WO2018205622A1 - 具有抑制肿瘤功能的peg化多肽及其制备方法与应用 - Google Patents

具有抑制肿瘤功能的peg化多肽及其制备方法与应用 Download PDF

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WO2018205622A1
WO2018205622A1 PCT/CN2017/116865 CN2017116865W WO2018205622A1 WO 2018205622 A1 WO2018205622 A1 WO 2018205622A1 CN 2017116865 W CN2017116865 W CN 2017116865W WO 2018205622 A1 WO2018205622 A1 WO 2018205622A1
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polypeptide
compound
pibc
tumor
pegylated
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French (fr)
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胡俊
孟颂东
杨哲
杨博
吴飚
丁筠
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北京康明海慧生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • the invention relates to a PEGylated polypeptide having the function of inhibiting tumor in the field of biotechnology, a preparation method and application thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • peptide drugs 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.
  • the technical problem to be solved by the present invention is how to inhibit tumors, especially breast cancer tumors.
  • 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.
  • 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.
  • the other end of the PEG i.e., the end to which the maleimide is not attached
  • the thiol-containing amino acid residue may be a cysteine residue.
  • the compound A can be as shown in 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:
  • 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.
  • 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.
  • the present invention also provides any of the following applications of the PEGylated polypeptide:
  • M3 use in the preparation of a product that inhibits tumor cell proliferation and/or growth and/or invasion
  • the gp96 protein may be B1) or B2):
  • 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.
  • 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.
  • the present invention also provides a product having any of the following functions N1) to N3), the product containing the PEGylated polypeptide;
  • N3 inhibits tumor growth.
  • the tumor cells described in N1) and N2) may be breast cancer tumor cells.
  • the tumor described in N3) may be breast cancer.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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;
  • n is a non-zero natural number
  • the molecular weight of PEG is 2000-5000
  • 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.
  • 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.
  • 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.
  • the thiol group is subjected to an addition reaction to obtain 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 ⁇ l 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.
  • 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, 5 ⁇ m, 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
  • 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%.
  • 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 ⁇ m, 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.
  • 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.
  • 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%.
  • 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.
  • 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.
  • 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.
  • 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%.
  • 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.
  • 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.
  • 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'.
  • 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.
  • PCR polymerase chain reaction
  • step 3 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ L/ 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 ⁇ L of 1 mg/mL gp96 protein was injected onto the activated surface to bind to the surface of the CM5 sensor chip; 35 ⁇ L of ethanolamine was injected to inactivate excess reactive groups; 10 ⁇ L 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 ⁇ L/min.
  • 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 ⁇ l of Examples 1-4 A PEGylated polypeptide fragment or PIBC (diluted with HBS buffer containing 1 mg/mL carboxymethyldextran); rapid injection of 10 ⁇ L 20 mmol/L HCl, regeneration of gp96 protein surface with Extraclean; re-injection of 10 ⁇ L 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.
  • PEGylated polypeptide inhibits proliferation of SKBr3 in breast cancer cells
  • 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.
  • the PEGylated polypeptide (final concentration 6 ⁇ M) was added as the experimental group, and three wells without the polypeptide were set as the control group.
  • 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.
  • PEGylated polypeptide inhibits the invasiveness of breast cancer cell SKBr3
  • Matrigel was frozen and thawed overnight on ice on the day before the experiment. 60 ⁇ l of Matrigel was added to the Transwell upper chamber, coated at 37 ° C for 1 hour, and washed twice with PBS buffer.
  • 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.
  • PEGylated polypeptide promotes apoptosis of breast cancer cell SKBr3
  • PIBC and the PEGylated polypeptide of Example 1-4 were added for further 24 hours as an experimental group; PBS solution was added as a negative control group.
  • the Apoptosis Assay kit stains the cells and analyzes the results by flow cytometry. The specific steps are as follows:
  • 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).
  • 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%
  • mPEG 2000 CY inhibits the growth of transplanted tumor of breast cancer cell MDA-MB-231
  • 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 ⁇ m/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 5 ⁇ m/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);
  • 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.
  • Test peptide PIBC, mPEG 2000 CY
  • 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 ⁇ g/mL. Take 20 ⁇ l of the prepared standard curve working solution, add 80 ⁇ l of blank mouse plasma, and prepare standard curve samples with peptide concentrations of 5, 7.5, 10, 15, 20, 30, 50 ⁇ g/mL. Add 20 ⁇ l 20% (quality) to the standard curve sample.
  • 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 ⁇ l of aprotinin and 5 ⁇ l 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 ⁇ l of plasma of the sample to be tested, add 20 ⁇ l of 20% phosphoric acid solution, 20 ⁇ l 50% Aqueous acetonitrile solution and 300 ⁇ l 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.
  • T 1/2 terminal elimination half-life
  • 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.
  • 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.

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Abstract

本发明提供了一种具有抑制肿瘤活性的PEG化多肽及其制备方法与应用。所述的PEG化多肽利用PEG修饰PIBC多肽（SEQ ID NO：1）而得到，该修饰通过利用化合物甲与化合物乙进行Michael加成反应形成碳硫单键实现，化合物甲为一端修饰有马来酰亚胺的PEG，化合物乙为N端或C端引入含有巯基的半胱氨酸残基的PIBC；Michael加成反应发生在马来酰亚胺与巯基间。本发明的PEG化多肽在保留了PIBC抑制热休克蛋白gp96活性的同时，延长了PIBC的体内半衰期。

Description

具有抑制肿瘤功能的PEG化多肽及其制备方法与应用 技术领域
本发明涉及生物技术领域中，具有抑制肿瘤功能的PEG化多肽及其制备方法与应用。
背景技术
根据全国肿瘤登记中心发表的《2015中国癌症统计数据报告》显示，乳腺癌是中国女性发病率最高的癌症类型，其发病率正以每年3％的速度递增，成为城市中死亡率增长最快的癌症。目前乳腺癌主要治疗方法包括手术、化疗、内分泌以及靶向治疗。靶向治疗具有定位准确，针对性强，毒副反应小等特点，能够明显地延长患者的生存期，提高患者的生存质量，尤其适于晚期病人或无法耐受放、化疗的患者。现有的乳腺癌的靶向治疗主要针对的是HER2阳性乳腺癌，其靶向药物Trastuzumab(即赫塞汀)是一种人源化的HER2单克隆抗体，对HER2基因扩增和过表达的乳腺癌病人，trastuzumab单独应用或与其它药物联合应用有效率可分别达到25％或50％，赫塞汀目前已成为乳腺癌等治疗的一线药物，销售额位居肿瘤生物治疗药物前列。而HER2阳性的乳腺癌患者仅占乳腺癌患者总数的20％左右，因此急需寻找新的乳腺癌靶点，开发出更为广谱的抗乳腺癌靶向药物。
热休克蛋白gp96属于HSP90家族的成员，在正常生理条件下定位于细胞内质网中，与细胞质HSP90高度同源。与其它热休克蛋白一样，gp96的主要生物学功能是分子伴侣。它参与新生蛋白的折叠，转运，降解及多聚体的组装，抑制错误折叠蛋白的分泌等。与其他分子伴侣不同，gp96能结合的蛋白、多肽链种类有限，更具选择性。目前已经报道的已有20多种蛋白的成熟需要gp96的参与，这些研究揭示gp96作为分子伴侣的调控作用，与多个客户蛋白形成蛋白质复合物，其介导的调控网络对于多个肿瘤蛋白发挥功能至关重要。除了定位于内质网中，gp96蛋白还存在于某些细胞表面。越来越多的研究发现，gp96在多种癌细胞的膜表面有过表达现象，暗示着其与癌症的发生发展存在某种联系。孟颂东等研究发现内质网中的gp96位移至细胞膜上发挥核心脚手架蛋白的调控作用，与多个客户蛋白形成蛋白质机器复合物，对于多个肿瘤蛋白发挥功能至关重要，与乳腺癌的恶性程度与不良预后密切相关。细胞膜上gp96可能是乳腺癌治疗的新靶点。
中国专利ZL 201110159487.4基于热休克蛋白gp96氨基酸序列及空间构象，通过一系列技术分析和手段，设计开发含有α-螺旋序列的多肽PIBC(Peptide Inhibitor for Breast Cancer)，该多肽能与热休克蛋白gp96结合，干扰gp96与肿瘤相关受体HER-2的相互作用并降低其稳定性、促进HER-2在肿瘤细胞中的降解，该多肽能够降低乳腺癌患者的肿瘤组织中HER2、uPAR、ER-a36的蛋白含量，从而实现抗乳腺癌的临床疗效。
虽然多肽类药物的作用位点专一、疗效确切，但溶解度低、免疫原性高、易被蛋白酶降解和被肾脏清除等缺陷严重地制约了其临床应用。
发明公开
本发明所要解决的技术问题是如何抑制肿瘤，尤其是乳腺癌肿瘤。
为解决上述技术问题，本发明首先提供了具有抑制肿瘤功能的PEG化多肽。
本发明所提供的PEG化多肽，为利用PEG修饰名称为PIBC的多肽得到的化合物；
PIBC为A1)或A2)所述的多肽：A1)序列表中SEQ ID No.1所示多肽；A2)将序列表中的SEQ ID NO.1中的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由A1)衍生的多肽。
上述PEG化多肽中，所述修饰可通过利用化合物甲与化合物乙进行Michael加成反应形成碳硫单键实现；
所述化合物甲为一端修饰有马来酰亚胺的PEG；
所述化合物乙为N端或C端引入含有巯基氨基酸残基的PIBC；
所述Michael加成反应发生在马来酰亚胺与巯基间。
所述化合物甲中，PEG的另一端(即非连接马来酰亚胺的一端)可连接有甲氧基。所述含有巯基氨基酸残基可为半胱氨酸残基。
所述化合物甲可如式V所示；
Figure PCTCN2017116865-appb-000001
式V中，n为非零自然数。
所述化合物乙可为SEQ ID NO.2或SEQ ID NO.3所示的多肽。
所述PEG化多肽具体可为式I所示化合物：
Figure PCTCN2017116865-appb-000002
式I中，n为非零自然数；
Cys与PIBC通过Cys的羧基与PIBC的N末端氨基形成的肽键或通过Cys的氨基与PIBC的C末端羧基形成的肽键相连。
PEG的分子式为HO(CH2CH2O)nH，PEG的分子量为2000～5000，式V和式I中的n与PEG分子式中的n的定义相同，即n满足使PEG的分子量为2000～5000的条件。
所述化合物甲具体可为北京键凯科技有限公司产品。
为解决上述技术问题，本发明还提供了所述PEG化多肽的制备方法。
本发明所提供的PEG化多肽的制备方法，包括：利用所述化合物甲与所述化合物乙进行Michael加成反应得到所述PEG化多肽；
所述Michael加成反应发生在马来酰亚胺与巯基间。
为解决上述技术问题，本发明还提供了所述PEG化多肽的下述任一应用：
M1)在制备与gp96蛋白结合的产品中的应用；
M2)在制备治疗和/或预防gp96蛋白所致疾病产品中的应用；
M3)在制备抑制肿瘤细胞增殖和/或生长和/或侵袭产品中的应用；
M4)在制备促进肿瘤细胞凋亡产品中的应用；
M5)在制备抑制肿瘤生长产品中的应用。
上述应用中，所述gp96蛋白可为下述B1)或B2)：
B1)序列表中SEQ ID No.4所示多肽；
B2)将序列表中的SEQ ID NO.4中的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由B1)衍生的多肽。
M2)中所述gp96蛋白所致疾病为肿瘤。
上述应用中，M2)中所述gp96蛋白所致疾病可为乳腺癌。M3)和M4)中所述肿瘤细胞均可为乳腺癌肿瘤细胞，M5)中所述肿瘤可为乳腺癌。
为解决上述技术问题，本发明还提供了具有如下N1)-N3)中任一功能的产品，所述产品含有所述PEG化多肽；
N1)抑制肿瘤细胞增殖和/或生长和/或侵袭；
N2)促进肿瘤细胞凋亡；
N3)抑制肿瘤生长。
上述产品中，N1)和N2)中所述肿瘤细胞均可为乳腺癌肿瘤细胞。N3)中所述肿瘤可为乳腺癌。
本发明中，所述乳腺癌细胞均可为乳腺癌细胞SKBr3。所述肿瘤均可为乳腺癌细胞MDA-MB-231引发的肿瘤。
附图说明
图1为PEG化多肽mPEG2000CY的质谱鉴定结果图。
图2为PEG化多肽mPEG5000CY的质谱鉴定结果图。
图3为PEG化多肽mPEG2000LC的质谱鉴定结果图。
图4为PEG化多肽mPEG5000LC的质谱鉴定结果图。
图5为昆虫细胞表达系统表达的分泌型人热休克蛋白gp96的鉴定图谱；1：分子量标准；2:纯化后的gp96蛋白；3:gp96蛋白的western blot结果。
图6为PIBC及mPEG2000CY处理组的肿瘤抑制率。
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂、仪器等，如无特殊说明，均可从商业途径得到。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
PBS缓冲液：8g NaCl、0.2g KCl、3.625g Na2HPO4·12H2O、0.24g KH2PO4,加水至1L，调pH7.3。5mM Na2HPO4：1.7907g Na2HPO4·12H2O，用去离子水定容至1L。5mM NaH2PO4：0.78g NaH2PO4·2H2O，用去离子水定容至1L。
质谱采用VG PLATFORM质谱仪，用MALDI-TOF技术进样。
下述实施例中如无特殊说明，所述液体与液体的比值为体积与体积比；所述固体与液体的比值为物质的量与体积比,所述物质的量以mmol计，所述体积以ml计；所述固体与固体的比值为质量与质量比。
下述实施例中如无特殊说明，所述室温反应具体为将反应温度控制在20-30℃范围内，包括20℃和30℃。
本发明的具有抑制肿瘤功能的化合物，为利用PEG修饰名称为PIBC的多肽得到的PEG化多肽，该修饰通过利用化合物甲与化合物乙进行Michael加成反应形成碳硫单键实现，化合物甲为一端连接有马来酰亚胺的PEG，化合物乙为N端或C端引入含有巯基氨基酸残基的PIBC，PIBC为序列表中SEQ ID No.1所示多肽，SEQ ID NO.1的第1位为PIBC的N端氨基酸残基；Michael加成反应发生在马来酰亚胺与巯基间；
本发明的PEG化多肽的结构式为式I：
Figure PCTCN2017116865-appb-000003
式I中，n为非零自然数；PEG的分子量为2000～5000，PEG的分子式为HO(CH2CH2O)nH；
Cys与PIBC通过Cys的羧基与PIBC的N端氨基形成的肽键或通过Cys的氨基与PIBC的C端羧基形成的肽键相连。
下面具体以PEG的分子量为2000和5000为例，具体阐述本发明的PEG化多肽的制备方法和功能。
实施例1、PEG化多肽mPEG2000CY的制备
(一)多肽的获得
将在序列表中SEQ ID NO.1所示PIBC的N端添加半胱氨酸得到的多肽记为CY，CY的氨基酸序列如序列表中SEQ ID NO.2所示，由吉尔生化(上海)有限公司合成SEQ ID NO.2所示的多肽CY。
(二)多肽的PEG化修饰
原料：mPEGxMal(甲氧基聚乙二醇马来酰亚胺,x为PEG的平均分子量；Mal表示马来酰亚胺，马来酰亚胺修饰在PEG的一端；m表示甲氧基，甲氧基连接在PEG的另一端)。其化学结构式如式V所示：
Figure PCTCN2017116865-appb-000004
PEG的分子式为HO(CH2CH2O)nH，式I、式V的n与PEG分子式中的n的定义均相同。将mPEGxMal(x＝2000，即PEG的平均分子量为2000，mPEG2000Mal，北京键凯科技有限公司产品)通过化学选择性极高的Michael加成反应与多肽CY的N端氨基酸半胱氨酸的巯基进行加成反应，得到mPEG2000CY。
1、mPEG2000CY的合成：
20毫克(0.01mmol)mPEG2000Mal与10毫克(0.002mmol)多肽CY的混合物溶于10毫升5mM的NaH2PO4缓冲溶液中，用5mM的Na2HPO4溶液调节反应溶液pH值至7.2，HPLC监测反应，直至多肽反应完全。
2、PEG化多肽mPEG2000CY的纯化及表征
采用HiTrap SP FF(1ml)大量纯化mPEG2000CY，洗脱液为流动相A1液和流动相B1液，A1液由溶质和溶剂组成，溶剂为20mM Tris-HCl(pH7.4)，溶质及其浓度为1mM EDTA·2Na和0.01％(质量百分比浓度)NaN3；B1液由溶质和溶剂组成，溶剂为20mM Tris-HCl(pH7.4)，溶质及其浓度为1000mM NaCl、1mM EDTA·2Na和0.01％(质量百分比浓度)NaN3。洗脱条件为：先用A1液和B1液的体积比分别为80％和20％的混合液冲平，再用A1液和B1液的体积比分别为67％和33％的混合液洗脱收集样品。当吸光值开始升高时则开始收集样品。将收集到的样品用Millipore超滤离心管(3KDa)4℃，3500r/min离心浓缩至500微升。浓缩后的样本用HiTrap Desalting(5ml)进行脱盐处理。冻干溶剂后得到的蓬松状态的PEG化多肽mPEG2000CY纯品。
mPEG2000CY化学结构由MALDI-TOF质谱进行表征，mPEG2000CY的质谱表征结果见 图1。mPEG2000CY的结构式如式I所示。此时，式I中Cys与PIBC通过Cys的羧基与PIBC的N端氨基酸残基的氨基形成的肽键相连。
mPEG2000CY纯度由分析型高效液相色谱仪(流速：1ml/min)给出。分析性高效液相色谱仪的型号：安捷伦1200，所采用色谱柱的型号：Angilent Eclipse XDB-C18Analytical,5μm,4.6×150006Dm。色谱操作条件：线性梯度洗脱，洗脱液由流动相A2液和流动相B2液组成。流动相A2液为三氟乙酸体积百分浓度为0.1％的三氟乙酸水溶液,流动相B2液为三氟乙酸体积百分浓度为0.1％的三氟乙酸乙腈溶液。线性梯度洗脱B2液的体积百分比由40％匀速升至65％，A2液的体积百分比由60％匀速降至35％，洗脱时间11分钟，洗脱流速为每分钟1毫升，紫外检测波长220纳米。分析型高效液相色谱仪检测结果显示，所得mPEG2000CY的纯度为93.4％。
实施例2、PEG化多肽mPEG5000CY的制备
将mPEGxMal(x＝5000，即PEG的平均分子量为5000，mPEG5000Mal，北京键凯科技有限公司产品)通过化学选择性极高的Michael加成反应与多肽CY的N端氨基酸半胱氨酸的巯基进行加成反应，得到mPEG5000CY。
mPEG5000CY按照实施例1中步骤(二)中1的方法制备，将mPEG2000Mal替换为mPEG5000Mal，进行反应，直至多肽反应完全。
采用安捷伦1200反相高效液相色潽仪对按照上述步骤所得的反应产物进行纯化。色谱柱型号：Angilent Eclipse XDB-C18Semi-Prep,5μm,9.4×250mm。色谱操作条件：线性梯度洗脱，洗脱液由实施例1的A2液和B2液组成。线性梯度洗脱B2液的体积百分比由30％匀速升至52％B，A2液的体积百分比由70％匀速降至48％，洗脱时间11分钟，洗脱流速为每分钟2.5毫升，紫外检测波长220纳米。冻干溶剂后得到的蓬松状态的PEG化多肽mPEG5000CY纯品。
mPEG5000CY的MALDI-TOF质谱表征结果见图2。mPEG5000CY的结构式如式I所示。此时，式I中Cys与PIBC通过Cys的羧基与PIBC的N端氨基酸残基的氨基形成的肽键相连。
mPEG5000CY纯度分析同实施例1所述,不同的是线性梯度洗脱B2液的体积百分比由20％匀速升至100％，A2液的体积百分比由80％匀速降至0，洗脱时间25分钟。分析型高效液相色谱仪检测结果显示，所得mPEG5000CY的纯度为95.3％。
实施例3、PEG化多肽mPEG2000LC的制备
将在序列表中SEQ ID NO.1所示PIBC的C端添加半胱氨酸得到的多肽记为LC，LC的氨基酸序列如序列表中SEQ ID NO.3所示，由吉尔生化(上海)有限公司合成SEQ ID NO.3所示的多肽LC。
将mPEGxMal(x＝2000，即PEG的平均分子量为2000，mPEG2000Mal，北京键凯科技有限公司产品)通过化学选择性极高的Michael加成反应与多肽LC的C端 氨基酸半胱氨酸的巯基进行加成反应，即得到mPEG2000LC，mPEG2000LC的制备、纯化和表征的具体过程同实施例1中步骤(二)所述，不同的是将实施例1中的多肽CY换成多肽LC，得到蓬松状态的PEG化多肽mPEG2000LC。mPEG2000LC的MALDI-TOF质谱表征结果见图3。分析型高效液相色谱仪检测结果显示，所得mPEG2000LC的纯度为94.7％。mPEG2000LC的结构式如式I所示。此时，式I中Cys与PIBC通过Cys的氨基与PIBC的C端氨基酸残基的羧基形成的肽键相连。
实施例4、PEG化多肽mPEG5000LC的制备
将mPEGxMal(x＝5000)(北京键凯科技有限公司)通过化学选择性极高的Michael加成反应与多肽LC的C端氨基酸半胱氨酸的巯基进行加成反应，即得PEG化多肽mPEG5000LC，mPEG5000LC的制备、纯化和表征的具体过程同实施例1中步骤(二)所述，不同的是将实施例1中的mPEGxMal(x＝2000)换成mPEGxMal(x＝5000)，多肽CY换成多肽LC，得到蓬松状态的PEG化多肽mPEG5000LC。mPEG5000LC的MALDI-TOF质谱表征结果见图4。分析型高效液相色谱仪检测结果显示，所得PEG5KDYC的纯度为92.2％。mPEG5000LC的结构式如式I所示。此时，式I中Cys与PIBC通过Cys的氨基与PIBC的C端氨基酸残基的羧基形成的肽键相连。
实施例5、gp96蛋白与PEG化多肽的相互作用
gp96蛋白(人热休克蛋白；GENBANK ACCESSION NO.AY040226)的氨基酸序列如序列表的SEQ ID NO.4所示，其编码序列如SEQ ID NO.5所示。
一、pFastBacTM1-gp96质粒的构建
1、gp96引物的设计与合成：以GenBank中人gp96基因的序列为模板，在gp96基因5’端加入BamHI酶切位点，3’端加入XbaI酶切位点。正向引物序列为：5’-CGggattcATGGACGATGAAGTTGATGTGGAT-3’；反向引物序列为：5’-GCTCTAGATTAGAATTCATCTTTTTCAGCTG-3’。
2、提取人肝癌细胞HepG2的mRNA,反转录合成cDNA。
3、以步骤1.2中获取的cDNA为模板，采用步骤1设计的引物通过聚合酶链式反应(PCR)扩增目的基因，得到PCR产物，即为gp96基因。
4、用EcoRI和XbaI双酶切步骤3获得的PCR产物，回收大小约为2400bp的酶切产物。
5、用EcoRI和XbaI双酶切pFastBacTM 1空载质粒(Invitrogen，产品目录号为10359-016)，回收回收得到大小约为4700bp的骨架载体。
6、将步骤4获得的大小约为2400bp的酶切产物和步骤5获得的大小约为4700bp的载体骨架连接，得到重组载体，将序列正确的重组载体命名为pFastBacTM1-gp96。重组载体pFastBacTM 1-gp96为将将pFastBacTM 1载体的EcoRI和XbaI识别序列间的DNA片段替换为序列表中序列2所示的DNA分子，且保持pFastBacTM 1载体的其他序列不变得到的载体。重组质粒pFastBacTM 1-gp96表达 gp96蛋白，gp96蛋白的氨基酸序列如序列表中SEQ ID NO.4所示。
二、昆虫细胞表达gp96重组蛋白及gp96蛋白的纯化
利用Cellfectin II reagent(Life technologies,货号：10362-100)将步骤一的pFastBacTM1-gp96转染到Sf9细胞(Invitrogen，产品目录号为11496-015)中。将转染阳性质粒的Sf9细胞培养72h，通过细胞病变情况表明含有重组的一代杆状病毒(P1)已经释放到培养基中，收取细胞上清获得P1毒。加适量P1到Sf9单层(1×106细胞/mL)细胞中，27℃，培养72h，4000rpm离心5min，收取上清获得二代毒(P2)。将适量P2毒加到100ml Sf9(1.6×106细胞/mL)的悬浮细胞中27℃，100-120rpm/min培养72h,扩增获得三代毒(P3)。将P3毒利用大鼠抗gp96抗体(santa cruz，产品号sc-56399)作为一抗进行western blotting，结果表明gp96蛋白在Sf9细胞中表达。
随后，在新鲜的Sf9细胞(1.5×106细胞/mL，300ml)中加入适量P3毒，27℃,100-120rpm/min在Insect-XPRESSTM Protein-free Insect Cells medium with L-Glutamine(Catalog No:12-730Q)培养基中进行悬浮培养。72小时后，将悬浮培养的培养液7000rpm离心20分钟得到清澈的上清液，将上清液经过0.22mm滤膜滤过后，经过HiTrap Q HP柱，Superdex 200 10/300GL离子柱纯化后得到纯化产物。将纯化产物经变性聚丙烯酰胺凝胶电泳和Western blot免疫印迹试验(所用一抗为大鼠抗gp96抗体(santa cruz，产品号sc-56399))进行鉴定(图5)，确定纯化产物中含有高纯度的gp96蛋白。利用超滤管将上述纯化产物中溶剂替换为PBS缓冲液，并浓缩，采用BCA法测定蛋白浓度，最后将蛋白分装，贮存于-80℃。
三、gp96蛋白与PEG化多肽片段的相互作用
通过Biacore方法分别检测实施例1-4制备的PEG化多肽片段与gp96蛋白之间的相互作用。检测所用仪器为Biacore3000系统，使用CM5传感芯片，按说明书将步骤二的gp96蛋白通过氨基偶联固化在CM5传感芯片上，具体方法如下：采用过滤并除气的HBS缓冲盐溶液(10mmol/L HEPES，0.15mol/L NaCl，3.4mol/L EDTA，0.05％P-20；pH7.4)作为流动相溶液，将CM5传感器芯片模块嵌入BIAcore系统；设定流过流动池的流速为5μL/min；用0.2mol/L的N-乙基-N-二甲基-氨丙基碳二亚胺和0.05mol/L的N-羟基琥珀酰亚胺等体积混合溶液活化CM5传感芯片表面7min；注射35μL 1mg/mL的gp96蛋白到活化表面，使之与CM5传感芯片表面结合；注射35μL乙醇胺使过量的反应基团失活；快速注射10μL 20mmol/L的HCl，然后用Extraclean除去非共价结合型材料；通过在开始注射gp96蛋白前放置第1个基线报道点,并在注射20mmol/L HCl结束后2min放置第2个报道点,来测定结合gp96蛋白的水平；设定结合gp96的流通池为检测通道，未结合gp96蛋白的流通池为参比通道，HBS缓冲溶液为流动相，流通池的流速为10μL/min；同时将待测物注入gp96蛋白流通池和参比流通池，使结合反应在22-24℃，pH7.4的条件下进行；注射10μl实施例1-4中 的一种PEG化多肽片段或PIBC(使用含1mg/mL羧甲基葡聚糖的HBS缓冲液稀释)检测；快速注射10μL 20mmol/L的HCl，用Extraclean再生gp96蛋白表面；再次注射10μL PEG化多肽片段，重复此循环，以测定其结合到gp96蛋白表面的重现性。按上述步骤，分别检测不同浓度级别(156，312，625，1250，2500nmol/L)的多肽片段，每一浓度级别重复测定1次。
实施例1-4的PEG化多肽片段与gp96蛋白的结合系数KD(mM/L)见表1。
表1 PEG化多肽与gp96结合反应的系数
多肽 结合系数KD(mM/L)
PIBC 0.633
mPEG2000CY 0.690
mPEG5000CY 0.732
mPEG2000LC 0.754
mPEG5000LC 0.823
结果表明，实施例1-4的PEG化多肽与gp96蛋白均有亲和力，其中mPEG2000CY结合能力最强。
实施例6、PEG化多肽对乳腺癌细胞SKBr3的作用效果
乳腺癌细胞SKBr3：ATCC(American type culture collection)产品，产品号为HTB-30。分别将实施例1-4制备的PEG化多肽片段进行如下实验：
一、PEG化多肽抑制乳腺癌细胞SKBr3增殖
通过CCK-8试剂盒(日本同仁化学研究所，货号CK04-05)分别检测各个PEG化多肽片段对乳腺癌细胞SKBr3增殖的抑制作用。具体操作步骤如下：
1、将SKBr3细胞铺到96孔板中，汇合度为50％左右。每组细胞设三个复孔。
2、待细胞贴壁后，加入PEG化多肽(终浓度6μM)做为实验组，设三个不加多肽的孔为对照组。
3、在不同的时间检测点(0，3，6，12小时)，每孔中加入CCK-8检测试剂10μl，37℃孵育2小时。
4、测定490nm的OD值。
细胞生长抑制率计算公式为：(对照组OD490平均值-实验组OD490平均值)/对照组OD490×100％。各个PEG化多肽片段处理组的细胞生长抑制率(平均值)见表2。
表2 PEG化多肽处理的细胞生长抑制率
多肽 抑制率
PIBC 72％
mPEG2000CY 61％
mPEG5000CY 32％
mPEG2000LC 46％
mPEG5000LC 23％
结果表明，实施例1-4的PEG化多肽能显著抑制乳腺癌细胞SKBr3增殖(生长)，其中mPEG2000CY抑制效果最明显。
二、PEG化多肽抑制乳腺癌细胞SKBr3的侵袭力
利用Tanswell plate(Corning公司，产品号#3422)和Matrigel(BD公司，产品号354234)分别检测实施例1-4的PEG化多肽对乳腺癌细胞SKBr3侵袭能力的影响。细胞侵袭实验根据Transwell和Matrigel说明书进行操作。主要操作步骤如下：
1、实验前一天在冰上过夜冻融Matrigel，在Transwell上层小室中加入60μl的Matrigel，37℃包被1小时，PBS缓冲液洗涤2次。
2、将己消化计数好的乳腺癌细胞SKBr3用PIBC及实施例1-4的PEG化多肽的无血清培养液(终浓缩为6μM)稀释到40万/ml，取100μl加入Transwell上层小室，Transwell下层小室加入600μl完全细胞培养液，作为实验组；用无血清培养液代替含多肽片段的无血清培养液，作为阴性对照组。
3、在CO2孵箱中37℃5％CO2继续培养24小时，用棉签刮去上层Transwell小室中的细胞，50％甲醇/50％丙酮固定15分钟后，再用PBS缓冲液洗3次，DAPI封片，荧光显微镜下计数侵袭的细胞数。
侵袭抑制率的计算公式为：(阴性对照组中侵袭的细胞数-实验组中侵袭的细胞数)/阴性对照组中侵袭的细胞数×100％。
各个PEG化多肽片段处理组相对阴性对照组对SKBr3的侵袭抑制率(平均值)见表3。
表3 PEG化多肽处理组相对阴性对照组对SKBr3侵袭的抑制率
多肽 抑制率
PIBC 61.3％
mPEG2000CY 53.8％
mPEG5000CY 41.2％
mPEG2000LC 36.3％
mPEG5000LC 28.5％
结果表明，实施例1-4的PEG化多肽能显著抑制乳腺癌细胞SKBr3侵袭功能，其中mPEG2000CY抑制效果最明显。
三、PEG化多肽促进乳腺癌细胞SKBr3的凋亡
分别检测实施例1-4的PEG化多肽对乳腺癌细胞SKBr3凋亡的促进作用。具体步骤如下：
1、乳腺癌细胞SKBr3接种于6孔细胞培养板，20万细胞/孔；
2、待细胞贴壁后，加入PIBC及实施例1-4的PEG化多肽(终浓度6μM)继续培养24小时，作为实验组；将加入PBS溶液的作为阴性对照组。
3、运用Invitrogen公司生产的试剂盒
Figure PCTCN2017116865-appb-000005
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：
(1)用胰酶常规消化细胞，PBS缓冲液洗两遍(细胞数量一般为6孔板的四分之一或一个24孔板的数量为宜)。
(2)用20μl 1x Annexin V Buffer将细胞轻轻悬起来，加入1μl的FITC annexin V后轻轻混匀，室温避光染色15分钟。
(3)向反应管中加入1x Annexin V Buffer使终体积为200μl。
(4)加入浓度为100μg/ml的PI，使其终浓度为1μg/ml，室温避光染色3分钟左右即可上机检测。
实施例1-4的PEG化多肽处理组相比阴性对照组增加的细胞凋亡率(平均值)见表4。
表4 PEG化多肽处理组比阴性对照组增加的细胞凋亡率
多肽 凋亡率
PIBC 55.5％
mPEG2000CY 44.3％
mPEG5000CY 37.3％
mPEG2000LC 36.4％
mPEG5000LC 28.8％
结果表明，实施例1-4的PEG化多肽能明显促进乳腺癌细胞SKBr3细胞凋亡。其中mPEG2000CY促进效果最明显。
四、mPEG2000CY抑制乳腺癌细胞MDA-MB-231的移植瘤生长
检测mPEG2000CY对乳腺癌细胞MDA-MB-231的移植瘤生长的抑制作用。具体步骤如下：
1、将培养至对数生长期的乳腺癌细胞MDA-MB-231皮下接种BALB/c裸鼠(北京维通利华实验动物技术有限公司)，每只接种1000万细胞，建立移植瘤模型，之后在裸鼠体内经过3次传代用于接瘤实验。
2、等到肿瘤生长到100mm3时将BALB/c裸鼠随机分为3组，每组5只，分别进行如下治疗处理，第1次处理当天记为第1天：
PIBC组：用PIBC溶液(多肽溶于0.9％的生理盐水)进行皮下注射治疗， 每次注射剂量为5μm/kg，每周治疗3次(分别于该周的第一天、第三天和第六天)；
mPEG2000CY组：用mPEG2000CY溶液(mPEG2000CY溶于0.9％的生理盐水)进行皮下注射治疗，每次注射剂量为5μm/kg，注射体积同PIBC治疗组，每周治疗3次(分别于该周的第一天、第三天和第六天)；
对照组：用PBS缓冲液进行皮下注射治疗，注射体积同PIBC治疗组，每周治疗3次(分别于该周的第一天、第三天和第六天)；
3、每周检测二次肿瘤体积，治疗1个月后处死裸鼠，称取肿瘤重量并计算肿瘤抑制率。
肿瘤抑制率的计算公式如下：(对照组小鼠的肿瘤体积-多肽组小鼠肿瘤的体积)/对照组小鼠的肿瘤体积×100％。
PIBC及mPEG2000CY治疗组的肿瘤抑制率结果如图6所示。结果表明PIBC及mPEG2000CY均能有效抑制乳腺癌细胞MDA-MB-231引起的乳腺癌肿瘤生长,且mPEG2000CY组比PIBC抑制效果更明显，两组之间具有显著性差异。
实施例7、PEG化多肽的SD大鼠体内血浆半衰期评价
供试多肽：PIBC、mPEG2000CY
1.标准曲线的制作：用硼砂缓冲液(PH 9.5)配制各供试多肽浓度为1mg/mL的储备液。取该储备液适量用乙腈体积百分比为50％的乙腈水溶液配制成多肽浓度为25、37.5、50、75、100、150、250μg/mL的标准曲线工作液。取准备好的标准曲线工作液20μl，加入空白鼠血浆80μl，配制多肽浓度为5、7.5、10、15、20、30、50μg/mL的标准曲线样品，标准曲线样品中加入20μl 20％(质量百分比)磷酸溶液和300μl甲醇-乙腈(甲醇与乙腈的体积比为1:1)，涡旋混匀约2min；4000rmp/min离心10分钟，取上清液上样分析，得各供试药物的标准曲线，并依该方法配置质控样品检测其精密度。
2.实验过程
药物配置：给药前配置，将供试多肽用等体积的0.9％氯化钠注射液和5mM Na2HPO4溶解成均一透明溶液，PIBC、mPEG2000CY的终浓度分别为8mg/ml和12mg/ml，用于皮下给药。
试验动物：雌雄SD大鼠，体重160～180克，来源：北京华阜康生物科技股份有限公司。
动物实验：给药：每种多肽处理四只SD大鼠，雌雄各两只。给药前称定体重，给药计量为8mg/kg。
样品采集：给药时记为零时刻，分别在零时刻和给药后30min、1h、2h、4h、6h、10h、12h、24h通过尾静脉取血0.3ml于装有6μl抑肽酶和5μl肝素钠的离心管中，4500rmp/min离心5min分离上层血浆置于-80℃冰箱保存。
样品处理：取100μl待测样本的血浆，加入20μl 20％磷酸溶液、20μl 50％ 乙腈水溶液和300μl甲醇-乙腈(1:1)溶液，涡旋混匀约2min，4000rmp/min离心10分钟，取上清液上样分析。
色谱条件：色谱柱：XSELECT CSH C18,4.6×150mm,5μm，流动相：A相：0.1％(体积百分比)TFA水溶液，B相：0.1％(体积百分比)TFA乙腈溶液，洗脱液由A相和B相组成，洗脱液中B相的体积百分比由20％匀速升至35％，A相的体积百分比80％匀速降至65％，洗脱时间10分钟，洗脱流速为每分钟1毫升，紫外检测波长：220nm，进样体积：20μL。
3.实验结果
1)由药物PIBC、mPEG2000CY的标准曲线所得的药物浓度与峰面积的关系式分别为y＝3.916x+8.23(R＝0.995)、y＝4.205x+1.23(R＝0.994)。其中y为峰面积，x为药物浓度。
2)各时间点各药物浓度依据标准曲线得到，药动学参数由WinNonlin药动学软件中的非房室模型计算得出，结果见表5。
表5药动学参数
Figure PCTCN2017116865-appb-000006
表5的结果表明：
1)mPEG2000CY在动物体内的末端消除半衰期(T1/2)明显延长。
2)mPEG2000CY的清除率(Cl_F_obs)显著低于多肽PIBC，平均滞留时间(MRTlast)显著延长。
以上结果可说明PEG化多肽mPEG2000CY在动物体的消除要比多肽PIBC慢。这种结构修饰除了能显著增强其水溶性外，还能达到延长药物作用时间的目的。
工业应用
实验证明，本发明的具有抑制肿瘤功能的PEG化多肽与gp96蛋白具有亲和力，能显著抑制肿瘤细胞增殖(生长)，能显著抑制肿瘤细胞侵袭功能，能明显促进肿瘤细胞凋亡，还能有效抑制肿瘤细胞引起的肿瘤生长,且mPEG2000CY比PIBC抑制效果更显著。另外，本发明的PEG化多肽在动物体内的末端消除半衰期(T1/2)明显延长，清除率(Cl_F_obs)显著低于多肽PIBC，平均滞留时间(MRTlast)显著延长。表明，本发明的具有抑制肿瘤功能的PEG化多肽可用于治疗肿瘤。

Claims (10)

  1. PEG化多肽，为利用PEG修饰名称为PIBC的多肽得到的化合物；
    PIBC为A1)或A2)所述的多肽：A1)序列表中SEQ ID No.1所示多肽；A2)将序列表中的SEQ ID NO.1中的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由A1)衍生的多肽。
  2. 根据权利要求1所述的PEG化多肽，其特征在于：所述修饰通过利用化合物甲与化合物乙进行Michael加成反应形成实现；
    所述化合物甲为一端修饰有马来酰亚胺的PEG；
    所述化合物乙为N端或C端引入含有巯基氨基酸残基的PIBC。
  3. 根据权利要求1或2所述的PEG化多肽，其特征在于：所述化合物甲中，PEG的另一端连接有甲氧基；和/或，所述含有巯基氨基酸残基为半胱氨酸残基。
  4. 根据权利要求3所述的PEG化多肽，其特征在于：所述化合物甲的结构式如式V所示；
    Figure PCTCN2017116865-appb-100001
    式V中，n为非零自然数；
    和/或，所述化合物乙为SEQ ID NO.2或SEQ ID NO.3所示的多肽。
  5. 根据权利要求1-4中任一所述的PEG化多肽，其特征在于：所述PEG化多肽的结构式如式I所示：
    Figure PCTCN2017116865-appb-100002
    式I中，n为非零自然数；
    进一步，PEG的分子式为HO(CH2CH2O)nH，PEG的的分子量为2000～5000，式V和式I中的n与PEG分子式中的n的定义相同。
  6. 权利要求1-5中任一所述PEG化多肽的制备方法，包括：利用化合物甲与化合物乙进行Michael加成反应得到所述PEG化多肽；
    所述化合物甲为一端连接有马来酰亚胺的PEG；
    所述化合物乙为N端或C端引入含有巯基氨基酸残基的PIBC。
  7. 权利要求1-5中任一所述PEG化多肽的下述任一应用：
    M1)在制备与gp96蛋白结合的产品中的应用；
    M2)在制备治疗和/或预防gp96蛋白所致疾病产品中的应用；
    M3)在制备抑制肿瘤细胞增殖和/或生长和/或侵袭产品中的应用；
    M4)在制备促进肿瘤细胞凋亡产品中的应用；
    M5)在制备抑制肿瘤生长产品中的应用。
  8. 根据权利要求7所述的应用，其特征在于：所述gp96蛋白为下述B1)或B2)：
    B1)序列表中SEQ ID No.4所示多肽；
    B2)将序列表中的SEQ ID NO.4中的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由B1)衍生的多肽；
    和/或，M2)中所述gp96蛋白所致疾病为肿瘤。
  9. 具有如下N1)-N3)中任一功能的产品，含有权利要求1-5中任一所述PEG化多肽；
    N1)抑制肿瘤细胞增殖和/或生长和/或侵袭；
    N2)促进肿瘤细胞凋亡；
    N3)抑制肿瘤生长。
  10. 根据权利要求7或8所述的应用或权利要求9所述的产品，其特征在于：权利要求7或8中，M2)中所述gp96蛋白所致疾病为乳腺癌；M3)和M4)中所述肿瘤细胞均为乳腺癌肿瘤细胞；M5)中所述肿瘤为乳腺癌；
    权利要求9中，N1)和N2)中所述肿瘤细胞均为乳腺癌肿瘤细胞；N3)中所述肿瘤为乳腺癌。
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