WO2018205622A1 - Pegylated polypeptide having tumor inhibition function, preparation method therefor and application thereof - Google Patents

Abstract

The present invention provides a PEGylated polypeptide having a tumor inhibition function, a preparation method therefor and application thereof. The PEGylated polypeptide is obtained by modifying a PIBC polypeptide (SEQ ID NO:1) with PEG. The modification is implemented by forming a carbon-sulfur single bond by subjecting a first compound and a second compound to Michael addition reaction. The first compound is PEG having one end modified with maleimide, and the second compound is a PIBC having an N-end or C-end into which a cysteine residue containing sulfydryl is introduced; the Michael addition reaction occurs between the maleimide and the sulfydryl. The PEGylated polypeptide of the present invention not only remains the PIBC to inhibit the activity of heat shock protein gp96, but also extends the in vivo half-life of the PIBC.

Description

PEGylated polypeptide with tumor suppressing function and preparation method and application thereof Technical field

The invention relates to a PEGylated polypeptide having the function of inhibiting tumor in the field of biotechnology, a preparation method and application thereof.

Background technique

According to the 2015 China Cancer Statistics Report published by the National Cancer Registry, breast cancer is the most common type of cancer among Chinese women, and its incidence is increasing at a rate of 3% per year, making it the fastest growing mortality rate in cities. cancer. Current treatments for breast cancer include surgery, chemotherapy, endocrine, and targeted therapy. Targeted therapy has the characteristics of accurate positioning, strong pertinence, and small side effects, which can significantly prolong the survival of patients and improve the quality of life of patients. It is especially suitable for patients with advanced patients or patients who cannot tolerate radiotherapy and chemotherapy. The existing targeted therapy for breast cancer is mainly targeted at HER2-positive breast cancer, and its targeted drug Trastuzumab (ie Herceptin) is a humanized HER2 monoclonal antibody that amplifies and overexpresses the HER2 gene. For breast cancer patients, trastuzumab alone or in combination with other drugs can achieve 25% or 50% efficiency respectively. Herceptin has become the first-line drug for breast cancer treatment, and its sales rank among the top biomedical drugs. While HER2-positive breast cancer patients account for only about 20% of the total number of breast cancer patients, it is urgent to find new breast cancer targets and develop a broader spectrum of anti-breast cancer targeted drugs.

The heat shock protein gp96 belongs to the HSP90 family and is localized to the endoplasmic reticulum under normal physiological conditions and is highly homologous to the cytoplasmic HSP90. Like other heat shock proteins, the main biological function of gp96 is molecular chaperones. It is involved in the folding, transport, degradation and assembly of nascent proteins, inhibiting the secretion of misfolded proteins. Unlike other molecular chaperones, gp96 can bind to a limited variety of proteins and peptide chains. It has been reported that the maturation of more than 20 proteins has been required for gp96, and these studies have revealed that gp96 acts as a molecular chaperone to form a protein complex with multiple client proteins, and its mediated regulatory network plays a role in multiple tumor proteins. Function is crucial. In addition to localization in the endoplasmic reticulum, gp96 protein is also present on certain cell surfaces. More and more studies have found that gp96 is overexpressed on the surface of many cancer cells, suggesting that it has some relationship with the development of cancer. Meng Yidong and other studies have found that gp96 in the endoplasmic reticulum shifts to the cell membrane to play a role in the regulation of core scaffolding proteins, forming protein machinery complexes with multiple client proteins, essential for the function of multiple tumor proteins, and the degree of malignancy of breast cancer It is closely related to poor prognosis. Gp96 on the cell membrane may be a new target for breast cancer treatment.

Chinese patent ZL 201110159487.4 based on the amino acid sequence and spatial conformation of heat shock protein gp96, through a series of technical analysis and means to design and develop a peptide-containing peptide PIBC (Peptide Inhibitor for Breast Cancer), which can bind to heat shock protein gp96 Interfering with the interaction of gp96 with the tumor-associated receptor HER-2 and reducing its stability, promoting the degradation of HER-2 in tumor cells, the polypeptide can reduce HER2, uPAR, ER-a36 in tumor tissues of breast cancer patients The protein content, thereby achieving clinical efficacy against breast cancer.

Although peptide drugs have specific action sites and exact curative effects, their solubility is low, their immunogenicity is high, they are easily degraded by proteases and cleared by the kidneys, which seriously restricts their clinical application.

Invention disclosure

The technical problem to be solved by the present invention is how to inhibit tumors, especially breast cancer tumors.

In order to solve the above technical problems, the present invention first provides a PEGylated polypeptide having a tumor suppressing function.

The PEGylated polypeptide provided by the present invention is a compound obtained by modifying a polypeptide named PIBC with PEG;

The polypeptide of PIBC is A1) or A2): A1) the polypeptide of SEQ ID No. 1 in the sequence listing; A2) the amino acid sequence of SEQ ID NO. 1 in the sequence listing is subjected to one or several amino acid residues. Substituted and/or deleted and/or polypeptides derived from A1) which have the same function.

In the above PEGylated polypeptide, the modification can be achieved by performing a Michael addition reaction of the compound A and the compound B to form a carbon sulfur single bond;

The compound A is a PEG modified with maleimide at one end;

The compound B is introduced into the N-terminal or C-terminal PIBC containing a mercapto amino acid residue;

The Michael addition reaction occurs between the maleimide and the thiol group.

In the compound A, the other end of the PEG (i.e., the end to which the maleimide is not attached) may be attached with a methoxy group. The thiol-containing amino acid residue may be a cysteine residue.

The compound A can be as shown in Formula V;

In the formula V, n is a non-zero natural number.

The compound B may be the polypeptide represented by SEQ ID NO. 2 or SEQ ID NO.

The PEGylated polypeptide may specifically be a compound of formula I:

In formula I, n is a non-zero natural number;

Cys and PIBC are linked by a peptide bond formed by a carboxyl group of Cys to an N-terminal amino group of PIBC or a peptide bond formed by an amino group of Cys to a C-terminal carboxyl group of PIBC.

The molecular formula of PEG is HO(CH ₂ CH ₂ O)nH, the molecular weight of PEG is 2000-5000, and the n in formula V and formula I is the same as the definition of n in the formula of PEG, that is, n satisfies the molecular weight of PEG of 2000~ 5000 conditions.

The compound A can be specifically a product of Beijing Key Kai Technology Co., Ltd.

In order to solve the above technical problems, the present invention also provides a method for preparing the PEGylated polypeptide.

The method for preparing a PEGylated polypeptide provided by the present invention comprises: using the compound A and the compound B to perform a Michael addition reaction to obtain the PEGylated polypeptide;

The Michael addition reaction occurs between the maleimide and the thiol group.

In order to solve the above technical problems, the present invention also provides any of the following applications of the PEGylated polypeptide:

M1) for use in the preparation of a product that binds to the gp96 protein;

M2) for the preparation of a product for treating and/or preventing diseases caused by gp96 protein;

M3) use in the preparation of a product that inhibits tumor cell proliferation and/or growth and/or invasion;

M4) application in the preparation of a product for promoting tumor cell apoptosis;

M5) Application in the preparation of products for inhibiting tumor growth.

In the above application, the gp96 protein may be B1) or B2):

B1) a polypeptide represented by SEQ ID No. 4 in the Sequence Listing;

B2) A polypeptide derived from B1) having the amino acid sequence of SEQ ID NO. 4 in the sequence listing subjected to substitution and/or deletion and/or addition of one or several amino acid residues and having the same function.

The disease caused by the gp96 protein in M2) is a tumor.

In the above application, the disease caused by the gp96 protein in M2) may be breast cancer. The tumor cells described in M3) and M4) may both be breast cancer tumor cells, and the tumor in M5) may be breast cancer.

In order to solve the above technical problems, the present invention also provides a product having any of the following functions N1) to N3), the product containing the PEGylated polypeptide;

N1) inhibiting tumor cell proliferation and/or growth and/or invasion;

N2) promotes tumor cell apoptosis;

N3) inhibits tumor growth.

In the above products, the tumor cells described in N1) and N2) may be breast cancer tumor cells. The tumor described in N3) may be breast cancer.

In the present invention, the breast cancer cells may all be breast cancer cells SKBr3. The tumor may be a tumor caused by breast cancer cell MDA-MB-231.

DRAWINGS

Figure 1 is a graph showing the results of mass spectrometry identification of the PEGylated polypeptide mPEG ₂₀₀₀ CY.

Figure 2 is a graph showing the results of mass spectrometry identification of the PEGylated polypeptide mPEG ₅₀₀₀ CY.

Figure 3 is a graph showing the results of mass spectrometry identification of the PEGylated polypeptide mPEG ₂₀₀₀ LC.

Figure 4 is a graph showing the results of mass spectrometry identification of the PEGylated polypeptide mPEG ₅₀₀₀ LC.

Figure 5 is a map of the secreted human heat shock protein gp96 expressed by the insect cell expression system; 1: molecular weight standard; 2: purified gp96 protein; 3: western blot results of gp96 protein.

Figure 6 shows the tumor inhibition rates of the PIBC and mPEG ₂₀₀₀ CY treated groups.

The best way to implement the invention

The present invention is further described in detail with reference to the preferred embodiments thereof. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. For the quantitative tests in the following examples, three replicate experiments were set, and the results were averaged.

PBS buffer: 8 g NaCl, 0.2 g KCl, 3.625 g Na ₂ HPO ₄ · 12H ₂ O, 0.24 g KH ₂ PO ₄ , add water to 1 L, adjust pH 7.3. 5 mM Na ₂ HPO ₄ : 1.7907 g Na ₂ HPO ₄ • 12H ₂ O, dilute to 1L with deionized water. 5 mM NaH ₂ PO ₄ : 0.78 g NaH ₂ PO ₄ · 2H ₂ O, made up to 1 L with deionized water.

Mass spectrometry was performed using a VG PLATFORM mass spectrometer using MALDI-TOF technique.

In the following examples, unless otherwise specified, the liquid to liquid ratio is a volume to volume ratio; the solid to liquid ratio is the amount to volume ratio of the substance, the amount of the substance being in mmol, the volume In terms of ml; the solid to solid ratio is the mass to mass ratio.

Unless otherwise specified in the following examples, the room temperature reaction is specifically controlled to be in the range of 20-30 ° C, including 20 ° C and 30 ° C.

The compound having tumor suppressing function of the present invention is a PEGylated polypeptide obtained by modifying a polypeptide named PIBC with PEG, and the modification is achieved by performing a Michael addition reaction of the compound A and the compound B to form a carbon sulfur single bond, and the compound A is one end. PEG attached to maleimide, compound B is introduced into the N-terminal or C-terminal PIBC containing a thiol amino acid residue, PIBC is the polypeptide represented by SEQ ID No. 1 in the sequence listing, and the first position of SEQ ID NO. Is the N-terminal amino acid residue of PIBC; the Michael addition reaction occurs between the maleimide and the thiol;

The structural formula of the PEGylated polypeptide of the present invention is Formula I:

In the formula I, n is a non-zero natural number; the molecular weight of PEG is 2000-5000, and the molecular formula of PEG is HO(CH ₂ CH ₂ O)nH;

Cys and PIBC are linked by a peptide bond formed by the carboxyl group of Cys to the N-terminal amino group of PIBC or by a peptide bond formed by the amino group of Cys with the C-terminal carboxyl group of PIBC.

The preparation method and function of the PEGylated polypeptide of the present invention are specifically described below by taking the molecular weights of PEG as 2000 and 5000 as examples.

Example 1. Preparation of PEGylated polypeptide mPEG ₂₀₀₀ CY

(1) Acquisition of peptides

The polypeptide obtained by adding cysteine to the N-terminus of PIBC shown in SEQ ID NO. 1 in the Sequence Listing is referred to as CY, and the amino acid sequence of CY is shown in SEQ ID NO. 2 in the Sequence Listing, by Jill Biochemical (Shanghai) The polypeptide CY shown in SEQ ID NO. 2 was synthesized.

(B) PEGylation of peptides

Starting material: mPEGxMal (methoxy polyethylene glycol maleimide, x is the average molecular weight of PEG; Mal represents maleimide, maleimide is modified at one end of PEG; m represents methoxy, A The oxy group is attached to the other end of the PEG). Its chemical structure is shown in formula V:

The molecular formula of PEG is HO(CH ₂ CH ₂ O)nH, and the definition of n in formula I and formula V is the same as the definition of n in the formula of PEG. mPEGxMal (x=2000, ie the average molecular weight of PEG is 2000, mPEG ₂₀₀₀ Mal, product of Beijing Keykai Technology Co., Ltd.) through the highly selective Michael addition reaction with the N-terminal amino acid cysteine of the polypeptide CY The thiol group is subjected to an addition reaction to obtain mPEG ₂₀₀₀ CY.

1. Synthesis of mPEG ₂₀₀₀ CY:

A mixture of 20 mg (0.01 mmol) of mPEG ₂₀₀₀ Mal and 10 mg (0.002 mmol) of polypeptide CY was dissolved in 10 ml of a 5 mM NaH ₂ PO ₄ buffer solution, and the pH of the reaction solution was adjusted to 7.2 with a 5 mM Na ₂ HPO ₄ solution. The reaction was monitored by HPLC until the polypeptide reaction was complete.

2. Purification and characterization of PEGylated peptide mPEG ₂₀₀₀ CY

The mPEG ₂₀₀₀ CY was purified by HiTrap SP FF (1ml). The eluent was mobile phase A1 and mobile phase B1. The A1 solution consisted of solute and solvent. The solvent was 20 mM Tris-HCl (pH 7.4). The concentration is 1 mM EDTA·2Na and 0.01% (mass percent concentration) NaN ₃ ; the B1 solution consists of a solute and a solvent, the solvent is 20 mM Tris-HCl (pH 7.4), the solute and its concentration are 1000 mM NaCl, 1 mM EDTA·2Na and 0.01% (mass percent concentration) NaN ₃ . The elution conditions were as follows: firstly, the mixture of A1 liquid and B1 liquid volume ratio of 80% and 20%, respectively, was used, and then the volume ratio of A1 liquid and B1 liquid was 67% and 33%, respectively. Collect samples. The sample collection begins when the absorbance begins to rise. The collected samples were concentrated to 500 μl by centrifugation at 4 ° C, 3500 r/min using a Millipore ultrafiltration centrifuge tube (3 KDa). The concentrated sample was desalted using HiTrap Desalting (5 ml). A fluffy PEGylated polypeptide mPEG ₂₀₀₀ CY pure product obtained after lyophilization of the solvent.

The chemical structure of mPEG ₂₀₀₀ CY was characterized by MALDI-TOF mass spectrometry. The mass spectrometric characterization of mPEG ₂₀₀₀ CY is shown in Figure 1. The structural formula of mPEG ₂₀₀₀ CY is as shown in Formula I. At this time, Cys and PIBC in the formula I are linked by a peptide bond formed by a carboxyl group of Cys and an amino group of an N-terminal amino acid residue of PIBC.

The purity of mPEG ₂₀₀₀ CY was given by analytical high performance liquid chromatography (flow rate: 1 ml/min). Analytical HPLC model: Agilent 1200, model number: Angilent Eclipse XDB-C18Analytical, 5μm, 4.6×150006Dm. Chromatographic operating conditions: linear gradient elution, the eluate consists of mobile phase A2 and mobile phase B2. The mobile phase A2 solution was a 0.1% trifluoroacetic acid aqueous solution of trifluoroacetic acid, and the mobile phase B2 solution was a trifluoroacetic acid acetonitrile solution having a trifluoroacetic acid volume percent concentration of 0.1%. The volume fraction of the linear gradient eluting B2 solution was increased from 40% to 65%, the volume percentage of the A2 solution was reduced from 60% to 35%, the elution time was 11 minutes, and the elution flow rate was 1 ml per minute. 220 nm. The analytical results of the analytical high performance liquid chromatography showed that the purity of the obtained mPEG ₂₀₀₀ CY was 93.4%.

Example 2. Preparation of PEGylated polypeptide mPEG ₅₀₀₀ CY

mPEGxMal (x=5000, ie the average molecular weight of PEG is 5000, mPEG ₅₀₀₀ Mal, product of Beijing Keykai Technology Co., Ltd.) through the highly selective Michael addition reaction with the N-terminal amino acid cysteine of the polypeptide CY The thiol group is subjected to an addition reaction to obtain mPEG ₅₀₀₀ CY.

mPEG ₅₀₀₀ CY was prepared according to the method of 1 in the step (2) of Example 1, and mPEG ₂₀₀₀ Mal was replaced with mPEG ₅₀₀₀ Mal, and the reaction was carried out until the polypeptide reaction was completed.

The reaction product obtained according to the above procedure was purified using an Agilent 1200 reversed-phase high performance liquid chromatography. Column type: Angilent Eclipse XDB-C18Semi-Prep, 5 μm, 9.4×250 mm. Chromatographic operating conditions: linear gradient elution, the eluate consisting of the A2 and B2 solutions of Example 1. The volume fraction of the linear gradient eluting B2 solution was increased from 30% to 52% B, the volume percentage of the A2 solution was reduced from 70% to 48%, the elution time was 11 minutes, and the elution flow rate was 2.5 ml per minute. UV detection The wavelength is 220 nm. A fluffy PEGylated polypeptide mPEG ₅₀₀₀ CY pure product obtained after lyophilization of the solvent.

The MALDI-TOF mass spectrometry results of mPEG ₅₀₀₀ CY are shown in Figure 2. The structural formula of mPEG ₅₀₀₀ CY is as shown in Formula I. At this time, Cys and PIBC in the formula I are linked by a peptide bond formed by a carboxyl group of Cys and an amino group of an N-terminal amino acid residue of PIBC.

mPEG ₅₀₀₀ CY purity analysis is the same as described in Example 1, except that the volume fraction of the linear gradient eluting B2 solution is increased from 20% to 100%, and the volume percentage of the A2 solution is reduced from 80% to 0, and the elution time is 25 minute. The analytical results of the analytical high performance liquid chromatograph showed that the purity of the obtained mPEG ₅₀₀₀ CY was 95.3%.

Example 3 Preparation of PEGylated polypeptide mPEG ₂₀₀₀ LC

The polypeptide obtained by adding cysteine to the C-terminus of PIBC shown in SEQ ID NO. 1 in the Sequence Listing is referred to as LC, and the amino acid sequence of LC is shown in SEQ ID NO. 3 in the Sequence Listing, by Jill Biochemical (Shanghai) The polypeptide LC shown in SEQ ID NO. 3 was synthesized.

mPEGxMal (x=2000, ie, the average molecular weight of PEG is 2000, mPEG ₂₀₀₀ Mal, product of Beijing Keykai Technology Co., Ltd.) through a highly chemically selective Michael addition reaction with the C-terminal amino acid cysteine of polypeptide LC The thiol group undergoes an addition reaction to obtain mPEG ₂₀₀₀ LC. The specific process for preparation, purification and characterization of mPEG ₂₀₀₀ LC is the same as that described in step (2) of Example 1, except that the polypeptide CY of Example 1 is replaced with a polypeptide. LC gave a fluffy PEGylated polypeptide mPEG ₂₀₀₀ LC. The MALDI-TOF mass spectrometry results of mPEG ₂₀₀₀ LC are shown in Figure 3. The analytical results of the analytical high performance liquid chromatography showed that the purity of the obtained mPEG ₂₀₀₀ LC was 94.7%. The structural formula of mPEG ₂₀₀₀ LC is as shown in Formula I. At this time, Cys and PIBC in the formula I are linked by a peptide bond formed by the amino group of Cys and the carboxyl group of the C-terminal amino acid residue of PIBC.

Example 4 Preparation of PEGylated polypeptide mPEG ₅₀₀₀ LC

mPEGxMal (x=5000) (Beijing Keykai Technology Co., Ltd.) was subjected to an addition reaction with a highly selective Michael addition reaction with a thiol group of the C-terminal amino acid cysteine of the polypeptide LC to obtain a PEGylated polypeptide mPEG. The specific procedure for the preparation, purification and characterization of ₅₀₀₀ LC, mPEG ₅₀₀₀ LC is the same as that described in step (2) of Example 1, except that the mPEGxMal (x=2000) in Example 1 is replaced by mPEGxMal (x=5000). The polypeptide CY was replaced with the polypeptide LC to obtain a PEGylated polypeptide mPEG ₅₀₀₀ LC in a fluffy state. The MALDI-TOF mass spectrometry results of mPEG ₅₀₀₀ LC are shown in Figure 4. The analytical results of the analytical high performance liquid chromatography showed that the purity of the obtained PEG5KDYC was 92.2%. mPEG ₅₀₀₀ LC structural formula as Formula I below. At this time, Cys and PIBC in the formula I are linked by a peptide bond formed by the amino group of Cys and the carboxyl group of the C-terminal amino acid residue of PIBC.

Example 5, interaction of gp96 protein with PEGylated polypeptide

The amino acid sequence of the gp96 protein (human heat shock protein; GENBANK ACCESSION NO. AY040226) is shown in SEQ ID NO. 4 of the Sequence Listing, and the coding sequence thereof is shown in SEQ ID NO.

I. Construction pFastBac ^TM 1-gp96 plasmid

1. Design and synthesis of gp96 primer: The sequence of human gp96 gene in GenBank was used as a template, BamHI restriction site was added to the 5' end of gp96 gene, and XbaI restriction site was added to the 3' end. The forward primer sequence was: 5'-CGggattcATGGACGATGAAGTTGATGTGGAT-3'; the reverse primer sequence was: 5'-GCTCTAGATTAGAATTCATCTTTTTCAGCTG-3'.

2. Extract the mRNA of human hepatoma cell HepG2 and reverse transcribe to synthesize cDNA.

3. Using the cDNA obtained in step 1.2 as a template, the target gene was amplified by polymerase chain reaction (PCR) using the primer designed in step 1, to obtain a PCR product, which is a gp96 gene.

4. The PCR product obtained in step 3 was double-digested with EcoRI and XbaI to recover a digested product having a size of about 2400 bp.

5, digested with EcoRI and XbaI pFastBac ^TM 1 empty vector (Invitrogen, catalog number 10359-016) was recovered was recovered in the backbone vector size of about 4700bp.

6, the size of the size of the step 4 of the cleavage product of about 2400bp obtained in step 5 is about 4700bp vector backbone connection, to obtain a recombinant vector, the correct sequence of the recombinant vector was named pFastBac ^TM 1-gp96. The recombinant vector pFastBac ^TM 1-gp96 of the DNA fragment between EcoRI and XbaI recognition sequence replacement vector pFastBac ^TM 1 DNA molecule shown in the Sequence Listing as sequences, and other sequences pFastBac ^TM 1 remains constant vector obtained Carrier. The recombinant plasmid pFastBac ^TM 1-gp96 gp96 protein expression, protein gp96 amino acid sequence shown in the Sequence Listing as SEQ ID NO.4.

2. Purification of gp96 recombinant protein and gp96 protein by insect cells

Using Cellfectin II reagent (Life technologies, NO: 10362-100) a step of pFastBac ^TM 1-gp96 transfected into Sf9 cells (Invitrogen, catalog number 11496-015) in. The Sf9 cells transfected with the positive plasmid were cultured for 72 hours, and the cytopathic condition indicated that the recombinant generation baculovirus (P1) had been released into the medium, and the cell supernatant was collected to obtain P1 toxicity. Add appropriate amount of P1 to Sf9 monolayer (1×10 ⁶ cells/mL) cells, incubate at 27 ° C for 72 h, centrifuge at 4000 rpm for 5 min, and collect the supernatant to obtain second-generation toxic (P2). An appropriate amount of P2 toxicity was added to suspension cells of 100 ml of Sf9 (1.6×10 ⁶ cells/mL) at 27 ° C, and cultured at 100-120 rpm/min for 72 hours, and amplification was carried out to obtain three generations of poison (P3). The P3 virus was subjected to western blotting using a rat anti-gp96 antibody (santa cruz, product number sc-56399) as a primary antibody, and the results showed that the gp96 protein was expressed in Sf9 cells.

Subsequently, an appropriate amount of drug in fresh P3 Sf9 cells (1.5 × 10 ⁶ cells / mL, 300ml) in, 27 ℃, 100-120rpm / min in ^{Insect-XPRESS TM Protein-free Insect} Cells medium with L-Glutamine (Catalog No :12-730Q) Suspension culture was carried out in the medium. After 72 hours, the suspension culture medium was centrifuged at 7000 rpm for 20 minutes to obtain a clear supernatant. The supernatant was filtered through a 0.22 mm filter and purified by HiTrap Q HP column and Superdex 200 10/300 GL ion column. product. The purified product was identified by denaturing polyacrylamide gel electrophoresis and Western blot (the primary antibody used was rat anti-gp96 antibody (santa cruz, product number sc-56399)) (Fig. 5), and it was confirmed that the purified product contained High purity gp96 protein. The solvent in the above purified product was replaced with a PBS buffer by an ultrafiltration tube, and concentrated, and the protein concentration was measured by a BCA method, and finally, the protein was dispensed and stored at -80 °C.

Interaction between gp96 protein and PEGylated polypeptide fragments

The interaction between the PEGylated polypeptide fragment prepared in Examples 1-4 and the gp96 protein was separately detected by the Biacore method. The instrument used for the detection was Biacore 3000 system. Using the CM5 sensor chip, the gp96 protein of step 2 was solidified on the CM5 sensor chip by amino coupling according to the instructions. The specific method was as follows: filtered and degassed HBS buffered saline solution (10 mmol/ L HEPES, 0.15 mol/L NaCl, 3.4 mol/L EDTA, 0.05% P-20; pH 7.4) As a mobile phase solution, the CM5 sensor chip module was embedded in the BIAcore system; the flow rate through the flow cell was set to 5 μL/ Min; activate the surface of CM5 sensor chip with a volume mixing solution of 0.2 mol/L N-ethyl-N-dimethyl-aminopropyl carbodiimide and 0.05 mol/L N-hydroxysuccinimide for 7 min. 35 μL of 1 mg/mL gp96 protein was injected onto the activated surface to bind to the surface of the CM5 sensor chip; 35 μL of ethanolamine was injected to inactivate excess reactive groups; 10 μL of 20 mmol/L HCl was quickly injected, and then non-co-extracted with Extraclean Valence-binding material; determination of the level of binding to gp96 protein by placing a first baseline reporter point prior to the start of injection of gp96 protein and placing a second reporter point 2 min after the end of injection of 20 mmol/L HCl; setting binding to gp96 The flow cell is the detection channel and is not combined with g The flow cell of p96 protein is the reference channel, the HBS buffer solution is the mobile phase, and the flow rate of the flow cell is 10 μL/min. At the same time, the analyte is injected into the gp96 protein flow cell and the reference flow cell, so that the binding reaction is at 22-24 ° C. , under conditions of pH 7.4; injection of 10 μl of Examples 1-4 A PEGylated polypeptide fragment or PIBC (diluted with HBS buffer containing 1 mg/mL carboxymethyldextran); rapid injection of 10 μL 20 mmol/L HCl, regeneration of gp96 protein surface with Extraclean; re-injection of 10 μL PEGylation The polypeptide fragment was repeated this cycle to determine its reproducibility of binding to the surface of the gp96 protein. According to the above steps, polypeptide fragments of different concentration levels (156, 312, 625, 1250, 2500 nmol/L) were detected, and the measurement was repeated once for each concentration level.

The binding coefficients K _D (mM/L) of the PEGylated polypeptide fragments of Examples 1-4 to the gp96 protein are shown in Table 1.

Table 1 Coefficients of binding reaction of PEGylated polypeptide to gp96

Peptide Binding coefficient K _D (mM/L) PIBC 0.633 mPEG ₂₀₀₀ CY 0.690 mPEG ₅₀₀₀ CY 0.732 mPEG ₂₀₀₀ LC 0.754 mPEG ₅₀₀₀ LC 0.823

The results showed that the PEGylated polypeptides of Examples 1-4 had affinity with gp96 protein, and mPEG ₂₀₀₀ CY had the strongest binding ability.

Example 6. Effect of PEGylated polypeptide on breast cancer cell SKBr3

Breast cancer cell SKBr3: ATCC (American type culture collection) product, product number is HTB-30. The PEGylated polypeptide fragments prepared in Examples 1-4 were subjected to the following experiments:

1. PEGylated polypeptide inhibits proliferation of SKBr3 in breast cancer cells

The inhibitory effect of each PEGylated polypeptide fragment on the proliferation of breast cancer cell SKBr3 was examined by CCK-8 kit (Japan Tongren Chemical Research Institute, catalog number CK04-05). The specific steps are as follows:

1. The SKBr3 cells were plated in 96-well plates at a confluence of about 50%. There are three duplicate wells in each group of cells.

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

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

4. Determine the OD value at 490 nm.

The cell growth inhibition rate was calculated as: (control group OD ₄₉₀ average - experimental group OD ₄₉₀ average) / control group OD ₄₉₀ × 100%. The cell growth inhibition rate (average value) of each PEGylated polypeptide fragment-treated group is shown in Table 2.

Table 2 Cell growth inhibition rate of PEGylated polypeptide treatment

Peptide Inhibition rate

PIBC 72% mPEG ₂₀₀₀ CY 61% mPEG ₅₀₀₀ CY 32% mPEG ₂₀₀₀ LC 46% mPEG ₅₀₀₀ LC twenty three%

The results showed that the PEGylated polypeptides of Examples 1-4 significantly inhibited the proliferation (growth) of SKBr3 in breast cancer cells, and the inhibition effect of mPEG ₂₀₀₀ CY was the most obvious.

2. PEGylated polypeptide inhibits the invasiveness of breast cancer cell SKBr3

The effect of the PEGylated polypeptides of Examples 1-4 on the invasive ability of breast cancer cells SKBr3 was examined using Tanswell plate (Corning, Inc., product number #3422) and Matrigel (BD, product number 354234), respectively. Cell invasion assays were performed according to the Transwell and Matrigel instructions. The main steps are as follows:

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

2. The breast cancer cells SKBr3, which have been digested and counted well, were diluted with DPBC and the serum-free medium of PEGylated polypeptide of Example 1-4 (final concentration to 6 μM) to 400,000/ml, and 100 μl was added to the Transwell upper chamber, Transwell. 600 μl of complete cell culture medium was added to the lower chamber as an experimental group; serum-free medium containing the polypeptide fragment was replaced with serum-free medium as a negative control group.

3. Incubate for 24 hours at 37 ° C 5% CO ₂ in a CO ₂ incubator, scrape off the cells in the upper Transwell chamber with a cotton swab, fix with 50% methanol/50% acetone for 15 minutes, then wash 3 times with PBS buffer. , DAPI was mounted, and the number of invading cells was counted under a fluorescence microscope.

The invasive inhibition rate was calculated as: (number of cells invaded in the negative control group - number of cells invaded in the experimental group) / number of cells invaded in the negative control group × 100%.

The inhibition inhibition rate (average value) of SKBr3 in each PEGylated polypeptide fragment-treated group versus the negative control group is shown in Table 3.

Table 3 Inhibition rate of SKBr3 invasion by PEGylated peptide-treated group versus negative control group

Peptide Inhibition rate PIBC 61.3% mPEG ₂₀₀₀ CY 53.8% mPEG ₅₀₀₀ CY 41.2% mPEG ₂₀₀₀ LC 36.3% mPEG ₅₀₀₀ LC 28.5%

The results showed that the PEGylated polypeptides of Examples 1-4 significantly inhibited the invasion of SKBr3 in breast cancer cells, and the inhibitory effect of mPEG ₂₀₀₀ CY was the most obvious.

3. PEGylated polypeptide promotes apoptosis of breast cancer cell SKBr3

The promotion of apoptosis of breast cancer cell SKBr3 by the PEGylated polypeptides of Examples 1-4 was examined, respectively. Specific steps are as follows:

1. Breast cancer cells SKBr3 were inoculated into 6-well cell culture plates at 200,000 cells/well;

2. After the cells were attached, PIBC and the PEGylated polypeptide of Example 1-4 (final concentration: 6 μM) were added for further 24 hours as an experimental group; PBS solution was added as a negative control group.

Apoptosis Assay kit对细胞染色后通过流式细胞仪分析结果，具体的操作步骤如下：3. Use the kit produced by Invitrogen

The Apoptosis Assay kit stains the cells and analyzes the results by flow cytometry. The specific steps are as follows:

(1) The cells are routinely digested with trypsin and washed twice with PBS buffer (the number of cells is generally one quarter of a 6-well plate or the number of a 24-well plate is preferably).

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

(3) Add 1x Annexin V Buffer to the reaction tube to a final volume of 200 μl.

(4) Add PI at a concentration of 100 μg/ml to a final concentration of 1 μg/ml, and stain at room temperature for about 3 minutes to perform on-machine detection.

The increased apoptosis rate (mean) of the PEGylated polypeptide treated group of Examples 1-4 compared to the negative control group is shown in Table 4.

Table 4 Increased apoptosis rate of PEGylated peptide treated group compared with negative control group

Peptide Apoptotic rate PIBC 55.5% mPEG ₂₀₀₀ CY 44.3% mPEG ₅₀₀₀ CY 37.3% mPEG ₂₀₀₀ LC 36.4% mPEG ₅₀₀₀ LC 28.8%

The results showed that the PEGylated polypeptides of Examples 1-4 can significantly promote the apoptosis of SKBr3 cells in breast cancer cells. Among them, mPEG ₂₀₀₀ CY promoted the most obvious effect.

IV. mPEG ₂₀₀₀ CY inhibits the growth of transplanted tumor of breast cancer cell MDA-MB-231

The inhibitory effect of mPEG ₂₀₀₀ CY on the growth of transplanted tumor of breast cancer cell MDA-MB-231 was examined. Specific steps are as follows:

1. Breast cancer cells MDA-MB-231 cultured in logarithmic growth phase were subcutaneously inoculated with BALB/c nude mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), each inoculated with 10 million cells, and a transplanted tumor model was established. After that, it was passaged in nude mice for 3 times for the tumor-bearing experiment.

2. When the tumor grows to 100 mm ³ , the BALB/c nude mice are randomly divided into 3 groups, 5 in each group, and the following treatments are performed respectively. The first treatment day is recorded as the first day:

PIBC group: subcutaneous injection with PIBC solution (polypeptide dissolved in 0.9% saline), Each injection dose was 5 μm/kg, and it was treated 3 times a week (on the first, third and sixth days of the week, respectively);

mPEG ₂₀₀₀ CY group: subcutaneous injection with mPEG ₂₀₀₀ CY solution (mPEG ₂₀₀₀ CY dissolved in 0.9% saline), each injection dose was 5μm/kg, injection volume was the same as PIBC treatment group, 3 times per week (respectively On the first, third and sixth days of the week);

Control group: subcutaneous injection in PBS buffer, injection volume and PIBC treatment group, 3 times a week (on the first, third and sixth days of the week);

3. The volume of the second tumor was measured weekly. After 1 month of treatment, the nude mice were sacrificed, the tumor weight was weighed and the tumor inhibition rate was calculated.

The tumor inhibition rate was calculated as follows: (tumor volume of control mice - volume of tumor mice of the polypeptide group) / tumor volume of control mice × 100%.

The tumor inhibition rate results of the PIBC and mPEG ₂₀₀₀ CY treatment groups are shown in Fig. 6. The results showed that both PIBC and mPEG ₂₀₀₀ CY could effectively inhibit the growth of breast cancer cells induced by MDA-MB-231 in breast cancer cells, and the mPEG ₂₀₀₀ CY group was more effective than PIBC. There was a significant difference between the two groups.

Example 7. Evaluation of plasma half-life in SD rats with PEGylated polypeptide

Test peptide: PIBC, mPEG ₂₀₀₀ CY

1. Preparation of standard curve: A stock solution having a test polypeptide concentration of 1 mg/mL was prepared using borax buffer (pH 9.5). The stock solution was prepared in an amount of 50% by volume of acetonitrile in an acetonitrile aqueous solution to prepare a standard curve working solution having a polypeptide concentration of 25, 37.5, 50, 75, 100, 150, 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 peptide concentrations of 5, 7.5, 10, 15, 20, 30, 50 μg/mL. Add 20 μl 20% (quality) to the standard curve sample. Percentage) phosphoric acid solution and 300 μl methanol-acetonitrile (methanol to acetonitrile volume ratio of 1:1), vortex mixing for about 2 min; centrifuge at 4000 rmp / min for 10 minutes, take the supernatant for sample analysis, to obtain each test drug The standard curve is configured and the quality control sample is configured to detect its precision.

2. Experimental process

Drug configuration: pre-dose configuration, the test peptide was dissolved into a uniform transparent solution with an equal volume of 0.9% sodium chloride injection and 5 mM Na ₂ HPO ₄ . The final concentrations of PIBC and mPEG ₂₀₀₀ CY were 8 mg/ml and 12 mg, respectively. /ml for subcutaneous administration.

Test animals: male and female SD rats weighing 160-180 g, source: Beijing Huakang Biotechnology Co., Ltd.

Animal experiments: Administration: Four SD rats were treated with each polypeptide, two males and two females. The body weight was weighed before administration, and the administration amount was 8 mg/kg.

Sample collection: At the time of administration, the time was zero, and 0.3 ml of blood was taken from the tail vein at time zero and 30 min, 1 h, 2 h, 4 h, 6 h, 10 h, 12 h, 24 h after administration, and 6 μl of aprotinin and 5 μl were placed. In the centrifuge tube of sodium heparin, the supernatant plasma was separated and stored in a refrigerator at -80 ° C for 5 min at 4500 rmp/min.

Sample processing: Take 100 μl of plasma of the sample to be tested, add 20 μl of 20% phosphoric acid solution, 20 μl 50% Aqueous acetonitrile solution and 300 μl of methanol-acetonitrile (1:1) solution were vortexed for about 2 min, centrifuged at 4000 rpm/min for 10 minutes, and the supernatant was loaded for analysis.

Chromatographic conditions: column: XSELECT CSH C18, 4.6 × 150 mm, 5 μm, mobile phase: phase A: 0.1% by volume of TFA in water, phase B: 0.1% by volume of TFA in acetonitrile, eluent from phase A Composition with phase B, the volume percentage of phase B in the eluent is increased from 20% to 35%, the volume percentage of phase A is 80% to 65%, the elution time is 10 minutes, and the elution flow rate is 1 ml per minute. , UV detection wavelength: 220 nm, injection volume: 20 μL.

3. Experimental results

1) The relationship between the drug concentration and the peak area obtained from the standard curve of the drug PIBC and mPEG ₂₀₀₀ CY was y=3.916x+8.23 (R=0.995) and y=4.205x+1.23 (R=0.994), respectively. Where y is the peak area and x is the drug concentration.

2) The concentration of each drug at each time point was obtained according to the standard curve, and the pharmacokinetic parameters were calculated from the non-compartment model in WinNonlin pharmacokinetic software. The results are shown in Table 5.

Table 5 pharmacokinetic parameters

The results in Table 5 indicate that:

1) The terminal elimination half-life (T _1/2 ) of mPEG ₂₀₀₀ CY in animals is significantly prolonged.

2) The clearance rate of mPEG ₂₀₀₀ CY (Cl_F_obs) was significantly lower than that of peptide PIBC, and the mean residence time (MRTlast) was significantly prolonged.

The above results indicate that the elimination of the PEGylated polypeptide mPEG ₂₀₀₀ CY in the animal is slower than the polypeptide PIBC. In addition to significantly enhancing its water solubility, this structural modification can also achieve the purpose of prolonging the action time of the drug.

Industrial application

The experiment proves that the PEGylated polypeptide with tumor suppressing function of the invention has affinity with gp96 protein, can significantly inhibit tumor cell proliferation (growth), can significantly inhibit tumor cell invasion function, can obviously promote tumor cell apoptosis, and can effectively inhibit Tumor cells caused tumor growth, and mPEG ₂₀₀₀ CY was more potent than PIBC inhibition. In addition, the terminal elimination half-life (T _1/2 ) of the PEGylated polypeptide of the present invention is significantly prolonged in animals, and the clearance rate (Cl_F_obs) is significantly lower than that of the polypeptide PIBC, and the mean residence time (MRTlast) is significantly prolonged. It is shown that the PEGylated polypeptide of the present invention having tumor suppressing function can be used for treating tumors.

Claims (10)

a PEGylated polypeptide, which is a compound obtained by modifying a polypeptide named PIBC with PEG; The polypeptide of PIBC is A1) or A2): A1) the polypeptide of SEQ ID No. 1 in the sequence listing; A2) the amino acid sequence of SEQ ID NO. 1 in the sequence listing is subjected to one or several amino acid residues. Substituted and/or deleted and/or polypeptides derived from A1) which have the same function. The PEGylated polypeptide according to claim 1, wherein the modification is achieved by a Michael addition reaction using a compound A and a compound B; The compound A is a PEG modified with maleimide at one end; The compound B is introduced into the PIBC containing a mercapto amino acid residue at the N-terminus or the C-terminus. The PEGylated polypeptide according to claim 1 or 2, wherein in the compound A, the other end of the PEG is linked to a methoxy group; and/or the thiol-containing amino acid residue is a cysteine residue. base. The PEGylated polypeptide according to claim 3, wherein the compound A has the structural formula shown in Formula V;

In the formula V, n is a non-zero natural number; And/or, the compound B is the polypeptide represented by SEQ ID NO. 2 or SEQ ID NO. The PEGylated polypeptide according to any one of claims 1 to 4, wherein the PEGylated polypeptide has the structural formula shown in Formula I:

In formula I, n is a non-zero natural number; Further, the molecular formula of PEG is HO(CH ₂ CH ₂ O)nH, the molecular weight of PEG is 2000-5000, and the formula n and the formula n are the same as the definition of n in the PEG formula. The method for preparing a PEGylated polypeptide according to any one of claims 1 to 5, comprising: performing a Michael addition reaction of the compound A with the compound B to obtain the PEGylated polypeptide; The compound A is a PEG having a maleimide attached to one end; The compound B is introduced into the PIBC containing a mercapto amino acid residue at the N-terminus or the C-terminus. Any of the following applications of the PEGylated polypeptide of any of claims 1-5: M1) for use in the preparation of a product that binds to the gp96 protein; M2) for the preparation of a product for treating and/or preventing diseases caused by gp96 protein; M3) use in the preparation of a product that inhibits tumor cell proliferation and/or growth and/or invasion; M4) application in the preparation of a product for promoting tumor cell apoptosis; M5) Application in the preparation of products for inhibiting tumor growth. The use according to claim 7, wherein the gp96 protein is B1) or B2): B1) a polypeptide represented by SEQ ID No. 4 in the Sequence Listing; B2) a polypeptide derived from B1) having the amino acid sequence of SEQ ID NO. 4 in the sequence listing subjected to substitution and/or deletion and/or addition of one or several amino acid residues and having the same function; And/or, the disease caused by the gp96 protein in M2) is a tumor. a product having any of the following functions N1) to N3, comprising the PEGylated polypeptide of any of claims 1-5; N1) inhibiting tumor cell proliferation and/or growth and/or invasion; N2) promotes tumor cell apoptosis; N3) inhibits tumor growth. The product according to claim 7 or 8, or the product according to claim 9, wherein the disease caused by the gp96 protein in M2) in claim 7 or 8 is breast cancer; M3) and M4) The tumor cells are all breast cancer tumor cells; the tumor in M5) is breast cancer; In claim 9, the tumor cells in N1) and N2) are breast cancer tumor cells; and the tumor in N3) is breast cancer.

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