSEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSDPNGRRLTES YCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCI ENSFMTASK

amino acid sequence 3 (SEQ ID NO: 3):

GGSHHHHHHSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCF QQARAVGLAGTFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLF PSWEALFSGSEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSDP NGRRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASC HHAYIVLCIENSFMTASK

amino acid sequence 4 (SEQ ID NO: 4):

MGGSHHHHHHSHRDFQPVLHLVALNSPLSGGMRGIRGADFQ CFQQARAVGLAGTFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDEL LFPSWEALFSGSEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGS DPNGRRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASCH HAYIVLCIENSFMTASK

3.modified reduced human endostatin or analogs thereof

As mentioned above, the reduced human endostatin or the analogue thereof of the present invention has higher activity than the oxidized human endostatin, but is insoluble in water at physiological pH value, and cannot meet the requirement of drug-forming property. In order to improve the solubility of the reduced human vascular endothelial inhibin or the analogue thereof, the invention tries to modify the reduced human vascular endothelial inhibin or the analogue thereof by using a modifier. The invention utilizes the modifier to modify the reduced human vascular endothelial inhibin or the analogues thereof, and obtains good solubility under the condition of physiological pH value while maintaining the high-activity structure of the reduced human vascular endothelial inhibin or the analogues thereof. The modification simultaneously improves the molecular weight of the medicine, reduces the speed of kidney filtration and prolongs the half-life period of the medicine.

Therefore, the invention provides a modified reduced human endostatin or an analogue thereof, wherein the modification is selected from any one of a high molecular polymer, a protein molecule or a fragment thereof, a small molecular substance or a combination thereof. "modification" refers to covalent modification or non-covalent combination of a modifier (such as a high molecular polymer or a small molecular substance) with reduced human endostatin or an analog thereof, or the modifier (such as a protein molecule or a fragment thereof) and the reduced human endostatin or the analog thereof form a fusion protein. In a preferred embodiment of the invention, the protein molecule or fragment thereof comprises albumin, an immunoglobulin, a cytokine or a fragment thereof and the like, preferably an immunoglobulin Fc fragment. The reduced human vascular endothelial inhibin or the analogue thereof modified by the immunoglobulin Fc fragment can be obtained by chemical modification or fusion expression. When the reduced human vascular endothelial inhibin modified by the immunoglobulin Fc fragment is expressed by fusion, the reduced human vascular endothelial inhibin modified by the immunoglobulin Fc fragment is a fusion protein formed by the immunoglobulin Fc fragment and the reduced human vascular endothelial inhibin.

Therefore, the invention provides a modified reduced human vascular endothelial inhibin or an analogue thereof, which can be dissolved in water under the condition of physiological pH value while keeping a high activity state, and the modified soluble reduced human vascular endothelial inhibin or the analogue thereof can measure the in vitro cell activity and can be further developed into a medicament for systemic administration.

Preferably, the reduced state human endostatin or its analog is modified by high molecular polymer. More preferably, the reduced form of human endostatin or its analogs is modified with polyethylene glycol (PEG). The polyethylene glycol molecule has amphipathy, can be dissolved in water and most of organic solvents, and has the properties of no toxicity, no immunogenicity, high solubility in aqueous solution and the like. Coupling proteins with hydrophilic macromolecules such as polyethylene glycol can increase protein stability, reduce non-specific adsorption and immunogenicity. When the conjugate reaches a certain molecular weight, the elimination efficiency of the kidney can be greatly reduced, and the method is an effective method for prolonging the in vivo half-life period of the protein drug. The initial polyethylene glycol modification has amino group as reaction site, and includes mainly the N terminal alpha-amino group of protein and the epsilon-amino group in the side chain of lysine residue. The product of this type of reaction is a protein molecule coupled to one or more polyethylene glycol molecules. Modifications to the side chain epsilon-amino groups of lysine residues also tend to produce modified isomers because the reaction sites are not specific. At present, aiming at the difference of isoelectric points of alpha-amino at the N end of protein and epsilon-amino at the side chain of lysine residue, a polyethylene glycol modification reagent which is only modified aiming at the N end of the protein is newly developed, so that the modification sites are consistent, and the composition of the modified product is uniform. In addition, the thiol group on cysteine can also serve as a specific modification site.

WO 2007082483A1 (title of the invention: a medicine for treating tumor and application thereof) discloses PEG modified Endostatin (Endostatin), but the Endostatin used in the document is renatured Endostatin (oxidized state human Endostatin), contains two pairs of disulfide bonds, and is water-soluble at physiological pH. The basis for this inference is as follows:

(1) The reference to "such introduction of cysteine as a modification site is also limited because some proteins with cysteine themselves may be mismatched or not renatured. "it is inferred that endostatin modified with polyethylene glycol in this document is renatured.

(2) In the literature, we mentioned that "in the invention, we surprisingly found that the inhibition rate of the product of the recombinant human vascular endothelial inhibin with the modified N-terminal to the migration of endothelial cells in vitro cytology experiments is 2 times or more than that of the unmodified protein, namely that the biological activity of the protein is greatly improved, and the phenomenon that the in vitro activity is obviously increased is not reported. "angiostatin for cell experiments must be soluble at physiological pH values or cannot be used to determine activity. Thus, the above expression indicates that the endostatin used in this document is soluble under physiological pH conditions, i.e., a so-called renaturation state with two disulfide bonds. Generally, the activity reduction after protein modification is a normal condition, because the connected polyethylene glycol can form a steric hindrance effect, and the activity is enhanced in the literature. It is concluded that the endostatin in the document is prepared by in vitro renaturation after the expression of inclusion bodies in E.coli, and a part of the endostatin does not form disulfide bonds or incomplete disulfide bond pairing, because the modification of polyethylene glycol enables the part of the endostatin in a reduced state to be retained instead of forming precipitates as in the normal case, and the activity of the part of the endostatin is improved due to the part of the non-renatured endostatin.

As can be seen from the above, the prior art does not disclose polyethylene glycol-modified reduced human endostatin. The inventor compares the activities of polyethylene glycol modified reduced human endostatin and polyethylene glycol modified saturation (oxidized human endostatin), and the experimental result proves that the inhibition rates of the polyethylene glycol modified reduced recombinant human endostatin (nES) on HMEC cell migration are respectively 28%, 76% and 90% under the administration concentrations of 1, 2 and 5 mug/ml, the inhibition rate of the polyethylene glycol modified saturation at the administration concentration of 10 mug/ml on HMEC cell migration is 48%, and the inhibition rate of the polyethylene glycol modified saturation at the administration concentration of 10 mug/ml on HMEC cell migration is 54%. This shows that when the administration concentration of the reduced recombinant human endostatin (nES) modified by polyethylene glycol is only 1/5 of the degree of modified by polyethylene glycol, the inhibition rate of inhibiting the migration of HMEC cells is much higher than that of the degree of modified by polyethylene glycol, and the activity of the reduced recombinant human endostatin (nES) modified by polyethylene glycol of the present invention is 5-10 times that of the degree of modified by polyethylene glycol (see example 1).

The embodiment of the invention has proved that the reduced state human vascular endothelial inhibin modified by polyethylene glycol has higher activity than the oxidized state human vascular endothelial inhibin modified by polyethylene glycol, and the activity of the former is 5-10 times of that of the latter. The examples of the present invention further demonstrate: the high-activity polyethylene glycol modified reduced human vascular endothelial inhibin can be further purified, and the activity of a specific component obtained after ion exchange purification can be continuously improved by about 10 times, namely the activity of the component is 50-100 times of that of oxidized human vascular endothelial inhibin modified by polyethylene glycol (see example 2).

4.Preparation method of modified reduced human endostatin or analogue thereof

In the invention, activated polyethylene glycol and reduced human vascular endothelial inhibin or analogues thereof are mixed and reacted to obtain modified reduced human vascular endothelial inhibin or analogues thereof. Preferably, monomethoxypolyethylene glycol propionaldehyde is mixed with reduced human vascular endothelial inhibin, and a reducing agent sodium cyanoborohydride is added for reaction; in the presence of sodium cyanoborohydride, monomethoxy polyethylene glycol propionaldehyde and primary amine of an N-terminal amino bond of the reduced human endostatin are subjected to reduction ammoniation reaction. More preferably, the mass ratio of the monomethoxypolyethylene glycol propionaldehyde to the reduced human vascular endothelial inhibin is 1:1, the pH value of the reaction is 4.5-5.5, the reaction time is 4-6 hours, and the reaction temperature is room temperature.

In a preferred embodiment of the invention, the reduced human endostatin in the highly active state is further enriched by purification, as indicated by in vitro activity. Therefore, the preparation method also comprises purifying the coupling product of the polyethylene glycol and the reduced human vascular endothelial inhibin by cation exchange chromatography (the medium is sulfopropyl dextran gel (SP for short)). The activity of a specific component (for example, a component eluted by an elution buffer with the conductivity of 25 mS/cm) obtained after purification by cation exchange chromatography (the medium is sulfopropyl dextran gel (SP for short)) is 50-100 times that of oxidized state human endostatin modified by polyethylene glycol.

5.Pharmaceutical composition

The invention provides a pharmaceutical composition, which comprises the reduced human vascular endothelial inhibin or the analogue thereof or the modified reduced human vascular endothelial inhibin or the analogue thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition comprises an effective amount (e.g. 0.01-99.9 wt%) of reduced human endostatin or an analog thereof or a modified reduced human endostatin or an analog thereof, and a pharmaceutically acceptable carrier. Such carriers are, for example, but not limited to, diluents (e.g., water), excipients, and the like; binders such as cellulose derivatives, gelatin, polyvinylpyrrolidone, etc.; fillers such as starch and the like; disintegrating agents such as calcium carbonate, sodium bicarbonate; lubricants such as calcium stearate or magnesium stearate, and the like. In addition, other adjuvants such as flavoring agents and sweetening agents may also be added to the composition. For oral administration, it can be prepared into conventional solid preparations such as tablet, powder or capsule; for injection, it can be prepared into injection. The specific dosage of the pharmaceutical composition can be determined by a physician according to the results of clinical trials and the condition, age, etc. of the patient.

The pharmaceutical composition can be prepared into common preparations and can also be sustained-release preparations. The sustained release preparation is selected from microcapsule, hydrogel, microsphere, micro osmotic pump or liposome, etc. The reduced human vascular endothelial inhibin or the analogue thereof or the modified reduced human vascular endothelial inhibin or the analogue thereof is prepared into a sustained release preparation, so that the half-life period of the reduced human vascular endothelial inhibin or the analogue thereof in vivo can be prolonged. When in use, the reduced human endostatin or the analogue thereof or the modified reduced human endostatin or the analogue thereof is put into a medicinal carrier (such as hydrogel, liposome and the like), so that the reduced human endostatin or the analogue thereof is slowly released from the medicinal carrier, and a stable concentration of the reduced human endostatin or the analogue thereof is maintained in vivo.

6.Use of

The examples of the invention have demonstrated that the reduced human endostatin or the analogues thereof and the polyethylene glycol modified reduced human endostatin or the analogues thereof have the activity of inhibiting the proliferation and migration of vascular endothelial cells. Therefore, the reduced human vascular endothelial inhibin or the analogues thereof, the polyethylene glycol modified reduced human vascular endothelial inhibin or the analogues thereof and the corresponding pharmaceutical compositions can be used for preparing antitumor drugs. Preferably, the tumor is selected from lung cancer, neuroendocrine tumor, colon cancer, bone cancer, liver cancer, stomach cancer, pancreatic cancer, oral cancer, breast cancer, prostate cancer, lymph cancer, esophageal cancer, oral cancer, nasopharyngeal cancer, cervical cancer, sarcoma, renal cancer, biliary cancer and malignant melanoma. In addition, the reduced human vascular endothelial inhibin or the analogues thereof and the polyethylene glycol modified reduced human vascular endothelial inhibin or the analogues thereof and the corresponding pharmaceutical composition can treat other diseases related to angiogenesis, such as macular degeneration and the like. Therefore, the reduced state human vascular endothelial inhibin or the analogue thereof, the polyethylene glycol modified reduced state human vascular endothelial inhibin or the analogue thereof and the corresponding pharmaceutical composition can be used for preparing anti-angiogenesis medicines.

7.Modified proteins or polypeptides

The invention provides a new idea that insoluble proteins (such as transmembrane proteins, collagen, fibronectin and the like) which account for a large proportion of proteins in a body locally play important biological functions, the structure and the function of the insoluble proteins are difficult to directly research by the prior art due to poor solubility under the condition of physiological pH value, and the insoluble proteins are difficult to develop into medicines for systemic administration. Therefore, the invention provides a modified protein or polypeptide, wherein the protein or polypeptide is not soluble in water under physiological pH value conditions, and the modified substance is selected from any one of high molecular polymer, protein molecule or fragment thereof, small molecular substance or combination thereof. In the present invention, a protein or polypeptide that is not soluble in water at physiological pH can be covalently modified or non-covalently bound using a modifier (e.g., a high molecular weight polymer or a small molecular weight substance), or a modifier (e.g., a protein molecule or a fragment thereof) can form a fusion protein with a protein or polypeptide that is not soluble in water at physiological pH. In a preferred embodiment of the present invention, the protein molecule or its fragment includes albumin, immunoglobulin, cytokine or their fragments, etc., preferably an immunoglobulin Fc fragment, and the protein or polypeptide modified by the immunoglobulin Fc fragment (insoluble in water at physiological pH) can be obtained by chemical modification or by fusion expression. When the protein or polypeptide modified by the immunoglobulin Fc fragment is expressed by fusion, the protein or polypeptide modified by the immunoglobulin Fc fragment is a fusion protein formed by the immunoglobulin Fc fragment and the protein or polypeptide.

Preferably, the high molecular weight polymer is insoluble in water at physiological pHIs/are as followsA protein or polypeptide is modified. More preferably, the polyethylene glycol is insoluble in water at physiological pHIs/are as followsThe protein or polypeptide is modified. In the prior art, polyethylene glycol is generally used to increase the half-life of the drug in vivo metabolism. The invention utilizes polyethylene glycol to modify water insolubility under physiological pH value conditionIs/are as followsA protein or polypeptide capable of increasing the solubility of a protein or polypeptide that is not soluble in water at physiological pH so that it becomes soluble in water at physiological pH. Book (notebook)Proteins or polypeptides that are not soluble in water at physiological pH of the invention include, but are not limited to, native proteins or polypeptides or analogs thereof.

The technical solution provided by the present invention is further described with reference to specific embodiments. The following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

Comparative example 1 polyethylene glycol modified degree of Endoku

The degree used in this comparative example was that produced by Shandong Xianshenmaedijin biopharmaceutical Co., ltd, and was commercially available. Dialyzing to 30mM HAc-NaAc, pH4.5-5.5 buffer solution, adding equal amount of monomethoxypolyethylene glycol propionaldehyde (mPEG-ALD, 20 kDa), and adding reducing agent sodium cyanoborohydride (NaBH) with final concentration of 20mM ₃ CN), stirring uniformly, standing for 4-6 hours at room temperature, and detecting by SDS-PAGE electrophoresis. SDS-PAGE electrophoresis detection confirms that the modified degree of the polyethylene glycol is successfully obtained.

Example 1 polyethylene glycol modified reduced recombinant human endostatin

1. Preparation of reduction state recombinant human vascular endothelial inhibin

The reduced recombinant human vascular endothelial inhibin in this embodiment comprises recombinant human vascular endothelial inhibin with "amino acid sequence 2" or "amino acid sequence 4". Cloning a DNA fragment containing a gene coding the 'amino acid sequence 1' or the 'amino acid sequence 3' into an escherichia coli expression vector pET30a, transforming the constructed vector into escherichia coli, and fermenting and expressing a target protein to be an inclusion body (methionine is automatically added to the N end of the escherichia coli expression protein). Dissolving the inclusion body expressed by the escherichia coli into 20mM Tris-HCl buffer solution containing 7M guanidine hydrochloride or 8M urea and 20mM Dithiothreitol (DTT), standing for 3-6 hours at room temperature, centrifuging to obtain supernatant, and purifying the reduced recombinant human vascular endothelial inhibin from the supernatant.

2. Polyethylene glycol modified reduced recombinant human vascular endothelial inhibin

Dialyzing the purified reduced recombinant human vascular endothelial inhibin into 30mM HAc-NaAc, pH4.5-5.5 buffer solution, adding equal mass of monomethylOxyethylenepropanal (mPEG-ALD, 20 kDa) was added to a final concentration of 20mM sodium cyanoborohydride (NaBH. RTM.) ₃ CN), stirring uniformly, standing for 4-6 hours at room temperature, and detecting by SDS-PAGE electrophoresis (figure 1). As shown in FIG. 1, compared with unmodified reduced recombinant human endostatin, the molecular weight of polyethylene glycol modified reduced recombinant human endostatin is larger, and the mobility thereof in SDS-PAGE electrophoresis is obviously smaller, which indicates that polyethylene glycol successfully modifies the reduced recombinant human endostatin.

Example 2 coupling of 20kDa polyethylene glycol to N-terminus of reduced recombinant human endostatin

The reduced recombinant human vascular endothelial inhibin of this example comprises recombinant human vascular endothelial inhibin with "amino acid sequence 2". The preparation method of the reduced recombinant human vascular endothelial inhibin is the same as that in example 1.

Dialyzing the reduced recombinant human endostatin into 30mM HAc-NaAc buffer solution with pH4.5-5.5, adding equal mass of monomethoxypolyethylene glycol propionaldehyde (mPEG-ALD, 20 kDa), adding a reducing agent sodium cyanoborohydride (NaBH 3 CN) with the final concentration of 20mM, uniformly stirring, standing at room temperature for 4-6 hours, and carrying out SDS-PAGE electrophoresis detection. One polyethylene glycol is coupled with one reduced endostatin molecule, and the coupling site is the alpha-amino at the N end of the reduced endostatin, and a small amount of the reduced endostatin is modified or unmodified by non-specific multiple sites. The reaction solution can be directly used for column purification to remove the multi-modified and unmodified reduced endostatin.

EXAMPLE 3 purification of polyethylene glycol-modified reduced recombinant human endostatin

The polyethylene glycol modified reduced recombinant human vascular endothelial inhibin obtained in example 1 was purified by cation exchange chromatography (medium is sulfopropyl dextran gel (abbreviated as SP)), and eluted with elution buffers (30mM NaAc, pH = 5.0) containing NaCl at different concentrations) of different conductivities to obtain FT (effluent) fractions, 25, 30 and 40mS/cm fractions, wherein the FT fraction is a liquid fraction eluted by directly sampling the polyethylene glycol modified reduced recombinant human vascular endothelial inhibin, the 25mS/cm fraction is a fraction eluted with an elution buffer having a conductivity of 25mS/cm, the 30mS/cm fraction is a fraction eluted with an elution buffer having a conductivity of 30mS/cm, and the 40mS/cm fraction is a fraction eluted with an elution buffer having a conductivity of 40 mS/cm.

Test example 1 determination of inhibitory Activity of polyethylene glycol-modified reduced recombinant human endostatin on migration of HMEC cells

The inhibitory activity of the polyethylene glycol modified degree and the polyethylene glycol modified reduced recombinant human vascular endothelial inhibin prepared respectively in comparative example 1 and example 1 on HMEC cell migration was determined by using HMEC cells.

For HMEC cells in logarithmic growth phase, a 24-well plate is taken, 800. Mu.l of cell culture solution is added into each well, then 200. Mu.l of drugs with different concentrations are added into each well, and the final volume is 1ml. Another 24-well plate was taken and the Transwell chamber was placed over the well. HMEC cells were digested with 0.25% trypsin-EDTA, centrifuged, resuspended in culture medium and counted, adjusting cell concentration to 10 ⁶ One per ml. Mu.l of the cell suspension was added to the upper Transwell chamber, 40. Mu.l of the drug was added at different concentrations, and the incubated Transwell chamber was placed in a drug-loaded 24-well plate. Putting 24-well plate at 37 deg.C, 5% CO ₂ Incubate in the incubator for 4 hours. The upper Transwell chamber was removed and cell migration in the 24-well plate was detected by fluorescence.

The activities of polyethylene glycol modified reduced recombinant human endostatin (nES, prepared in example 1) in inhibiting the migration of HMEC cells under the conditions of administration concentrations of 1, 2 and 5 μ g/ml were measured, and the inhibition rates were respectively 28%, 76% and 90%, the inhibition rate of positive control 10 μ g/ml in saturation (Endostar) was 48%, the inhibition rate of 10 μ g/ml polyethylene glycol modified saturation (PEG-Endostar, prepared in comparative example 1) was 54%, the blank control was Buffer solution (30 mM HAc-NaAc, ph5.2 Buffer), the inhibition rate of each administration group was calculated on the basis of the blank control, and the inhibition rate of each administration group was calculated on the assumption that the inhibition rate of the blank control group was 0 (fig. 2). As shown in FIG. 2, when the administration concentration of the reduced recombinant human endostatin (nES) modified by polyethylene glycol is only 1/5 of the degree of modification by polyethylene glycol, the inhibition rate of inhibiting the migration of HMEC cells is much higher than that of the degree of modification by polyethylene glycol or that of the degree of inhibition by HMEC cells. Therefore, the activity of the reduced recombinant human endostatin modified by polyethylene glycol is 5-10 times of that of Endostar or polyethylene glycol-modified Endostar.

Test example 2 measurement of inhibitory Activity of purified fractions on migration of HMEC cells

The FT (effluent) prepared in example 3, 25, 30, 40mS/cm fractions, and polyethylene glycol-modified enrichments prepared in comparative example 1 were assayed for HMEC cell migration inhibitory activity.

For HMEC cells in logarithmic phase, a 24-well plate is taken, 800 μ l of cell culture solution is added into each well, then 200 μ l of drugs with different concentrations are added into each well, and the final volume is 1ml. Another 24-well plate was taken and the Transwell chamber was placed over the well. HMEC cells were digested with 0.25% trypsin-EDTA, centrifuged, resuspended in culture medium and counted, adjusting cell concentration to 10 ⁶ Each/ml. Mu.l of the cell suspension was added to the upper Transwell chamber, 40. Mu.l of the drug was added at different concentrations, and the incubated Transwell chamber was placed in a drug-loaded 24-well plate. Putting 24-well plate at 37 deg.C, 5% CO ₂ Incubate in the incubator for 4 hours. The Transwell upper chamber was removed and cell migration in 24-well plates was detected by fluorescence.

The activity of inhibiting HMEC cell migration under the condition of 2ug/ml administration concentration is measured, the inhibition rates of FT (effluent), 25, 30 and 40mS/cm components are respectively-6%, 94%, 80% and 15%, the inhibition rate of a positive control 10 ug/ml component (Endostar) is 53%, the inhibition rate of a 10 ug/ml polyethylene glycol modified component (PEG-Endostar prepared in comparative example 1) is 58%, the inhibition rate of a blank control is buffer solution (30 mM HAc-NaAc, pH 5.2), the inhibition rate of each administration group is calculated on the basis of the blank control, and the inhibition rate of the blank control is assumed to be 0 (FIG. 3). As can be seen from FIG. 3, the 25mS/cm fraction was the most active.

The activities of 25mS/cm fractions at administration concentrations of 0.1, 0.2, 0.5, and 1.0. Mu.g/ml for inhibiting the migration of HMEC cells were measured, and the inhibition rates were 35%, 73%, 81%, and 92%, respectively, the inhibition rate of the positive control 10. Mu.g/ml for Endocar (Endocar) was 43%, the inhibition rate of the 10. Mu.g/ml for PEG-Endocar (prepared in comparative example 1) was 41%, the blank control was Buffer (30 mM HAc-NaAc, pH5.2 Buffer), the inhibition rate of each administration group was calculated based on the blank control, and the inhibition rate of each administration group was calculated assuming that the inhibition rate of the control group was 0 (FIG. 4). As can be seen from FIG. 4, the inhibition rate of the 25mS/cm fraction on HMEC cell migration was much higher than that of the polyethylene glycol-modified degree or degree at the administration concentration of only 1/50 of the polyethylene glycol-modified degree. Therefore, the activity of the 25mS/cm component (polyethylene glycol modified reduced state recombinant human vascular endostatin) is 50-100 times of that of Endostar or PEG-Endostar, and the component has obvious activity at the concentration of 100-200 ng/ml.

Test example 3 measurement of inhibitory Activity of polyethylene glycol-modified reduced recombinant human endostatin on proliferation of HMEC or HUVEC cells

HUVEC cells were cultured for passage to 3-5 passages and were ready for inoculation when the cell status was good. The polyethylene glycol modified reduced recombinant human vascular endothelial inhibin is diluted by 20mM PB buffer solution, three parallel holes are arranged in each gradient, and 40 mu l of the polyethylene glycol modified reduced recombinant human vascular endothelial inhibin is added into each hole in a 96-hole plate. The cell density was 6000 cells/ml, and 160. Mu.l of cell suspension was added to each well of the former 96-well plate at 37 ℃ in 5% CO ₂ Culturing in an incubator for 48-72h, and determining the proliferation activity by an MTT method.

Adding 0.5 μ g/ml polyethylene glycol modified reduced recombinant human endostatin into HMEC cell line culture medium, and adding 5% CO at 37 deg.C ₂ The growth in the incubator for 24h is shown in FIG. 5a, and the growth in the blank Buffer (30 mM HAc-NaAc, pH5.2 Buffer) is shown in FIG. 5b. As can be seen from the comparison between FIG. 5a and FIG. 5b, the reduced recombinant human vascular endostatin modified by polyethylene glycol can significantly inhibit the proliferation of HMEC cells.

Sequence listing

<110> Liu Peng

<120> an anti-angiogenic drug

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<220>

<223> amino acid sequence of endostatin

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His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn

1 5 10 15

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20 25 30

Cys Phe Gln Gln Ala Arg Ala Val Gly Leu Ala Gly Thr Phe Arg Ala

35 40 45

Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser Ile Val Arg Arg Ala

50 55 60

Asp Arg Ala Ala Val Pro Ile Val Asn Leu Lys Asp Glu Leu Leu Phe

65 70 75 80

Pro Ser Trp Glu Ala Leu Phe Ser Gly Ser Glu Gly Pro Leu Lys Pro

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Gly Ala Arg Ile Phe Ser Phe Asp Gly Lys Asp Val Leu Arg His Pro

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Arg Leu Thr Glu Ser Tyr Cys Glu Thr Trp Arg Thr Glu Ala Pro Ser

130 135 140

Ala Thr Gly Gln Ala Ser Ser Leu Leu Gly Gly Arg Leu Leu Gly Gln

145 150 155 160

Ser Ala Ala Ser Cys His His Ala Tyr Ile Val Leu Cys Ile Glu Asn

165 170 175

Ser Phe Met Thr Ala Ser Lys

180

<210> 2

<211> 184

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> amino acid sequence of endostatin

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Met His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu

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Asn Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe

20 25 30

Gln Cys Phe Gln Gln Ala Arg Ala Val Gly Leu Ala Gly Thr Phe Arg

35 40 45

Ala Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser Ile Val Arg Arg

50 55 60

Ala Asp Arg Ala Ala Val Pro Ile Val Asn Leu Lys Asp Glu Leu Leu

65 70 75 80

Phe Pro Ser Trp Glu Ala Leu Phe Ser Gly Ser Glu Gly Pro Leu Lys

85 90 95

Pro Gly Ala Arg Ile Phe Ser Phe Asp Gly Lys Asp Val Leu Arg His

100 105 110

Pro Thr Trp Pro Gln Lys Ser Val Trp His Gly Ser Asp Pro Asn Gly

115 120 125

Arg Arg Leu Thr Glu Ser Tyr Cys Glu Thr Trp Arg Thr Glu Ala Pro

130 135 140

Ser Ala Thr Gly Gln Ala Ser Ser Leu Leu Gly Gly Arg Leu Leu Gly

145 150 155 160

Gln Ser Ala Ala Ser Cys His His Ala Tyr Ile Val Leu Cys Ile Glu

165 170 175

Asn Ser Phe Met Thr Ala Ser Lys

180

<210> 3

<211> 191

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<213> Artificial Sequence (Artificial Sequence)

<220>

<223> amino acid sequence of endostatin

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Gly Gly Ser His His His His His His Ser His Arg Asp Phe Gln Pro

1 5 10 15

Val Leu His Leu Val Ala Leu Asn Ser Pro Leu Ser Gly Gly Met Arg

20 25 30

Gly Ile Arg Gly Ala Asp Phe Gln Cys Phe Gln Gln Ala Arg Ala Val

35 40 45

Gly Leu Ala Gly Thr Phe Arg Ala Phe Leu Ser Ser Arg Leu Gln Asp

50 55 60

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65 70 75 80

Asn Leu Lys Asp Glu Leu Leu Phe Pro Ser Trp Glu Ala Leu Phe Ser

85 90 95

Gly Ser Glu Gly Pro Leu Lys Pro Gly Ala Arg Ile Phe Ser Phe Asp

100 105 110

Gly Lys Asp Val Leu Arg His Pro Thr Trp Pro Gln Lys Ser Val Trp

115 120 125

His Gly Ser Asp Pro Asn Gly Arg Arg Leu Thr Glu Ser Tyr Cys Glu

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Thr Trp Arg Thr Glu Ala Pro Ser Ala Thr Gly Gln Ala Ser Ser Leu

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Leu Gly Gly Arg Leu Leu Gly Gln Ser Ala Ala Ser Cys His His Ala

165 170 175

Tyr Ile Val Leu Cys Ile Glu Asn Ser Phe Met Thr Ala Ser Lys

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<220>

<223> amino acid sequence of vascular endostatin

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1 5 10 15

Pro Val Leu His Leu Val Ala Leu Asn Ser Pro Leu Ser Gly Gly Met

20 25 30

Arg Gly Ile Arg Gly Ala Asp Phe Gln Cys Phe Gln Gln Ala Arg Ala

35 40 45

Val Gly Leu Ala Gly Thr Phe Arg Ala Phe Leu Ser Ser Arg Leu Gln

50 55 60

Asp Leu Tyr Ser Ile Val Arg Arg Ala Asp Arg Ala Ala Val Pro Ile

65 70 75 80

Val Asn Leu Lys Asp Glu Leu Leu Phe Pro Ser Trp Glu Ala Leu Phe

85 90 95

Ser Gly Ser Glu Gly Pro Leu Lys Pro Gly Ala Arg Ile Phe Ser Phe

100 105 110

Asp Gly Lys Asp Val Leu Arg His Pro Thr Trp Pro Gln Lys Ser Val

115 120 125

Trp His Gly Ser Asp Pro Asn Gly Arg Arg Leu Thr Glu Ser Tyr Cys

130 135 140

Glu Thr Trp Arg Thr Glu Ala Pro Ser Ala Thr Gly Gln Ala Ser Ser

145 150 155 160

Leu Leu Gly Gly Arg Leu Leu Gly Gln Ser Ala Ala Ser Cys His His

165 170 175

Ala Tyr Ile Val Leu Cys Ile Glu Asn Ser Phe Met Thr Ala Ser Lys

180 185 190

Claims

1. A reduced form of human endostatin or an analog thereof, wherein the reduced form of human endostatin comprises at most one pair of disulfide bonds formed from thiol groups on two cysteine residues of a human endostatin molecule.

2. The reduced human endostatin or analog thereof of claim 1, wherein the reduced human endostatin analog comprises at least one or a combination of:

-a partial peptide stretch of native vascular endostatin; or

-altering the amino acid sequence of native endostatin;

3. The reduced human endostatin or an analog thereof according to claim 1 or 2, wherein the amino acid sequence of the reduced human endostatin is shown in SEQ ID NO 1, 2, 3 or 4.

4. The reduced human endostatin or an analog thereof according to claim 3, wherein the amino acid sequence of the reduced human endostatin is shown in SEQ ID NO 1, and the disulfide bond formation comprises:

None of the above two disulfide bonds are formed.

5. A modified reduced form of human endostatin or an analog thereof, wherein the reduced form of human endostatin comprises at most one pair of disulfide bonds formed from thiol groups on two cysteine residues of a human endostatin molecule.

6. The reduced human endostatin or analog thereof of claim 5, wherein the reduced human endostatin analog comprises at least one or a combination of:

-a partial peptide stretch of native vascular endostatin; or alternatively

-altering the amino acid sequence of native endostatin;

7. The modified reduced human endostatin or the analog thereof according to claim 5 or 6, wherein the amino acid sequence of the reduced human endostatin is shown in SEQ ID NO 1, 2, 3 or 4.

8. The modified reduced human endostatin or an analog thereof according to claim 7, wherein the amino acid sequence of the reduced human endostatin is shown in SEQ ID NO 2 or 4.

9. The reduced human endostatin or the analog thereof of claim 7, wherein the amino acid sequence of the reduced human endostatin is shown in SEQ ID NO. 1, and the disulfide bond formation comprises:

None of the above two disulfide bonds are formed.

10. A modified reduced human endostatin or its analog according to claim 5, wherein the modifier is selected from any one of high molecular polymer, protein molecule or its fragment, small molecule substance or their combination.

11. The modified reduced human endostatin or the analog thereof of claim 10, wherein the high molecular polymer is polyethylene glycol.

12. The modified reduced human endostatin or the analog thereof of claim 11, wherein the amino acid sequence of the reduced human endostatin is shown in SEQ ID NO 2 or 4.

13. The modified reduced human endostatin or analog thereof of claim 11, wherein one reduced human endostatin or analog molecule is coupled to one or more polyethylene glycol molecules.

14. The modified reduced human endostatin or its analog of claim 13, wherein the site at which the reduced human endostatin or its analog is coupled to the polyethylene glycol is one of the N-terminal α -amino group of the reduced human endostatin or its analog, the e-amino group of the lysine residue side chain, the thiol group of the cysteine residue side chain, the carboxyl group of the aspartic acid residue side chain, the carboxyl group of the glutamic acid residue side chain, or a combination thereof.

15. The modified reduced human endostatin or analog thereof of claim 14, wherein the site at which the reduced human endostatin or analog thereof is coupled to the polyethylene glycol is an α -amino group at the N-terminus of the reduced human endostatin or analog thereof.

16. A modified reduced human endostatin or an analog thereof according to claim 11, wherein the polyethylene glycol molecule has an average molecular weight of between 1,000 and 100,000 daltons.

17. A modified reduced human endostatin or an analog thereof according to claim 16, wherein the polyethylene glycol molecule has an average molecular weight of between 5,000 and 40,000 daltons.

18. A pharmaceutical composition comprising a reduced form of human endostatin or an analog thereof of any one of claims 1-4 or a modified reduced form of human endostatin or an analog thereof of any one of claims 5-17, and a pharmaceutically acceptable carrier.

19. The pharmaceutical composition of claim 18, wherein the pharmaceutical composition is a sustained release formulation.

20. A modified protein or polypeptide, wherein said protein or polypeptide is not soluble in water at physiological pH, and is modified to be soluble in water at physiological pH; the modifier is selected from any one of high molecular polymer, protein molecule or fragment thereof, small molecular substance or combination thereof.