WO2015000181A1 - Novel recombinant fusion protein, preparation method therefor and use thereof - Google Patents

Abstract

The present invention provides a recombinant fusion protein, preparation method therefor and use thereof. The fusion protein comprises a component A comprising VEGFR1 extracellular domain 2 and an immunoglobulin component B, the two components being linked together in tandem. The present invention also provides use of the novel recombinant fusion protein medicament in the treatment of diseases such as tumor, etc.

Description

 Novel recombinant fusion protein and preparation method and use thereof

 Technical field

 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.

At present, 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, and 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.

 Therefore, the development of new structural recombinant fusion protein drugs is of great significance in the study of VEGF targeted therapeutic drugs. Summary of the invention

 The object of the present invention is to provide a novel recombinant fusion protein, a preparation method and use thereof.

 In a first aspect of the invention, there is provided a fusion protein having the structure of formula la or formula lb:

 C-A-B (la), or

 C-B-A (lb)

 among them,

 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;

 B is an immunoglobulin element;

 C is an optional signal peptide sequence;

"-" indicates a peptide bond or a peptide linker that links each of the above elements. In another preferred embodiment, 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.

 In another preferred embodiment, the element A is 100 amino acids in length.

 In another preferred embodiment, the 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,).

 In another preferred embodiment, the amino acid sequence of said element A is shown in positions 20-119 of SEQ ID NO.: 1. In another preferred embodiment, the D2 has a flanking sequence, and the flanking sequence includes:

 a first flanking sequence at the amino terminus of D2; and/or

 A second flanking sequence at the carboxy terminus of D2.

 In another preferred embodiment, the first flanking sequence consists of 1-5 amino acid residues.

 In another preferred embodiment, the second flanking sequence consists of 1-2 amino acid residues.

 In another preferred embodiment, the 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.

 In another preferred embodiment, the first flanking sequence is SDTGR.

 In another preferred embodiment, the second flanking sequence is NT.

 In another preferred embodiment, said element B is an Fc fragment of human immunoglobulin IgG1.

 In another preferred embodiment, 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).

 In another preferred embodiment, the fusion protein further comprises a signal peptide element (:.

 In another preferred embodiment, the fusion protein does not contain a signal peptide, and the structural formula is

 A-B (I l ia) , or

 B-A (I I lb)

 Where A, B and "-" are as defined above.

 In another preferred embodiment, the amino acid sequence of the signal peptide is shown as position 1-19 of SEQ ID NO: 1. In another preferred embodiment, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 1.

 In another preferred embodiment, the fusion protein has the following characteristics:

 a) binding activity to VEGF ED50 = 50-200 pM (preferably ED50 = 60_80 pM);

 b) blocking VEGF-induced VEGFR2 phosphorylation;

 c) blocking VEGF-induced angiogenesis in vitro or in vivo;

d) inhibits migration and invasion of tumor cells; e) Plasma half-life is 5 days.

In a second aspect of the invention, a protein dimer is provided, the dimer being formed from any of the fusion proteins of the first aspect.

 In another preferred embodiment, the dimer has the structure of Formula Ila or Formula lib:

C-A-B C-B-A

 II or II

 C-A-B (Ila) C-B-A (lib)

 among them,

 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;

 "-" means a peptide bond or a peptide linker connecting the above elements;

 "II" indicates a disulfide bond.

 In another preferred embodiment, the fusion protein does not contain a signal peptide, and the structural formula is

A-B B-A

 II or II

 A-B (Ila) B-A (lib)

 among them,

 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;

 "-" means a peptide bond or a peptide linker connecting the above elements;

"II" indicates a disulfide bond. In a third aspect of the invention, an isolated polynucleotide encoding the fusion protein of claim 1 is provided. In a fourth aspect of the invention, a vector comprising the polynucleotide of the third aspect is provided. According to a fifth aspect of the invention, a host cell comprising the vector or genome in the fourth aspect is provided A polynucleotide according to the third aspect.

 In another preferred embodiment, the host cell is a prokaryotic cell or a eukaryotic cell (e.g., CH0 cell, NSO cell, or 293 cell). In a sixth aspect of the invention, 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;

 The fusion protein or a dimer formed from the fusion protein is isolated. In a seventh aspect of the invention, a pharmaceutical composition is provided, the 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.

 In another preferred embodiment, the disease is selected from the group consisting of: tumor, wet macular degeneration or liver fibrosis.

 In another preferred embodiment, the tumor comprises: colorectal cancer, lung cancer.

 In another preferred embodiment, the disease is an angiogenesis-related disease. In a ninth aspect of the invention, there is provided 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.

 In another preferred embodiment, the fusion protein is administered as a monomer and/or a dimer.

 In another preferred embodiment, 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.

 Figure 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.

 Figure 4 shows that VEGFR1D2-FC blocks VEGF-induced VEGFR2 phosphorylation.

 Figure 5 shows that VEGFR1D2-FC blocks vascular endothelial cell tubular formation induced by VEGF.

 Figure 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.

 Figures 8 and 9 show that VEGFR1D2-FC has an activity of inhibiting the growth of lung cancer cells (A549).

 Figures 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.

 Taking VEGFR1D2-FC as an example, it 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.

 In one embodiment, the inventors confirmed by experiments that the expressed VEGFR1D2-FC has a strong VEGF-binding activity, ED50 = 60-80 pM. Due to certain disease states (such as tumors, age-related macular degeneration, liver fibrosis), excessive secretion of VEGF in the body leads to abnormal proliferation of blood vessels, thereby inducing disease or aggravating the condition. Therefore, VEGFR1D2-FC can be used to treat tumors, wet macular degeneration or liver fibers. VEGFR1 and its extramembrane region

The 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. When combined with its corresponding ligand, VEGFR proteins induce a range of different biological functional responses based on their corresponding receptor properties. VEGFR proteins include: VEGFR1 (Flt-1), VEGFR2 (KDR/FLk-1), VEGFR3 (Flt_4), or a combination thereof. In the present invention, 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. Studies have shown that neither VEGFR2 (KDR/FLk-Ι) nor VEGFR3 (Fit-4) D2 binds to VEGF, and the prior art (The EMBO Journal vol. 15 no. 18 pp. 4919-4927, 1996) shows that only The polypeptide consisting of the D2 region does not have VEGF-binding activity, for example, a fusion protein composed only of the D2 region and Fc does not have VEGF-binding activity (The EMBO Journal vol. 15 no. 18 pp. 4919-4927, 1996). Immunoglobulin G element

 In the present invention, 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. Under normal physiological conditions, 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

 In the present invention, "recombinant 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 fusion protein of a protein element and an immunoglobulin element, preferably Fc. 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. Furthermore, it should be understood that the term also encompasses active fragments and derivatives of fusion proteins.

 As used herein, "isolated" means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment). For example, 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.

As used herein, "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. As is known in the art, 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.

 As used herein, the term "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. For example, 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. As long as there are sufficiently long primers to bind well 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. For PCR amplification, 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.

 Once the relevant sequences have been obtained, 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.

 In addition, synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short. Usually, 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:

 (1) using a polynucleotide (or variant) of the present invention encoding a protein of the present invention, or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide;

 (2) a host cell cultured in a suitable medium;

 (3) Separating and purifying proteins from the culture medium or cells.

 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.

 Furthermore, 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.

 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. 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. When 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. When 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. After the host cell has grown to the appropriate cell density, 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. antibody

 In the present invention, "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. Here, "specificity" means that an antibody can bind to a protein of the present invention or a fragment thereof, respectively. Preferably, those antibodies which bind to a protein or fragment of the invention but which do not recognize and bind to other unrelated antigen molecules. 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. Peptide linker

 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. In general, 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 α-helix or β-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. Pharmaceutical composition and method of administration

 The invention also provides a composition comprising an effective amount of a fusion protein of the invention, and a pharmaceutically acceptable carrier. In general, the fusion proteins of the invention may be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the ρ Η is typically from about 5 to about 8, preferably, ρ Η is from about 6 to about 8.

As used herein, 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. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents. The pharmaceutical 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. In general, 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. In the case of a tumor patient, generally, when 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. . For example, 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:

 1) has a strong binding activity of VEGF, ED50=60_80pM;

 2) Blocking VEGF-induced VEGFR2 phosphorylation;

 3) Blocking VEGF-induced angiogenesis in vitro or in vivo;

 4) It can inhibit the migration and invasion of tumor cells.

 5) Block VEGF-induced liver fibrosis.

 6) Compared with the existing targeted fusion recombinant protein for VEGF, it has the advantages of small molecular weight and simple structure. The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually produced according to the conditions described in the conventional conditions, for example, Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions. The conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are percentages by weight and parts by weight. Example 1

Construction of 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 "Kozack" sequence and the "Hindl ll" gene cloning site were added to the amino terminus of the 357 nucleotides, and the "EcoRI" gene cloning site was added to the carboxy terminus to form a 378 nucleotide. Gene fragment.

 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. Human cytomegalovirus (HCMV) promoter sequence

(Accession#: X17403), after the ampicillin resistance gene, the Chinese hamster glutamine synthetase gene (Accession^: X03495) was added in front of the HCMV promoter. After the sequence design, the company will be commissioned by Shanghai Jierui Biological Engineering Co., Ltd. for synthesis and transformation. 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.

 Figure 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

 Expression of VEGFR1D2-FC

 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.

Using this cell, 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. Take out the electric rotor, let stand for 5 minutes, add 0. 6 ml of cell culture medium, mix it, suck it out, transfer it to the culture dish, and put it into the incubator. Protein expression was checked after 24-48 hours. If protein expression is successful, the gene is successfully transferred. At this point, the cells are diluted with medium and then transferred to 20 96-well cell culture plates at 3000-5000 cells per well. The cells are screened by a series of pressures (glutamine synthetase inhibitors), and finally a cell line capable of highly expressing VEGFR1D2-FC is screened.

At the time of protein production, 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. When the cell density reaches 5-6x 10 ⁶ cel ls/ml, 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

 Detection of binding activity of VEGFR1D2-FC to target (VEGF)

 The binding properties of the fusion protein to the target (VEGF) were determined by enzyme-linked immunosorbent assay (ELISA). Specific steps are as follows:

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 μl 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. After incubating for one hour at room temperature, the sample was discarded, washed 5 times with 0.05% PBS-T, and then 100 μl of diluted (1: 20000) HRP-Rabbit Ant i-Human IgG Fc (Luoyang Aotong, Cat#: C030222), incubate for one hour at room temperature, wash the washing solution 5 times, add HRP substrate, avoid color development for 10-20 minutes, then stop the color reaction with 2N 3⁄450 ₄ and read 0D450 value on the microplate reader.

The results showed that D2-Fc has a strong VEGF-binding activity (as shown in Figure 3), and the ED50 is below nanomolar (ED50 = ~ 67pM). Example 4

 VEGFR1D2-Fc inhibits VEGF-induced VEGFR2 phosphorylation and HUVEC cell proliferation

 Since VEGF binds to VEGFR2, 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 specific steps of the VEGFR2 phosphorylation experiment are as follows:

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. Minutes, remove the 6-well plate, discard the culture solution, add 150 ul of lysate (100 mM PMSF, Protease Inhibitor cocktail lOOmM Sodium Orthovanadete solution, the three protease inhibitors are added to the lysate in a ratio of 1:100), ice After the reaction was carried out for 2 min, the cells were gently pipetted, and the suspension was collected, centrifuged at 13,000 rpm for 1.5 min, and the supernatant was taken. The cell lysate was separated by 8% SDS-PAGE separation gel and then transferred to a PVDF membrane (Merck & Co., IPVDF., Cat No: IPVH00010). 5小时。 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. Mix the two reagents of liquid A and liquid B of ECL kit in the same volume in the test tube, then add it to the front side of the PVDF membrane, incubate for about 2 minutes, cover the PVDF membrane with a layer of plastic wrap, and wipe off the excess luminescent agent. Finally, by detecting the development tape _T anO n ^¾ -4200 automated chemiluminescence image analysis system.

 The results showed that 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 specific steps of HUVEC cell proliferation and tubular formation experiments are as follows:

 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.

The results showed that VEGFR1D2-Fc can inhibit the proliferation of HUVEC cells and the tube of HUVEC cells in gel medium. Formed as shown in Figure 5. Example 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.

1) Zebrafish preparation method:

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 卩 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 μΞ / οιιΐ; ρ Η is 6. 9~7. 2; 5:7~71. 6 mg/L CaC0 ₃ ). Because the embryo can obtain nutrients from its own yolk sac, no feeding is required within 9 days (9dpf) after fertilization. After the experiment was completed, the zebrafish at various developmental stages were overexposed with tricaine methanesulfonic acid to anesthetize the zebrafish. The procedure for anesthesia is in accordance with the specifications of the American Veterinary Medical Association (AVMA) for anesthesia.

 2) Experimental method:

 A. Determine the maximum non-lethal concentration of the sample to be tested (MNLC)

 - Using a microinjector, 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;

 - Three initial test concentrations of the sample to be tested.

 - negative control group: solvent group;

 - A blank control group is used to demonstrate that the solvent does not adversely affect the zebrafish;

 - all experimental groups (except the blank control group) contained the same concentration of solvent;

 - After the sample treatment, the number of zebrafish deaths in each experimental group was counted, and the optimal concentration effect curve was drawn using JMP statistical software, and MNLC was calculated;

 - If MNLC is not available in the initial concentration test, 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.

 B. Quantitative evaluation of the inhibitory effect of samples on angiogenesis

 - According to the concentration lethal curve, each sample to be tested is selected for 3 concentrations (usually the maximum non-lethal concentration (MNLC), 1/3 MNLC and 1/10 MNLC);

 - treatment of each concentration (blood circulation injection) 30-tail vascular fluorescent transgenic zebrafish;

- 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;

 - all experimental groups (except the blank control group) contained the same concentration of solvent;

 - After the sample treatment, 10 zebrafish were randomly selected from each group to observe, photograph and save the pictures under a fluorescence microscope;

- Image analysis using image analysis software to calculate vascular fluorescence signal intensity (s) for quantitative analysis, and statistical processing results are indicated by ^ SE;

 - The formula for calculating the efficacy of the sample to inhibit angiogenesis is as follows:

Angiogenesis inhibition rate (3⁄4) = ί - ^合合^ ^圼 group)) _xlW3⁄4

 Sc 赫服)

 - Statistical analysis was performed using analysis of variance and Dunnett's T-test, p < 0.05 indicates significant differences; - Provides a representative experimental map. Vascular Fluorescent Transgenic Zebrafish Embryos Injected Different Doses of VEGFR1D2-Fc (4.4, 14.7, 44 ng) and Positive Control Drugs (44, 100 ng) and Lovastatin (4 ng) by Microinjection 48 hours after fertilization The zebrafish blood circulation, 72 hours microscopic observation of the zebrafish intestinal blood vessels, and photographed, recorded under different conditions

3) Experimental results:

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, ρ<0·001). ). Two different positive control compounds have significant inhibitory effects on zebrafish blood vessels, so this evaluation model is reliable.

 The 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).

 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

 VEGFR1D2-FC inhibits tumor cells in a dose-dependent manner

 The following components were mixed using conventional techniques well known to those skilled in the art to prepare a final concentration of 1 wt% recombinant protein solution, which was formulated as follows:

Recombinant protein 10m _g

 Normal saline added to 10ml

 Adjust pH to 6. 8-7. 1

Balb/c nude mice were injected subcutaneously with 5x10 ⁶ lung cancer (A549) or colorectal cancer (C0L0-205) cells until the tumor grew to

130-170 were randomly divided into ³ groups. 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. For injection, 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.

 The results showed that the inhibition rate of colorectal cancer and lung cancer was more than 90% at high dose, while the inhibition rate of colorectal cancer at low dose was still above 94% (as shown in Figures 10 and 11). More than 85% (as shown in Figures 8 and 9). Example 7

 Pharmacokinetic analysis of VEGFR1D2-FC

 Sixteen normal Balb/c mice were injected subcutaneously with 50 ug of VEGFRlD2-Fc, respectively, and 2, 2 after injection.

4 8 24 48 96, and 144 hours of plasma collection through the female eye vein, ELISA method to detect the content of VEGFR1D2_Fc in plasma. The results indicate that the plasma half-life of VEGFR1D2-FC is at least 5 days (120 hours) (see Figure 12). All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

Rights request

A fusion protein, characterized in that the fusion protein has the structure of formula la or formula lb:

 C-A-B (la), or

 C-B-A (lb)

 among them,

 A is a protein element comprising a second extramembranous region D2 of vascular endothelial growth factor receptor 1 (VEGFRl), and element A is 94-103 amino acids in length;

 B is an immunoglobulin element;

 C is an optional signal peptide sequence;

 "-" indicates a peptide bond or a peptide linker that links each of the above elements.

 2. The fusion protein of claim 1, wherein the D2 has a flanking sequence, and the flanking sequence comprises:

 a first flanking sequence at the amino terminus of D2; and/or

 A second flanking sequence at the carboxy terminus of D2.

 The fusion protein according to claim 1, wherein the fusion protein has the following characteristics: a) binding activity to VEGF ED50 = 50-200 pM;

 b) blocking VEGF-induced VEGFR2 phosphorylation;

 c) blocking VEGF-induced angiogenesis in vitro or in vivo;

 d) inhibits migration and invasion of tumor cells;

 e) Plasma half-life of 5 days.

 A protein dimer characterized in that the dimer is formed from the fusion protein of any one of claims 1-3.

 The dimer according to claim 4, wherein the dimer has the structure of the formula Ila or lib:

 C-A-B C-B-A

 II or II

 C-A-B (Ila) C-B-A (lib)

 among them,

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;

 "-" means a peptide bond or a peptide linker connecting the above elements;

 "II" indicates a disulfide bond.

 6. An isolated polynucleotide, characterized in that the polynucleotide encodes the fusion protein of claim 1.

 7. A vector comprising the polynucleotide of claim 6.

 A host cell comprising the vector of claim 7 or the polynucleotide of claim 6 in which the polynucleotide of claim 6 is integrated.

 9. A method of producing a protein, characterized in that it comprises the steps of:

 The host cell of claim 8 is cultured under conditions suitable for expression, thereby expressing the fusion protein of claim 1;

 The fusion protein or a dimer formed from the fusion protein is isolated.

 10. A pharmaceutical composition, characterized in that the composition comprises:

 The fusion protein of claim 1 and/or the protein dimer of claim 4, and a pharmaceutically acceptable carrier.

 The use of the fusion protein of claim 1 and/or the protein dimer of claim 4, for use in the preparation of a medicament for treating a disease.