WO2005121176A1 - Proteine chimerique inhibitrice d'angiogenese et utilisation associee - Google Patents

Proteine chimerique inhibitrice d'angiogenese et utilisation associee Download PDF

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WO2005121176A1
WO2005121176A1 PCT/CN2005/000802 CN2005000802W WO2005121176A1 WO 2005121176 A1 WO2005121176 A1 WO 2005121176A1 CN 2005000802 W CN2005000802 W CN 2005000802W WO 2005121176 A1 WO2005121176 A1 WO 2005121176A1
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immunoglobulin
fusion protein
region
flt
kdr
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PCT/CN2005/000802
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English (en)
French (fr)
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Zheng Liu
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Chengdu Kanghong Biotechnologies Co. Ltd
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Priority to EP05752254A priority Critical patent/EP1767546B1/en
Priority to JP2007526176A priority patent/JP4680997B2/ja
Priority to AT05752254T priority patent/ATE548384T1/de
Priority to BRPI0512286A priority patent/BRPI0512286B8/pt
Priority to PL05752254T priority patent/PL1767546T3/pl
Priority to ES05752254T priority patent/ES2381014T3/es
Priority to US11/628,735 priority patent/US7750138B2/en
Priority to CA2569108A priority patent/CA2569108C/en
Priority to DK05752254.2T priority patent/DK1767546T3/da
Publication of WO2005121176A1 publication Critical patent/WO2005121176A1/zh
Priority to US12/773,315 priority patent/US20100215655A1/en

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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
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    • A—HUMAN NECESSITIES
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to the technical field of genetic engineering, and in particular, to a DNA sequence encoding a recombinant fusion protein capable of inhibiting angiogenesis, a fusion protein encoded by the same, a pharmaceutical use of the fusion protein, a medicine containing the fusion protein, and a preparation thereof.
  • Angiogenesis refers to the process of growing new blood vessels from existing blood vessels. Most of the blood vessels in adults are at rest, and angiogenesis is only seen in a few pathological or physiological states, such as tumors, diabetic retinopathy, arthritis, anemia organs, and proliferative endometrium. In the occurrence of tumors, angiogenesis plays a critical role in the rapid tumor growth (Hanahan and Folkman:. Patterns and emerging mechanisms of the angiogenic switch during tumor i genes is Cell.1996, 86: 353-364) 0 Animal The study of tumor models and human clinical trials have shown that inhibiting the formation of new blood vessels in tumors can effectively prevent the growth and development of tumors, thereby prolonging the life of patients.
  • Angiogenesis is regulated and controlled by a variety of biologically active substances.
  • the main cells that dominate the angiogenesis process are the vascular endothelial cells that constitute the innermost layer of the vascular wall.
  • a variety of growth factors can bind to the corresponding receptors on the surface of vascular endothelial cells, and regulate the activity of vascular endothelial menstruation via the intracellular signal transmission system, thereby regulating angiogenesis.
  • Vascular endothelial cell growth factor (abbreviated as VEGF) is the most important factor that regulates angiogenesis (Ferrara: VEGF and the quest for tumor angiogenesis factor. Nat. Rev. Cancer, 2002, 10: 795-803. Ferrara: Role of vascular endothelial growth factor in physiologic and pathologic angiogenesis: therapeutic implications. Semin. Oncol. 2002, 29 (6 sumppl): 10-14). Vascular endothelial cell growth factor can be secreted by a variety of cells, but it is often overexpressed in tumor cells. Vascular endothelial cell growth factor works by binding to the corresponding receptor.
  • the first functional region is an extracellular part located outside the cell, and it consists of seven immunoglobulin-like regions (dl-d7) This part has specific affinity for VEGF and is a key part of the binding of VEGF and receptors.
  • the second functional region is a transcellular membrane part composed of hydrophobic amino acids.
  • the third functional region is an intracellular part, including Tyrosine kinase group, after the receptor is activated by VEGF, the tyrosine kinase group is phosphorylated, thereby activating the internal signal transmission system of the cell, and ultimately causing changes in the function of endothelial cells, resulting in angiogenesis.
  • VEGF has a highly specific regulatory effect on vascular endothelial cells.
  • VEGF has the functions of promoting endothelial cell division, guiding endothelial cell migration, inhibiting cell death, and inducing vascular morphogenesis, etc., and is a highly efficient i-shower for angiogenesis.
  • VEGF is a key factor leading to angiogenesis in tumors. Many animal tests have shown that blocking the binding of VEGF to its receptor can effectively inhibit the angiogenesis in tumors, thereby preventing tumor growth.
  • VEGF is also closely related to the development of these diseases (Ferrara: Role of vascular endothel ial growth factor in phys iologic and pathologic angiogenes is: therapeut ic impl icat ions. Semin. Oncol. 2002, 29 (6 sumppl): 10-14).
  • VEGF vascular endothel ial growth factor
  • Soluble KDR can also inhibit the proliferation of vascular endothelial cells in vitro, but its antitumor effect is not good in animal experiments (Yoko Hasumi: Soluble FLT-1 Expres s ion Suppresses Carcinomatous Asci tes in Nude Mice Bear ing Ovar ian Cancer. Cancer Research 62, 2002: 2019-2023) 0
  • the present invention provides a new fusion protein composed of different fragments from FLT-1 and DR to effectively block the biological effects of VEGF and inhibit angiogenesis.
  • One of the objects of the present invention is to provide a novel recombinant fusion protein that blocks the biological effect of VEGF and thereby inhibits angiogenesis.
  • Another object of the present invention is to provide a nucleotide sequence encoding the fusion protein.
  • a third object of the present invention is to provide a vector containing a nucleotide sequence encoding the fusion protein and a recombinant transformed with the vector.
  • the fourth object of the present invention is to provide the application of the fusion protein in the preparation of a drug that blocks the biological effect of vascular endothelial cell growth factor and inhibits angiogenesis, and also provides a pharmaceutical composition containing the fusion protein and a pharmaceutically acceptable carrier and a dosage form thereof, Application of the pharmaceutical composition in treating diseases.
  • the key of the present invention is to design and construct a series of fusion proteins composed of different FLT-1 fragments and KDR fragments according to the structure of FLT-1 and KDR.
  • the fusion protein further includes human immunoglobulin Fc ( The construction method is shown in Figure 1), and then the fusion protein with high affinity for VEGF was screened by the VEGF binding test and the like to obtain a suitable VEGF blocker.
  • the construction technology of the fusion protein is based on a conventional molecular cloning method, and specific experimental steps are described in the second and third editions of ⁇ Molecular Cloning> (Joseph Sambrook, Science Press) and similar experimental manuals.
  • a fusion protein prepared by genetic recombination technology is composed of different fragments of FLT-1 and KDR from the VEGF receptor, and is characterized in that the fusion protein is selected from the following group: a is composed of the first immunoglobulin-like region of KDR, the second immunoglobulin-like region of FLT-1 and the third immunoglobulin-like region of KDR, expressed as: DRdl-FLTd2-KDRd3;
  • d. is composed of the first immunoglobulin-like region of FLT-1 and the 3-5 immunoglobulin-like region of KDR, expressed as: FLTd2- KDRd3, 4, 5;
  • the amino acid sequence of FLTd2 is described in SEQ ID NO. 1
  • the amino acid sequence of FLTd4 is described in SEQ ID NO. 2
  • the amino acid sequence of KDRdl is described in SEQ ID NO. 3
  • the amino acid sequence of KDRd3 is described in SEQ ID NO. 4.
  • the amino acid sequence of KDRd4 is as described in SEQ ID NO.
  • FLT stands for FLT-1 sequence
  • KDR stands for KDR sequence
  • di stands for the i-th immunoglobulin-like region (doma in) of FLT-1 or KDR.
  • fusion protein further includes human immunoglobulin FC, and the fusion protein is selected from the following group:
  • FP3 expressed as: FLTd2- KDRd3, 4- Fc;
  • FP5 expressed as: FLTd2-KDRd3, 4, 5- Fc;
  • FP6 expressed as: FLTci2-KDR (i3-FLTd4, 5- Fc.
  • Fc represents a human immunoglobulin Fc fragment, which is selected from human immunoglobulin FC such as IgG, IgM, IgA or subtype I g Gl, IgG2, IgG3 or I g G4, and the immunoglobulin FC fragment may be full-length FC Or a partial FC sequence, such as selected from a CH2 fragment, a CH3 fragment, or a hinge region fragment.
  • the fusion protein disclosed in the prior art herein referred to as FP1 is composed of the sequence of the second immunoglobulin region (FLTd2) of FLT-1 and the third immunoglobulin region (KDRd3) of KDR Sequence and human immunoglobulin Fc.
  • the FP2 ′ fusion protein provided by the present invention is based on FP1, and the amino acid sequence of the first immunoglobulin-like region (KDRdl) from KDR is added. These sequences can increase the site of binding to VEGF, thereby increasing the effect on VEGF. Affinity.
  • FP3, and FP4, fusion proteins are the sequences of the fourth immunoglobulin-like region of KDR (KDRd4) or the sequence of the fourth immunoglobulin-like region of FLT-1 (FLTd4) in addition to FP1, respectively.
  • FP5 and FP6 are the fourth and fifth immunoglobulin-like regions (KDRd4, 5) of KDR or the fourth and fifth immunoglobulin-like regions of FLT-1 (FLT- ld4, 5). These new sequences will be more conducive to the coupling between the fusion proteins, thereby further forming a spatial structure conducive to binding to VEGF, and increasing the affinity for binding to VEGF.
  • fusion protein FP3 whose amino acid sequence is as described in SEQ ID NO.
  • the fusion protein of the present invention can be obtained by conventional genetic recombination technology.
  • a recombinant encoding the above-mentioned fusion protein was obtained.
  • the DNA sequences encoding FLT-1 and KDR were recorded in GenBank of NCBI (Nation Center for Biotechnology Information).
  • the DNA sequence encoding the fusion protein was then cloned into a vector after synthesis by PCR.
  • the vector used may be a shield particle, virus or DNA fragment commonly used in molecular biology.
  • a protein secretion signal sequence is added to the end of the DNA sequence encoding the fusion protein to ensure that the protein is secreted from the cell.
  • the vector sequence includes a promoter for driving gene expression, protein translation initiation and termination signals, and a polyadenylic acid (PolyA) sequence.
  • Antibiotic resistance genes are included in the vector to facilitate vector propagation in host cells, such as bacteria.
  • the vector also includes eukaryotic cell-selective genes for stable selection of host cell lines.
  • the length of the amino acid sequence of each immunoglobulin-like region can vary. Therefore, the amino acid sequence of the fusion protein according to the present invention may also have certain changes, and they all belong to the scope of the present invention.
  • the recombinant vector is used to transfect or transform host cells and express the corresponding protein.
  • expression systems that can be used to express these fusion proteins, which can be eukaryotic cells or prokaryotic cells, and they include (but are not limited to) mammalian cells, bacteria, yeast, insect cells, and so on. Proteins expressed from mammalian cells have glycosyl modifications. Since the amino acid sequence of the fusion protein of the present invention includes glycosylated amino acids, mammalian cells are the best cells to express these proteins.
  • the recombinant plasmid containing the above-mentioned fusion protein can be transfected into a host cell.
  • methods for transfecting cells including but not limited to: electroporation (el ec tropora ti on), liposome-mediated methods, calcium-mediated methods, and the like.
  • mammalian cells In addition to mammalian cells, other expression systems such as bacteria, yeast or insect cells can also be used to express these fusion proteins, and they are also included in the cells that can be used in the present invention.
  • the protein shield production of these expression systems is higher than that of mammalian cells, however, the expressed protein lacks glycosylation or the sugar chains formed are different from mammalian cells.
  • the concentration of the fusion protein in the cell culture medium can be measured by an enzyme-linked immunosorbent assay (ELISA) or other methods. Since these fusion proteins have an immunoglobulin Fc fragment, the expressed fusion protein can be extracted by Protein A affinity chromatography.
  • ELISA enzyme-linked immunosorbent assay
  • Various fusion protein shields were obtained from the recombinant culture medium.
  • the VEGF binding assay was then used to detect and compare the affinity of various proteins for VEGF.
  • VEGF-induced human vascular endothelial cell division test was used to detect and compare the blocking effect of each fusion protein on VEGF.
  • the experimental results prove that compared with the FP1 and protein disclosed in the prior art, the various fusion proteins constructed by the present invention have a high affinity for VEGF (see Figure 2), and they can effectively block VEGF from vascular endothelial cells Stimulate and inhibit endothelial cell division. Further tests found that FP3 has the best blocking effect on VEGF, and is the most effective fusion protein in the present invention to block VEGF.
  • the fusion protein constructed by the present invention has a good blocking effect on VEGF, and both have anti-angiogenesis biological properties, so they can be used to treat angiogenesis or VEGF-related diseases.
  • diseases may include but are not limited to Various tumors, retinal angiopathy, joints Inflammation, anemia or endometrial hyperplasia, etc.
  • the present invention also provides animal experiments to further prove the anti-angiogenesis effect of the fusion protein provided by the present invention in vivo.
  • the fusion proteins provided by the present invention are very effective in inhibiting tumor growth, prolonging the life of an individual, and the effect is significantly better than the FP1 and protein of the prior art. Therefore, the fusion protein constructed by the present invention has high efficiency Anti-cancer ability.
  • the invention also provides a pharmaceutical composition containing the fusion protein of the invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be made into various forms of pharmaceutical preparations according to conventional techniques of formulation, preferably an injection, and most preferably a freeze-dried injection.
  • Figure 1 shows the structural composition of five fusion proteins and the FP1 protein of the prior art in a preferred embodiment of the present invention, which are constructed by genetic engineering methods with different FLT-1 and KDR fragments and immunoglobulin Fc fragments. .
  • FIG. 2 shows the results of a trial of five types of fusion proteins provided by the present invention and VEGF in a preferred embodiment of the present invention using the FP1 protein as a control, where the 0D reading represents the signal of binding of the fusion protein to VEGF
  • the results show that the five fusion proteins provided by the present invention have strong binding ability to VEGF, and all of them are significantly higher than FP1, and in particular, FP3 has the strongest binding ability.
  • Figure 3 shows that the fusion protein of the present invention effectively inhibits the division of human vascular endothelial cells in vitro relative to FP1.
  • Figure 4 shows that the fusion protein FP3 effectively inhibits the growth of mouse B16F10 melanoma in vivo.
  • Figure 5 shows that the fusion protein FP3 effectively inhibits the growth of human PC-3 prostate cancer in mice.
  • Figure 6 shows a comparative study of the fusion protein FP3 of the present invention and the prior art FP1 in effectively inhibiting the growth of mouse tumors.
  • Example 1 Cloning of DM sequence encoding fusion protein and construction of recombinant vector
  • the original DNA sequences encoding various fusion proteins of the present invention are derived from the corresponding cDNAs of FLT-1 and KDR. Since the expression of FLT-1 and KDR is mainly found in vascular endothelial cells, the present invention uses RNA purification kits (QIAGEN) to extract total RNA from human umbilical vein vascular endothelial cells (HUVEC); (iptese Pr omega) to synthesize cDNA from RNA; then use different primers to amplify by polymerase chain reaction (PCR) to obtain the required FLT-1 and KDR fragments; finally, PCR from FLT-1, KDR and human immunoglobulins Protein Fc (IgGl Fc) sequences are fused to construct recombinant DM sequences encoding different fusion proteins.
  • the structures of the six fusion proteins (including the prior art FP1) constructed in this preferred embodiment are shown in Figure 1 of the accompanying drawings.
  • Human umbilical vein vascular endothelial cells (HUVEC cells) (Clonet ics) were cultured in T-175 culture flasks with EGM-2 medium (Clonet ics), and about 1 x 10e7 cells were collected and extracted using Qiagen's RNA extraction kit. Total RNA of cells, and cDNA was synthesized by Invi trogen cDNA kit, frozen at -80 ° C until use. The following specific primers were used to amplify FLT-1 and KDR gene fragments from HUVEC cDNA.
  • Human IgGl Fc specific primers were used to amplify IgGl Fc gene fragments from cDNA from lymph nodes (BD Clcmtech).
  • the primers are:
  • FLT-1 d2 forward primer 5'-cctttcgtagagatgtacagtga-3 '
  • FLT-1 d2 reverse primer 5'-tatga t tgta t tggt t tgtccat-3 '
  • KDR d3-4 Reverse Primer 5 '-cggtgggacatacacaaccaga-3' Human IgGl Fc forward primer: 5'-gacaaactcacacatgcccact-3 'Human IgGl Fc reverse primer: 5'-tcat t tacccggagacagggagag-3' at denaturation 95 ° C, 30 minutes; annealing 56 ° C, 45 seconds; extension 72 ° C, PCR was performed under the conditions of 2 minutes, and 30 cycles were performed to obtain PCR products of IgG-like domains of FLT-1 and KDR and PCR products of human IgGl Fc fragments.
  • the PCR product was cloned into the pCR2.1 plasmid (Invi trogen), and transfected with E. coli i (JM109). White colonies were selected, added to LB medium, and cultured overnight. The plasmid was extracted by Qiangen plasmid extraction kit and digested by sequencing.
  • the confirmed plasmid was transfected into 293 or CH0 cells to obtain a cell line stably expressing FP3.
  • the specific amino acid sequence of FP3 is as described in SEQ ID NO.
  • Example 2 Constructing a gene and a recombinant vector
  • the target recombinant DNA was obtained by splicing genes encoding the first immunoglobulin-like region of FLT-1 and the third immunoglobulin-like region of KDR, and the same human IgG1 Fc fragment as in Example 1. The specific operation steps are the same as in Example 1.
  • Example 3 Construction of FP2 gene and recombinant vector
  • Example 4 Construction of FP4 gene and recombinant vector
  • Example 5 Construction of FP5 gene and recombinant vector
  • the target recombinant DNA was obtained by splicing the gene encoding the second immunoglobulin-like region of FLT-1 and the immunoglobulin-like region of 3-5 of KDR, and the same human IgG1 Fc fragment as in Example 1. Other than that, the specific operation steps are the same as in Example 1.
  • Example 6 Construction of FP6 gene and recombinant vector
  • Example 2 Expression of fusion proteins in cells
  • a plasmid DNA purification kit QIAGEN
  • the FUGEN6 plasmid transfection kit ROCHE
  • two different plasmid transfection methods were used to express the fusion protein.
  • the first method is the transient transfection method, which can be used to obtain a small amount of fusion protein.
  • 293 cells were cultured in cell culture with DMEM complete medium containing 10% fetal bovine serum. When the cells grow to cover 60-80 ° /.
  • the complex of the recombinant plasmid DM and FUGEN6 reagent was added to the cell culture medium. The next day, the medium was changed with serum-free DMEM medium. The culture was continued for another three days, and then the supernatant was collected. These supernatants The solution contains a fusion protein expressed from 293 cells after transfection. The concentration of the fusion protein was determined quantitatively by ELISA.
  • the second method is to use stable transfection to establish stable cells to express a large number of fusion protein polypeptides.
  • the cells used were also 293 cells (ATCC).
  • the transfection method of this recombinant plasmid is the same as the transient transfection method described above, but on the second day of transfection, 293 cells after transfection were completely cultured in EM containing neoenzyme, and cloned by limiting density dilution . After approximately 21 days, the neoenzyme-resistant clones were picked and expanded for cell culture. Finally, the transfected 293 cells were cultured in a drum culture flask to produce fusion proteins. The fusion protein concentration was determined quantitatively by ELISA.
  • Broth was purified using affinity chromatography methods, gel-filtration and the like to give FP3, Expression 8 having a molecular weight of 140kD o other embodiments fusion proteins
  • the corresponding fusion proteins FP1, FP2, FP4, and FP5T were prepared by the same method as in Example 7 above.
  • the binding ability of each fusion protein to VEGF is measured using a VEGF binding test.
  • a recombinant VEGF protein (Chemicom) was first coated on a 96-well ELISA plate. Non-specific protein binding sites were then blocked with a 5% milk powder solution. Various fusion proteins with different concentrations were added to each well and incubated at 37 ° C for two hours. After washing, rabbit anti-human Ig antibody-HRP (Sigma) was added. The color was finally developed with a peroxidase substrate. The OD value of each well of the 96-well plate was measured with an ELISA reader. A higher OD value indicates a stronger signal for binding the fusion protein to VEGF.
  • the five fusion proteins constructed and expressed by the present invention all have the ability to bind to VEGF, and have better binding ability than FP1 in the prior art.
  • their binding signal to VEGF can be detected.
  • FP3 has the largest binding capacity with VEGF, and is the most powerful VEGF blocker among which the half maximal binding concentration (Half maximal binding concentrat ion) is about 5 times lower than that of FP1.
  • FP5 and VEGF The binding ability is slightly weaker than FP3. This conclusion indicates that the amino acid sequence of the fourth immunoglobulin-like region of KDR can enhance the blocking ability of the fusion protein to VEGF.
  • Another key embodiment of the present invention is to demonstrate that the constructed fusion protein can effectively block vascular endothelial cell division induced by VEGF.
  • umbilical vein vascular endothelial cells (HUVEC cells, Clonetics) were seeded on a 96-well cell culture plate.
  • the cell culture medium was EBM basic medium.
  • HUVEC cell division test shows that compared with the FP1 of the prior art, the five fusion proteins constructed by the present invention can more effectively inhibit the division of vascular endothelial cells (as shown in Figure 3).
  • these five proteins can effectively block the stimulation of VEGF on its receptors, and all have the function of inhibiting angiogenesis.
  • FP3 is used to inhibit HUVEC cells.
  • the strongest inhibitory effect of division is their half inhibitory concentration (IC50) of about 3 ng / ral.
  • the IC50 of the prior art FP1 is about 12 ng / ml.
  • the IC50 of FP2, FP4, FP5 and FP6 is 5-8 ng / ml.
  • Example 5 Fusion peptide can effectively inhibit tumor growth in mice
  • Example 9 Preparation of injection containing fusion protein
  • the preparation of the injection is as follows: 24mg / ml of FP3, 5Mm PB, 100M NaCl and 20% sucrose are prepared according to the conventional preparation method of the injection.
  • Example 10 The fusion protein can effectively inhibit the growth of mouse B16F10 melanoma as a VEGF blocker.
  • One of the applications of the fusion protein constructed by the present invention is for the treatment of tumors.
  • the present invention selects FP3 to enhance the antitumor effect of animals.
  • the mouse tumor model tested by the present invention is B16F10 melanoma. This is a rapidly growing malignancy.
  • Purified fusion protein was injected via tail vein. The injection dose was 400 micrograms per mouse (the average weight of the mice was about 22 grams) twice a week.
  • the control group was injected with the same dose of purified human immunoglobulin Fc.
  • Figure 4 shows the tumor growth curve, indicating that the fusion protein FP3 very effectively inhibited the growth of the melanoma (P ⁇ 0.01).
  • Example 11 Fusion protein can effectively inhibit the growth of mouse xenograft prostate cancer PC-3 cells
  • the xenograf t model that allows human tumor cells to grow in mice is the closest animal tumor model to human tumors.
  • the rat lacks immunity, so many tumor cell lines from humans can grow in the rat and form tumors.
  • the invention tested the inhibitory effect of the fusion protein FP3 on the growth of human prostate cancer PC-3 cells (ATCC) in BALB / C mice.
  • cells were first PC- 3 1 10 5, 0. 05ml, injected BALB / C mice dorsal skin of trees, from the tail vein injection of purified fusion proteins. 400 micrograms per mouse, twice a week.
  • the control group was injected with the same dose of human immunoglobulin Fc.
  • the experimental results are shown in Figure 5.
  • FP1 and FP 3 are selected as a comparative test for inhibiting tumor growth. Selection of good growth BALB / C mice trees, injection each rat dorsal shield glial tumor cells C6 ⁇ ⁇ ⁇ ⁇ 5, 0. 05ml , purified were injected in the tail vein Fusion protein FP1 and FP3, the dosage is 2.5 mg / kg, twice a week, to 31 days. The control group was injected with the same dose of human immunoglobulin Fc. The experimental results are shown in Figure 6. FP1 and FP3 have a very significant therapeutic effect on the tumor.
  • the fusion protein constructed by the present invention has a high affinity for VEGF, can inhibit the proliferation of vascular endothelial cells in vitro, and can significantly inhibit tumor growth in vivo.
  • the fusion protein according to the present invention can be used for the treatment of various tumors.

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Description

抑制血管新生的融合蛋白质及其用途 技术领域
本发明涉及基因工程技术领域， 具体地说， 涉及编码能抑制血管新 生的重组融合蛋白的 DNA序列、 其所编码的融合蛋白、 该融合蛋白的药 物用途、 含有该融合蛋白的药物及其制剂。 背景技术
血管新生（angiogenesis)是指从已经存在的血管生长出新的血管 的过程。 成人体内的血管绝大多数处于静止状态， 血管新生只见于少数 病理或生理状态， 例如肿瘤、 糖尿病视网膜病变、 关节炎、 贫血器官、 增生期子宫内膜等。 在肿瘤的发生过程中， 血管新生对肿瘤的快速生长 起到关键的作用 (Hanahan and Folkman: Patterns and emerging mechanisms of the angiogenic switch during tumor i genes is. Cell.1996, 86: 353-364) 0 动物肿瘤模型的研究和人体临床试验已经证 明， 抑制肿瘤内新生血管的形成可以有效地阻止肿瘤的生长和发展， 从 而延长病人的生命。 血管新生受到多种生物活性物质的调节和控制。 主 导血管新生过程的主要细胞是构成血管壁最内层的血管内皮细胞。 多种 生长因子能与血管内皮细胞表面相应的受体结合， 经细胞内的信号传递 系统调节血管内皮细月包的活动， 从而调控血管的新生。
在各种生长因子中， 血管内皮细胞生长因子（Vascular endothelial cell growth factor, 简称为 VEGF) 是调节血管新生的最重要的因子 (Ferrara: VEGF and the quest for tumor angiogenesis factor. Nat. Rev. Cancer , 2002 , 10: 795-803. Ferrara: Role of vascular endothelial growth factor in physiologic and pathologic angiogenesis: therapeutic implications. Semin. Oncol. 2002, 29 (6 sumppl): 10 - 14)。 血管内皮细胞生长因子可由多种细胞分泌， 但它在肿 瘤细胞常常过量表达。 血管内皮细胞生长因子通过与相应的受体结合而 起作用。 与 VEGF相结合的受体主要有两种： FLT-l(fms-like tyrosine kinase)和 KDR。在分子结构上，这两种受体均由三个不同的功能区组成： 第一个功能区是位于细胞之外的细胞外部分， 它由七个免疫球蛋白样区 域 （dl-d7)组成， 该部分对 VEGF具有特异亲和性， 是 VEGF与受体相结 合的关键部位； 第二个功能区是由疏水性氨基酸组成的跨细胞膜部分； 第三个功能区是细胞内部分， 其中包括酪氨酸激酶基团， 在受体被 VEGF 激活后， 酪氨酸激酶基团发生磷酸化， 从而启动细胞内部的信号传递系 统， 最终造成内皮细胞的功能变化， 导致血管新生。
FLT-1和 KDR主要分布于血管内皮细胞。 因此， VEGF对于血管内皮 细胞具有高度专一性的调节作用。 VEGF具有促进内皮细胞分裂， 引导内 皮细胞迁移， 抑制细胞调亡， 和诱导血管形态发生等等功能， 是血管新 生的高效 i秀导剂。
在肿瘤组织中， VEGF的表达水平比正常组织为高。 另外， 肿瘤的快 速生长常常导致肿瘤内部的缺氧。 而氧分压的降低又导致 VEGF表达的增 高。 因此， VEGF是导致肿瘤中血管新生的关键因子。 许多动物试臉表明， 阻断 VEGF与其受体的结合能有效地抑制肿瘤中血管的新生， 从而阻止肿 瘤的生长。 在其他与血管新生相关的疾病中， 例如糖尿病视网膜病变和 关节炎的关节病变等等， VEGF 也与这些疾病的发展有密切的关系 (Ferrara: Role of vascular endothel ial growth factor in phys iologic and pathologic angiogenes is: therapeut ic impl icat ions. Semin. Oncol. 2002, 29 (6 sumppl) : 10-14)。
鉴于 VEGF在肿瘤及其他疾病中所起到的关键作用， 能特异性地阻断 VEGF的蛋白质或化合物将具有治疗这些疾病的可能。 例如， 研究证明， 抗 VEGF的中和抗体能有效地抑制肿瘤的生长（Jain: Tumor angiogenes is and access ibi l i ty: role of vascular endothel ial growth factor. Semin. Oncol. 2002 , 29 (6 suppl) : 3-9)。 因此， 寻找新的更为有效 的 VEGF阻断剂在临床上具有重大的意义。 由于 FLT- 1和 KDR对 VEGF有 天然的亲和性， 已有研究探讨可溶性 FLT- 1 (FLT-1的细胞外部分）或可溶 性 KDR (KDR 的细胞外部分）的抗血管新生作用（Yoko Hasumi: Soluble FLT-1 Express ion Suppresses Carcinomatous Asci tes in Nude Mice Bear ing Ovar ian Cancer. Cancer Research 62, 2002: 2019-2023)。 可 溶性 FLT-1 可以在体外有效地抑制血管内皮细胞的增生， '但其在血清中 的半衰期 4艮短， 不能达到有效的血清浓度。 可溶性 KDR在体外也具有抑 制血管内皮细胞的增生的作用 , 但在动物试验中其抗肿瘤效果不佳（Yoko Hasumi: Soluble FLT-1 Expres s ion Suppresses Carcinomatous Asci tes in Nude Mice Bear ing Ovar ian Cancer. Cancer Research 62, 2002: 2019-2023) 0
为克服现有技术中存在的缺陷， 本发明提供新的、 由来自 FLT- 1 和 DR的不同片段组成的融合蛋白， 以有效地阻断 VEGF生物作用、 抑制血 管新生。 发明内容
本发明目的之一是提供新的、 阻断 VEGF生物作用、 从而抑制血管新 生的重组融合蛋白。
本发明目的之二是提供编码所述融合蛋白的核苷酸序列。
本发明目的之三是提供含有编码所述融合蛋白的核苷酸序列的载体 和由该载体转化的重组体。
本发明的目的之四是提供该融合蛋白在制备阻断血管内皮细胞生长 因子生物作用、 抑制血管新生药物中的应用， 还提供含上述融合蛋白及 可药用载体的药物组合物及其剂型、 该药物组合物在治疗疾病上的应用。
本发明的关键在于， 根据 FLT- 1和 KDR的结构， 设计并构建了一系 列由不同的 FLT- 1 片段和 KDR片段构成的融合蛋白， 优选地， 该融合蛋 白还包括人免疫球蛋白 Fc (构建方式见图 1 )， 然后再用 VEGF结合试验 等方法筛选对 VEGF具有高亲和性的融合蛋白， 从而得到适用的 VEGF阻 断剂。 所述融合蛋白的构建技术基于常规的分子克隆方法， 具体实验步 骤如 <<分子克隆 >〉第二版和第三版（Joseph Sambrook, 科学出版社）及类 似的实验手册所记载。
按照本发明， 通过基因重组技术制备的融合蛋白， 由来自 VEGF受体 的 FLT-1和 KDR的不同片段构成， 其特征在于， 该融合蛋白选自以下组： a.是由 KDR的第 1免疫球蛋白样区域、 FLT- 1的第 2免疫球蛋白样区 域和 KDR 的第 3 免疫球蛋白样区域組成， 表示为： DRdl-FLTd2-KDRd3;
b.是由 FLT-1的第 1免疫球蛋白样区域和 KDR的第 3和第 4免疫球蛋 白样区域組成， 表示为： FLTd2-KDRd3， 4;
c.是由编码 FLT-1的第 2免疫球蛋白样区域、 KDR的第 3免疫球蛋白 样区域和 FLT-1 的第 4 免疫球蛋白样区域组成， 表示为： FLTd2-KDRd3-FLTd4;
d.是由 FLT- 1的第 1免疫球蛋白样区域和 KDR的第 3-5免疫球蛋白样 区域组成， 表示为： FLTd2- KDRd3，4，5;
e.是由 FLT-1的第 2免疫球蛋白样区域、 KDR的第 3免疫球蛋白样区 域和 FLT-1 的第 4-5 免疫球蛋白样区域組成， 表示为： FLTd2- DRd3-FLTd4) 5 ;
FLTd2氨基酸序列如 SEQ ID NO. 1所述， FLTd4的氨基酸序列如 SEQ ID NO. 2所述， KDRdl的氨基酸序列如 SEQ ID NO. 3所述， KDRd3的氨基酸序 列如 SEQ ID NO. 4所述， KDRd4的氨基酸序列如 SEQ ID NO. 5所述。
其中 FLT代表 FLT-1序列， KDR代表 KDR序列， di代表 FLT-1或 KDR 的第 i个免疫球蛋白样区域（doma in)。
按照本发明， 提供一种优选的融合蛋白， 其特征在于该融合蛋白还 包括人免疫球蛋白 FC, 该融合蛋白选自以下组：
FP2，表示为： KDRdl- FLTd2-KDRd3-Fc;
FP3，表示为： FLTd2- KDRd3, 4- Fc;
FP4，表示为： FLTd2-KDRd3- FLTd4- Fc;
FP5，表示为： FLTd2-KDRd3，4， 5- Fc;
FP6，表示为： FLTci2-KDR(i3-FLTd4, 5- Fc。
其中， Fc代表人免疫球蛋白 Fc片段，选自人免疫球蛋白 FC如 IgG、 IgM、 IgA或亚型 IgGl、 IgG2、 IgG3或 IgG4， 该免疫球蛋白 FC片段可 以为 FC全长或部分 FC序列， 如选自 CH2片断、 CH3片断或绞合区域片 段。 如附图 1 所示， 现有技术公开的融合蛋白（在此标记为 FP1，）是由 FLT-1的第二免疫球蛋白区域 (FLTd2)序列、 KDR的第三免疫球蛋白区域 (KDRd3)序列和人免疫球蛋白 Fc组成的。本发明提供的 FP2'融合蛋白是 在 FP1，的基础上增加了来自 KDR的第一免疫球蛋白样区域（KDRdl)的氨 基酸序列， 这些序列可以增加与 VEGF相结合的位点， 从而增加对 VEGF 的亲和力。 FP3，和 FP4，融合蛋白是分别在 FP1，的基础上增加了 KDR的第 四免疫球蛋白样区域的序列（KDRd4)或 FLT-1的第四免疫球蛋白样区域 的序列（FLTd4) 。 FP5，和 FP6，是在 FP1，的基础上分别增加了 KDR的第四 和第五免疫球蛋白样区域 (KDRd4 , 5)或 FLT- 1的第四和第五免疫球蛋白 样区域 (FLT-ld4 , 5)。这些新增的序列将更有利于融合蛋白之间的偶联， 从而进一步形成有利于和 VEGF相结合的空间结构，增加与 VEGF结合的 亲和力。
按照本发明，更优选地提供一种融合蛋白 FP3 ,其氨基酸序列如 SEQ ID NO. 7所述。
本发明的融合蛋白可通过常规的基因重组技术获得。 首先获得编码 上述融合蛋白的重组 其中， 编码 FLT-1和 KDR的 DNA序列记载在 NCBI (Nat iona l Center for Biotechno logy Informat ion)的 GenBank中。 然后将编码有上述融合蛋白的 DNA序列在用 PCR合成后分别被克隆到载 体中。 所用载体可以是分子生物学所常用的盾粒、 病毒或 DNA片段。 在 编码上述融合蛋白的 DNA序列的末端前加上蛋白分泌信号序列， 以保证 蛋白质从细胞中分泌出来。 载体序列中包括用于驱动基因表达的启动子， 蛋白质翻译起始和终止信号， 以及多聚腺苷酸 (PolyA)序列。 载体中有抗 菌素抗性基因， 以利于载体在宿主细胞， 如细菌中繁殖。 另外， 载体中 还包括真核细胞选择性基因， 用于稳定转染宿主细胞株的选择。
由于 FLT-1和 KDR中各个免疫球蛋白样区域的氨基酸序列之间并没 绝对的分界， 因此各免疫球蛋白样区域的氛基酸序列的长度可以有一定 的变化。 所以， 本发明所涉及的融合蛋白质的氨基酸序列也可以有一定 的变化， 它们都属于本发明的范围。
在完成含编码上述各种融合蛋白的 DNA序列的质粒构建以后， 即可 用该重组载体转染或转化宿主细胞， 表达相应的蛋白质。 能够用于表达 这些融合蛋白的表达系统有多种， 可以是真核细胞也可以是原核细胞， 它们包括（但不限于）哺乳动物细胞、 细菌、 酵母、 昆虫细胞等等。 从哺 乳动物细胞所表达的蛋白质具有糖基修饰。 由于本发明的融合蛋白质的 氨基酸序列中包括可糖基化的氨基酸， 因此， 哺乳动物细胞是表达这些 蛋白质的最佳细胞。 可用于蛋白质大规模表达的哺乳动物细胞有多种， 例如 293细胞、 CH0细胞、 SP20细胞、 NS0细胞、 COS细胞、 BHK细胞或 PerC6细胞等等。许多其他细胞也可用于这些蛋白盾的表达和生产， 因此 都包括在本发明所能使用的细胞之列。 含有编码上述融合蛋白的重组质 粒可经转染进入宿主细胞。 转染细胞的方法有多种， 其中包括但不限于： 电钻孔法（e l ec tropora t i on)、 脂质体介导法、 钙介导法等等。
除了哺乳动物细胞， 其他表达系统， 例如细菌、 酵母或昆虫细胞等 等也能用于表达这些融合蛋白， 它们也包括在本发明所能使用的细胞之 列。 这些表达系统的蛋白盾产量比哺乳动物细胞的为高， 但是， 所表达 的蛋白质缺乏糖基化或者所形成的糖链与哺乳动物细胞不同。
融合蛋白质表达后， 可用酶联免疫吸附试验 (ELISA)或其他方法测定 细胞培养液中融合蛋白质的浓度。 由于这些融合蛋白质具有免疫球蛋白 Fc片断， 因此可用蛋白 A亲和层析法提取所表达的融合蛋白质。
从重组体培养液中获得相应的各种融合蛋白盾。 然后利用 VEGF结合 试验来检测和比较各种蛋白质对 VEGF的亲和力。 进而， 利用 VEGF诱导 下的人血管内皮细胞分裂试验， 检测和比较各融合蛋白对 VEGF的阻断作 用。 实验结果证明， 与现有技术公开的 FP1，蛋白相比， 本发明所构建的 各种融合蛋白质对 VEGF具有高度的亲和力（见图 2)， 而且， 它们能有效 地阻断 VEGF对血管内皮细胞刺激、 抑制内皮细胞的分裂。 进一步的试验 发现， FP3对 VEGF的阻断效果最好，是本发明中阻断 VEGF最有效的融合 蛋白。
因此， 本发明所构建的融合蛋白对 VEGF具有很好的阻断作用， 都具 有抗血管新生的生物学特性， 从而可以用于治疗与血管新生或者 VEGF相 关的疾病，这些疾病可能包括但不限于各种肿瘤、 视网膜血管病变、 关节 炎、 贫血或子宫内膜增殖症等等。
本发明还提供了动物实验， 以进一步证明本发明提供的融合蛋白质 在活体内的抗血管新生作用， 实验证明， 对于 BALB/C裸鼠 B16F10黑色 素瘤和异种移植 (Xenograf t)模型人 PC- 3前列腺癌， 本发明提供的融合 蛋白都非常有效地抑制了肿瘤的生长， 延长了个体的生命， 而且效果明 显好于现有技术的 FP1，蛋白， 因此， 本发明所构建的融合蛋白具有高效 的抗癌能力。
本发明还提供了含有本发明融合蛋白和药用载体的药物组合物。 该 药物组合物可以按照制剂学常规技术制成各种形式的药物制剂 , 优选的 是注射剂， 最优选的是冷冻干燥注射剂。 附图说明
图 1 显示了本发明一较佳实施例中五种融合蛋白及现有技术的 FP1 蛋白的结构组成， 它们由不同的 FLT-1和 KDR片段以及免疫球蛋白 Fc片 段用基因工程方法构建而成。
图 2 显示了本发明一较佳实施例中，以现有技术的 FP1蛋白为对照， 本发明提供的五种融合蛋白与 VEGF结合的试险结果， 其中 0D读数代表 融合蛋白质与 VEGF的结合信号， 结果显示， 本发明提供的五种融合蛋白 与 VEGF具有艮强的结合能力， 而且都明显高于 FP1 , 尤其以 FP3的结合 能力最强。
图 3 显示了相对于 FP1 ,本发明的融合蛋白有效地抑制人血管内皮细 胞在体外的分裂。
图 4显示融合蛋白 FP3有效地抑制小鼠 B16F10黑色素瘤在体内的生 长。
图 5 显示融合蛋白 FP3有效地抑制人 PC-3前列腺癌在小鼠体内的生 长。
图 6 显示本发明的融合蛋白 FP3与现有技术的 FP1在有效地抑制小 鼠肿瘤的生长方面的比较研究。 具体实施方式
以下实施例对本发明所涉及的融合蛋白构建，试验和应用作了详细说 明。 但是本发明的内容及用途并不限制于实例的范围。 实施例 1 : 克隆编码融合蛋白的 DM序列及构建重组载体
除了编码免疫球蛋白 Fc的 DNA片断， 本发明编码各种融合蛋白的原 始 DNA序列来自 FLT- 1和 KDR相应的 cDNA。 由于 FLT-1和 KDR的表达主 要见于血管内皮细胞， 故本发明用 RNA提纯药盒（QIAGEN公司）从人脐带 静脉血管内皮细胞（HUVEC)提取了总 RNA; 然后用逆转录酶 (AMV Reverse Transcr iptese Pr omega)从 RNA合成 cDNA;再用不同的引物通过聚合酶 链反应 (PCR)扩增， 获得所需要的 FLT-1和 KDR片断； 最后用 PCR将来自 FLT- 1、 KDR和人免疫球蛋白 Fc (IgGl Fc)序列相融合， 从而构建成编码 不同融合蛋白的重组 DM序列。本优选实施例所构建的六种融合蛋白（包 括现有技术 FP1 ) 的结构见附图 1。 例 1 构建 FP3基因及重组载体
用 EGM-2培养基 ( Clonet ics )在 T - 175培养瓶中培养人脐带静脉 血管内皮细胞（HUVEC细胞） ( Clonet ics ), 收集大约 1 x 10e7个细胞， 利 用 Qiagen公司的 RNA提取试剂盒提取细胞总 RNA,并用 Invi trogen cDNA 试剂盒合成 cDNA, -80°C冻存备用。 采用下列特异引物从 HUVEC cDNA 中 扩增得到 FLT - 1和 KDR基因片段。
用人 IgGl Fc 特异引物从来自淋巴结（BD Clcmtech)的 cDNA中扩增 到 IgGl Fc 基因片段。
引物为：
FLT-1 d2正向引物： 5'-cctttcgtagagatgtacagtga-3'
FLT-1 d2反向引物： 5'-tatga t tgta t tggt t tgtccat-3'
KDR (13-4正向引物： 5'-gatgtggt tctgagtccgtctca-3'
KDR d3— 4反向引物： 5 '-cggtgggacatacacaaccaga-3' 人 IgGl Fc正向引物： 5'-gacaaaactcacacatgcccact-3' 人 IgGl Fc反向引物： 5'-tcat t tacccggagacagggagag-3' 在变性 95 °C , 30分； 退火 56 °C , 45秒； 延伸 72 °C , 2分钟的条件 下，进行 PCR扩增， 30个循环，获得 FLT-1和 KDR的 IgG样结构域的 PCR 产物及人 IgGl Fc片段的 PCR产物。 用 TA cloning 试剂盒， 把 PCR产 物克隆入 pCR2. 1质粒（ Invi trogen ), 并转染 Ε· col i ( JM109 ), 选取白 色菌落， 加入 LB培养基， 培养过夜。 Qiangen质粒提取试剂盒提取质粒 后酶切， 及测序鉴定。
采用拼接 PCR (sewing PCR)方法， 把 FLT-1、 KDR和 IgG Fc的 cDNA 连接在一起， 在引物中设计有 EcoRI 的酶切位点， 用 EcoRI 酶切后， Q iangen纯化试剂盒纯化 DNA片段并插入 pcDNA3. 1质粒， 将重组质粒转 染 E. co l i ( JM109 ), 选取阳性菌落， 加入 LB培养基， 培养过夜。 Qiagen 质粒提取试剂盒提取质粒后进行酶切及测序鉴定，所获得的编码 FP3 的 DNA序列如 SEQ ID NO. 6所述。 将已获证实的质粒再转染 293 或 CH0细 胞获得稳定表达 FP 3的细胞系。 FP3的具体氨基酸序列如序列表 SEQ ID NO. 7所述。 例 2 构¾ ??1基因及重組载体
除了所要获得的目的重组 DNA是由编码 FLT-1的第 1免疫球蛋白样 区域和 KDR的第 3免疫球蛋白样区域的基因、以及与例 1中相同的人 IgGl Fc片段拼接而成之外， 具体操作步骤同例 1。 例 3 构建 FP2基因及重组载体
除了所要获得的目的重组 DNA是由编码 KDR的第 1免疫球蛋白样区 域、 FLT- 1 的第 2免疫球蛋白样区域和 KDR的第 3免疫球蛋白样区的基 因、 以及与例 1 中相同的人 IgGl Fc片段拼接而成之外， 具体操作步驟 同例 1。 例 4 构建 FP4基因及重组载体
除了所要获得的目的重组 DM是由编码 FLT-1的第 2免疫球蛋白样 区域、 KDR的第 3免疫球蛋白样区域和 FLT- 1的第 4免疫球蛋白样区域的 基因、 以及与例 1 中相同的人 IgGl Fc片段拼接而成之外， 具体操作步 骤同例 1。 例 5 构建 FP5基因及重组载体
除了所要获得的目的重组 DNA是由编码 FLT-1的第 2免疫球蛋白样 区域和 KDR的第 3-5免疫球蛋白样区域的基因、 以及与例 1中相同的人 IgGl Fc片段拼接而成之外， 具体操作步骤同例 1。 例 6 构建 FP6基因及重组载体
除了所要获得的目的重组 DNA是由编码 FLT- 1的第 1免疫球蛋白样 区域、 KDR的第 3免疫球蛋白样区域和 FLT-1的第 4-5免疫球蛋白样区域 的基因、 以及与例 1 中相同的人 IgGl Fc片段拼接而成之外， 具体操作 步驟同例 1。 实施例 2 融合蛋白在细胞中的表达
例 7 融合蛋白 FP3的表达
在完成上述重組质粒的构建以后，用质粒 DNA提纯药盒（QIAGEN公司） 提取高纯度质粒 DNA。 然后， 利用 FUGEN6质粒转染药盒 (ROCHE公司）将 该重组质粒 DNA导入 293细胞（ATCC ) 中。 按所需蛋白质量的多少， 采 用了两种不同的质粒转染的方法表达融合蛋白。
第一种方法是瞬时转染法（Trans ient transfect ion) , 用这一方法 可以得到小规模量的融合蛋白。 首先用含 10%胎牛血清的 DMEM完全培养 基将 293细胞在细胞培 内培养。 当细胞生长至覆盖 60-80°/。面积时， 将重组质粒 DM与 FUGEN6试剂的复合物加入细胞培养液中。 第二天， 用 无血清 DMEM培养基换液。 再继续培养三天， 然后收集上清液。 这些上清 液中含有从转染后的 293细胞表达的融合蛋白。融合蛋白的浓度用 ELISA 法定量确定。
第二种方法是用稳定转染法（Stable transfect ion)建立稳定细胞， 以表达大量的融合蛋白多肽。 所用细胞亦是 293细胞（ATCC )。 该重組质 粒的转染方法与上述瞬时转染法相同， 但是， 在转染第二天， 转染后的 293细胞在含有新酶素的匪 EM完全培养中， 用有限密度稀释法进行克隆 培养。 大约 21天后， 挑取新酶素抗性克隆， 进行细胞的扩大培养。 最后， 转染后的 293 细胞在转鼓培养瓶中培养生产融合蛋白。 融合蛋白的浓度 用 ELISA法定量确定。
获得的培养液采用亲和层析、 凝胶过滤等方法纯化得到 FP3，其分子 量为 140kDo 例 8 其他融合蛋白的表达
采用与上述例 7相同的方法制备相应的融合蛋白 FP1、FP2、FP4、FP5T
FP6。 实施例 3 融合蛋白与 VEGF的结合实验
本发明用 VEGF结合试验测定了各融合蛋白与 VEGF的结合能力。 在 这一试猃中， 首先将重组 VEGF蛋白（Chemicom公司）包被在 96孔 ELISA 板上。 然后用百分之五的牛奶粉溶液阻断非特异性蛋白结合位点。 再向 各孔加入含有不同浓度的各种融合蛋白质， 在 37°C培养两个小时。 清洗 以后， 加入兔抗人 I g抗体 -HRP ( Sigma )。 最终用过氧化物酶底物显色。 用 ELISA阅读仪测量 96孔板各孔的 0D值。 0D值越高代表融合蛋白质与 VEGF 的结合信号越强。
如图 2所示， 本发明所构建和表达的五种融合蛋白都具有与 VEGF结 合的能力， 而且与现有技术的 FP1相比具有更好的结合能力。在每毫升 1 毫微克的浓度，便能检测到它们与 VEGF的结合信号。优选地， FP3与 VEGF 的结合能力最大， 是其中最强大的 VEGF 阻断剂， 其半数最大结合浓度 (Half maximal binding concentrat ion)比 FP1低约 5倍。 FP5 与 VEGF 的结合能力比 FP3稍弱。这一结杲说明， KDR的第四免疫球蛋白样区域的 氨基酸序列可增进融合蛋白对 VEGF的阻断能力。 但是， 在融合蛋白中进 一步增加 KDR的序列， 例如第五免疫球蛋白样区域， 并不进一步增加对 VEGF的抑制能力。 其他三个融合蛋白对 VEGF的抑制能力比 FP3和 FP5 为弱， 但比 FP1强。 实施例 4 融合蛋白有效地抑制人血管内皮细胞的在体外的分裂 本发明另一关键实施例是证明了所构建的融合蛋白能有效地阻断 VEGF所诱导的血管内皮细胞的分裂。 在这一实验中， 将脐带静脉血管内 皮细胞（HUVEC细胞， CL0NETICS公司）接种于 96孔细胞培养板。 细胞培 养液为 EBM基本培养基. (CL0NETICS 公司）， 含 2%胎牛血清和 15ng/ml VEGF。 在实验组的细胞培养液中， 加入含不同浓度融合蛋白的 293 细胞 上清液。 在阴性对照组的细胞培养液中， 加入未经重组质粒转染的 293 细胞上清液（不含融合蛋白）。 将经过不同处理的 HUVEC细胞在 37 °C继续 培养。 三天后， 运用细胞计数确定各孔中的 HUVEC密度。
HUVEC细胞分裂试验显示， 与现有技术的 FP1相比， 本发明所构建的 五种融合蛋白质均能更有效地抑制血管内皮细胞的分裂（如图 3所示）。 鉴于在这个实验中 HUVEC细胞的分裂由 VEGF刺激造成， 因此， 这五种蛋 白质都能有效的阻断 VEGF对其受体的刺激作用， 都有抑制血管新生的功 能， 其中， 以 FP3对 HUVEC细胞分裂的阻抑作用最强， 它们的半数抑制 浓度 (IC50) 在 3 ng/ral左右。 现有技术 FP1的 IC50为 12 ng/ml左右。 FP2、 FP4、 FP5和 FP6的 IC50在 5— 8 ng/ml。 实施例 5 融合多肽能有效地抑制小鼠肿瘤的生长 例 9 制备含融合蛋白的注射剂
注射剂的制备方法为： 将 24mg/ml 量的 FP3、 5Mm PB、 l OOMm NaCl 和 20%的蔗糖， 按照注射剂的常规制备方法制成。 例 10 融合蛋白能有效地抑制小鼠 B16F10黑色素瘤的生长 作为 VEGF阻断剂， 本发明所构建的融合蛋白的应用之一是用于肿瘤 的治疗。 鉴于 FP3融合蛋白对 VEGF有高效的阻断作用， 本发明选择 FP3 进亍了动物体内抗肿瘤效果的 H
本发明所试验的小鼠肿瘤模型是 B16F10黑色素瘤。 这是一种快速生 长的恶性肿瘤。 在这个试验中， 先将 B16F10细胞 1 X 105, 0. 05ml , 注入 BALB/C棵鼠背部皮下。 经尾静脉注射提纯的融合蛋白质。 注射剂量为每 小鼠每次 400微克（小鼠平均体重约 22克）， 每周两次。 对照组注射同剂 量的提纯人免疫球蛋白 Fc。图 4显示肿瘤的生长曲线，表明融合蛋白 FP3 非常有效地抑制了该黑色素瘤的生长（P<0. 01) 。 例 11 融合蛋白能有效地抑制小鼠异种移植前列腺癌 PC-3细胞的生 长
让人类肿瘤细胞在棵鼠体内生长的异种移植（Xenograf t)模型是与 人体肿瘤最为接近的动物肿瘤模型。 棵鼠缺乏免疫排斥能力， 因此， 许 多来自人的肿瘤细胞系能在棵鼠体内生长， 形成肿瘤。 本发明试验了融 合蛋白 FP3对人前列腺癌 PC- 3细胞（ ATCC )在 BALB/C棵鼠体内生长的 抑制作用。在这一模型中，首先将 PC- 3细胞 1 105, 0. 05ml ,注入 BALB/C 棵鼠背部皮下， 从尾静脉注射提纯的融合蛋白。 每个小鼠每次 400微克， 每周两次。 对照组注射相同剂量的人免疫球蛋白 Fc。 实验结果如图 5所 示。 在对照组， 肿瘤细胞接种后 45天， 肿瘤已长至大于 1000立方毫米。 而在用融合蛋白盾注射的动物， 融合蛋白 FP3几乎完全抑制了 PC- 3肿瘤 的生长（P<0. 01) , 对肿瘤具有非常^著的治疗作用。 实施例 6 融合蛋白 FP3与现有技术 FP1蛋白在有效地抑制小鼠肿瘤 的生长方面的比较研究
为更好地说明 FP3具有较好的肿瘤抑制作用， 本实施例选用了 FP1 同 FP 3做抑制肿瘤生长的比较试验。 选用生长良好的 BALB/C棵鼠， 每只 背部注射鼠神经胶盾瘤 C6细胞 Ι χ Ι Ο5, 0. 05ml , 在尾静脉分别注射提纯 的融合蛋白 FP1和 FP3 , 用量为 2. 5mg/kg，每周两次， 到 31天。 对照组 注射相同剂量的人免疫球蛋白 Fc。 实验结果如图 6所示， FP1和 FP3对 肿瘤具有非常显著的治疗作用， 到第 35天， FP1组肿瘤体积是 1167. 3, 而 FP3组是 557. 6, 对照组再第 24天已到 1312. 3。 上述数据表明， 本发 明的 FP 3蛋白的作用更明显高于现有技术的 FP1蛋白 （P<0. 05. )0
综上所述， 本发明所构建的融合蛋白质对 VEGF具有高度的亲和性， 能在体外抑制血管内皮细胞的增生， 并能在体内非常显著地抑制肿瘤的 生长。 鉴于血管新生为所有肿瘤增生之必需， 本发明所涉及的融合蛋白 质可用于多种肿瘤的治疗。

Claims

权 利 要 求
1.一种融合蛋白质， 由来自 VEGF受体的 FLT- 1和 KDR的不同片段构 成， 其特征在于， 该融合蛋白选自以下组：
a .是由 KDR的第 1免疫球蛋白样区域、 FLT-1的第 2免疫球蛋白样区 域和 KDR 的第 3 免疫球蛋白样区域组成， 表示为： DRdl-FLTd2- DRd3;
b.是由 FLT-1的第 2免疫球蛋白样区域和 KDR的第 3和第 4免疫球蛋 白样区域組成， 表示为： FLTd2-KDRd3, 4;
c.是由编码 FLT- 1的第 2免疫球蛋白样区域、 KDR的第 3免疫球蛋白 样区域和 FLT-1 的第 4 免疫球蛋白样区域组成， 表示为： FLTd2-KDRd3-FLTd4;
d.是由 FLT-1的第 1免疫球蛋白样区域和 KDR的第 3-5免疫球蛋白样 区域组成， 表示为: FLTd2-KDRd3, 4, 5 ;
e.是由 FLT-1的第 2免疫球蛋白样区域、 KDR的第 3免疫球蛋白样区 域和 FLT-1 的第 4-5 免疫球蛋白样区域组成， 表示为： FLTd2-KDRd3-FLTd4, 5。
2.—种融合蛋白质， 由来自权利要求 1 所述的融合蛋白与人免疫球 蛋白 FC构成， 该融合蛋白选自以下组：
FP2，表示为： KDRcU- FLTd2-KDRd3-Fc;
FP3，表示为： FLTd2- KDRd3， 4-Fc;
FP4，表示为： FLTd2-0)Rd3- FLTd4- Fc;
FP5，表示为： FLTd2- KDRd3， 4， 5-Fc;
FP6，表示为： FLTd2- KDRd3 - FLTd4， 5-Fc。
3.如权利要求 2所述的融合蛋白^ , 其中该免疫球蛋白 FC来自人免 疫球蛋白型或 型。
4.如权利要求 3所述的融合蛋白质， 其中该免疫球蛋白 FC片段是编 码 FC全长序列或部分 FC序列。
5.如权利要求 2所述的融合蛋白质 FP3 ,其氨基酸序列如 SEQ ID NO. 7 所述。
6.编码权利要求 1-4的融合蛋白质的重组 DNA。
7.编码权利要求 5所述融合蛋白质的重组 DNA, 其核苷酸序列如 SEQ ID NO. 6所述。
8.包含权利要求 6或 7所述重组 DNA的重组载体， 该载体选自质粒、 病毒或 DM片段。
9.包含权利要求 8 所述重组载体的重组体， 其中宿主细胞是真核细 胞或原核细胞。
10.如权利要求 1-5中任一项所迷融合蛋白质在制备抑制血管新生药 物中的用途。
11.一种药物組合物， 包括如权利要求 1-5中任一项所述融合蛋白质 及药学上可接受的载体。
12.如权利要求 11 所述的药物組合物， 其特征在于该药物组合物为 注射剂。
13.如权利要求 11所述的药物组合物在治疗血管新生疾病中的应用， 该疾病包括肿瘤、 糖尿病视网膜病变、 关节炎、 贫血或子宫内膜增殖症。
PCT/CN2005/000802 2004-06-08 2005-06-08 Proteine chimerique inhibitrice d'angiogenese et utilisation associee WO2005121176A1 (fr)

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WO2018229034A1 (en) 2017-06-14 2018-12-20 Bayer Pharma Aktiengesellschaft Non-antibody vegf antagonists for the treatment of neovascular glaucoma
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WO2019217927A1 (en) 2018-05-10 2019-11-14 Regeneron Pharmaceuticals, Inc. High concentration vegf receptor fusion protein containing formulations
WO2021108530A1 (en) * 2019-11-26 2021-06-03 University Of Massachusetts Recombinant adeno-associated virus for delivery of kh902 (conbercept) and uses thereof
WO2021119544A1 (en) 2019-12-12 2021-06-17 Novartis Ag Injection device and injection solution transferring system
US12103960B2 (en) 2020-05-08 2024-10-01 Regeneron Pharmaceuticals, Inc. VEGF traps and mini-traps and methods for treating ocular disorders and cancer
WO2022013787A1 (en) 2020-07-16 2022-01-20 Novartis Ag Anti-betacellulin antibodies, fragments thereof, and multi-specific binding molecules
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