WO2015149708A1 - Nouvelle protéine de fusion bifonctionnelle recombinante, son procédé de préparation et son utilisation - Google Patents

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

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WO2015149708A1
WO2015149708A1 PCT/CN2015/075759 CN2015075759W WO2015149708A1 WO 2015149708 A1 WO2015149708 A1 WO 2015149708A1 CN 2015075759 W CN2015075759 W CN 2015075759W WO 2015149708 A1 WO2015149708 A1 WO 2015149708A1
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protein
fusion protein
tgf
tβrii
vegf
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PCT/CN2015/075759
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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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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the invention relates to the field of biomedicine. More specifically, the present invention relates to a novel recombinant bifunctional fusion protein, a process for its preparation and use.
  • TGF- ⁇ and VEGFR receptors on the surface of cell membrane and the corresponding ligands is an important cause of the development and development of such diseases. Therefore, the development of corresponding protein ligand drugs has a good application prospect.
  • protein drugs include growth factors, hormone proteins, enzyme proteins, cytokines, interferons, erythropoietin, and fusion proteins.
  • other protein drugs are homogeneous proteins, that is, contain only one protein component.
  • Existing fusion protein drugs (such as Avastin and Aflibercept) are composed of the extracellular domain of the receptor protein and the human IgG Fc segment. Although they contain two or more protein components, they still Only one function is to block the binding of one cell membrane endogenous receptor to the corresponding ligand.
  • bifunctional fusion protein drugs which have hitherto been able to bind to both TGF- ⁇ 1 and VEGF and simultaneously block the biological activities induced by these two targets have not been reported. Therefore, the development of such novel bifunctional fusion protein drugs is of great significance for prolonging the survival time of patients, improving the quality of life of patients and reducing mortality.
  • the object of the present invention is to provide a novel recombinant bifunctional fusion protein, a preparation method and use thereof.
  • a fusion protein comprising the following elements fused together:
  • signal peptide is operably linked to the fusion element consisting of (ii), (iii) and (iv);
  • the first protein element is a TGF- ⁇ receptor extramembrane region protein element;
  • the second protein element is a protein element comprising a second extramembranous region D2 of the vascular endothelial growth factor receptor VEGFR1.
  • operably linked means that the signal peptide can direct expression or transmembrane transfer (localization) of the fusion element.
  • the fusion protein has a structure selected from the group consisting of:
  • A is a TGF- ⁇ receptor extracellular domain protein element
  • B is a protein element comprising a second extramembranous region D2 of the vascular endothelial growth factor receptor VEGFR1;
  • C is an immunoglobulin element
  • D is an optional signal peptide sequence
  • the element A is selected from the group consisting of T ⁇ RI, T ⁇ RII, T ⁇ RIII.
  • the element A is a protein element of the T ⁇ RII extramembranous region.
  • the element A has the amino acid sequence shown in positions 24-186 of SEQ ID NO.: 1.
  • the element B has the amino acid sequence (core sequence) shown at positions 190-282 of SEQ ID NO.: 1 and is 94, 95, 96, 97, 98, 99, or 100 in length. Amino acids.
  • amino acid sequence flanking the amino acid sequence (core sequence) shown in positions 190-282 of SEQ ID NO.: 1 in element B is derived from the second extramembranous region of native VEGFR1, respectively. Amino acid sequence on both sides of D2 (Domain 2,).
  • the D2 has a flanking sequence, and the flanking sequence comprises:
  • flanking sequence at the amino terminus of D2; and/or a second flanking sequence at the carboxy terminus of D2.
  • 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 190-282 of SEQ ID NO.: 1) of native VEGFR1, respectively.
  • the first flanking sequence is SDTGR.
  • the second flanking sequence is NT.
  • the element C is an Fc fragment of human immunoglobulin IgG1.
  • the peptide linker is 0-10 amino acids in length, preferably 0-5 amino acids.
  • the fusion protein further comprises a signal peptide element D.
  • amino acid sequence of the signal peptide element D is shown as positions 1-23 of SEQ ID NO: 1.
  • amino acid sequence of the fusion protein is set forth in SEQ ID NO: 1.
  • the fusion protein does not contain a signal peptide and the structural formula is selected from the group consisting of:
  • the fusion protein has the following functions:
  • a) binding activity to VEGF EC 50 is 0.6-2 nM;
  • binding activity to TGF- ⁇ 1 EC 50 is 1.5-2.5 nM
  • c) can simultaneously bind to both VEGF and TGF- ⁇ 1 ligands
  • e inhibits migration and invasion of tumor cells induced by TGF- ⁇ 1.
  • a protein dimer consisting of two fusion proteins according to any one of the first aspects of the invention.
  • the dimer has a structure selected from the group consisting of:
  • A is a TGF- ⁇ receptor extracellular domain protein element
  • B is a protein element comprising a second extramembranous region D2 of VEGFR;
  • C is an immunoglobulin element
  • D is an optional signal peptide sequence
  • ⁇ means a disulfide bond
  • the fusion protein does not contain a signal peptide and has a structure selected from the group consisting of:
  • an isolated polynucleotide is provided, the polynucleotide encoding according to The fusion protein of the first aspect of the invention.
  • a vector comprising the polynucleotide of the third aspect of the invention is provided.
  • a host cell comprising the vector of the fourth aspect of the invention or the polynucleotide of the third aspect of the invention.
  • the host cell is a prokaryotic cell or a eukaryotic cell (eg, CHO cell, NSO cell, 293 cell).
  • a prokaryotic cell e.g, CHO cell, NSO cell, 293 cell.
  • a method of producing a protein comprising the steps of:
  • composition comprising:
  • fusion protein according to the first aspect of the invention and/or a protein dimer according to the second aspect of the invention
  • a pharmaceutically acceptable carrier is selected from:
  • a fusion protein according to the first aspect of the invention and/or a protein dimer according to the second aspect of the invention for the preparation of a medicament for the treatment of a disease.
  • the disease is a disease associated with TGF- ⁇ and VEGF.
  • the disease is selected from the group consisting of: tumor, liver fibrosis.
  • the tumor comprises: a colorectal cancer tumor, a lung cancer tumor, a liver cancer tumor, a breast cancer tumor, a gastric cancer tumor, and a pancreatic cancer tumor.
  • a method of inhibiting a disease associated with TGF- ⁇ and VEGF comprising the step of administering the fusion protein of the first aspect to a subject in need thereof.
  • the fusion protein is administered as a monomer and/or a dimer.
  • the object is a human.
  • FIG. 1 is a schematic diagram showing the molecular structure of a recombinant fusion protein T ⁇ RII-D2-Fc, which shows that T ⁇ RII-D2-Fc contains three components, the extramembranous end of TGF- ⁇ type receptor (T ⁇ RII), VEGF receptor 1 Membrane The second region of the outer end (VEGFR1-D2), and the Fc fragment of human IgG1.
  • Figure 2A shows the nucleotide sequence of T ⁇ RII-D2-Fc, in which 69 nucleotides of red are signal peptide coding sequences, 483 nucleotides of blue are coding sequences of the outer end of T ⁇ RII membrane, 300 reddish
  • the nucleotide is the coding sequence of VEGFR1-D2; the first 6 of the 702 nucleotides in black are EcoRI cleavage sites, and the last 696 nucleotides are coding sequences of the human IgG1 Fc fragment.
  • Figure 2B shows the amino acid sequence of T ⁇ RII-D2-Fc, in which the red 23 amino acids are signal peptides, the blue 161 amino acids are T ⁇ RII membrane outer ends, the light red 100 amino acids are VEGFR1-D2, and the black 234 The amino acids are the EcoRI site (2 amino acids "EF") and the human IgG1 Fc fragment (232 amino acids).
  • Fig. 3A shows the SDS-PAGE electrophoresis pattern of each fusion protein.
  • the R lane is an electrophoresis band under reducing conditions
  • the NR lane is an electrophoresis band under non-reducing conditions, from T ⁇ RII-
  • the electropherogram of D2-Fc shows that under reducing conditions, the molecular size of T ⁇ RII-D2-Fc is about 80-90 kDa, and under non-reducing conditions is greater than 170 kDa, which is larger than the theoretical value (57 kDa, 120 kDa) because there are many molecules.
  • FIG. 3B shows the HPLC analysis of the T ⁇ RII-D2-Fc protein. It can be seen from the figure that the T ⁇ RII-D2-Fc protein has a high purity and a polymer content of less than 2%.
  • FIG. 4A shows the results of the TGF- ⁇ 1 binding activity test of each fusion protein and the target.
  • the fusion proteins of the three different combinations have the binding activity to TGF- ⁇ 1, wherein the activity of T ⁇ RII-D2-Fc
  • D2-T ⁇ RII-Fc is less active and T ⁇ RII-Fc-D2 is the weakest.
  • the positive control protein T ⁇ RII-Fc also had good TGF- ⁇ 1 binding activity with an EC 50 of 2.30 nM. It should be emphasized that at the same concentration, the binding activity of T ⁇ RII-D2-Fc to the target is about 34% higher than that of T ⁇ RII-Fc. Both D2-Fc and the negative control protein IgG-Fc were inactive.
  • FIG. 4B shows the results of the test for binding activity of each fusion protein to the target VEGF-165.
  • the fusion proteins of the three different combinations have the binding activity to VEGF-165, wherein D2-T ⁇ RII-Fc has the best activity, T ⁇ RII-Fc-D2 has the second activity, and T ⁇ RII-D2-Fc.
  • the activity had an EC 50 of 0.14 nM; neither T ⁇ RII-Fc nor the negative control IgG-Fc had activity.
  • the fusion protein of the present invention has excellent simultaneous binding to the target TGF- ⁇ 1 and target VEGF activity.
  • Figure 5 shows that T ⁇ RII-D2-Fc inhibits TGF- ⁇ 1-induced tumor cell invasion
  • Figure A is a control (no additional addition of T ⁇ RII-D2-Fc and TGF- ⁇ 1 in the medium)
  • Figure B adds TGF- ⁇ 1 10ng/ml, TGF- ⁇ 1 10ng/ml, T ⁇ RII-D2-Fc 1 ⁇ g/ml was added to Figure C
  • TGF- ⁇ 110ng/ml and T ⁇ RII-D2-Fc 10 ⁇ g/ml were added to Figure D, added in Figure E.
  • TGF- ⁇ 1 10 ng/ml, T ⁇ RII-D2-Fc 50 ⁇ g/ml, and Figure F added TGF- ⁇ 1 10 ng/ml and hIgG 50 ⁇ g/ml.
  • TGF- ⁇ 1 significantly induced the invasion of tumor cells from the upper layer of the chamber to the lower layer (Invasion), but after the addition of T ⁇ RII-D2-Fc, Tumor cell invasion was significantly inhibited and the inhibitory effect was dose dependent.
  • the negative control protein hIgG did not inhibit TGF- ⁇ 1-induced tumor cell invasion (Invasion).
  • T ⁇ RII-D2-Fc binds to TGF- ⁇ 1 and blocks the binding of TGF- ⁇ 1 to the TGF- ⁇ receptor on the tumor cell membrane, thereby inhibiting downstream signaling and ultimately inhibiting tumor cell invasion.
  • FIG. 6 shows that T ⁇ RII-D2-Fc blocks vascular endothelial cell tubular formation induced by VEGF.
  • Panel A is a control (only VEGF-165 is added to the medium, no additional T ⁇ RII-D2-Fc is added), and Figure B is added with VEGF-165 20 ng/ml, T ⁇ RII-D2-Fc 20 ⁇ g/ml, and Figure C is added with VEGF- 165 20 ng/ml, T ⁇ RII-D2-Fc 50 ⁇ g/ml, Figure D was added with VEGF-165 20 ng/ml, T ⁇ RII-D2-Fc 100 ⁇ g/ml, and Figure E was added with VEGF-165 20 ng/ml and hIgG 100 ⁇ g/ml.
  • VEGF-165 induces tubular formation of vascular endothelial cells (HUVEC), but if T ⁇ RII-D2-Fc is simultaneously added, the tubular formation of HUVEC cells can be significantly inhibited, and the inhibitory effect is dose-dependent.
  • the negative control protein hIgG could not inhibit the vascular formation of HUVEC cells induced by VEGF-165.
  • Figure 7A shows the inhibitory effect of the fusion protein of the present invention on the growth of mouse breast cancer cells (4T1). It can be seen from the figure that if the tumor cells are not treated after subcutaneous inoculation, the tumor volume grows to 600 mm 3 (mm 3 ) after 20 days, and after treatment with the fusion protein, the two doses (5 mg/kg, 10 mg/kg) Both T ⁇ RII-D2-Fc and high dose (10 mg/kg) D2-Fc significantly inhibited tumor growth, and the tumor volume after 20 days was only half that of the negative control group, which was 300 mm 3 (mm 3 ). T ⁇ RII-Fc has a certain inhibitory effect, but it is not significant.
  • Figure 7B shows the inhibitory effect of the fusion protein of the present invention on the metastasis of mouse breast cancer cells (4T1).
  • both doses of T ⁇ RII-D2-Fc significantly inhibited tumor metastasis to the lungs, and the inhibition rate was 60-70% compared with the negative control group.
  • T ⁇ RII-Fc inhibited tumor metastasis by 61%, while D2-Fc inhibition was only 50%.
  • the inventors have for the first time established a genetic engineering technology platform through extensive and in-depth research, which can be used to produce recombinant bifunctional fusion protein drugs, such as T ⁇ RII-D2-Fc.
  • the present invention has been completed on this basis.
  • T ⁇ RII-D2-Fc has the following functions: 1) can bind to both VEGF and TGF- ⁇ 1 ligands; 2) block VEGF-induced in vitro or in vivo angiogenesis; 3) inhibit TGF - ⁇ 1 induced migration and invasion of tumor cells.
  • the T ⁇ RII-D2-Fc of the present invention not only has the binding activity to VEGF and TGF- ⁇ 1 at the same time, but has an EC 50 of 0.60 and 1.53, respectively, and can treat certain diseases synergistically and more effectively, especially such as tumors and old age. Eye macular degeneration, liver fibrosis and other diseases.
  • VEGFR and its extramembrane region are VEGFR and its extramembrane region
  • VEGFR protein belongs to the receptor tyrosine kinase superfamily and is a membrane mosaic protein.
  • the extramembranous portion of VEGFR has approximately 750 amino acid residues and consists of seven Ig domains that are structurally similar to immunoglobulins.
  • VEGFR proteins can induce a range of different biological functional responses based on their corresponding receptor properties.
  • VEGFR proteins of the invention 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 position 190-282 of SEQ ID NO.:1.
  • TGF- ⁇ receptor and its extramembranous region
  • the TGF-beta receptor is a single transmembrane protein receptor with serine/threonine protein kinase activity in the intracellular region, which functions as a heterodimer.
  • TGF- ⁇ receptors There are currently 1 to 5 types of TGF- ⁇ receptors, and there are 3 types of TGF- ⁇ glycoprotein receptors (ie, type I, type II, and type III receptors) on the surface of various human cells.
  • Type and type II receptors act as signal transduction.
  • Type I and type II receptors are single transmembrane serine/threonine protein kinase receptors.
  • the extracellular zone is shorter and the cytoplasmic zone is longer.
  • the extracellular domain is rich in cysteine.
  • the cytoplasmic domain contains a serine/threonine protein kinase domain.
  • the TGF-beta receptor protein of the present invention comprises: T ⁇ RI, T ⁇ RII, T ⁇ RIII or a combination thereof.
  • T ⁇ RII is preferred, and preferably the natural T ⁇ RII is wild type.
  • a preferred class of TGF-beta receptor extramembranous regions of the invention refers to the extramembranous region of T ⁇ RII.
  • a representative extracellular domain sequence of T ⁇ RII is position 24-184 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 includes four subclasses: IgG1, IgG2, IgG3, IgG4.
  • the protein structures of these four subclasses have great similarities, with 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 IgG1, IgG2 is second, and IgG3 and IgG4 concentrations are lower.
  • a preferred G element is a human IgGl Fc fragment, or a mutant or derivative thereof.
  • bifunctional fusion protein in the present invention, “recombinant bifunctional fusion protein”, “protein of the invention”, “fusion protein of the invention”, “bifunctional fusion protein” are used interchangeably and mean having the structure of formula Ia or Ib, or IIIa or The structure described in IIIb, that is, a fusion protein comprising a protein element comprising a TGF- ⁇ receptor extramembrane region protein element, a second extramembranous region D2 of VEGFR, and an immunoglobulin element.
  • a representative example is T ⁇ RII-D2-Fc.
  • the protein of the invention may be a monomer or a multimer (e.g., a dimer) formed from 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 Other related 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 variants of the above polynucleotides which encode protein fragments, analogs and derivatives 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 that may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby.
  • the term "primer” refers to a generic term for a oligodeoxynucleotide that, in pairing with a template, can be used to synthesize a DNA strand complementary to a template under the action of a DNA polymerase.
  • the primer may be native RNA, DNA, or any form of natural nucleotide.
  • the primer may even be a non-natural nucleotide such as LNA or ZNA.
  • the primer is “substantially” (or “substantially") 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 the 3' end, and such primer is 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 element of the fusion protein of the present invention (e.g., VEGFR1D2 or T ⁇ RII extramembranous region) or a fragment thereof can be generally 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 used as commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art.
  • the template is amplified to obtain the relevant 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 recombinant sequence 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 performing the ligation.
  • a method of amplifying DNA/RNA using PCR technology is preferably used to obtain the gene of the present invention.
  • the 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 conventional methods such as by gel electrophoresis.
  • the invention also relates to vectors comprising the polynucleotides of the invention, as well as 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.
  • 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 recombinant 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 can 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 CHO, COS, 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 (such as 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 such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and
  • 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 the antibody can bind to the protein of the present invention or a fragment thereof, respectively.
  • 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 bifunctional 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 an alpha helix or a beta 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 0 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 pH is usually from about 5 to about 8, preferably from about 6 to about 8.
  • the term "effective amount” or “effective amount” refers to an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals, such as from 0.001 to 99% by weight; preferably 0.01-95 wt%; more preferably, 0.1-90 wt%.
  • a "pharmaceutically acceptable” ingredient is one that is suitable for use in humans and/or mammals without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, having 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.
  • 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.
  • 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 is particularly suitable for the treatment of diseases in which VEGF and TGF- ⁇ 1 are excessively secreted, or diseases characterized by abnormal vascular proliferation and invasion of tumor cells.
  • Representative diseases include, but are not limited to, tumors, wet macular degeneration, or liver fibers.
  • the T ⁇ RII extramembrane region gene coding sequence consists of 483 nucleotides, as shown at positions 70-552 of SEQ ID NO.: 2.
  • the VEGFR1-D2 gene coding sequence consists of 300 nucleotides, as shown at positions 553-852 of SEQ ID NO.: 2, including 279 nucleotides of the D2 coding sequence, 15 nucleotides of the upstream flanking sequence, The downstream flanking sequence is 6 nucleotides.
  • a signal peptide coding sequence derived from T ⁇ RII i.e., positions 1-69 in SEQ ID NO.: 2 was added to the 5' end of the T ⁇ RII extramembrane region to constitute 852 nucleotides.
  • the synthesized product (synthesized by Nanjing Kingsray Biotech Co., Ltd.) was digested with HindIII/EcoRI and cloned into the pHB-Fc plasmid vector to form a pHB-TbRII-D2-Fc protein expression vector.
  • the pHB-Fc plasmid vector was prepared by using the pcDNA/HA-FLAG (Accession #: FJ524378) vector as the starting plasmid, and the endozyme EcoRI was followed by the Fc sequence of human IgG1, which was preceded by the endonuclease HindIII.
  • HCMV human cytomegalovirus
  • the sequence SEQ ID NO: 2 is the nucleotide sequence encoding the recombinant bifunctional fusion protein, as shown in Figure 2A.
  • the full length is 1554 bp, of which 1-69 bp is the signal peptide coding sequence, 70-552 bp is the T ⁇ RII extramembrane region coding sequence, 553-852 bp is the VEGFR1D2 coding sequence, 853-858EcoRI cleavage site GAATTC, and 859-1554 bp is the Fc fragment.
  • TGA is the termination password.
  • Figure 1 is a schematic diagram showing the molecular structure of the recombinant bifunctional fusion protein T ⁇ RII-D2-Fc. This schematic diagram is for illustrative purposes only and does not represent the specific actual structure of the bifunctional fusion protein of the invention.
  • the sequence SEQ ID NO: 1 is the amino acid sequence encoding the recombinant bifunctional fusion protein, as shown in Figure 2B. Full length 518 amino acids. Among them, 1-23 amino acids are signal peptides, 24-184 amino acids are T ⁇ RII extramembrane regions, 185-284 are VEGFR1D2 fragments containing flanking sequences (underlined), and 285-286 are EcoRI cleavage sites 2 The amino acids, amino acids 287-518 are Fc fragments.
  • the host cell used for protein expression was CHO-K1 cells (Cat# CCL-61) purchased from ATCC. The cells were acclimated into CHO-K1 cells that were cultured in suspension in serum-free medium (EX-CELLTM 302) after a series of domestication steps.
  • plasmid pHB-T ⁇ RII-D2-Fc, pHB-D2-T ⁇ RII-Fc, and pHB-T ⁇ RII-Fc-D2 were separately transferred into cells by electroporation. Specifically, the cells in the logarithmic growth phase were collected under aseptic conditions, centrifuged (1200 rpm x 5 min), resuspended in complete medium, and the cell density was adjusted to 1 x 10 7 cells/ml. Transfer 350 ul of cell suspension to a 0.4 cm electric rotor and pulse once under set electrical conditions (voltage range 200 to 350 V, generally 260 V, time 20 ms or so).
  • cells of the cell line with high expression of the protein were inoculated into a cell reactor containing 3 liters of EX-CELLTM 302 medium at a cell density of 3 x 10 5 cells/ml, and culture conditions were 37 ° C, 5% CO 2 .
  • the cells are tested by pH, glucose, glutamine, etc. during the cultivation process, and supplemented with nutrients according to various indicators.
  • the culture temperature was lowered from 37 ° C to 33 ° C, and the culture was continued until the cell viability reached 60-70%.
  • the harvested cell culture supernatant was concentrated by ultrafiltration and purified by Protein A affinity chromatography.
  • the purified protein was quantitatively determined by the Lowry method (refer to the 2010 edition of the Chinese Pharmacopoeia), and the protein quantification standard was 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, and the endotoxin content is lower than the standard requirement.
  • T ⁇ RII-D2-Fc was at the position of ⁇ 80kDa (monomer), and under non-reducing conditions, it was larger than 170kDa (dimer).
  • the protein size is similar to T ⁇ RII-D2-Fc.
  • the measured molecular weight of the recombinant protein of the three structural combinations suggests that the recombinant protein has a certain degree of glycosylation (the theoretical molecular weight of the dimer is 114 kDa).
  • HPLC analysis of protein purity was greater than 98%.
  • VEGF and TGF- ⁇ 1 The binding properties of the fusion protein to the target (VEGF and TGF- ⁇ 1) were determined by enzyme-linked immunosorbent assay (ELISA). Specific steps are as follows:
  • TGF- ⁇ 1 (Cat: 10804-HNAH, Sino biological Inc.) and VEGF-165 (Cat: 11066-HNAH, Sino biological) were coated with CBS (Sigma-Aldrich Co., Product code: 1001329288 C3041-100CAP). Inc.) were diluted to 500ng / ml, was added to the ELISA plates take 100ul (Nunc TM, Cat: 442404) per well 50ng. The coated plates were placed in a refrigerator at 4 ° C overnight. The test was washed once with 0.05% PBS-T and then with 3% skim milk for 1 hour at room temperature.
  • TbRII-D2-Fc protein 100 nM, 50 nM, 25 nM, up to 0.0244 nM
  • 100 ul per well 100 ul per well.
  • HRP-Rabbit Anti-Human IgG Fc 100 ul of diluted (1:20000) HRP-Rabbit Anti-Human IgG Fc (Luoyang Aotong, Cat#: C030222) was added.
  • HRP-Rabbit Anti-Human IgG Fc (Luoyang Aotong, Cat#: C030222) was added.
  • Incubate for one hour at room temperature wash the washing solution 5 times, add HRP substrate, and darken the color for 10-20 minutes, then stop the color reaction with 2N H 2 SO 4 and read the 0D450 value on the microplate reader.
  • T ⁇ RII-D2-Fc had binding activities to TGF- ⁇ 1 (Fig. 4A) and VEGF-A (Fig. 4B), respectively, and the corresponding EC 50 reached 1.53 nM and 0.6 nM, respectively.
  • the other two structurally combined proteins also have strong binding activity to the two targets, with EC 50 of about 2.5 nM (TGF- ⁇ 1) and 0.16 nM (VEGF-165), respectively.
  • T ⁇ RII-D2-Fc inhibits tumor cell invasion induced by TGF- ⁇ 1
  • Tumor cell invasion experiments were performed using a 24-well cell chamber plate.
  • the method comprises the following steps: adding a medium containing TGF- ⁇ 1 to the chamber, adding tumor cells (PC-3) and a certain concentration of T ⁇ RII-D2-Fc to the upper layer of the filter, and incubating for 24 hours in the incubator, filtering with crystal violet The membrane was stained and the density of cells at the bottom of the photographed filter was observed.
  • TGF- ⁇ 1-induced tumor cells was analyzed using PC-3 cells.
  • the specific procedure was as follows: A Matrigel-containing cell sieve was placed in a 24-well culture plate containing cell culture medium (BD Bioscience), and the culture solution contained 10 ng/ml of TGF- ⁇ 1. Add 1 ⁇ 10 5 PC-3 cells to the cell sieve, then add different concentrations of T ⁇ RII-D2-Fc and hIgG to the corresponding sieve according to the grouping requirements, and place the 24-well cell culture plate in the cell culture incubator. The medium was cultured at 30 ° C under 5% CO 2 for 24 hours.
  • T ⁇ RII-D2-Fc significantly inhibited the invasion of PC-3 cells from the upper layer to the lower layer of the chamber, and the inhibitory effect was dose-dependent.
  • T ⁇ RII-D2-Fc blocks vascular endothelial cell tubular formation induced by VEGF
  • the HUVEC cells were adjusted to a concentration of 3 ⁇ 10 5 /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 various concentrations of T ⁇ RII-D2-Fc (20, 50, 100 ug/ml) was then added to the culture plate at 50 ul per well.
  • the culture plates were placed in an incubator and photographed and archived under different microscopes at different time points (0h, 2h, 4h, 6h, 8h, 24h).
  • HUVEC cells formed a vascular pattern under the microscope when cultured in a gel in the presence of VEGF, similar to the formation of blood vessels in vivo.
  • This experiment is often used to verify the effects of a drug on angiogenesis.
  • the inventors analyzed the effect of T ⁇ RII-D2-Fc on angiogenesis in vitro. The results indicate that (Fig. 6), T ⁇ RII-D2-Fc can significantly inhibit the tubular formation of HUVEC cells.
  • T ⁇ RII-D2-Fc inhibits tumor growth and tumor metastasis
  • 1x10 5 mouse breast cancer cells (4T1) were subcutaneously injected into the mammary gland of normal female Balb/c mice, and were randomly divided into 5 groups on the second day.
  • the first group was intraperitoneally injected with 5 mg/kg T ⁇ RII-D2-Fc and the second group.
  • 10 mg/kg T ⁇ RII-D2-Fc was intraperitoneally injected
  • 10 mg/kg D2-Fc (control) was injected intraperitoneally
  • 10 mg/kg T ⁇ RII-Fc (control) was injected intraperitoneally in the fourth group twice a week for 6 times.
  • the fifth group was a negative control and PBS was injected intraperitoneally. Tumor volume was measured three times a week. On the 21st day after the treatment, the mice were sacrificed, the tumors were harvested, the lungs were taken, and the lung metastases were observed under a microscope.
  • T ⁇ RII-D2-Fc significantly inhibited tumor growth at both doses (Fig. 7A), and the inhibition rates were all greater than 50%.
  • the tumor inhibition rate of D2-Fc is also very good, reaching ⁇ 55%, while the tumor inhibition rate of T ⁇ RII-Fc is only 26%.
  • the negative control group had an average of 11.4 metastases, and the T ⁇ RII-D2-Fc treatment group had 4.2 (5 mg/kg) and 3.56 (10 mg/kg), respectively.
  • T ⁇ RII-Fc could not effectively inhibit tumor growth, it significantly inhibited tumor metastasis in the lungs (4.4 of metastases).
  • D2-Fc inhibited tumor growth well, the inhibition of tumor metastasis was significantly weaker than that of T ⁇ RII-D2-Fc and D2-Fc.
  • the bifunctional fusion protein T ⁇ RII-D2-Fc has a stronger synergistic inhibitory effect on tumor growth and tumor metastasis, while a fusion protein targeting only one target can only have a significant inhibitory effect, that is, it can only inhibit tumor growth. (blocking VEGF), either only inhibit tumor metastasis (blocking TGF- ⁇ 1).
  • the inhibitory effect of the experimental group of 10 mg/kg T ⁇ RII-D2-Fc was also significantly better than that of the experimental group of 5 mg/kg D2-Fc+5 mg/kg T ⁇ RII-Fc.

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Abstract

L'invention concerne une protéine de fusion bifonctionnelle recombinante, son procédé de préparation et son utilisation. Plus particulièrement, l'invention concerne une protéine de fusion ayant un élément A comprenant un domaine extracellulaire du récepteur du TGF-bêta, un élément B comprenant un deuxième domaine extracellulaire D2 du VEGFR1 et un élément immunoglobuline C qui sont connectés en série. La protéine peut se lier simultanément à deux types de ligands : VEGF et TGF-bêta 1, et inhiber l'activité biologique des ligands. L'invention concerne également l'utilisation de la nouvelle protéine de fusion bifonctionnelle recombinante dans le traitement de maladies.
PCT/CN2015/075759 2014-04-04 2015-04-02 Nouvelle protéine de fusion bifonctionnelle recombinante, son procédé de préparation et son utilisation WO2015149708A1 (fr)

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WO2017065559A1 (fr) 2015-10-15 2017-04-20 (주)알테오젠 Procédé de production de protéine de fusion présentant un domaine fc d'igg
CN113698493A (zh) * 2021-08-09 2021-11-26 北京东方百泰生物科技股份有限公司 一种针对VEGF和TGF-β的双功能蛋白及其应用
EP3363811B1 (fr) * 2015-10-15 2023-04-19 Alteogen, Inc. Procédé de production de protéine de fusion présentant un domaine fc d'igg
US11912754B2 (en) 2017-10-12 2024-02-27 Immunowake Inc. VEGFR-antibody light chain fusion protein

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CN110050000B (zh) * 2017-05-12 2022-07-26 苏州盛迪亚生物医药有限公司 含有TGF-β受体的融合蛋白及其医药用途
CN108671229B (zh) * 2018-05-08 2022-03-25 华博生物医药技术（上海）有限公司 一种重组人血管内皮生长因子受体-抗体融合蛋白的药物组合制剂

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WO2010003118A1 (fr) * 2008-07-02 2010-01-07 Trubion Pharmaceuticals, Inc. Protéines de liaison multi-cibles antagonistes du tgf-b
CN102850458A (zh) * 2011-06-28 2013-01-02 华博生物医药技术（上海）有限公司 新型重组双功能融合蛋白及其制法和用途
CN103319610A (zh) * 2013-07-05 2013-09-25 华博生物医药技术（上海）有限公司 新型重组融合蛋白及其制法和用途

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WO2010003118A1 (fr) * 2008-07-02 2010-01-07 Trubion Pharmaceuticals, Inc. Protéines de liaison multi-cibles antagonistes du tgf-b
CN102850458A (zh) * 2011-06-28 2013-01-02 华博生物医药技术（上海）有限公司 新型重组双功能融合蛋白及其制法和用途
CN103319610A (zh) * 2013-07-05 2013-09-25 华博生物医药技术（上海）有限公司 新型重组融合蛋白及其制法和用途

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017065559A1 (fr) 2015-10-15 2017-04-20 (주)알테오젠 Procédé de production de protéine de fusion présentant un domaine fc d'igg
EP3363811B1 (fr) * 2015-10-15 2023-04-19 Alteogen, Inc. Procédé de production de protéine de fusion présentant un domaine fc d'igg
US11912754B2 (en) 2017-10-12 2024-02-27 Immunowake Inc. VEGFR-antibody light chain fusion protein
CN113698493A (zh) * 2021-08-09 2021-11-26 北京东方百泰生物科技股份有限公司 一种针对VEGF和TGF-β的双功能蛋白及其应用
CN113698493B (zh) * 2021-08-09 2022-05-31 北京东方百泰生物科技股份有限公司 一种针对VEGF和TGF-β的双功能蛋白及其应用

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