WO2005007701A1 - Proteine de fusion intensifiee fv-lpd-ae ayant une action antitumorale et d'inhibition de l'angiogenese et son utilisation - Google Patents

Proteine de fusion intensifiee fv-lpd-ae ayant une action antitumorale et d'inhibition de l'angiogenese et son utilisation Download PDF

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WO2005007701A1
WO2005007701A1 PCT/CN2004/000842 CN2004000842W WO2005007701A1 WO 2005007701 A1 WO2005007701 A1 WO 2005007701A1 CN 2004000842 W CN2004000842 W CN 2004000842W WO 2005007701 A1 WO2005007701 A1 WO 2005007701A1
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ldp
fusion protein
cancer
tumor
ldm
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PCT/CN2004/000842
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Chinese (zh)
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Liang Li
Yongsu Zhen
Qingfang Miao
Boyang Shang
Xiujun Liu
Min Jiang
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Institute Of Medicinal Biotechnology, Chinese Academy Of Medical Sciences
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/515—Angiogenesic factors; Angiogenin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the invention relates to a novel antibody-directed drug with an anti-angiogenic effect, strong killing of tumor cell activity and anti-tumor treatment effect, and strengthens a fusion protein.
  • Matrix metalloproteinases play an important role in the invasion and metastasis of cancer cells, especially type IV collagenases MMP-2 and MMP-9 can degrade the extracellular base shield components such as type IV collagen and destroy the integrity of the basement membrane and extracellular matrix It is helpful for tumor cell invasion and metastasis, and its expression in tumor neovascular endothelial cells is higher than that in normal tissue vascular endothelial cells. Inhibition of matrix metalloproteinase activity can inhibit tumor cell invasion and metastasis and tumor angiogenesis.
  • matrix metalloproteinase MMP-2 / MMP 9-monoclonal antibody as a drug carrier guide may be provided not only targeted tumor, and which itself has an anti-anti-tumor effect ⁇ present invention
  • ⁇ -2 / ⁇ Li P-9 mAb 3G11 is immunologically positive for a variety of tumor cells and has specific binding ability to a variety of human tumor tissues 9 especially digestive tract tumor tissues
  • Single-chain antibody is typically a length of flexible peptide chain connecting the V H and the minimum antibody fragment having functional complete antigen binding site thereof.
  • the scFv is more stable than the B fragment, and the scFv molecule has a better ability to penetrate solid tumors than intact antibodies, so it is more suitable as a carrier for monoclonal antibody-directed drugs.
  • LDM highly active "warhead” drug Lidamycin
  • C-1027 or C1027 The highly active "warhead” drug Lidamycin (hereinafter referred to as LDM, also known as C-1027 or C1027) is isolated from the soil of Qianjiang County, Hubei province, China. ⁇ iw ⁇ cesg / s wras, the strain deposit number is: CGMCC No. 0135)
  • the enediyne antibiotics are the killing tumor cells reported to date.
  • LDM has a very significant effect on colon cancer 26 in mice, and it has significant effects on human transplanted tumors such as human liver cancer Bel-7402 and cecum cancer Hce-8693 transplanted in mice (Chinese Journal of Antibiotics 1994 , 19 ⁇ 2>: 164-168 ⁇
  • the molecule of LDM is composed of two parts: one is a chromophore with an enediyne structure, which has a cytotoxic effect, but is unstable; the other is a prosthetic protein (LDP) composed of 110 amino acid residues.
  • LDP prosthetic protein
  • the stability of the chromophore is protective.
  • the chromophore in the LDM molecule exists in two forms: active chromophore (ie active enediyne, AE) and inactivated chromophore.
  • AE active enediyne
  • the structure is extremely unstable, and almost all are inactivated after about 1 hour, that is, AE undergoes an irreversible aromatization reaction and is converted into a structurally stable inactivated chromophore.
  • AE is the active part of LDM, and the only co-protein is To protect the function of AE, the level of AE content in the LDM molecule determines the strength of its effect. Therefore, in order for LDM to have high-strength biological activity, the high percentage of AE in the chromophore of LDM must be guaranteed.
  • the invention is based on the newly constructed fusion protein Fv-LDP, and strictly controls the quality of the chromophore used.
  • the active enediyne chromophore (AE) is used to perform molecular strengthening to obtain a strengthened fusion protein Fv. -LDP-AE, as a new type of antibody-targeting drug with better anti-tumor activity. Summary of the invention
  • an immuno-directed fusion protein prepared by using single-chain antibody scFv constructed with genetic engineering technology and lidamycin can effectively direct an effector molecule to a specific tumor target site, compared with an immunocouple obtained by chemical coupling technology.
  • the complex has the advantages of molecular uniformity and efficient miniaturization.
  • One aspect of the present invention relates to the enhanced fusion protein Fv-LDP-AE, which is a single-chain antibody scFv (3G11) against matrix metalloproteinase MMP-2 / MMP-9, a lipamycin co-protein LDP, and two The fusion protein Fv-LDP formed by the flexible peptide spacer (GGGGS) and the histidine hexamer tail (His 6 -tag) at the carboxyl group, and the daptomycin-active diene acetylene chromophore AE (molecular weight 843 kDa).
  • GGGGS flexible peptide spacer
  • His 6 -tag histidine hexamer tail
  • daptomycin-active diene acetylene chromophore AE molecular weight 843 kDa
  • the full-length encoding gene of the fusion protein Fv-LDP according to the present invention is 1119 bp (as shown in SEQ ID NO: 1), encodes 372 amino acids (as shown in SEQ ID NO: 2), and has a molecular weight of approximately 38.7 kDa. .
  • the monoclonal antibody scFv (3G11) of the present invention is derived from mouse anti-type IV collagenase (including 92 kDa and 72 kDa) hybridoma cell line 3D6, and the assembled fusion protein LDM-Fv (Acta Pharmica Sinica 2000, 35 ⁇ 7>: 488-491) and the derived scFv-M97 monoclonal antibody anti-mouse type IV collagenase (92 kDa) of the hybridoma cell lines of both C2H5 0 scFv gene sequences by homologous than the current 91 %,
  • the full-length 741 eFv (3G11) encoding gene of the present invention encodes 741, and the full-length 732 bp gene of the seFv-M97 encoding gene; especially the single-chain antibody ⁇ FY (3G11) and scFv-M97 of the present invention Nucleotide sequences of two in the CDR2 region
  • scFv (3G11) of the present invention is significantly different from scFv-M97 in specific antigen recognition and antigen-antibody binding characteristics. difference.
  • the parental monoclonal antibody 3G11 of scFv (3G11) in the present invention has high immunocompatibility with its antigen MMP-2 / 9, and it has been confirmed by Western-blotting analysis; moreover, it is confirmed by experiments that it is derived from the hybridoma cell line 3D6 MAb 3G11 shows immunological activity in a variety of human tumor tissues (Li Liang, Liu Xiujun, Zhen Yongsu. Antibodies Study on the tumor targeting distribution of type IV collagenase mAb and its antitumor effect.
  • the newly constructed fusion protein Fv-LDP of the present invention inserts a small stretch of flexible peptide spacer (GGGGS) between the single chain antibody scFv (3G11) and lidamycin co-protein LDP, so that scFv and LDP can be correctly folded, respectively. Form their original spatial conformations so as not to affect their respective biological activities.
  • GGGGS flexible peptide spacer
  • the molecular weight of LDM is known to be 11349.1120 Da.
  • the molecular weight of the co-protein LDP is 10505.7830 Da, and the molecular weight of the chromophore is 843,3295 Da.
  • the chemical structure and molecular formula of the active and inactive chromophores of LDM are as follows:
  • Another aspect of the present invention relates to the preparation of a strengthened fusion protein Fv-LDP-AE.
  • the quality of the chromophore used is strictly controlled, and the active enediyne chromophore (AE) with strong activity is used to perform molecular strengthening to obtain strengthening.
  • the fusion protein Fv-LDP-AE is a novel antibody-directed drug with better antitumor effect.
  • the fusion protein utilizes the binding specificity of an anti-MMP-2 / 9 single-chain antibody scFv (3G11) to target a highly effective adamycin chromophore (AE) to a tumor tissue site where an antigen is highly expressed, and exerts its effect on tumor cells.
  • the high cytotoxic effect shows strong angiogenesis inhibitory activity, and has significant effects in in vivo animal experiments, showing a good application prospect.
  • the preparation technology route includes: For the preparation method of the LDM used in the present invention, refer to the Chinese patent (patent application number: 00121527.2) filed with the Chinese Patent Office on August 10, 2000 and granted in October 2003.
  • the activity of LDM products can be determined by measuring the relative content of AE in the total chromophore.
  • HPLC analysis is usually used to determine the percentage of AE in the total chromophore in LDM products.
  • AE is greater than or equal to 80% of the total chromophore as the quality standard for qualified LDM products.
  • the molecular strengthening is performed by the following steps: Fv-LDP / 0.01 M PBS (pH 7.0) and AE methanol solution are mixed and stirred at a molecular ratio of 1: 5 and a volume ratio of 1:50. Reaction at room temperature for 12 h to obtain the enhanced fusion protein Fv-LDP-AE 0
  • an LDM qualified product with an AE percentage value greater than 80% is preferably used, and LM with an AE percentage value greater than the nutrient% is preferably used for molecular strengthening to prepare a reinforced fusion protein ⁇ .
  • AE in high-quality LDM The percentage value can be up to 90.63%.
  • the chromophore required to prepare the assembled fusion protein LDM-Fv in this laboratory was extracted from the low-activity LDM obtained by the old preparation method, of which the proportion of AE was only 60%, and a stronger tumor could not be obtained.
  • Product of cellular cytotoxic activity was extracted from the low-activity LDM obtained by the old preparation method, of which the proportion of AE was only 60%, and a stronger tumor could not be obtained.
  • the cytotoxic activity of the assembled fusion protein LDM-Fv was determined by an in vitro clone formation method, and its IC 5 () value was 9.5 ⁇ 10 15 M, which was higher than the IC 50 value of the enhanced fusion protein Fv-LDP-AE according to the present invention (1.65 ⁇ 10 16 M) 58 times lower; no immune binding activity was observed in human colon cancer tissues, no strong angiogenesis inhibitory effect was seen, and its efficacy was not demonstrated in animal experiments.
  • Another aspect of the present invention relates to the use of the enhanced fusion protein Fv-LDP-AE for the preparation and treatment of human malignant tumors, such as colon cancer, rectal cancer, esophageal cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, lung cancer and kidney cancer
  • human malignant tumors such as colon cancer, rectal cancer, esophageal cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, lung cancer and kidney cancer
  • Yet another aspect of the present invention relates to a pharmaceutical composition containing a therapeutically effective amount of the fortified fusion protein Fv-LDP-AE of the present invention.
  • the pharmaceutical composition further comprises administration with the pharmaceutical composition.
  • Modes and dosage forms are compatible with pharmaceutically acceptable carriers and excipients.
  • Yet another aspect of the present invention relates to a method for treating a malignant tumor, which comprises administering a therapeutically effective amount of a fortified fusion protein or a pharmaceutical composition of the present invention to a patient with a malignant tumor.
  • the research of the present invention proves that the enhanced fusion protein is selectively distributed in human colon cancer tissues with high expression of type IV collagenase, shows strong killing activity on tumor cells, and has a high inhibitory effect on angiogenesis. Animal experiments have very significant treatment effect. After a search, there have been no reports of similar enhanced fusion proteins at home and abroad, and it is currently known as the smallest molecular weight immune-directed fusion protein that has proven to have significant anti-cancer efficacy in vivo. BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure 1 PCR amplification of scFv and LDP genes and restriction endonuclease analysis of recombinant cloning vectors. Among them: 1- DNA molecular weight standard; 2- scFv PCR product; 3- LDP PCR product; 4- Recombinant plasmid pGEM-T-scFv / Nde l + EcoR I; 5- Recombinant shield particle pGEM-T-LDP / EcoR I + Xho I.
  • Figure 2 Restriction endonuclease analysis of recombinant pEFL in the transformed strain CAMS / FLDFP.
  • 1- DNA molecular weight standard 2-plasmid pET-30a (+); 3-recombinant plasmid pEFL; 4- pET-30a (+) / Nde I + EcoR I; 5- pEFL / Nde I + EcoR I; 6 -pET-30a (+) / EcoR I + Xho I; 7- pEFL / EcoR I + Xho I; 8- pET-30a (+) flat e I + Xho I; 9- pEFL / Nde I + Xho l
  • FIG. 3 SDS-PAGE analysis of the fusion protein Fv-LDP expression product. among them:
  • FIG. 4 Western-blot analysis of the fusion protein Fv-LDP.
  • A Lidamycin co-protein monoclonal antibody F9 as the primary antibody
  • B Antihistidine-labeled tail monoclonal antibody as the primary antibody
  • Figure 5 SDS-PAGE analysis of fusion protein purified by metal chelation chromatography. Among them: 1-protein molecular weight standard; 2 3 ⁇ 4 5- sample before affinity column was not loaded ; 4- liquid collected after column washing with binding buffer; 5- liquid collected after column washing with 20 mM imidazole washing buffer 6-10- Protein fractions collected every 1 h after washing the column with 1 M imidazole elution buffer.
  • Figure 6 Isolation and purification of the enhanced fusion protein Fv-LDP-AE.
  • Analytical column PD-10; Mobile phase: PBS, pH 7.0; Peak 1- Purified fortified fusion protein Fv-LDP-AE; Peak 2- Unreacted excess AE.
  • Figure 7 ELISA analysis of the immunoreactivity of the fusion protein Fv-LDP with type IV collagenase and different tumor cells. Among them: AIV collagenase; ⁇ HT-29 cells; ⁇ HT-1080 cells.
  • Figure 8 Immunohistochemical analysis of the immunological activity of the fusion protein Fv-LDP with human colon cancer tissues. Among them: A: immunohistochemical staining of Fv-LDP in human colon cancer tissue; B: negative control, PBS instead of Fv-LDP as the primary antibody; magnification is 200 times, the scale in the figure is 20 j3 ⁇ 4m.
  • Figure 9 Analysis of type IV collagenase enzyme i of HT-1080 cells by fusion protein Fv-LDP.
  • 1-PBS 2-empty vector obtained by IPTG induction of whole bacterial protein
  • 3-complete monoclonal antibody 3G11 (6 ⁇ )
  • 4-fusion protein Fv-LDP (30 fM)
  • Figure 10 Inhibition of enhanced fusion protein Fv-LDP-AE on bFGF-stimulated angiogenesis in chicken embryos.
  • A chicken embryo urinary diaphragm blood vessels treated with PBS only
  • B bFGF as stimulant, chicken embryo urinary diaphragm blood vessels treated with PBS
  • C bFGF as stimulant, LDM (0.1 / ig / chicken (Embryo) treated chicken embryo with urinary membrane blood vessels
  • D bvGF as stimulant, Fv-LDP-AE (0.4] 3 ⁇ 4g / chicken embryo) treated chicken embryo urine diaphragm blood vessel.
  • Figure 11 Enhanced killing activity of the fusion protein Fv-LDP-AE on tumor cells HT-29.
  • Fv-LDP-AE fusion protein Fv-LDP-AE
  • LDM fusion protein
  • Figure 12 The inhibitory effect of the enhanced fusion protein Fv-LDP-AE on the growth of liver cancer H22 in mice: ⁇ Control; ⁇ Fv-LDP group; National LDM group; ⁇ Fv-LDP-AE3, 2 group; ⁇ Fv-IJDP -AE1,6 groups; ⁇ Fv-LDP-AEi group
  • Figure 14 Mass spectrometric analysis of lidamycin active enediyne chromophore AE.
  • Test instrument Quattro LC tandem mass spectrometer; equipped with an electrospray ion source (UK MICROMASS company); the sample was dissolved in 50% acetonitrile at a concentration of 0.5mg / ml, and injected directly into the electrospray ion source for mass spectrometry through a syringe pump analysis.
  • Recombinant plasmids pCANscFv or PIC-9kFvl027 and pIJ1027GRGDS contain scFv gene and LDP gene, respectively, which are stored in our laboratory.
  • the GEM-T vector is a product of the American Promega company.
  • the E. coli strain R coli DH5a was stored in this laboratory.
  • the PCR primers were synthesized by Cyperion, and the corresponding restriction enzymes (TaKaRa products) restriction sites were introduced.
  • end primer (PL2, SEQ ID NO: 4):
  • the recombinant plasmid pCANscFv or pKFvl027 was used as a template, PHI was 5, primers, PL2 was 3, and the primers were PCR amplified to obtain a single-chain antibody scFv gene fragment with a small peptide spacer at the C-terminus; and the recombinant plasmid IJ1027GRGDS was used as a template , PLD1 is 5, primers, PLD2 is 3, primers are PCR amplified to obtain LDP gene fragments.
  • the PCR reaction system was pre-denatured at 94 ° C for 2 minutes, and then subjected to 25 PCR cycles: Denaturation at 94 ° C for 1 minute, 55 ° C (scFv gene amplification) or 58 ° C (LDP gene amplification) annealing for 1 minute, 72 ° C extension for 1 minute, and 72 after the last cycle. C was held for 10 minutes.
  • the two PCR products were purified and recovered using DNA fragment glass milk recovery kit (product of BioDev), and were connected to Promage's pGEM-T vector according to the method provided by Promage's kit to transform E. coli DH5, and the recombinant T vector pGEM- was selected.
  • T-Fv and pGEM-T-LDP were identified by enzymatic digestion ( Figure 1), and sequenced by Shanghai Bio-Biotech, respectively.
  • the scFv (3G11) gene was 741 bp in length, encoding 247 acids, and the LDP gene was 342 bp in length.
  • the two fragments were cloned into the expression vector pET30a (+) digested with Nde I and Xho I, and transformed into competent cells of the host strain BL21 (DE3) star TM (product of Invitrogen). Extract the recombinant plasmid.
  • the above transformants were picked from the LB plate and inoculated into an LB medium containing 50 ⁇ m of kanamycin, and shaken at 37 ° C overnight; the next day, the seed was transfected at 1:50, and cultured at 37 ⁇ with shaking to OD 6 . .
  • IPTG isopropylthio-3-D-galactoside
  • One of these strains contained a pEFL plasmid capable of expressing the fusion protein Fv-LDP, named CAMS / FLDFP, and was deposited on June 24, 2003 at the General Microbiology Center of the China Microbial Species Collection Management Committee in Beijing. . 0961
  • urea lx binding buffer (20 mM Tris-HCl, 0.5M NaCl, 5 mM imidazole, 6M urea, H 7.9)
  • 6 volumes of urea lx washing buffer 20mM Tris-HCl, 0.5M NaCl, 60 mM imidazole, 6M urea, pH 7.9
  • 1x elution buffer containing 6M urea (20 mM Tris-HCl, 0.5M aCI, 1 M Imidazole, 6M urea, pH 7.9) was eluted, and the eluted fractions were collected to obtain a purified fusion protein Fv-LDP ( Figure 5).
  • the protein samples were sequentially renatured with buffer I (20 mM Tris-HCl, 0.5M NaCl, 3M urea, SmMEDTA, pH 8.0), renatured buffer 11 (20 mM Tris-HCl, 0.5 M NaCl, 1M urea, 5 mM EDTA, 0.2 mM GSSG, 2 mM GSH, 0.4 M L-Arg, pH 8.0) and renaturing buffer III (20 mM Tris-HCl, O.SMNaCl, SmMEDTA, p. 0), respectively Perform dialysis for 12-24 h, and then dialyze for 24 h with PBS (pH 7.4).
  • buffer I 20 mM Tris-HCl, 0.5M NaCl, 3M urea, SmMEDTA, pH 8.0
  • renatured buffer 11 (20 mM Tris-HCl, 0.5 M NaCl, 1M urea, 5 mM EDTA, 0.2 mM
  • the obtained precipitate was dissolved by adding 200 ml of cold water, dialyzed, and then centrifuged to remove insoluble matter. Apatite column adsorption, 0.001 M phosphate buffer (pH 6.8) eluted, the active part was freeze-dried to obtain the crude product 1500 mgo. The crude product was dissolved in water, and the active part was lyophilized by Sephadex G-75 column chromatography. Obtained 145 mg antitumor high activity lidamycin white powder refined product o
  • the chromophore Compared with the LDM protein part, the chromophore has a smaller molecular weight, and its theoretical content is only 7.4% of lidamycin. Since the active portion LDM AE is functioning, apoprotein AE only protection function, it is generally by the relative content of AE in the total amount of chromophoric 5 Determination i.e., may determine the level of activity of article LDM
  • the LDM product prepared as above was dissolved in an HPLC mobile phase (acetonitrile: water 23: 77), and separated on a FPLC fast protein chromatography instrument by Waters radial pressure C4 semi-preparative column, and the eluent was acetonitrile: water ( 23: 77), collected by an automatic collector, and the collected components were detected by an HPLC C4 analytical column.
  • Quattro LC tandem mass spectrometer was used for mass spectrometry analysis of the chromophore (Figure 14).
  • the main peak of the active chromophore AE component m / z was 844.4 (M + 1), and its molecular weight was 843 kDa.
  • the molecular weight of its aromatization product is 845 kDa.
  • the fusion protein Fv-LDP was incubated at 37 ° C for 1 hour, and then 100 ⁇ 1 ⁇ ⁇ ⁇ anti-LDM monoclonal antibody F9 was used as the HPR-labeled goat anti-mouse IgG antibody and incubated at 37 ° C for 1 hour. Wash 3-4 times, and then add 100 ⁇ OPD substrate reaction solution to each well for color reaction. The microplate reader measures the absorbance at 490 legs. The results showed that the fusion protein prepared as described in Example 4 was positive for type IV collagenase, HT-29 and HT-1080 tumor cells. The results are shown in FIG. 7.
  • the streptavidin-biotin-enzyme-linked complex (SABC) staining method provided by immunohistochemistry kit (Boster) was added dropwise. Goat serum blocking solution, incubate at room temperature for 20 minutes; aspirate excess liquid, without washing, directly add the appropriately diluted fusion protein Fv-LDP, and incubate at room temperature; and then add the appropriately diluted F9 monoclonal antibody and biotinylated goat anti-mouse in order. IgG antibody, and finally SABC reagent was added dropwise. The color was developed with DAB kit at room temperature, the conventional hematoxylin was slightly counterstained, and the dehydrated transparent mount was used. Observe the staining results ( Figure 8).
  • the supernatant was determined by the conventional protein quantification method (Bradford method) to determine the protein content, draw a standard curve and determine the concentration of the sample, and take the supernatant at the corresponding volume for non-denaturing PAGE electrophoresis.
  • Electrophoresis completed Rinse in 100 ml of 2.5% Triton X-100 solution for 30 minutes, repeat once, rinse with distilled water twice, and add 100 ml of type IV collagenase buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 10 mM CaCl 2 , 1 ⁇ M ZnCl 2 ), 37. C was incubated for 16 h; Coomassie Brilliant Blue R250 staining was used to observe the negatively stained gelatin hydrolysis bands.
  • Example 10 Inhibition of angiogenesis by the enhanced fusion protein Fv-LDP-AE
  • the inhibition of angiogenesis by chicken embryo urinary membrane method was examined. Freshly fertilized white-skinned chicken eggs with the air chamber side facing up, incubate in a constant temperature room at 37 ° C, 60% ⁇ degrees, and then sterilize the egg shells later, pierce the needle with a needle, and inhale 2 ml of air into the air. Separate the chicken embryo urinary membrane from the vascular egg membrane in the room while grinding the shell with a grinding wheel.
  • mice with a body weight of 18-22 g were collected and randomly divided into a group of 10 each.
  • mice with liver cancer ⁇ 22 ascites were taken, diluted with physiological saline to a cell number of 1.5 ⁇ 10 6 / ml, and inoculated subcutaneously in the armpits of Kunming mice at 0.2 ml / only.
  • mice Twenty-four hours after the mice were inoculated with H22 tumors subcutaneously, normal saline, Fv-LDP, free LDM, and three doses of Fv-LDP-AE were administered on the first and tenth days of the experiment, respectively.
  • Fv-LDP-AE inhibits the growth of mouse transplanted liver cancer H22

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Abstract

L'invention porte sur un nouveau médicament ciblé contre les anticorps ayant un effet inhibiteur de l'angiogenèse, un effet de suppression de l'intensité sur les cellules tumorales et un effet thérapeutique antitumoral, sur une protéine de fusion intensifiée Fv-LDP-AE, sur son procédé de préparation et sur l'utilisation de ladite protéine en vue de la préparation d'un médicament antitumoral.
PCT/CN2004/000842 2003-07-22 2004-07-21 Proteine de fusion intensifiee fv-lpd-ae ayant une action antitumorale et d'inhibition de l'angiogenese et son utilisation WO2005007701A1 (fr)

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