WO2015000181A1 - Nouvelle protéine de fusion recombinée, son procédé de préparation et utilisation - Google Patents

Nouvelle protéine de fusion recombinée, son procédé de préparation et utilisation Download PDF

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Publication number
WO2015000181A1
WO2015000181A1 PCT/CN2013/078922 CN2013078922W WO2015000181A1 WO 2015000181 A1 WO2015000181 A1 WO 2015000181A1 CN 2013078922 W CN2013078922 W CN 2013078922W WO 2015000181 A1 WO2015000181 A1 WO 2015000181A1
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Prior art keywords
fusion protein
protein
dimer
vegfr1d2
sequence
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PCT/CN2013/078922
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English (en)
Chinese (zh)
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田文志
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华博生物医药技术（上海）有限公司
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Priority to PCT/CN2013/078922 priority Critical patent/WO2015000181A1/fr
Publication of WO2015000181A1 publication Critical patent/WO2015000181A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor

Definitions

  • the invention relates to the field of biomedicine. More specifically, the present invention relates to a novel recombinant fusion protein, a process for its preparation and use. Background technique
  • Angiogenesis is a new type of angiogenesis induced by vascular endothelial growth factor VEGF, which induces phosphorylation of VEGFR2 by binding VEGF to VEGF receptors (VEGFR1, VEGFR2) on the surface of vascular endothelial cells. Signaling leads to proliferation of vascular endothelial cells. If VEGF is over-secreted under certain disease conditions (such as tumor, wet age-related macular degeneration), it may induce abnormal proliferation of blood vessels. The result of hyperplasia is to promote the proliferation and metastasis of tumor cells, promote choroidal vascular proliferation and lead to wetness. Macular degeneration.
  • targeted therapeutic drugs for VEGF have been extensively studied, and approved targeted therapeutic drugs for VEGF include monoclonal antibodies and recombinant fusion proteins.
  • the former include Avastin (Av aS tin), injection ranibizumab (Lucentis), the latter aflibercept (Zaltrap, Aflibercept, also known as VEGF-Trap).
  • Avastin is used to treat colorectal cancer and lung cancer
  • ranibizumab injection is used to treat wet age-related macular degeneration
  • aboxicept is used to treat colorectal cancer and wet age-related macular degeneration.
  • the genetic recombination fusion protein KH902 developed by a domestic company has also completed the clinical trial of treating wet age-related macular degeneration, waiting for the new drug approval stage.
  • the object of the present invention is to provide a novel recombinant fusion protein, a preparation method and use thereof.
  • a fusion protein having the structure of formula la or formula lb:
  • A is a protein element comprising a second extramembranous region D2 of vascular endothelial growth factor receptor 1 (VEGFR1), and element A is 94-103 amino acids in length;
  • VEGFR1 vascular endothelial growth factor receptor 1
  • B is an immunoglobulin element
  • C is an optional signal peptide sequence
  • the element A has the amino acid sequence (core sequence) shown at positions 25-117 of SEQ ID NO.: 1 and is 94, 95, 96, 97, 98, 99, or 100 in length. Amino acids.
  • the element A is 100 amino acids in length.
  • amino acid sequence flanking the amino acid sequence (core sequence) shown in positions 25-117 of SEQ ID NO.: 1 in the element A is derived from the second extramembranous region of native VEGFR1, respectively. Amino acid sequence on both sides of D2 (Domain 2,).
  • the amino acid sequence of said element A is shown in positions 20-119 of SEQ ID NO.: 1.
  • the D2 has a flanking sequence, and the flanking sequence includes:
  • the first flanking sequence consists of 1-5 amino acid residues.
  • the second flanking sequence consists of 1-2 amino acid residues.
  • first and second flanking sequences are derived from the amino acid sequences flanking the second extramembranous region D2 (positions 25-117 of SEQ ID NO.: 1) of native VEGFR1, respectively.
  • the first flanking sequence is SDTGR.
  • the second flanking sequence is NT.
  • said element B is an Fc fragment of human immunoglobulin IgG1.
  • the peptide linker is 0-10 amino acids in length, preferably 1-5 amino acids. More preferably, the peptide linker is EF (positions 120-121 in SEQ ID NO.: 1).
  • the fusion protein further comprises a signal peptide element (:.
  • the fusion protein does not contain a signal peptide, and the structural formula is
  • amino acid sequence of the signal peptide is shown as position 1-19 of SEQ ID NO: 1.
  • amino acid sequence of the fusion protein is shown in SEQ ID NO: 1.
  • the fusion protein has the following characteristics:
  • Plasma half-life is 5 days.
  • a protein dimer is provided, the dimer being formed from any of the fusion proteins of the first aspect.
  • the dimer has the structure of Formula Ila or Formula lib:
  • A is a protein element comprising a second extramembranous region D2 of VEGFR1, and the length of the element A is 94-103 amino acids;
  • B is an immunoglobulin element;
  • c is an optional signal peptide sequence
  • the fusion protein does not contain a signal peptide, and the structural formula is
  • A is a protein element comprising a second extramembranous region D2 of VEGFR1, and the length of the element A is 94-103 amino acids;
  • B is an immunoglobulin element;
  • an isolated polynucleotide encoding the fusion protein of claim 1 is provided.
  • a vector comprising the polynucleotide of the third aspect is provided.
  • a host cell comprising the vector or genome in the fourth aspect is provided A polynucleotide according to the third aspect.
  • the host cell is a prokaryotic cell or a eukaryotic cell (e.g., CH0 cell, NSO cell, or 293 cell).
  • a method of producing a protein comprising the steps of:
  • the host cell of the fifth aspect is cultured under conditions suitable for expression, thereby expressing the fusion protein of the first aspect
  • a pharmaceutical composition comprising:
  • the fusion protein of the first aspect and/or the protein dimer of the second aspect and
  • a pharmaceutically acceptable carrier According to an eighth aspect of the invention, there is provided the use of the fusion protein of the first aspect of the invention and/or the protein dimer of the second aspect, for the preparation of a medicament for treating a disease.
  • the disease is selected from the group consisting of: tumor, wet macular degeneration or liver fibrosis.
  • the tumor comprises: colorectal cancer, lung cancer.
  • the disease is an angiogenesis-related disease.
  • a method of inhibiting angiogenesis or treating an angiogenesis-related disease comprising the steps of: administering to a subject in need thereof the fusion protein of the first aspect.
  • the fusion protein is administered as a monomer and/or a dimer.
  • the object is a human.
  • Other aspects of the invention will be apparent to those skilled in the art from this disclosure. It is to be understood that within the scope of the present invention, the above-described various technical features of the present invention and the technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here. DRAWINGS
  • Figure 1A is a schematic view showing the structure of the recombinant fusion protein VEGFR1D2-FC.
  • Figure IB shows the nucleotide sequence of a recombinant fusion protein VEGFR1D2-FC of the present invention.
  • Figure 1C shows the amino acid sequence of a recombinant fusion protein VEGFR1D2-FC of the present invention.
  • FIG. 2 shows the SDS-PAGE electrophoresis pattern of VEGFR1D2-Fc protein.
  • Each lane is as follows: 1 is a non-reducing condition, 2 is a Marker, and 3 is a reducing condition.
  • Figure 3 shows the results of the VEGFR1D2-FC protein and target VEGF binding activity test.
  • FIG. 4 shows that VEGFR1D2-FC blocks VEGF-induced VEGFR2 phosphorylation.
  • FIG. 5 shows that VEGFR1D2-FC blocks vascular endothelial cell tubular formation induced by VEGF.
  • FIG. 6 shows that VEGFR1D2-FC has an inhibitory effect on angiogenesis in zebrafish.
  • Different doses of VEGFR1D2-Fc (4.4, 14.7, 44 ng) were injected into the blood circulation of zebrafish, and intestinal blood vessels under different conditions were recorded. quantity.
  • Figure 7 shows a comparison of the experiments with both Endo and Lovastatin for inhibition of angiogenesis in zebrafish. Endo (44, lOOng) and lovastatin (4 ng) were injected into the blood circulation of zebrafish and the number of intestinal blood vessels under different conditions was recorded.
  • FIGS 8 and 9 show that VEGFR1D2-FC has an activity of inhibiting the growth of lung cancer cells (A549).
  • FIGS 10 and 11 show that VEGFR1D2-FC has an activity of inhibiting the growth of colorectal cancer cells (C0L0-205).
  • Figure 12 shows the results of pharmacokinetic experiments with VEGFR1D2-FC. detailed description
  • the inventors have extensively and intensively studied and unexpectedly found that the fusion protein composed of the F2 extracellular region D2 (Domain 2) increases the flanking sequence and binds it to the Fc fragment of I gG1 to have strong VEGF binding. Activity, thereby developing a new class of recombinant fusion protein drugs, such as VEGFR1D2-Fc.
  • the present invention has been completed on this basis.
  • VEGFR1D2-FC has the following functions: 1) blocking VEGF-induced VEGFR2 phosphorylation; 2) blocking VEGF-induced angiogenesis in vitro or in vivo; 3) dose-dependent inhibition of tumor cells Migration and invasion.
  • VEGFR protein belongs to the receptor tyrosine kinase superfamily and is a membrane mosaic protein.
  • the extramembranous portion of VEGFR is approximately There are 750 amino acid residues consisting of 7 Ig domains with similar immunoglobulin structures.
  • VEGFR proteins include: VEGFR1 (Flt-1), VEGFR2 (KDR/FLk-1), VEGFR3 (Flt_4), or a combination thereof.
  • VEGFR1 (Flt-1) is preferred, and preferably native VEGFR1 is wild type.
  • D2 of the present invention refers to the second extramembranous region (Domain 2) of VEGFR1 (Flt-1).
  • a representative D2 sequence is positions 25-117 of SEQ ID NO.: 1.
  • a suitable immunoglobulin G element is not particularly limited and may be an immunoglobulin element derived from a human or other mammal, or a mutant and a derivative thereof. An element of human immunoglobulin is preferred.
  • Human immunoglobulin G comprises four subclasses: IgGl, IgG2, IgG3, I g G4.
  • the protein structure of these four subclasses has great similarity and has four regions: one variable region (VH) and three constant regions (CH1, CH2, CH3).
  • the Fc fragment consists of two constant regions (CH2-CH3) with a disulfide bond in the CH2 region such that the two Fc fragment monomers constitute a covalently bound homodimer.
  • the concentration of IgG in human plasma is highest in IgGl, followed by IgG2, and lower in IgG3 and IgG4.
  • a preferred G element is a human IgGl Fc fragment, or a mutant or derivative thereof. Fusion protein and preparation thereof
  • fusion protein protein of the present invention
  • fusion protein of the present invention are used interchangeably, and mean a structure having the formula la or lb, that is, containing the second extramembranous region D2 including VEGFR1.
  • a representative example is VEGFR1D2-Fc.
  • the protein of the present invention may be a monomer or a multimer (e.g., a dimer) formed of a monomer.
  • the term also encompasses active fragments and derivatives of fusion proteins.
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is isolated and purified, as separated from other substances present in the natural state.
  • isolated recombinant fusion protein means that the recombinant fusion protein is substantially free of natural and related thereto. Other proteins, lipids, sugars or other substances.
  • One skilled in the art can purify recombinant fusion proteins using standard protein purification techniques. Substantially pure proteins produce a single major band on a non-reducing polyacrylamide gel.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • the present invention also relates to a variant of the above polynucleotide which encodes a proteinaceous fragment, analog and derivative having the same amino acid sequence as the present invention.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of its encoded polypeptide.
  • primer refers to a generic term for a oligodeoxynucleotide that, in pair with a template, can be used to synthesize a DNA strand complementary to a template under the action of a DNA polymerase.
  • Primers can be native RNA, DNA, or can be any form of natural nucleotide.
  • the primer may even be a non-natural nucleotide such as LNA or ZNA.
  • the primer “substantially” (or “substantially”) is complementary to a particular sequence on a strand on the template.
  • the primer must be sufficiently complementary to a strand on the template to initiate extension, but the sequence of the primer need not be fully complementary to the sequence of the template.
  • a sequence that is not complementary to the template is added to the 5' end of the primer complementary to the template at a 3' end, and such primers are still substantially complementary to the template.
  • the non-fully complementary primers can also form a primer-template complex with the template for amplification.
  • the full-length nucleotide sequence of the fusion protein of the present invention or its element (e.g., VEGFR1D2) or a fragment thereof can usually be obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed according to published nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art.
  • the template is amplified and related to the sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.
  • the recombination method can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then connecting them.
  • a method of amplifying DNA/RNA using a PCR technique is preferably used to obtain the gene of the present invention.
  • Primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method.
  • the amplified DNA/RNA fragment can be isolated and purified by a conventional method such as gel electrophoresis.
  • the invention also relates to vectors comprising the polynucleotides of the invention, and host cells genetically engineered using the vector or fusion protein coding sequences of the invention, and methods of producing the proteins of the invention by recombinant techniques.
  • the polynucleotide sequences of the present invention can be utilized to express or produce recombinant proteins by conventional recombinant DNA techniques. Generally there are the following steps:
  • Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequences of the proteins of the invention and suitable transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; animal cells of CH0, NS0, C0S7, or 293 cells, and the like.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2.
  • Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (e.g., temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the protein in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If desired, the protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation, use Protein precipitant treatment (salting method), centrifugation, osmotic bacteria, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and others Various liquid chromatography techniques and combinations of these methods.
  • conventional renaturation use Protein precipitant treatment (salting method), centrifugation, osmotic bacteria, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and others Various liquid
  • antibody and “ligand” are used interchangeably and refer to polyclonal antibodies and monoclonal antibodies, particularly monoclonal antibodies, which are specific for the protein of the present invention.
  • specificity means that an antibody can bind to a protein of the present invention or a fragment thereof, respectively.
  • the antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art.
  • the invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab) 2 fragments; antibody heavy chains; antibody light chains; genetically engineered single chain Fv molecules; Or chimeric antibodies.
  • immunologically active antibody fragments such as Fab' or (Fab) 2 fragments; antibody heavy chains; antibody light chains; genetically engineered single chain Fv molecules; Or chimeric antibodies.
  • the invention provides a fusion protein which optionally contains a peptide linker.
  • the size and complexity of the peptide linker may affect the activity of the protein.
  • the peptide linker should be of sufficient length and flexibility to ensure that the two proteins attached have sufficient freedom in space to perform their function. At the same time, the effect of the formation of ⁇ -helix or ⁇ -sheet in the peptide linker on the stability of the fusion protein is avoided.
  • the length of the linker peptide is generally from 0 to 10 amino acids, preferably from 1 to 5 amino acids.
  • the invention also provides a composition comprising an effective amount of a fusion protein of the invention, and a pharmaceutically acceptable carrier.
  • the fusion proteins of the invention may be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the ⁇ ⁇ is typically from about 5 to about 8, preferably, ⁇ ⁇ is from about 6 to about 8.
  • the term "effective amount” or “effective amount” refers to an amount that can produce a function or activity to a human and/or animal and can be accepted by a human and/or an animal, such as from 0.001 to 99 wt%; Preferably, 0. 01_95 wt%; more preferably, as used herein, a "pharmaceutically acceptable” ingredient is suitable for use in humans and/or mammals without excessive adverse side effects (eg, toxicity, irritation, and allergies), That is, a substance with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • compositions of the present invention comprise a safe and effective amount of a fusion protein of the invention and a pharmaceutically acceptable carrier.
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration, and the pharmaceutical composition of the present invention can be prepared into an injection form, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition is preferably manufactured under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount.
  • the pharmaceutical preparation of the present invention can also be formulated into a sustained release preparation.
  • the effective amount of the fusion protein of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the fusion protein of the invention such as bioavailability, metabolism, half-life, etc.; severity of the disease to be treated by the patient, body weight of the patient, immune status of the patient, administration Ways, etc.
  • the fusion protein of the present invention is administered at a dose of about 0.5 mg to 5 g g / k g of animal body weight per day (preferably 2 mg to 4 mg / kg of animal body weight), a satisfactory effect can be obtained.
  • a satisfactory effect can be obtained.
  • several separate doses may be administered per day, or the dose may be proportionally reduced, as is critical to the condition of the treatment.
  • the fusion protein of the present invention and its dimer or multimer mainly include the following advantages:
  • VEGFR1D2-FC expression vector The VEGFR1-D2 gene coding sequence consists of 300 nucleotides, as shown at position 58_357 of SEQ ID NO.: 2, including 279 nucleotides of the D2 coding sequence, 15 nucleotides of the upstream flanking sequence, and downstream flanking The sequence is 6 nucleotides. At the 5' end, 57 signal peptide coding sequences derived from the mouse IgG heavy chain (i.e., positions 1-57 of SEQ ID NO.: 2) were added to constitute 357 nucleotides.
  • the synthesized product (synthesized by Nanjing Kingsray Biotech Co., Ltd.) was digested (Hindl l l/EcoRI) and cloned into pHB_Fc plasmid vector to form pHB-VEGFR1D2-Fc protein expression vector.
  • the pHB_Fc plasmid vector was prepared as follows: The pcDNA/HA-FLAG (Accession#: FJ524378) vector was used as the starting plasmid, and the Fc sequence of human IgG1 was added after the endonuclease EcoRI, and the endonuclease Hindl ll was added.
  • Sequence SEQ ID NO: 2 is the nucleotide sequence encoding the recombinant fusion protein, as shown in Figure 1B.
  • the full length is 1059 bp, wherein l_57bp is the signal peptide coding sequence, 58_357bp is the VEGFR1D2 coding sequence, 358_363bp is the EcoRI restriction site GAATTC, 364-1059bp is the Fc fragment, and TGA is the stop code.
  • FIG. 1A is a schematic view showing the structure of the recombinant fusion protein VEGFR1D2-FC. This schematic diagram is for illustrative purposes only and does not represent the specific actual structure of the fusion protein of the present invention.
  • Sequence SEQ ID NO: 1 is the amino acid sequence encoding the recombinant fusion protein, as shown in Figure 1C. It has a total length of 353 amino acids and a molecular weight of approximately 80kDa. Among them, amino acids 1-19 are signal peptides, 20_119 are VEGFR1D2 fragments containing flanking sequences (underlined), 120-121 are 2 amino acids of EcoRI restriction site, and amino acids 122-353 are Fc fragments.
  • Example 2
  • the host cell used for protein expression was CH0-K1 cells (Cat# CCL-61) purchased from ATCC. The cells were acclimated into a CH0-K1 cell that can be cultured in suspension in serum-free medium (EX-CELLTM 302) after a series of domestication steps.
  • the pHB-VEGFR1D2-Fc plasmid was transferred into cells by electroporation.
  • the specific method is: Collect cells in logarithmic growth phase under aseptic conditions, centrifuge (1200 rpm x 5 min) and resuspend in complete culture. Base, and adjust the cell density to lx 10 7 cel ls/ml. Transfer 350 ul of cell suspension to a 0.4 cm electro-rotary cup and pulse once under set electrical conditions (voltage range 200 to 350 V, generally 260 V, time 20 ms or so). Add 10 - 30 ug of plasmid DNA to the electro-conductor containing the cells, mix gently, place the electro-conical cup into the electro-rotator, and pulse.
  • cells of the cell line with high expression of VEGFR1D2-FC were inoculated into a cell reactor containing 3 liters of EX-CELLTM 302 medium at a cell density of 3 ⁇ 10 5 cel ls/ml, and the culture condition was 37° (:, 5% C02.
  • the cells are tested by pH, glucose, glutamine, etc. during the culture, and supplemented with nutrients according to various indicators.
  • the culture temperature will be from Harvesting was continued at 37 ° C to 33 ° C and continued until the cell viability reached 60-70%.
  • the harvested cell culture supernatant was concentrated by ultrafiltration and purified by Protein A affinity chromatography.
  • Purified protein utilized Lowry The method is quantitatively determined (refer to the 2010 edition of the Chinese Pharmacopoeia), and the protein quantification standard is bovine serum albumin (batch number 140619-201120, China National Institute for Drug Control).
  • the size of the produced protein by SDS-PAGE analysis is basically consistent with the theoretical value. , endotoxin content is lower than the standard requirements.
  • Figure 2 shows the SDS-PAGE electropherogram of the VEGFR1D2-Fc protein.
  • Example 3 shows the SDS-PAGE electropherogram of the VEGFR1D2-Fc protein.
  • VEGFR1D2-FC VEGFR1D2-FC to target (VEGF)
  • VEGF vascular endothelial growth factor
  • ELISA enzyme-linked immunosorbent assay
  • VEGF-165 (Cat: 11066-HNAH, Sino biological Inc.) was diluted to 500 ng/ml with coating buffer CBS (Sigma_Aldrich Co., Product code: 1001329288 C3041-100CAP), and 100 ⁇ l was added to the ELISA plate (NuncTM). , Cat : 442404), 50ng per hole.
  • the coated plates were placed in a refrigerator at 4 ° C overnight. The test was washed once with 0. 05% PBS-T, and then blocked with 3% skim milk for 1 hour at room temperature.
  • the diluted D2-Fc protein (50, 25 0. 0244 nM) was added to the coated plates at 100 ul per well.
  • VEGFR1D2-Fc inhibits VEGF-induced VEGFR2 phosphorylation and HUVEC cell proliferation
  • VEGF vascular endothelial growth factor
  • the receptor first autophosphorylates, which in turn activates the signal transduction pathway of phosphatidylinositol metabolism and mitogen-activated protein kinase, exhibiting mitogenic properties of VEGF, and inducing human vascular endothelial cells (HUVEC) ) proliferation. Therefore, inhibition of VEGF-induced phosphorylation of VEGFR2 inhibits proliferation of vascular endothelial cells.
  • the HUVEC cells were adjusted to a concentration of 2 X 107 ml with a culture solution (Oxels Biotechnology (Shanghai) Co., Ltd., product number: HUVEC-004), and the cells were added to a 6-well cell culture plate, and 4 ml of cell suspension was added to each well. The solution was incubated overnight in an incubator. On the next day, discard the culture solution, gently wash twice with PBS, then add 4 ml of medium containing VEGF (R&D, Cat#293_VE/CF) and different concentrations of VEGFR1D2-Fc protein, incubate in a 37 °C incubator.
  • VEGF R&D, Cat#293_VE/CF
  • the PVDF membrane was incubated in PBST for 1 minute, and then blocked with a blocking solution at room temperature for 0.5 hours.
  • the diluted (1:750) anti-VEGFR2 phosphorylated antibody (Cell Signaling Technology, Cat# 3770S) was incubated with the PVDF membrane overnight (4 °C) and washed 3 times with PBST for 10 minutes each time.
  • the PVDF membrane was incubated with secondary antibody (1:2000) (HRP-Goat Anti-Rabbit IgG, Luoyanghaotong, Cat No: C030212) at low temperature (4 °C) for 2 hours. Wash 3 times with PBST for 10 minutes each time.
  • VEGFR1D2-FC significantly inhibited the phosphorylation of VEGFR2 induced by VEGF, and the inhibitory activity was dose-dependent (as shown in Figure 4), and significantly inhibited the phosphorylation of VEGFR2 even at the minimum dose (InM).
  • the HUVEC cells were adjusted to a concentration of 3 X 107 ml, and the cells were added to a 96-well culture plate containing Matrigel at 50 ul per well.
  • the prepared culture medium containing VEGF (20 ng/ml) and different concentrations of VEGFR1D2-Fc (1, 5, 10, 20 ug/ml) was then added to the culture plate at 50 ul per well.
  • the culture plates were placed in an incubator and photographed at different time points (0h, 2h, 4h, 6h, 8h, 24h) under a microscope.
  • VEGFR1D2-Fc can inhibit the proliferation of HUVEC cells and the tube of HUVEC cells in gel medium. Formed as shown in Figure 5.
  • VEGFR1D2-FC dose-dependently inhibits angiogenesis in vivo
  • the angiogenic transgenic zebrafish model was used to evaluate the effect of the test sample VEGFR1D2-FC on angiogenesis.
  • vascular fluorescent transgenic zebrafish embryos The propagation of vascular fluorescent transgenic zebrafish embryos is carried out in a natural paired mating manner. Prepare 4 to 5 pairs of adult zebrafish for each mating, with an average of 200 to 300 embryos per pair. Embryos are cleaned 6 hours (g ⁇ 6 hpf) and 24 hpf (removed dead embryos) and appropriate embryos are selected based on the embryo's developmental stage. The oil is incubated with water at 28 ° C (fish culture water quality: 200 mg of instant sea salt per 1 L of reverse osmosis water, the conductivity is 480 ⁇ 510 ⁇ / ⁇ ; ⁇ ⁇ is 6. 9 ⁇ 7. 2; 5:7 ⁇ 71. 6 mg/L CaC0 3 ).
  • the sample is injected into a fluorescent transgenic zebrafish (blood circulation injection), and each sample to be tested is set to a plurality of different concentrations, each of which treats 30 zebrafish;
  • the concentration range of the sample to be tested will be expanded to the maximum solubility or stock solution of the sample to be tested.
  • each sample to be tested is selected for 3 concentrations (usually the maximum non-lethal concentration (MNLC), 1/3 MNLC and 1/10 MNLC);
  • - positive control group Endo and lovastatin
  • - negative control group solvent group
  • a blank control group is used to demonstrate that the solvent does not have a detrimental effect on the zebrafish;
  • Angiogenesis inhibition rate (3 ⁇ 4) ⁇ - ⁇ ⁇ group)) xlW3 ⁇ 4
  • the solvent group (1 XPBS) was not statistically significant compared with the blank control (p>0.05), indicating that the injection solvent had no effect on angiogenesis of zebrafish.
  • the angiogenesis rate of 4 ng of lovastatin was (45.6 ⁇ 2.2)% (4 ng was the maximum non-lethal dose of lovastatin, so the angiogenesis rate was the maximum inhibition of lovastatin at this time.
  • the rate was statistically significant (p ⁇ 0.001) compared with the solvent group.
  • the angiogenesis rates of 44 ng and 100 ng Endo were (9.7 ⁇ 2.8)% and (20.1 ⁇ 2.6)o/ 0 , respectively, which were statistically significant compared with the solvent group (p ⁇ 0.05, ⁇ 0 ⁇ 001). ).
  • Two different positive control compounds have significant inhibitory effects on zebrafish blood vessels, so this evaluation model is reliable.
  • VEGFR1D2-FC angiogenesis inhibition rate of 4.4 ng was 7.5 ⁇ 3.5%, but it was not statistically significant compared with the solvent group (p>0.05); the dose was 14.7 ng and 44 ng of VEGFR1D2_Fc angiogenesis The inhibition rates were (15.2 ⁇ 3.3)% and (21.4 ⁇ 2.4)%, respectively, compared with the solvent group (p ⁇ 0.01, p ⁇ 0.001), indicating that VEGFR1D2-FC significantly inhibited angiogenesis. .
  • the dose was in the range of 4.4 ng to 44 ng, and the inhibition rate of VEGFR1D2-FC on zebrafish angiogenesis increased with the amount of administration, showing a dose-dependent VEGFR1D2-Fc administration.
  • Normal zebrafish intestines have 6-8 intact intestine vessels. If angiogenesis is inhibited, intact intestinal vessels The number is reduced. 4. 4 ng VEGFR1D2-Fc zebrafish complete intestine blood vessel number is 5-6, 14. 7 ng VEGFR1D2-Fc zebrafish complete intestinal vascular number is 3-4, 44 ng VEGFR1D2-Fc zebrafish complete intestine The number of lower vessels is 2_3; this also qualitatively demonstrates that VEGFR1D2-FC inhibits angiogenesis (Figure 6).
  • VEGFR1D2-Fc The angiogenesis inhibition rate of 44 ng VEGFR1D2-Fc was (21. 4 ⁇ 2. 4)%, and the angiogenesis inhibition rate of 44 ng Endo was (9.7 ⁇ 2. 8)%; At (44 ng), the inhibitory angiogenesis effect of VEGFR1D2_Fc was significantly better than Endo (P ⁇ 0.01) (Fig. 7).
  • Example 6 The angiogenesis inhibition rate of 44 ng VEGFR1D2-Fc was (21. 4 ⁇ 2. 4)%, and the angiogenesis inhibition rate of 44 ng Endo was (9.7 ⁇ 2. 8)%; At (44 ng), the inhibitory angiogenesis effect of VEGFR1D2_Fc was significantly better than Endo (P ⁇ 0.01) (Fig. 7).
  • VEGFR1D2-FC inhibits tumor cells in a dose-dependent manner
  • mice were injected subcutaneously with 5x10 6 lung cancer (A549) or colorectal cancer (C0L0-205) cells until the tumor grew to
  • the treatment group was intraperitoneally injected with high (20 mg/k g ) and low dose (5 mg/k g ) VEGFR1D2-FC protein twice a week for 6 consecutive doses.
  • the negative control was PBS.
  • the positive control was doxorubicin (3 mg/kg) (colorectal cancer) or Avastin (5 mg/kg 20 mg/kg) (lung cancer). Tumor volume was measured twice a week.
  • mice Sixteen normal Balb/c mice were injected subcutaneously with 50 ug of VEGFRlD2-Fc, respectively, and 2, 2 after injection.

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Abstract

Cette invention concerne une protéine de fusion recombinée, son procédé de préparation et utilisation, la protéine de fusion comprenant un composant A comprenant le domaine extracellulaire 2 du VEGFR1 et un composant B d'immunoglobuline, les deux composants étant liés ensemble en tandem. Cette invention concerne également l'utilisation d'un médicament de type nouvelle protéine de fusion recombinée dans le traitement de maladies telles qu'une tumeur, etc.
PCT/CN2013/078922 2013-07-05 2013-07-05 Nouvelle protéine de fusion recombinée, son procédé de préparation et utilisation WO2015000181A1 (fr)

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CN108623694A (zh) * 2017-03-24 2018-10-09 北京市肿瘤防治研究所 血管内皮细胞生长因子受体拮抗肽f56的衍生物及应用
JP2020114266A (ja) * 2015-10-15 2020-07-30 アルテオジェン・インコーポレイテッド ＩｇＧ Ｆｃドメインを有する融合タンパク質の生産方法
EP3743091A4 (fr) * 2018-01-26 2021-12-15 The Regents of the University of California Méthodes et compositions pour le traitement de troubles angiogéniques à l'aide d'agents anti-vegf
US11382955B2 (en) 2019-11-25 2022-07-12 The Regents Of The University Of California Long-acting VEGF inhibitors for intraocular neovascularization
EP3954712A4 (fr) * 2019-04-01 2022-12-07 Huabo Biopharm (Shanghai) Co., Ltd. Anticorps anti-pd-l1/vegf bifonctionnel et son utilisation

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2020114266A (ja) * 2015-10-15 2020-07-30 アルテオジェン・インコーポレイテッド ＩｇＧ Ｆｃドメインを有する融合タンパク質の生産方法
CN108623694A (zh) * 2017-03-24 2018-10-09 北京市肿瘤防治研究所 血管内皮细胞生长因子受体拮抗肽f56的衍生物及应用
CN108623694B (zh) * 2017-03-24 2021-09-17 北京市肿瘤防治研究所 血管内皮细胞生长因子受体拮抗肽f56的衍生物及应用
EP3743091A4 (fr) * 2018-01-26 2021-12-15 The Regents of the University of California Méthodes et compositions pour le traitement de troubles angiogéniques à l'aide d'agents anti-vegf
US11524053B2 (en) 2018-01-26 2022-12-13 The Regents Of The University Of California Methods and compositions for treatment of angiogenic disorders using anti-VEGF agents
EP3954712A4 (fr) * 2019-04-01 2022-12-07 Huabo Biopharm (Shanghai) Co., Ltd. Anticorps anti-pd-l1/vegf bifonctionnel et son utilisation
US11382955B2 (en) 2019-11-25 2022-07-12 The Regents Of The University Of California Long-acting VEGF inhibitors for intraocular neovascularization
US11433118B2 (en) 2019-11-25 2022-09-06 The Regents Of The University Of California Long-acting VEGF inhibitors for intraocular neovascularization
US11576948B2 (en) 2019-11-25 2023-02-14 The Regents Of The University Of California Long-acting VEGF inhibitors for intraocular neovascularization

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