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WO2018205622A1 - Polypeptide pegylé ayant une fonction inhibitrice de tumeur, son procédé de préparation et son application - Google Patents

Polypeptide pegylé ayant une fonction inhibitrice de tumeur, son procédé de préparation et son application Download PDF

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Publication number
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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胡俊
孟颂东
杨哲
杨博
吴飚
丁筠
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Beijing Cominghealth Bio Tec Co Ltd
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Beijing Cominghealth Bio Tec Co Ltd
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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

Definitions

  • 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

La présente invention concerne un polypeptide pegylé ayant une fonction d'inhibition de tumeur, son procédé de préparation et son application. Le polypeptide pegylé est obtenu par modification d'un polypeptide PIBC (SEQ ID NO : 1) avec du PEG. La modification est mise en oeuvre par formation d'une liaison simple carbone-soufre par la soumission d'un premier composé et d'un second composé à une réaction d'addition de Michael. Le premier composé est un PEG ayant une extrémité terminale modifiée par maléimide, et le second composé est un PIBC ayant une extrémité N-terminale ou une extrémité C-terminale dans laquelle un résidu de cystéine contenant du sulfydryle est introduit; la réaction d'addition de Michael se produit entre le maléimide et le sulfydryle. Le polypeptide pegylé de la présente invention ne se limite pas seulement au PIBC pour inhiber l'activité de la protéine de choc thermique gp96, mais s'étend également à la demi-vie in vivo du PIBC.
PCT/CN2017/116865 2017-05-12 2017-12-18 Polypeptide pegylé ayant une fonction inhibitrice de tumeur, son procédé de préparation et son application Ceased WO2018205622A1 (fr)

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CN118290405A (zh) * 2024-04-03 2024-07-05 江苏省人民医院（南京医科大学第一附属医院） 一种靶向slc7a5高表达肿瘤的荧光探针及其制备方法和应用

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CN113121668A (zh) * 2020-01-10 2021-07-16 北京康明海慧生物科技有限公司 PEG修饰的可抑制gp96的多肽、其制备方法及用途
CN114686428B (zh) * 2020-12-30 2024-04-02 浙江康明海慧生物科技有限公司 细胞-多肽偶联物及其制备方法和应用

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CN102827257A (zh) * 2011-06-14 2012-12-19 中国科学院微生物研究所 一组gp96蛋白的多肽片段及其应用
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WO2016054315A1 (fr) * 2014-10-01 2016-04-07 Medimmune, Llc Méthode de conjugaison d'un polypeptide

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CN102827257A (zh) * 2011-06-14 2012-12-19 中国科学院微生物研究所 一组gp96蛋白的多肽片段及其应用
CN104684546A (zh) * 2012-06-07 2015-06-03 哈佛大学校长及研究员协会 用于药物靶向的纳米疗法
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* Cited by examiner, † Cited by third party
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CN118290405A (zh) * 2024-04-03 2024-07-05 江苏省人民医院（南京医科大学第一附属医院） 一种靶向slc7a5高表达肿瘤的荧光探针及其制备方法和应用

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