Peptides capable of inhibiting gp96, methods of making and uses thereof

Abstract

The present application relates to the field of biological medicine, in particular to PEG modified polypeptide capable of inhibiting gp96, its preparation method and use. Specifically, the present application relates to PEGylated polypeptide, comprising PIBC and PEG with an average molecular weight of 20000-40000 connected by covalent bond. The present application also relates to the method for preparing the PEGylated polypeptide, the medicine or preparation comprising the PEGylated polypeptide, and the use of the PEGylated polypeptide.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a PEG modified polypeptide capable of inhibiting gp96, a preparation method and use thereof. BACKGROUND

[0002] Breast cancer is the highest incidence of cancer in Chinese women, and its incidence is increasing at a rate of 3% per year, becoming the fastest growing cancer in mortality in the city. Although there are many drugs for breast cancer at present, there are certain limitations, of which the most significant is the lack of therapeutic drugs for triple negative breast cancer. Triple negative breast cancer (TNBC) refers to breast cancer with negative estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her-2), accounting for about 15%-20% of pathological types of breast cancer, which is a highly malignant tumor. At present, the treatment means and drugs are extremely limited, and there is a lack of therapeutic targets and targeted drugs, and new potential targets are urgently needed to be found.

[0003] Through immunohistochemical detection of tumor tissues of 80 breast cancer patients, it was found that about 70% of the patients had high expression of heat shock protein gp96 on the membrane of breast cancer cells, including most of the triple negative breast cancer patients, while the normal cell membrane surface did not express gp96, so that the membrane gp96 can be used as a molecular marker and a potential therapeutic target for triple negative breast cancer. On this basis, based on the amino acid sequence and spatial conformation of gp96, a polypeptide containing an alpha-helix sequence was designed, which was designated as PIBC. The polypeptide can specifically bind to gp96, block the rearrangement and conformational change of the internal motif of gp96 molecules, and then interfere with the interaction of membrane gp96 with HER-2, uPAR and ER-a36 and cause the endocytosis and degradation of these tumor proteins (Chinese patent ZL201110159487.4). In vitro cell experiments and in vivo tumor-bearing mouse experiments found that the polypeptide drug effectively inhibited the growth of triple negative breast cancer, promoted cell apoptosis, and inhibited tumor invasion and metastasis, indicating that the polypeptide can be used as a candidate drug for targeting triple negative breast cancer.

[0004] Although the action site of the polypeptide is specific, it has strong stimulation, low solubility, high immunogenicity, easy degradation by proteases and clearance by the kidney, which seriously restricts its clinical application. SUMMARY

[0005] One of the technical problems to be solved by the present application is at least how to reduce the toxicity and stimulation of PIBC, improve its hemolytic property, and prolong the half-life of PIBC, so that PIBC is safer and more effective. The inventors found that modifying PIBC with polyethylene glycol (PEG) can achieve the above effects, thereby providing the following application.

[0006] PEGylated polypeptides

[0007] In one aspect, the present application provides a PEGylated polypeptide comprising a PIBC and a PEG having an average molecular weight (number average molecular weight (Mn)) of about 20,000-40,000, and the PIBC is covalently linked to the PEG, wherein the PIBC is selected from the group consisting of:

[0008] A1): a polypeptide having an amino acid sequence as set forth in SEQ ID NO. 1; and

[0009] A2): a polypeptide derived from A1) and having the same function as A1), having an amino acid sequence with one or more (e.g., 1-10 or 1-5 or 1-3) substitutions (e.g., conservative substitutions) and / or deletions and / or additions of amino acid residues compared to the amino acid sequence as set forth in SEQ ID NO. 1, or having at least 60%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0010] In certain embodiments, the PEG is linked to the N-terminus or C-terminus of the PIBC.

[0011] In certain embodiments, the PEG is a linear PEG or a branched PEG.

[0012] In certain embodiments, the PEG has an average molecular weight of about 20,000-25,000, 25,000-30,000, 30,000-35,000 or 35,000-40,000, e.g., about 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000, 33,000, 34,000, 35,000, 36,000, 37,000, 38,000, 39,000 or 40,000.

[0013] In the PEGylated polypeptide of the present application, the PIBC can be covalently linked to the PEG through a linker, or directly linked to the PEG through a covalent bond.

[0014] In certain embodiments, the PEGylated polypeptide has the following structure:

[0015] R-(CH2CH2O) n -linker-PIBC;

[0016] wherein n is the degree of polymerization of the PEG, and n satisfies the condition that the PEG has a molecular weight of about 20,000-40,000, R is an end group of the PEG, e.g., a methoxy group; and linker is a linker, e.g., The amino acid residue is a thiol group-bearing amino acid (e.g., cysteine (Cys)) residue.

[0017] In some embodiments, the PEGylated polypeptide has a structure as shown in Formula I:

[0018]

[0019] In Formula I, n is the degree of polymerization of PEG, and n satisfies the condition that the molecular weight of PEG is about 20,000-40,000.

[0020] In some embodiments, in Formula I, Cys is connected to PIBC via a peptide bond formed between the carboxyl group of Cys and the N-terminal amino group of PIBC, or via a peptide bond formed between the amino group of Cys and the C-terminal carboxyl group of PIBC.

[0021] In some embodiments, the covalent linkage is achieved by Michael addition reaction using a reactant 1 and a reactant 2, wherein the reactant 1 is a PEG having a maleimide group at one end, and the reactant 2 is a PIBC having a thiol group-bearing amino acid residue introduced at the N- or C-terminus. The Michael addition reaction occurs between the maleimide group and the thiol group.

[0022] In some embodiments, the reactant 1 has a structure as shown in Formula II:

[0023]

[0024] In Formula II, n is the degree of polymerization of PEG, and n satisfies the condition that the molecular weight of PEG is 20,000-40,000. The reactant 1 can be referred to as methoxypolyethylene glycol maleimide (mPEGxMal, where x is the average molecular weight of PEG; Mal represents maleimide, which is modified at one end of PEG; and m represents methoxy group).

[0025] In some embodiments, in the reactant 2, the thiol group-bearing amino acid residue is a cysteine residue. In some embodiments, the reactant 2 is a polypeptide having an amino acid sequence as shown in SEQ ID NO. 2 or SEQ ID NO. 3.

[0026] Methods of making pegylated polypeptides

[0027] In one aspect, the present application provides a method for preparing the PEGylated polypeptide of the present application, comprising the step of covalently linking a PIBC to a PEG having an average molecular weight of about 20,000-40,000.

[0028] In certain embodiments, the method comprises the step of performing a Michael addition reaction between a reactant 1 and a reactant 2; the reactant 1 is a PEG having a maleimide attached at one end; the reactant 2 is a PIBC having a thiol-containing amino acid residue introduced at the N- or C-terminus. The Michael addition reaction occurs between the maleimide and the thiol.

[0029] In certain embodiments, the reactant 1 or reactant 2 is as defined above.

[0030] In certain embodiments, the Michael addition reaction is performed at a pH of about 7.2-7.6. In certain embodiments, the Michael addition reaction is performed in a NaH2P04buffer solution.

[0031] In certain embodiments, the Michael addition reaction is performed at room temperature (e.g., 20-30 °C).

[0032] In certain embodiments, reactant 1 is in excess relative to reactant 2. In certain embodiments, the starting molar ratio of reactant 1 to reactant 2 is about 2-10: 1.

[0033] In certain embodiments, the method further comprises the step of purifying the reaction product. In certain embodiments, the purification is achieved by chromatography (e.g., ion exchange chromatography or high performance liquid chromatography).

[0034] Pharmaceutical compositions, formulations, uses

[0035] In one aspect, the present application provides a pharmaceutical composition comprising a PEGylated polypeptide of the present application. In certain embodiments, the pharmaceutical composition is used for treating and / or preventing a disease associated with overexpression of gp96 protein in a subject.

[0036] The pharmaceutical composition of the present application can further comprise one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers that can be used in the present application include, but are not limited to, fillers, diluents, binders, wetting agents, disintegrants, lubricants, surfactants, preservatives, colorants, flavoring agents, fragrances, effervescent agents, emulsifiers, flocculants, deflocculants, bacteriostatic agents, solubilizers. In certain embodiments, the pharmaceutically acceptable carrier is selected from the group consisting of ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), glycerol, sorbic acid, potassium sorbate, water, protamine sulfate, sodium phosphate (dibasic), potassium phosphate (dibasic), sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, beeswax, polyethylene-polyoxypropylene block polymers, lanolin, and any combination thereof.

[0037] The pharmaceutical compositions of the present application can be prepared in various suitable dosage forms, including, but not limited to, oral dosage forms, injectable dosage forms (e.g., dosage forms suitable for subcutaneous injection, intramuscular injection, or intravenous injection), inhalable dosage forms, mucosally administrable dosage forms, or topically administrable dosage forms. In certain embodiments, the pharmaceutical compositions are prepared in oral dosage forms, such as tablets, capsules, granules, oral solutions, oral suspensions, pellets, or microtablets.

[0038] In one aspect, the present application provides use of a PEGylated polypeptide of the present application in the manufacture of a medicament for treating and / or preventing a disease associated with overexpression of gp96 protein (e.g., a tumor) in a subject.

[0039] In one aspect, the present application provides a formulation comprising a PEGylated polypeptide of the present application. In certain embodiments, the formulation is used to bind to gp96 protein, inhibit tumor cell proliferation and / or growth and / or invasion, promote tumor cell apoptosis, and / or, inhibit tumor growth.

[0040] In one aspect, the present application provides use of a PEGylated polypeptide of the present application in the manufacture of a formulation for binding to gp96 protein, inhibiting tumor cell proliferation and / or growth and / or invasion, promoting tumor cell apoptosis, and / or, inhibiting tumor growth.

[0041] The formulations of the present application can be administered in vivo or in vitro; for example, the formulations are administered to a subject in vivo to bind to gp96 protein in the subject, to inhibit tumor cell proliferation and / or growth and / or invasion in the subject, to promote tumor cell apoptosis in the subject, and / or, to inhibit tumor growth in the subject; or, the formulations are administered to gp96 protein in vitro to bind to gp96 protein in vitro; or, the formulations are administered to cells (e.g., cell lines or cells from a subject, such as tumor cells) in vitro to inhibit tumor cell proliferation and / or growth and / or invasion in vitro, and / or, to promote tumor cell apoptosis in vitro.

[0042] In one aspect, the present application provides a method of treating and / or preventing a disease associated with overexpression of gp96 protein (e.g., a tumor) in a subject, comprising administering to a subject in need thereof a therapeutically and / or prophylactically effective amount of a PEGylated polypeptide or a pharmaceutical composition of the present application.

[0043] In one aspect, the present application provides a method of inhibiting proliferation and / or growth and / or invasion of tumor cells, promoting apoptosis of tumor cells, and / or inhibiting growth of a tumor, comprising administering to a tumor cell or a tumor a PEGylated polypeptide or a formulation of the present application. In certain embodiments, the tumor cell is present in a subject and the method is performed in vivo in the subject. In certain embodiments, the tumor cell is present in vitro and the method is performed in vitro. The method can be used for prophylactic or therapeutic purposes, or for non-prophylactic or non-therapeutic purposes (e.g., scientific research).

[0044] In the above aspects of the present application, the gp96 protein can be present in vitro or in vivo in a subject.

[0045] In the above aspects of the present application, the tumor includes, but is not limited to, brain tumor, lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, female genital tract cancer, carcinoma in situ, lymphoma, neurofibroma, thyroid cancer, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, prostate tumor, mast cell tumor, multiple myeloma, melanoma, glioma, or sarcoma. In certain embodiments, the tumor is breast cancer, e.g., triple negative breast cancer.

[0046] In the above aspects of the present application, the tumor cell includes, but is not limited to, brain tumor cell, lung cancer cell, squamous cell carcinoma cell, bladder cancer cell, gastric cancer cell, ovarian cancer cell, peritoneal cancer cell, pancreatic cancer cell, breast cancer cell, head and neck cancer cell, cervical cancer cell, endometrial cancer cell, rectal cancer cell, liver cancer cell, kidney cancer cell, esophageal adenocarcinoma cell, esophageal squamous cell carcinoma cell, prostate cancer cell, female genital tract cancer cell, carcinoma in situ cell, lymphoma cell, neurofibroma cell, thyroid cancer cell, bone cancer cell, skin cancer cell, brain cancer cell, colon cancer cell, testicular cancer cell, gastrointestinal stromal tumor cell, prostate tumor cell, mast cell tumor cell, multiple myeloma cell, melanoma cell, glioma cell, or sarcoma cell.

[0047] In certain embodiments, the breast cancer cell is SKBr3 or MDA-MB-231.

[0048] In the above aspects of the present application, the subject can be a mammal, e.g., a bovine, equine, porcine, canine, feline, rodent, primate; wherein, particularly preferred subjects are humans.

[0049] Definitions of terms

[0050] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person of ordinary skill in the art. Also, the laboratory procedures referred to herein are conventional procedures used in the relevant art. In addition, in order to better understand the present application, the definitions and explanations of the relevant terms are provided below.

[0051] In the present application, gp96 protein refers to a heat shock protein (also known as GRP94) with a molecular weight of about 96KD present in the endoplasmic reticulum of eukaryotic cells. The amino acid sequence of gp96 protein is known to those skilled in the art and is found in various public databases (e.g., GenBank database, Genbank Accession NO. AY040226). An exemplary amino acid sequence of wild-type gp96 protein is shown in SEQ ID NO. 4. Thus, in the present application, when referring to the sequence of gp96 protein, it is described using the sequence shown in SEQ ID NO. 4. However, those skilled in the art understand that mutations or variations (including but not limited to, substitutions, deletions, and / or additions) can be naturally produced or artificially introduced in SEQ ID NO. 4 without affecting the biological properties of gp96 protein. Thus, in the present application, the term "gp96 protein" is intended to include all such polypeptides and variants, including the polypeptide shown in SEQ ID NO. 4 and its natural or artificial variants that retain the biological properties of gp96 protein.

[0052] In the present application, PIBC (Peptide Inhibitor for Breast Cancer) is a polypeptide that can bind to gp96 protein. The polypeptide contains an alpha-helix sequence, is capable of specifically binding to gp96, blocks the rearrangement of the internal motif and conformational changes of gp96 molecule, and further interferes with the interaction of membrane gp96 with HER-2, uPAR or ER-a36. An exemplary amino acid sequence of PIBC can be shown in SEQ ID NO. 1. In the present application, the term "PIBC" is intended to include variants of PIBC, which means a polypeptide having one or more (e.g., 1-10 or 1-5 or 1-3) amino acids different (e.g., conservative amino acid substitutions) from the amino acid sequence of PIBC or having at least 60%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, and having the same function as PIBC, which can be one or more of the following functions: (i) capable of specifically binding to gp96, (ii) capable of blocking the rearrangement of the internal motif and conformational changes of gp96 molecule, (iii) capable of interfering with the interaction of membrane gp96 with HER-2, uPAR or ER-a36.

[0053] As used herein, the term "identity" is used in reference to the matching of sequences between two polypeptides or between two nucleic acids. When a position in each of two sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percentage of identity" between two sequences is a function of the number of matching positions shared by the sequences divided by the number of positions compared x 100. For example, if 6 of 10 positions in two sequences are matched then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of 6 positions are matched). Typically, the comparison is made over the full length of the sequences being compared. Such a comparison can be conveniently accomplished by use of the algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443-453, as implemented in the computer program ALIGN available from DNAstar, Inc., using, for example, a program parameter of an affine gap cost of 1 for the gap open penalty and 0.5 for the gap extension penalty. Percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) as integrated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, percentage identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) as implemented in the GAP program, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a gap length weight of 1, 2, 3, 4, 5, or 6, as incorporated in the GCG software package (available at www.gcg.com).

[0054] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the biological activity of a protein / polypeptide comprising the amino acid sequence. Conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis, for example. Conservative amino acid substitutions include substitutions of an amino acid residue for another with similar side chains, e.g., substitutions that take place within a family of amino acid residues that are physicochemically or functionally similar (e.g., have similar size, shape, charge, chemical properties, including ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a conservative substitution is one in which the replacement amino acid residue has a side chain that is similar in size, charge, and / or polarity to the side chain of the original amino acid residue. Methods of identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0055] As used herein, "about" should be understood by those skilled in the art to be a degree of variability acceptable by the skilled artisan, and to vary to some degree depending on the context in which the term is used. If the context of use indicates, the term "about" means plus or minus 10% of the particular numerical value or range.

[0056] As used herein, "effective amount" means an amount that is sufficient to achieve or at least partially achieve a desired effect. For example, a therapeutically effective amount means an amount that is sufficient to cure or at least partially arrest a disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight, and gender, the mode of administration of the drug, and other therapies that the patient is receiving, among other things.

[0057] The amount of the drug administered to the subject depends on the type and severity of the disease or condition, and on the characteristics of the subject, such as general health, age, sex, body weight, and tolerance to the drug, as well as on the type of preparation and the mode of administration of the drug, and on the period or interval of administration, and the like. Those skilled in the art are able to determine the appropriate dose according to these factors and other factors.

[0058] Beneficial effects

[0059] Experiments show that the PEG-modified PIBC of the present application has affinity with gp96 protein, can significantly inhibit tumor cell proliferation (growth), invasion, significantly promote tumor cell apoptosis, effectively inhibit tumor growth caused by tumor cells, and more importantly, can significantly reduce the irritability of PIBC, prolong the half-life of polypeptide in vivo, and ultimately significantly improve the drugability of PIBC. The above results show that the PEG-modified PIBC of the present application can be used as a drug for treating tumors. BRIEF DESCRIPTION OF DRAWINGS

[0060] Figure 1 PEGylated polypeptide mPEG 20000 CY mass spectrum identification result graph.

[0061] Figure 2 PEGylated polypeptide mPEG 40000 CY mass spectrum identification result graph.

[0062] Figure 3 PEGylated polypeptide mPEG 20000 LC mass spectrum identification result graph.

[0063] Figure 4 PEGylated polypeptide mPEG 40000 LC mass spectrum identification result graph.

[0064] Figure 5 For example 5, the identification spectrum of secreted human heat shock protein gp96 expressed by insect cell expression system; 1: molecular weight standard; 2: gp96 protein after purification; 3: western blot result of gp96 protein.

[0065] Figure 6 PIBC and mPEG 20000 CY treatment group tumor inhibition rate. DETAILED DESCRIPTION

[0066] The application will be further described in conjunction with the specific embodiments. The examples are only for illustrating the application, but not for limiting the scope of the application. The experimental methods in the following examples are all conventional methods, unless otherwise specified. The materials, reagents and instruments used in the following examples are commercially available, unless otherwise specified. The quantitative experiments in the following examples are all set in triplicate, and the results are averaged.

[0067] PBS buffer: 8 g NaCl, 0.2 g KCl, 3.625 g Na2HPO4·12H2O, 0.24 g KH2PO4, add water to 1 L, adjust pH to 7.3.

[0068] 5 mM Na2HPO4: 1.7907 g Na2HPO4·12H2O, dilute with deionized water to 1 L.

[0069] 5 mM NaH2PO4: 0.78 g NaH2PO4·2H2O, dilute with deionized water to 1 L.

[0070] Mass spectrometry was performed using a VG PLATFORM mass spectrometer with MALDI-TOF technology.

[0071] In the following examples, the ratio of liquid to liquid is volume to volume, unless otherwise specified; the ratio of solid to liquid is amount of substance to volume, the amount of substance is in mmol, and the volume is in ml; the ratio of solid to solid is mass to mass.

[0072] In the following examples, the room temperature reaction specifically refers to controlling the reaction temperature in the range of 20-30°C, including 20°C and 30°C, unless otherwise specified.

[0073] Example 1, PEGylated polypeptide mPEG 20000 Preparation of CY

[0074] (I) Obtaining of polypeptide

[0075] The polypeptide obtained by adding cysteine to the N-terminus of PIBC shown in SEQ ID NO. 1 in the sequence listing is named CY, and the amino acid sequence of CY is shown in SEQ ID NO. 2 in the sequence listing. The polypeptide CY shown in SEQ ID NO. 2 was synthesized by GenScript Biotech (Shanghai) Co., Ltd.

[0076] (II) PEGylation modification of polypeptide

[0077] Raw material: mPEGxMal (methoxypolyethylene glycol maleimide, x is the average molecular weight of PEG; Mal represents maleimide, which is modified at one end of PEG; m represents methoxyl), the chemical structural formula is shown as formula II:

[0078]

[0079] mPEGxMal (x = 20000, i.e. the average molecular weight of PEG is 20000, mPEG 20000 Mal, product of Beijing KeyGene Technology Co., Ltd.) is added to the thiol group of the N-terminal amino acid cysteine of the polypeptide CY through Michael addition reaction to obtain mPEG 20000 CY.

[0080] 1. Synthesis of mPEG 20000 CY:

[0081] 80 mg (0.004 mmol) of mPEG 20000 Mal is dissolved in 10 ml of 5 mM NaH2PO4buffer solution, and 10 mg (0.002 mmol) of polypeptide CY is added. The pH value of the reaction solution is adjusted to 7.2 with 5 mM Na2HPO4solution. The reaction is monitored by HPLC until the polypeptide reaction is complete.

[0082] 2. Purification and characterization of PEGylated polypeptide mPEG 20000 CY

[0083] mPEG 20000 CY is purified in large quantities by HiTrap SP FF (1 ml). The eluent is mobile phase A1 and mobile phase B1. A1 is composed of a solvent and a solute. The solvent is 20 mM Tris-HCl (pH 7.4), and the solute and its concentration are 1 mM EDTA·2Na and 0.01% (mass percentage) NaN3. B1 is composed of a solvent and a solute. The solvent is 20 mM Tris-HCl (pH 7.4), and the solute and its concentration are 1000 mM NaCl, 1 mM EDTA·2Na and 0.01% (mass percentage) NaN3. The elution conditions are as follows: first, the mixed solution of A1 and B1 with a volume ratio of 80% and 20% is used for flat washing, and then the mixed solution of A1 and B1 with a volume ratio of 70% and 30% is used for elution and sample collection. The collected sample is concentrated to 500 microliters by centrifugation at 4°C and 3500 r / min using a Millipore ultrafiltration centrifuge tube (10KD). The concentrated sample is desalted by HiTrap Desalting (5 ml). After the solvent is freeze-dried, the fluffy PEGylated polypeptide mPEG 20000 CY is obtained.

[0084] mPEG 20000 The chemical structure of CY was characterized by MALDI-TOF mass spectrometry, mPEG 20000 The mass spectrometry characterization results of CY are shown in Table 1. Figure 1 mPEG 20000 The structural formula of CY is shown in Formula I. At this time, the Cys in Formula I is connected to PIBC through a peptide bond formed by the carboxyl group of Cys and the amino group of the N-terminal amino acid residue of PIBC.

[0085] mPEG 20000 The purity of CY was given by an analytical high-performance liquid chromatograph (flow rate: 1 ml / min). The model of the analytical high-performance liquid chromatograph was Agilent 1200, and the model of the chromatographic column used was Angilent Eclipse XDB-C18 Analytical, 5 μm, 4.6 x 1500 6D m. The chromatographic operating conditions were as follows: linear gradient elution, eluent consisting of mobile phase A2 liquid and mobile phase B2 liquid. The mobile phase A2 liquid was trifluoroacetic acid aqueous solution with a trifluoroacetic acid volume percentage of 0.1%, and the mobile phase B2 liquid was trifluoroacetic acid acetonitrile solution with a trifluoroacetic acid volume percentage of 0.1%. The volume percentage of the linear gradient elution B2 liquid was uniformly increased from 40% to 65%, and the volume percentage of the A2 liquid was uniformly decreased from 60% to 35%, the elution time was 11 minutes, the elution flow rate was 1 ml / min, and the ultraviolet detection wavelength was 220 nm. The analytical high-performance liquid chromatograph detection results showed that the obtained mPEG 20000 The purity of CY was 94.5%.

[0086] Example 2, PEGylated polypeptide mPEG 40000 Preparation of CY

[0087] mPEG x Mal (x = 40000, i.e., the average molecular weight of PEG is 40000, MPEG 40000 Mal, product of Beijing Joinnoble Biotech Co., Ltd.) was subjected to addition reaction with the thiol group of the N-terminal amino acid cysteine of polypeptide CY through Michael addition reaction, to obtain mPEG 40000 CY.

[0088] mPEG 40000 CY was prepared according to the method of 1 in step (two) in Example 1, mPEG 20000 Mal was replaced by mPEG 40000 Mal, and the reaction was carried out until the polypeptide reaction was complete.

[0089] The reaction product obtained according to the above procedure was purified by Agilent 1200 reverse phase high performance liquid chromatography. The column type was Angilent Eclipse XDB-C18 Semi-Prep, 5 μm, 9.4 x 250 mm. The chromatographic operating conditions were linear gradient elution, the eluent was composed of A2 and B2 of Example 1. The volume percentage of B2 was linearly increased from 30% to 52% B, and the volume percentage of A2 was linearly decreased from 70% to 48%, the elution time was 11 minutes, the elution flow rate was 2.5 mL / min, and the UV detection wavelength was 220 nm. After lyophilization of the solvent, the fluffy PEGylated polypeptide mPEG 40000 CY was obtained.

[0090] mPEG 40000 The MALDI-TOF mass spectrometry characterization results of CY are shown in Figure 2 mPEG 40000 The structural formula of CY is shown in Formula I. At this time, the Cys in Formula I is connected to PIBC through a peptide bond formed between the carboxyl group of Cys and the amino group of the N-terminal amino acid residue of PIBC.

[0091] mPEG 40000 The purity analysis of CY was the same as described in Example 1, except that the volume percentage of B2 was linearly increased from 20% to 100%, and the volume percentage of A2 was linearly decreased from 80% to 0, and the elution time was 25 minutes. The analytical high performance liquid chromatography detection results showed that the purity of the obtained mPEG 40000 CY was 95.3%.

[0092] Example 3, PEGylated polypeptide mPEG 20000 Preparation of LC

[0093] The polypeptide obtained by adding a cysteine at the C-terminus of PIBC shown in SEQ ID NO. 1 in the Sequence Listing was named LC, and the amino acid sequence of LC is shown in SEQ ID NO. 3 in the Sequence Listing. The polypeptide LC shown in SEQ ID NO. 3 was synthesized by GenScript (Shanghai) Biotech Co., Ltd.

[0094] mPEGxMal (x = 20000, i.e., the average molecular weight of PEG is 20000, mPEG 20000 Mal, product of Beijing KeyGene Technology Co., Ltd.) was added to the thiol group of the C-terminal amino acid cysteine of the polypeptide LC through Michael addition reaction, to obtain mPEG 20000 LC, mPEG 20000 The specific process of preparation, purification and characterization of LC was the same as described in step (ii) of Example 1, except that the polypeptide CY in Example 1 was replaced by the polypeptide LC, to obtain the fluffy PEGylated polypeptide mPEG20000 LC. mPEG 20000 LC. mPEG Figure 3 LC. mPEG 20000 LC. mPEG 20000 LC. mPEG LC. mPEG

[0095] LC. mPEG 40000 LC. mPEG LC. mPEG

[0096] LC. mPEG 40000 LC. mPEG 40000 LC. mPEG 40000 LC. mPEG 40000 LC. mPEG 40000 LC. mPEG Figure 4 LC. mPEG 40000 LC. mPEG 40000 LC. mPEG LC. mPEG

[0097] LC. mPEG LC. mPEG

[0098] LC. mPEG LC. mPEG

[0099] LC. mPEG TM LC. mPEG LC. mPEG

[0100] 1. Design and synthesis of gp96 primers: The sequence of human gp96 gene in GenBank was used as a template to design forward and reverse primers. The forward primer sequence was 5'-CGggattcATGGACGATGAAGTTGATGTGGAT-3' (SEQ ID NO. 6), and the reverse primer sequence was 5'-GCTCTAGATTAGAATTCATCTTTTTCAGCTG-3' (SEQ ID NO. 7). The forward primer contained a BamHI restriction site at the 5' end, and the reverse primer contained an XbaI restriction site at the 5' end.

[0101] 2. Extract mRNA from human hepatoma cells HepG2 and synthesize cDNA by reverse transcription.

[0102] 3. Use the cDNA obtained in step 2 as a template and the primers designed in step 1 to amplify the target gene by polymerase chain reaction (PCR) to obtain the PCR product, which is the gp96 gene.

[0103] 4. Double enzyme digest the PCR product obtained in step 3 with EcoRI and XbaI, and recover the enzyme digestion product with a size of about 2400 bp.

[0104] 5. Double enzyme digest pFastBac TM 1. Empty plasmid (Invitrogen, product catalog number 10359-016), recover the backbone vector with a size of about 4700 bp.

[0105] 6. Connect the enzyme digestion product with a size of about 2400 bp obtained in step 4 and the vector backbone with a size of about 4700 bp obtained in step 5 to obtain a recombinant plasmid. After sequencing verification, the recombinant plasmid with correct sequence is named pFastBac TM 1-gp96. The recombinant plasmid pFastBac TM 1-gp96 encodes and can express gp96 protein. The amino acid sequence of gp96 protein is shown in SEQ ID NO. 4 in the sequence listing.

[0106] II. Insect cell expression of gp96 recombinant protein and purification of gp96 protein

[0107] Use Cellfectin II reagent (Life technologies, catalog number: 10362-100) to transfect the pFastBac TM1- gp96 was transfected into Sf9 cells (Invitrogen, Catalog No: 11496-015). The Sf9 cells transfected with the plasmid were cultured for 72 hours. The cytopathic effect observed indicated that the recombinant baculovirus (P1) had been released into the culture medium, and the P1 virus was obtained from the cell supernatant. An appropriate amount of P1 was added to the Sf9 monolayer (1 x 10 6 cells / mL) cells, and the cells were cultured at 27°C for 72 hours. The supernatant was obtained by centrifugation at 4000 rpm for 5 minutes, and the second generation virus (P2) was obtained from the supernatant. An appropriate amount of P2 virus was added to 100 ml of Sf9 (1.6 x 10 6 cells / mL) suspension cells, and the cells were cultured at 27°C at 100-120 rpm / min for 72 hours. The third generation virus (P3) was obtained by amplification. Western blotting was performed using a rat anti-gp96 antibody (Santa Cruz, Product No: sc-56399) as the primary antibody, and the results showed that the gp96 protein was expressed in the Sf9 cells.

[0108] Subsequently, an appropriate amount of P3 virus was added to fresh Sf9 cells (1.5 x 10 6 cells / mL, 300 ml), and the cells were cultured at 27°C at 100-120 rpm / min in Insect-XPRESS TM Protein-free Insect Cells medium with L-Glutamine (Catalog No: 12-730Q). After 72 hours, the suspension culture was centrifuged at 7000 rpm for 20 minutes to obtain a clear supernatant. The supernatant was filtered through a 0.22 mm filter membrane, and then purified through a HiTrap Q HP column and a Superdex 200 10 / 300 GL ion column to obtain the purified product. The purified product was identified by denaturing polyacrylamide gel electrophoresis and Western blotting (the primary antibody used was a rat anti-gp96 antibody (Santa Cruz, Product No: sc-56399)). The identification results are shown in Figure 5 FIG. 2, which shows that the purified product contains high-purity gp96 protein. The solvent in the above purified product was replaced with PBS buffer using an ultrafiltration tube, and the protein was concentrated. The protein concentration was determined by the BCA method, and finally the protein was aliquoted and stored at -80°C.

[0109] III. Interaction of gp96 protein with PEGylated polypeptide fragments

[0110] The interaction between the PEGylated polypeptide fragments prepared in Examples 1-4 and gp96 protein was detected by Biacore method. The instrument used for detection was Biacore 3000 system, and CM5 sensor chip was used. The gp96 protein of Step 2 was immobilized on the CM5 sensor chip by amino coupling according to the instruction, and the specific method was as follows: the filtered and degassed HBS buffer salt solution (10 mmol / L HEPES, 0.15 mol / L NaCl, 3.4 mol / L EDTA, 0.05% P-20; pH 7.4) was used as the mobile phase solution, and the CM5 sensor chip module was inserted into the BIAcore system; the flow rate through the flow cell was set to 5 μL / min; the CM5 sensor chip surface was activated with 0.2 mol / L N-ethyl-N-dimethyl-aminopropyl carbodiimide and 0.05 mol / L N-hydroxysuccinimide mixed solution with equal volume for 7 min; 35 μL of 1 mg / mL gp96 protein was injected to the activated surface to bind with the CM5 sensor chip surface; 35 μL of ethanolamine was injected to inactivate the excess reactive groups; 10 μL of 20 mmol / L HCl was rapidly injected, and then the non-covalently bound materials were removed by Extraclean; the level of gp96 protein binding was determined by placing the first baseline report point before starting the injection of gp96 protein and the second report point 2 min after the end of the injection of 20 mmol / L HCl; the gp96 protein binding flow cell was set as the detection channel, and the flow cell not binding gp96 protein was set as the reference channel, and the HBS buffer solution was used as the mobile phase, and the flow rate of the flow cell was 10 μL / min; the sample to be detected was injected into the gp96 protein flow cell and the reference flow cell at the same time, so that the binding reaction was carried out at 22-24 °C and pH 7.4; 10 μL of one of the PEGylated polypeptide fragments in Examples 1-4 or PIBC (diluted with HBS buffer containing 1 mg / mL carboxymethyl dextran) was injected for detection; 10 μL of 20 mmol / L HCl was rapidly injected to regenerate the gp96 protein surface by Extraclean; 10 μL of the PEGylated polypeptide fragment was injected again, and the cycle was repeated to determine the reproducibility of binding to the gp96 protein surface. According to the above steps, the polypeptide fragments at different concentration levels (156, 312, 625, 1250, 2500 nmol / L) were detected respectively, and each concentration level was determined repeatedly for 1 time.

[0111] The binding coefficient K of the PEGylated polypeptide fragments of Examples 1-3 and gp96 protein D (mM / L) are shown in Table 1.

[0112] Table 1 Binding coefficient of PEGylated polypeptide and gp96

[0113] Polypeptides Binding coefficient K D (mM / L) mPEG 20000 CY]]> 4.562 mPEG 40000 CY]]> 50.41 mPEG 20000 LC]] 10.31

[0114] The results show that the PEGylated polypeptides of Examples 1-3 have affinity with gp96 protein, and the mPEG with N-terminal modification of PEG molecular weight of 20000 20000 CY has the strongest binding ability.

[0115] Example 6, Test of irritation at injection site of PEGylated polypeptide

[0116] The control solvent group, PIBC group, mPEG 2000 CY group (PEG molecular weight of 2000, polypeptide with cysteine modification added at N-terminal, prepared according to Reference Example 1), mPEG 5000 CY group (PEG molecular weight of 5000, polypeptide with cysteine modification added at N-terminal, prepared according to Reference Example 1), mPEG 20000 CY group, mPEG 20000 LC group, mPEG 40000 CY group and mPEG 40000 LC group, the control solvent group was injected with solvent (normal saline), and the rest of the groups were injected with PIBC at three concentrations, at a dose of 125, 250 and 500 mg / kg (based on the amount of PIBC), respectively, by single subcutaneous injection to SD rats, 5 rats in each group. The clinical manifestations of the rats and the changes at the injection site were observed daily, and the rats were euthanized after 7 days, and the pathological changes at the injection site were observed by gross anatomy and microscopy.

[0117] During the test, compared with the solvent group, the PIBC group, mPEG 2000 CY group and mPEG 5000 In the CY group, the rats had reduced spontaneous activity, body weight and appropriate amount, and the reduction was related to the dose, and the local administration had strong skin irritation. When the SD rats were given a single subcutaneous injection at a dose of 125, 250 and 500 mg / kg, the skin at the administration site was abnormal (crusting or ulceration), and the irritation became more obvious as the dose increased. Gross anatomy showed that the skin at the administration site and its adjacent site was crusted, and microscopic examination showed corresponding histological changes, such as local reaction of degeneration or necrosis at the administration site.

[0118] mPEG 20000 CY group, mPEG 20000 LC group, mPEG 40000 CY group and mPEG 40000 The clinical manifestations of the LC group were normal, and the skin irritation was significantly reduced. At the highest dose of 500 mg / kg, no erythema and edema appeared at the injection site, and no obvious abnormalities were found under dissection and microscopic observation.

[0119] Compared with PIBC, mPEG 2000 CY and mPEG 5000CY shows strong skin irritation which greatly affects its clinical application. The skin irritation of PEG modified polypeptide with molecular weight of 20,000-40,000 is obviously improved.

[0120] Example 7, hemolysis test of PEGylated polypeptide

[0121] Take 20 ml of rabbit blood, stir the blood with a glass rod, remove fibrinogen to make the blood defibrinated. Add about 10 times the amount of 0.9% sodium chloride solution, shake well, centrifuge at 1000-1500 r / min for 15 minutes, remove the supernatant, and wash the precipitated red blood cells with 0.9% sodium chloride solution according to the above method for 2-3 times until the supernatant is not red. The obtained red blood cells are made into a 2% suspension with 0.9% sodium chloride solution for use in the test.

[0122] Take 9 clean test tubes and number them. Tubes 1-7 are test product tubes (PIBC, mPEG 2000 CY, mPEG 5000 CY, mPEG 20000 CY, mPEG 20000 LC, mPEG 40000 CY and mPEG 40000 LC, at a concentration of 5 mg / ml calculated as PIBC), tube 8 is the negative control tube, and tube 9 is the positive control tube. Add 2% red blood cell suspension, 0.9% sodium chloride solution or distilled water according to the table, mix well, and immediately place in a constant temperature incubator at 37°C±0.5°C for incubation. Observe every 15 minutes at the beginning, then every 1 hour after 1 hour, and observe for 3 hours.

[0123]

[0124] In the test, PIBC, mPEG 2000 CY, mPEG 5000 CY, 3 hours later the solution was clear red, with a small amount of red blood cells remaining at the bottom of the tube, and hemolysis occurred, so PIBC, mPEG 2000 CY, mPEG 5000 CY is not suitable for injection; while adding the test substance mPEG 20000 CY, mPEG 20000 LC, mPEG 40000 CY and mPEG 40000 LC, 3 hours later, the red blood cells in the solution all settled, the supernatant was colorless and clear, and no hemolysis occurred, indicating that PIBC modified by PEG with a molecular weight of 20,000-40,000 can be used for injection.

[0125] Example 8, repeated intravenous infusion of PIBC or mPEG 20000Toxicological test of CY given to SD rats for 14 days

[0126] 30 rats (15 / sex) were divided into 3 groups according to body weight, and were given intravenous infusion of vehicle control (sodium chloride injection, group 1), 10 mg / kg / day of PIBC (group 2) and 10 mg / kg / day of mPEG (group 3) respectively. 20000 CY. The administration volume was 10 mL / kg, and the administration speed was 2 mL / kg / min. After repeated administration for 14 days, the animals were euthanized on D15. The death, clinical symptoms, body weight, food intake of the animals were observed and recorded, and the clinical pathology (blood cell count, blood coagulation function index, blood biochemical index) detection, organ weight, gross observation were performed, and the abnormal tissues observed in gross observation were subjected to histopathological examination.

[0127] Vehicle control group: no abnormality was observed in the animals.

[0128] PIBC group: from the third day to the end of administration, local irritation reactions were successively observed at the administration site, including mild / moderate swelling of the tail (M: 5 / 5, F: 5 / 5), discoloration (purple) at the distal end of the tail (M: 5 / 5, F: 4 / 5), mild / moderate / severe ulceration at the injection site (M: 5 / 5, F: 2 / 5). In addition, 3 / 5 female animals showed reduced spontaneous activity from D1 to D3, and 1 / 5 female animals showed abnormal movement (slow walking) from D1 after administration to D2 before administration. The body weight and body weight gain of male animals decreased on D7. Blood cell count showed that WBC, Neut, Baso, Retic, PLT and Mono of male and female animals increased, and RBC, HGB and HCT decreased. In addition, Lymph (10^9 / L) and Eos (10^9 / L) of male animals and Mono of female animals increased. Blood coagulation function index showed that PT of male and female animals prolonged and FIB increased. Blood biochemical index showed that Alb, A / G and Na+ of male and female animals decreased, CK and LDH increased, and in addition, Cre of male animals and AST and ALT of female animals increased. Gross anatomy showed that (8 / 10) animals had enlarged inguinal lymph nodes, (10 / 10) animals had swelling at the injection site (tail), (9 / 10) animals had discoloration, and (5 / 10) animals had skin ulceration at the injection site (tail). Microscopic examination showed that the cortical lymphoid tissue of the inguinal lymph nodes proliferated, the medullary sinus tissue proliferated and bled; the injection site (tail) formed a scab and / or ulcer, the epidermis necrosed, proliferated and keratinized excessively, the dermis necrosed, edema and inflammatory cell infiltration, the subcutaneous tissue necrosed, edema, fibrous tissue and / or fibroblast proliferation, neutrophil-dominant inflammatory cell infiltration, thrombosis, muscle fiber atrophy / necrosis in the deep layer of subcutaneous tissue, and osteoblast reactive proliferation.

[0129] mPEG 20000CY group: no death or near-death was observed in each animal, and no obvious abnormality was observed in the body weight and food intake, coagulation function, and blood biochemical indicators. Macroscopic examination showed that no obvious abnormality was observed in each organ.

[0130] The results show that intravenous injection of PIBC has strong irritability and toxicity, while mPEG-PIBC has no obvious toxicity. 20000 CY can significantly reduce its toxicity.

[0131] Example 9, Effect of PEGylated polypeptide on breast cancer cell SKBr3

[0132] Breast cancer cell SKBr3: ATCC (American type culture collection) product, product number HTB-30. Breast cancer cell MDA-MB-231 ATCC (American type culture collection) product, product number HTB-26. The PEGylated polypeptide fragments prepared in Examples 1-4 were subjected to the following experiments, respectively.

[0133] I. Inhibition of breast cancer cell SKBr3 proliferation by PEGylated polypeptide

[0134] The CCK-8 kit (Japan Tongren Chemical Research Institute, item number CK04-05) was used to detect the inhibition of each PEGylated polypeptide fragment on the proliferation of breast cancer cell SKBr3. The specific operation steps are as follows:

[0135] 1. SKBr3 cells were plated in a 96-well plate with a confluence of about 50%. Three replicate wells were set for each group of cells.

[0136] 2. After the cells adhered, PEGylated polypeptide (final concentration 6 μM) was added as the experimental group, and three wells without polypeptide were set as the control group.

[0137] 3. At different time points (0, 3, 6, and 12 hours), 10 μl of CCK-8 detection reagent was added to each well, and incubated at 37°C for 2 hours.

[0138] 4. The OD value at 490 nm was measured.

[0139] The cell growth inhibition rate calculation formula is: (control group OD 490 average - experimental group OD 490 average) / control group OD 490 × 100%. The cell growth inhibition rate (average) of each PEGylated polypeptide fragment treatment group is shown in Table 2.

[0140] Table 2 Cell growth inhibition rate of PEGylated polypeptide treatment

[0141]

[0142]

[0143] The results show that the PEGylated polypeptides of Examples 1-4 can all inhibit the proliferation (growth) of breast cancer cells SKBr3, wherein mPEG 20000 CY has the most obvious inhibition effect.

[0144] II. Inhibition of the Invasion of Breast Cancer Cells MDA-MB-231 by PEGylated Polypeptides

[0145] The Tanswell plate (Corning Company, product number #3422) and Matrigel (BD Company, product number 354234) were used to detect the influence of the PEGylated polypeptides of Examples 1-4 on the invasion ability of breast cancer cells MDA-MB-231. The cell invasion experiment was operated according to the Transwell and Matrigel instructions. The main operation steps are as follows:

[0146] 1. The Matrigel was frozen and thawed on ice overnight one day before the experiment, 60 μl of Matrigel was added to the upper chamber of the Transwell, and coated at 37°C for 1 hour, and washed twice with PBS buffer.

[0147] 2. The breast cancer cells MDA-MB-231 which had been digested and counted were diluted to 4 million / ml with serum-free culture medium containing PIBC and the PEGylated polypeptides of Examples 1-4 (the final concentration was 10 μM), 100 μl was taken and added to the upper chamber of the Transwell, 600 μl of complete cell culture medium was added to the lower chamber of the Transwell as the experimental group; the serum-free culture medium was used instead of the serum-free culture medium containing the polypeptide fragments as the negative control group.

[0148] 3. Continue to cultivate in the CO2 incubator at 37°C and 5% CO2 for 24 hours, scrape off the cells in the upper Transwell chamber with a cotton swab, fix with 50% methanol / 50% acetone for 15 minutes, then wash with PBS buffer for 3 times, DAPI mounting, and count the number of invaded cells under the fluorescence microscope.

[0149] The calculation formula of the invasion inhibition rate is: (the number of invaded cells in the negative control group - the number of invaded cells in the experimental group) / the number of invaded cells in the negative control group x 100%.

[0150] The invasion inhibition rates (average values) of the PEGylated polypeptide fragment treatment groups relative to the negative control group on MDA-MB-231 are shown in Table 3.

[0151] Table 3 Inhibition rates of PEGylated polypeptide treatment groups relative to the negative control group on the invasion of MDA-MB-231

[0152]

[0153]

[0154] Results show that the PEGylated polypeptides of Examples 1-4 can all inhibit the invasion of breast cancer cells MDA-MB-231, wherein mPEG 20000 CY has the most obvious inhibitory effect.

[0155] III. PEGylated polypeptides promote the apoptosis of breast cancer cells SKBr3

[0156] The PEGylated polypeptides of Examples 1-4 were respectively detected for their promotion of the apoptosis of breast cancer cells SKBr3. The specific steps are as follows:

[0157] 1. Breast cancer cells SKBr3 were inoculated in 6-well cell culture plates, 200,000 cells per well.

[0158] 2. After the cells adhered, PIBC and the PEGylated polypeptides of Examples 1-4 (final concentration 6 μM) were added for further culture for 24 hours, serving as the experimental group; PBS solution was added to serve as the negative control group.

[0159] 3. After the cells were stained by Apoptosis Assay kit produced by Invitrogen Company, the results were analyzed by flow cytometry. The specific operation steps are as follows: (1) The cells were routinely digested with trypsin and washed twice with PBS buffer (the number of cells is generally one-fourth of a 6-well plate or one 24-well plate).

[0160] (2) The cells were gently suspended with 20 μl of 1x Annexin V Buffer, 1 μl of FITC annexin V was added and mixed gently, and the staining was performed at room temperature for 15 minutes in the dark.

[0161] (3) 1x Annexin V Buffer was added to the reaction tube to make the final volume 200 μl.

[0162] (4) PI with a concentration of 100 μg / ml was added to make the final concentration 1 μg / ml, and the staining was performed at room temperature for about 3 minutes in the dark before the machine detection.

[0163] The increased apoptosis rate (average value) of the PEGylated polypeptide treatment groups of Examples 1-4 compared with the negative control group is shown in Table 4.

[0164] Table 4 Increased apoptosis rate of PEGylated polypeptide treatment groups compared with the negative control group

[0165] ​

[0166] Polypeptides Apoptosis rate mPEG 20000 CY]] 32.1％ mPEG 40000 CY]] 11.1％ mPEG 20000 LC]] 21.1％ mPEG 40000 LC]] 3.8％

[0167] Results show that the PEGylated polypeptides of Examples 1-4 can all promote the apoptosis of breast cancer cells SKBr3 cells, wherein mPEG 20000 CY has the most obvious promotion effect.

[0168] Four, mPEG 20000 CY inhibits the growth of breast cancer cell MDA-MB-231 transplanted tumor

[0169] Detection of mPEG 20000 CY on the growth of breast cancer cell MDA-MB-231 transplanted tumor. The specific steps are as follows:

[0170] 1. Subcutaneously inoculate breast cancer cells MDA-MB-231 cultured to logarithmic growth phase into BALB / c nude mice (Beijing Vantoll Life Experimental Animal Technology Co., Ltd.), each with 10 million cells, to establish a transplanted tumor model, and then passaged in nude mice for three times for tumor inoculation experiment.

[0171] 2. When the tumor grows to 100mm 3 , randomly divide the BALB / c nude mice into 3 groups, 5 in each group, and perform the following treatment, with the first treatment day recorded as day 1:

[0172] PIBC group: intravenous injection treatment with PIBC solution (polypeptide dissolved in 0.9% physiological saline), with a dose of 5mg / kg per injection, once a day, for a total of two weeks of treatment;

[0173] mPEG 20000 CY group: intravenous injection treatment with mPEG 20000 CY solution (mPEG 20000 CY dissolved in 0.9% physiological saline), with a dose of 5mg / kg per injection (calculated based on the amount of PIBC), with the same injection volume as the PIBC treatment group, once every two days, for a total of two weeks of treatment;

[0174] Control group: intravenous injection treatment with PBS buffer, with the same injection volume as the PIBC treatment group, once a day, for a total of two weeks.

[0175] 3. Detect the tumor volume twice a week, sacrifice the nude mice after two weeks of treatment, weigh the tumor and calculate the tumor inhibition rate.

[0176] The calculation formula of tumor inhibition rate is as follows: (tumor volume of control group mice - tumor volume of polypeptide group mice) / tumor volume of control group mice x 100%.

[0177] PIBC and mPEG20000 The tumor inhibition rate results of the CY treatment group are shown in Table 2. Figure 6 The results show that PIBC and mPEG 20000 CY can effectively inhibit the growth of breast cancer tumors caused by breast cancer cells MDA-MB-231.

[0178] Example 10: In vivo plasma half-life evaluation of PEGylated polypeptides in SD rats

[0179] Tested polypeptides: PIBC, mPEG 20000 CY

[0180] 1. Preparation of standard curve: Prepare PIBC and mPEG 20000 CY stock solutions at a concentration of 1 mg / mL using borax buffer (pH 9.5). Take an appropriate amount of the stock solution and dilute it with 50% acetonitrile water solution to prepare standard curve working solutions with polypeptide concentrations of 25, 37.5, 50, 75, 100, 150, and 250 μg / mL. Take 20 μl of the prepared standard curve working solution, add 80 μl of blank mouse plasma, and prepare standard curve samples with polypeptide concentrations of 5, 7.5, 10, 15, 20, 30, and 50 μg / mL. Add 20 μl of 20% (mass percentage) phosphoric acid solution and 300 μl of methanol-acetonitrile (volume ratio of methanol to acetonitrile is 1:1) to the standard curve samples, vortex for about 2 min, centrifuge at 4000 rpm / min for 10 min, and take the supernatant for analysis. Obtain the standard curve of each tested drug, and prepare quality control samples according to this method to detect their precision.

[0181] 2. Experimental process

[0182] Drug preparation: Prepare before administration. Dissolve the tested polypeptides with an equal volume of 0.9% sodium chloride injection and 5 mM Na2HPO4 into a uniform transparent solution. The final concentrations of PIBC, mPEG 20000 CY are 8 mg / ml and 12 mg / ml, respectively, for intravenous administration.

[0183] Test animals: Male and female SD rats, weighing 160-180 grams, from Beijing Huafukang Biotechnology Co., Ltd.

[0184] Animal experiment: Administration: Four SD rats, two male and two female, were treated with each polypeptide. The body weight was determined before administration, and the administration dose was 8 mg / kg.

[0185] Sample collection: At the time of administration, the zero time point was recorded. The PIBC administration group took blood from the tail vein at the zero time point and 2 min, 10 min, 20 min, 40 min, 60 min, 90 min, and 120 min after administration. The mPEG 20000The CY administration group was taken blood from the tail vein at zero time and 30 min, 1 h, 2 h, 4 h, 6 h, 10 h, 12 h, 24 h, 36 h, 48 h and 72 h after administration, 0.3 ml of blood was taken each time and placed in a centrifuge tube containing 6 μl of aprotinin and 5 μl of heparin sodium, centrifuged at 4500 rmp / min for 5 min, the upper plasma was separated and stored in a-80℃ refrigerator.

[0186] Sample processing: 100 μl of the plasma of the sample to be tested was taken, 20 μl of 20% phosphoric acid solution, 20 μl of 50% acetonitrile aqueous solution and 300 μl of methanol-acetonitrile (1:1) solution were added, vortexed for about 2 min, centrifuged at 4000 rmp / min for 10 min, and the supernatant was taken for analysis.

[0187] Chromatographic conditions: chromatographic column: XSELECT CSH C18, 4.6x150mm, 5 μm, mobile phase: A phase: 0.1% (volume percent) TFA aqueous solution, B phase: 0.1% (volume percent) TFA acetonitrile solution, eluent composed of A phase and B phase, volume percent of B phase in the eluent increased from 20% to 35% at a constant rate, volume percent of A phase decreased from 80% to 65% at a constant rate, elution time 10 min, elution flow rate 1 ml / min, ultraviolet detection wavelength: 220 nm, sample injection volume: 20 μL.

[0188] 3. Experimental results

[0189] 1) PIBC, mPEG 20000 The relationship between the drug concentration and the peak area obtained from the standard curve of CY was y=2.879x+4.12 (R=0.996) and y=3.742x+0.98 (R=0.991), wherein y is the peak area and x is the drug concentration.

[0190] 2) The drug concentration at each time point was obtained according to the standard curve, and the results are shown in Table 5.

[0191] Table 5 Blood drug concentration at different administration times

[0192]

[0193] The results of Table 5 show that: mPEG 20000 The half-life (T 1 / 2 ) of CY in the animal body is about 30.1 h, which is more than 80 times longer than the half-life of PIBC of 22 min.

[0194] The above only describes the preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application. SEQUENCE LISTING <110> Beijing Kangminghai Wisdom Biotechnology Co., Ltd. <120> PEG-modified polypeptides capable of inhibiting gp96, methods of making and uses thereof <130> IDC190318 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide: polypeptide with N-terminal added cysteine of PIBC <400> 1 Leu Asn Val Ser Arg Glu Thr Leu Gin Gin His Lys Leu Leu Lys Val 1 5 10 15 Ile Arg Lys Lys Leu Val Arg Lys Thr Leu Asp Met Ile Lys Lys Ile 20 25 30 Ala Asp Asp Lys Tyr 35 <210> 2 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide: polypeptide with N-terminal added cysteine of PIBC <400> 2 Cys Leu Asn Val Ser Arg Glu Thr Leu Gin Gin His Lys Leu Leu Lys 1 5 10 15 Val Ile Arg Lys Lys Leu Val Arg Lys Thr Leu Asp Met Ile Lys Lys 20 25 30 Ile Ala Asp Asp Lys Tyr 35 <210> 3 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide: Polypeptide resulting from C-terminal addition of cysteine to PIBC <400> 3 Leu Asn Val Ser Arg Glu Thr Leu Gin Gin His Lys Leu Leu Lys Val 1 5 10 15 Ile Arg Lys Lys Leu Val Arg Lys Thr Leu Asp Met Ile Lys Lys Ile 20 25 30 Ala Asp Asp Lys Tyr Cys 35 <210> 4 <211> 782 <212> PRT <213> Homo sapiens <400> 4 Asp Asp Glu Val Asp Val Asp Gly Thr Val Glu Glu Asp Leu Gly Lys 1 5 10 15 Ser Arg Glu Gly Ser Arg Thr Asp Asp Glu Val Val Gin Arg Glu Glu 20 25 30 Glu Ala Ile Gin Leu Asp Gly Leu Asn Ala Ser Gin Ile Arg Glu Leu 35 40 45 Arg Glu Lys Ser Glu Lys Phe Ala Phe Gin Ala Glu Val Asn Arg Met 50 55 60 Met Lys Leu Ile Ile Asn Ser Leu Tyr Lys Asn Lys Glu Ile Phe Leu 65 70 75 80 Arg Glu Leu lie Ser Asn Ala Ser Asp Ala Leu Asp Lys lie Arg Leu 85 90 95 lie Ser Leu Thr Asp Glu Asn Ala Leu Ser Gly Asn Glu Glu Leu Thr 100 105 110 Val Lys lie Lys Cys Asp Lys Glu Lys Asn Leu Leu His Val Thr Asp 115 120 125 Thr Gly Val Gly Met Thr Arg Glu Glu Leu Val Lys Asn Leu Gly Thr 130 135 140 lie Ala Lys Ser Gly Thr Ser Glu Phe Leu Asn Lys Met Thr Glu Ala 145 150 155 160 Gln Glu Asp Gly Gin Ser Ser Ser Glu Leu lie Gly Gin Phe Gly Val 165 170 175 Gly Phe Tyr Ser Ala Phe Leu Val Ala Asp Lys Val lie Val Thr Ser 180 185 190 Lys His Asn Asn Asp Thr Gin His lie Trp Glu Ser Asp Ser Asn Glu 195 200 205 Phe Ser Val lie Ala Asp Pro Arg Gly Asn Thr Leu Gly Arg Gly Thr 210 215 220 Thr lie Thr Leu Val Leu Lys Glu Glu Ala Ser Asp Tyr Leu Glu Leu 225 230 235 240 Asp Thr Ile Lys Asn Leu Val Lys Lys Tyr Ser Gln Phe Ile Asn Phe 245 250 255 Pro Ile Tyr Val Trp Ser Ser Lys Thr Glu Thr Val Glu Glu Pro Met 260 265 270 Glu Glu Glu Glu Ala Ala Lys Glu Glu Lys Glu Glu Ser Asp Asp Glu 275 280 285 Ala Ala Val Glu Glu Glu Glu Glu Glu Lys Lys Pro Lys Thr Lys Lys 290 295 300 Val Glu Lys Thr Val Trp Asp Trp Glu Leu Met Asn Asp Ile Lys Pro 305 310 315 320 Ile Trp Gln Arg Pro Ser Lys Glu Val Glu Glu Asp Glu Tyr Lys Ala 325 330 335 Phe Tyr Lys Ser Phe Ser Lys Glu Ser Asp Asp Pro Met Ala Tyr Ile 340 345 350 His Phe Thr Ala Glu Gly Glu Val Thr Phe Lys Ser Ile Leu Phe Val 355 360 365 Pro Thr Ser Ala Pro Arg Gly Leu Phe Asp Glu Tyr Gly Ser Lys Lys 370 375 380 Ser Asp Tyr Ile Lys Leu Tyr Val Arg Arg Val Phe Ile Pro Asp Asp 385 390 395 400 Phe His Asp Met Met Pro Lys Tyr Leu Asn Phe Val Lys Gly Val Val 405 410 415 Asp Ser Asp Asp Leu Pro Leu Asn Val Ser Arg Glu Thr Leu Gln Gln 420 425 430 His Lys Leu Leu Lys Val Ile Arg Lys Lys Leu Val Arg Lys Thr Leu 435 440 445 Asp Met Ile Lys Lys Ile Ala Asp Asp Lys Tyr Asn Asp Thr Phe Trp 450 455 460 Lys Glu Phe Gly Thr Asn Ile Lys Leu Gly Val Ile Glu Asp His Ser 465 470 475 480 Asn Arg Thr Arg Leu Ala Lys Leu Leu Arg Phe Gln Ser Ser His His 485 490 495 Pro Thr Asp Ile Thr Ser Leu Asp Gln Tyr Val Glu Arg Met Lys Glu 500 505 510 Lys Gln Asp Lys Ile Tyr Phe Met Ala Gly Ser Ser Arg Lys Glu Ala 515 520 525 Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys Lys Gly Tyr Glu Val 530 535 540 Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys Ile Gln Ala Leu Pro 545 550 555 560 Glu Phe Asp Gly Lys Arg Phe Gin Asn Val Ala Lys Glu Gly Val Lys 565 570 575 Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg Glu Ala Val Glu Lys 580 585 590 Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp Lys Ala Leu Lys Asp 595 600 605 Lys lie Glu Lys Ala Val Val Ser Gin Arg Leu Thr Glu Ser Pro Cys 610 615 620 Ala Leu Val Ala Ser Gin Tyr Gly Trp Ser Gly Asn Met Glu Arg lie 625 630 635 640 Met Lys Ala Gin Ala Tyr Gin Thr Gly Lys Asp lie Ser Thr Asn Tyr 645 650 655 Tyr Ala Ser Gin Lys Lys Thr Phe Glu lie Asn Pro Arg His Pro Leu 660 665 670 lie Arg Asp Met Leu Arg Arg lie Lys Glu Asp Glu Asp Asp Lys Thr 675 680 685 Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr Ala Thr Leu Arg Ser 690 695 700 Gly Tyr Leu Leu Pro Asp Thr Lys Ala Tyr Gly Asp Arg Ile Glu Arg 705 710 715 720 Met Leu Arg Leu Ser Leu Asn Ile Asp Pro Asp Ala Lys Val Glu Glu 725 730 735 Glu Pro Glu Glu Glu Pro Glu Glu Thr Ala Glu Asp Thr Thr Glu Asp 740 745 750 Thr Glu Gln Asp Glu Asp Glu Glu Met Asp Val Gly Thr Asp Glu Glu 755 760 765 Glu Glu Thr Ala Lys Glu Ser Thr Ala Glu Lys Asp Glu Phe 770 775 780 <210> 5 <211> 2349 <212> DNA <213> Homo sapiens <400> 5 gacgatgaag ttgatgtgga tggtacagta gaagaggatc tgggtaaaag tagagaagga 60 tcaaggacgg atgatgaagt agtacagaga gaggaagaag ctattcagtt ggatggatta 120 aatgcatcac aaataagaga acttagagag aagtcggaaa agtttgcctt ccaagccgaa 180 gttaacagaa tgatgaaact tatcatcaat tcattgtata aaaataaaga gattttcctg 240 agagaactga tttcaaatgc ttctgatgct ttagataaga taaggctaat atcactgact 300 gatgaaaatg ctctttctgg aaatgaggaa ctaacagtca aaattaagtg tgataaggag 360 aagaacctgc tgcatgtcac agacaccggt gtaggaatga ccagagaaga gttggttaaa 420 aaccttggta ccatagccaa atctgggaca agcgagtttt taaacaaaat gactgaagca 480 caggaagatg gccagtcgtc ttctgaattg attggccagt ttggtgtcgg tttctattcc 540 gccttccttg tagcagataa ggttattgtc acttcaaaac acaacaacga tacccagcac 600 atctgggagt ctgactccaa tgaattttct gtaattgctg acccaagagg aaacactcta 660 ggacggggaa cgacaattac ccttgtctta aaagaagaag catctgatta ccttgaattg 720 gatacaatta aaaatctcgt caaaaaatat tcacagttca taaactttcc tatttatgta 780 tggagcagca agactgaaac tgttgaggag cccatggagg aagaagaagc agccaaagaa 840 gagaaagaag aatctgatga tgaagctgca gtagaggaag aagaagaaga aaagaaacca 900 aagactaaaa aagttgaaaa aactgtctgg gactgggaac ttatgaatga tatcaaacca 960 atatggcaga gaccatcaaa agaagtagaa gaagatgaat acaaagcttt ctacaaatca 1020 ttttcaaagg aaagtgatga ccccatggct tatattcact ttactgctga aggggaagtt 1080 accttcaaat caattttatt tgtacccaca tctgctccac gtggtctgtt tgacgaatat 1140 ggatctaaaa agagcgatta cattaagctc tatgtgcgcc gtgtattcat cccagacgac 1200 ttccatgata tgatgcctaa atacctcaat tttgtcaagg gtgtggtgga ctcagatgat 1260 ctccccttga atgtttcccg cgagactctt cagcaacata aactgcttaa ggtgattagg 1320 aagaagcttg ttcgtaaaac gctggacatg atcaagaaga ttgctgatga taaatacaat 1380 gatacttttt ggaaagaatt tggtaccaac atcaagcttg gtgtgattga agaccactcg 1440 aatcgaacac gtcttgctaa acttcttagg ttccagtctt ctcatcatcc aactgacatt 1500 actagcctag accagtatgt ggaaagaatg aaggaaaaac aagacaaaat ctacttcatg 1560 gctgggtcca gcagaaaaga ggctgaatct tctccatttg ttgagcgact tctgaaaaag 1620 ggctatgaag ttatttacct cacagaacct gtggatgaat actgtattca ggcccttccc 1680 gaatttgatg ggaagaggtt ccagaatgtt gccaaggaag gagtgaagtt cgatgaaagt 1740 GAGAAAACCA AGAAGAATTC GAGCCTCTGC TGAACTGG 60 ATGAAAGATA AAGCCCTTAA GGACAAGATT GAAAAGGCTG TGGTGTCTCA 60 GAATCTCCGT GTGCTTTGGT GGCCAGCCAG TACGGATGGT CTGGCAACAT 120 GGAGAAGAAG AAGAAGAAGA AGAAGAAGAA GAAGAAGAAG AAGAAGAAGA 60 AAGAAAACAT TTGAAATTAATCCCAGACACC CGCTGATCAG AGACATGCTT 60 CGGTTGTTTT GTTTGAAACAG CAACCTGAAA GATGAAAGAT GATGAAACAG 60 TTGATGATGA ATAGAAAGAA GATGATGATG ATGATGATGA ATAGAAAGAA 60 ATGCTTCGCC TCAGTTTGAA CATTGACCC 60 GAAGAAGAAG AAGAAGAAGA AGAAGAAGAA GAAGAAGAAG AAGAAGAAGA 60 ATGGATGTGG GAACAGATGA AGAAGAAGAA ACAGCAAAGG AATCTACAGC 60 GAATTCTAA 60 <210> 6 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> PRIMER <400> 6 cgggattcat ggacgatgaa gttgatgtgg at 32 <210> 7 <211> 31 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 7 gctctagatt agaattcatc tttttcagct g 31

Claims

1. A PEGylated polypeptide comprising PIBC and linear PEG with an average molecular weight of 20,000 to 40,000, wherein the PIBC and the PEG are covalently linked by a linker group, wherein... PIBC is a polypeptide with the amino acid sequence shown in SEQ ID NO. 1; the PEG is attached to the N-terminus of PIBC; The PEGylated polypeptide has the following structure: Formula I Where n is the degree of polymerization of PEG, and n satisfies the condition that the molecular weight of PEG is 20,000 to 40,000.

2. The PEGylated polypeptide of claim 1, wherein the covalent linkage is achieved by a Michael addition reaction of reactant 1 and reactant 2, wherein, The reactant 1 is a PEG with maleimide attached to one end, and the reactant 2 is a PIBC with an amino acid residue containing a thiol group introduced at the N-terminus. The reactant 1 has the structure shown in Formula II: Formula II; In Formula II, n is the degree of polymerization of PEG, and n satisfies the condition that the molecular weight of PEG is 20,000 to 40,000.

3. The PEGylated polypeptide of claim 2, wherein reactant 2 is a polypeptide having the amino acid sequence shown in SEQ ID NO.

2.

4. A PEGylated polypeptide comprising PIBC and linear PEG with an average molecular weight of 20,000-25,000, wherein the PIBC and the PEG are covalently linked by a linker group, wherein... PIBC is a polypeptide with the amino acid sequence shown in SEQ ID NO. 1; the PEG is attached to the C-terminus of PIBC; The PEGylated polypeptide has the following structure: Formula I Where n is the degree of polymerization of PEG, and n satisfies the condition that the molecular weight of PEG is 20,000 to 25,000.

5. The PEGylated polypeptide of claim 4, wherein the covalent linkage is achieved by a Michael addition reaction of reactant 1 and reactant 2, wherein, The reactant 1 is a PEG with maleimide attached to one end, and the reactant 2 is a PIBC with an amino acid residue containing a thiol group introduced at the C-terminus. The reactant 1 has the structure shown in Formula II: Formula II; In Formula II, n is the degree of polymerization of PEG, and n satisfies the condition that the molecular weight of PEG is 20,000 to 25,000.

6. The PEGylated polypeptide of claim 5, wherein reactant 2 is a polypeptide having the amino acid sequence shown in SEQ ID NO.

3.

7. A method for preparing the PEGylated polypeptide of any one of claims 1-3, comprising the step of covalently linking PIBC with PEG having an average molecular weight of 20,000 to 40,000; The method includes: The steps for a Michael addition reaction using reactant 1 and reactant 2; The reactant 1 is a PEG with maleimide attached to one end; The reactant 2 is a PIBC with an N-terminus containing an amino acid residue containing a thiol group.

8. A method for preparing the PEGylated polypeptide of any one of claims 4-6, comprising the step of covalently linking PIBC with PEG having an average molecular weight of 20,000 to 25,000; The method includes: The steps for a Michael addition reaction using reactant 1 and reactant 2; The reactant 1 is a PEG with maleimide attached to one end; The reactant 2 is a PIBC with an amino acid residue containing a thiol group introduced at its C-terminus.

9. A pharmaceutical composition comprising the PEGylated polypeptide of any one of claims 1-6.

10. A formulation comprising the PEGylated polypeptide of any one of claims 1-6.

11. Use of the PEGylated polypeptide of any one of claims 1-6 in the preparation of a medicament for treating and / or preventing a subject from having a disease associated with overexpression of gp96 protein, said disease being selected from breast cancer, lung cancer, liver cancer, colon cancer, rectal cancer, and ovarian cancer.

12. The use of claim 11, wherein the disease is triple-negative breast cancer.

13. Use of the PEGylated polypeptide of any one of claims 1-6 in the preparation of a formulation for binding to gp96 protein, inhibiting tumor cell proliferation and / or growth and / or invasion, and promoting tumor cell apoptosis, wherein the tumor cells are selected from breast cancer cells, lung cancer cells, liver cancer cells, colon cancer cells, rectal cancer cells, and ovarian cancer cells.

14. The use of claim 13, wherein the tumor cells are triple-negative breast cancer cells.

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