WO2012062072A1 - Anticorps monoclonal chimère de l'homme et de la souris contre le récepteur de l'angiostatine et son utilisation - Google Patents

Anticorps monoclonal chimère de l'homme et de la souris contre le récepteur de l'angiostatine et son utilisation Download PDF

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WO2012062072A1
WO2012062072A1 PCT/CN2011/071723 CN2011071723W WO2012062072A1 WO 2012062072 A1 WO2012062072 A1 WO 2012062072A1 CN 2011071723 W CN2011071723 W CN 2011071723W WO 2012062072 A1 WO2012062072 A1 WO 2012062072A1
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antibody
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human
monoclonal antibody
cell
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倪健
朱向玲
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苏州工业园区晨健抗体组药物开发有限公司
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • the present invention relates to the field of biotechnology, and in particular to the preparation and application of an anti-angiostatin receptor human mouse chimeric monoclonal antibody.
  • ATP synthase is not only present in the mitochondrial inner membrane, but also expressed on the surface of plasma membranes of endothelial cells and tumor cells.
  • Mozer and Pizzo identified the binding site of angiostatin (ring cake domain 1_3) on the surface of endothelial cells. Proteins of approximately 55 kD in size bound to angiostatin were successfully isolated by ligand hybridization of human umbilical vein endothelial cells (HUVEC) membrane components. The amino terminal sequencing, peptide fingerprinting and immunological analysis of the protein indicated that the receptor for the anti-tumor angiogenesis angiostatin on the cell surface is the ⁇ / ⁇ subunit of human F1F0-ATP synthase (gene name is ATP5A1, respectively). And ⁇ 5 ⁇ ). The presence of this receptor on the cell surface was confirmed by flow cytometry and immunofluorescence analysis.
  • angiostatin binds to recombinant ATP5A1, and anti-ATP5A1 antibodies inhibit the anti-cell proliferation of angiostatin by 90%.
  • ATP5A1/ATP5B on the cell surface is a receptor for angiostatin on the surface of endothelial cells.
  • Angiostatin may inhibit the proliferation and migration of endothelial cells by binding to the ATP5A1/ATP5B subunit, thereby inhibiting angiogenesis.
  • F1F0-ATP synthase may be detected as a tumor marker, which is of great significance for the early diagnosis and prognosis of tumors.
  • human F1F0-ATP synthase expressed by membrane may have proliferative effect, not only a tumor marker, but also It is the cause of cancer. If the membrane-expressed human F1F0-ATP synthase can successfully verify the tumor's promoting effect, it will further consolidate its tumor target, and design a corresponding drug for its proliferative function to suppress tumors in order to treat tumors with high efficiency.
  • a chimeric antibody is an antibody that splicing a murine monoclonal antibody variable region and a human antibody constant region by genetic engineering.
  • the advantage over other humanized antibodies is that the technical route is simple; the antibody has good integrity and a long retention period in the body.
  • Rituximab was approved by the FDA in 1997 and is the world's first marketed anti-tumor mAb for the treatment of CD20-positive B-lymphocytic non-Hodgkin's lymphoma. antibody.
  • the first chimeric antibody for clinical use is alemtuzumab, which is used to treat non-Hodgkin's lymphoma and rheumatoid arthritis.
  • trastuzumab approved in 1998, is a mAb drug that inhibits HER-2/neu-positive breast cancer, a humanized antibody.
  • Gemuzumab ozogamicin was approved in 2000 for the treatment of CD33-positive acute relapsed myeloid leukemia, which is also a humanized antibody.
  • the antibody drugs approved for the treatment of tumors are also 90Y-ibritumomab, 131I_tositumomab, cetuximab and bevacizumab, among which the proportion of chimeric or humanized antibodies has increased year by year, and fully human antibodies have become therapeutic antibodies in antibody types. The inevitable trend. More than 20 chimeric antibodies have been in clinical trials, and a large number of chimeric or humanized antibodies are in preclinical studies.
  • the chimeric antibody constant region is determined by the antibody subtype, and different subtypes have different immune effects, so that different subtype constant regions can be selected for amplification according to the desired immunological effect.
  • IgG1 is suitable for mediating antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), while IgG2 and IgG4 are suitable for activating or blocking a receptor.
  • the constant region of the antibody not only provides an effector function, but also protects against the degradation of proteases by binding to the IgG Fc receptor Fc Y Rn on the surface of the tissue cells, maintaining a longer half-life in vivo (more than 20 days).
  • the heavy light chain expression unit of the chimeric antibody can be ligated in tandem to one expression vector, or can be co-transfected on two vectors, respectively, and it has been reported in the literature that the proportion of clones producing antibodies in the transformants of the tandem expression vector is significantly higher.
  • the expression vector is co-transfected.
  • the screening markers currently used to increase the amplification of the gene of interest are mainly dihydrofolate reductase (DHFR) and glutamine synthetase (GS).
  • DHFR dihydrofolate reductase
  • GS glutamine synthetase
  • the system for expression of antibodies for clinical therapy is primarily the CH0 cell system, which provides efficient glycosylation for the constant regions of chimeric antibodies. It has been reported in the literature that the antibody expression can be increased to 300-900 mg/L by selecting an effective expression vector and screening system.
  • the object of the present invention is to disclose an anti-angiostatin receptor humanized chimeric antibody, and a DNA sequence encoding the same, constructing a eukaryotic expression vector containing the DNA sequence, screening and preserving stable high expression
  • the host cell strain of the antibody establishes a process for purifying the antibody.
  • the detection results of the above antibodies and similar products are disclosed.
  • the first aspect of the invention discloses an anti-angiostatin receptor human mouse chimeric monoclonal antibody, comprising an antibody light chain element and an antibody heavy chain element, wherein the light chain amino acid sequence is SEQ ID NO: 1 or a conservative variant thereof The sequence, the heavy chain amino acid sequence thereof is SEQ ID NO: 2 or a conservative variant thereof, and the heavy and light chains are linked by a disulfide bond.
  • a second aspect of the invention discloses an isolated DNA molecule encoding the variable or full length amino acid of the heavy and/or light chain of the aforementioned anti-angiostatin receptor human murine chimeric monoclonal antibody.
  • a third aspect of the invention discloses a vector comprising the aforementioned isolated DNA molecule.
  • the expression vector in the present invention refers to a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus or other vector well known in the art.
  • the carrier may be pCI-neo or the like.
  • a fourth aspect of the invention discloses a host cell comprising the aforementioned vector.
  • the host cell in the present invention may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf 9; animal cells of CH0, or Bowes melanoma cells, and the like.
  • the host cell is CH0 cells and the storage number is 20089278.
  • a method for producing the anti-angiostatin receptor human mouse chimeric monoclonal antibody wherein the host cell is cultured under the condition that the antibody is expressed, thereby expressing the antibody, and The antibody is isolated by purification.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (e.g., temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If desired, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art.
  • Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant [salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the present invention screens and obtains the gene sequence of the antibody of interest from the monoclonal cultured cell line, and constructs the eukaryotic expression vector, and can reconstitute the activity of the antibody after expression, and obtain the anti-angiostatin receptor human mouse chimeric monoclonal antibody.
  • the use of the above anti-angiostatin receptor human mouse chimeric monoclonal antibody for the preparation of an antitumor drug is disclosed.
  • a pharmaceutical composition comprising a therapeutically effective amount of the aforementioned anti-angiostatin receptor human murine chimeric monoclonal antibody is disclosed.
  • compositions can be used by formulating pharmaceutical compositions by any means known in the art.
  • Such compositions comprise the active ingredient in association with one or more pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients depending upon the mode of administration and the dosage form employed.
  • Therapeutic inert inorganic or organic carriers known to those skilled in the art include, but are not limited to, lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, and polysaccharides.
  • polyethylene glycol water, sucrose, ethanol, glycerin, and the like, various preservatives, lubricants, dispersants, flavoring agents.
  • Moisturizing hydrazines, antioxidants, sweeteners, coloring agents, stabilizers, salts, buffers, and the like may also be added, which are used as needed to aid in the stability of the formulation or to enhance activity or its bioavailability or An acceptable mouthfeel or odor is produced in the case of oral administration, and the inhibitor may be used in the form of the original compound itself, or optionally in the form of a pharmaceutically acceptable salt thereof, in the form of the present invention.
  • the cloned antibodies can be administered alone, or in various combinations, and administered in combination with other therapeutic agents.
  • the composition so formulated can be administered as appropriate in any suitable manner known to those skilled in the art.
  • the present invention further investigates the therapeutic effectiveness of the antibody against an angiostatin receptor monoclonal antibody in an animal by in vitro activity assay and animal experiment.
  • the monoclonal antibody against the angiostatin receptor provided by the present invention is a chimeric antibody, and it is more suitable for in vivo application while maintaining its anti-angiostatin receptor activity.
  • the monoclonal antibody has the activity of binding to the natural antigen on the surface of the tumor cell membrane, and can significantly inhibit the ATP synthesis on the surface of the tumor cell membrane, and has an important significance for anti-tumor treatment.
  • Figure 1 Example 3 test results of A549 cells inhibiting proliferation for 24 hours
  • FIG. 1 Example 3 test results of A549 cells inhibiting proliferation for 48 hours
  • Figure 3 Example 3 test results of A549 cells inhibiting proliferation for 48 hours
  • Figure 4 Example 3 Results of inhibition of proliferation of A549 cells for 72 hours
  • Figure 6 Example 3 Results of inhibition of ATPase synthesis activity by A549 cells against antibodies of the present invention
  • Figure 7 Example 3 Test results of the antibody of the present invention against 95-D cells inhibiting ATPase synthesis activity
  • Example 1 Preparation of chimeric antibody A hybridoma cell line was obtained by conventional fusion of mouse spleen cells immunized with a human ATP synthase antigen and mouse myeloma cells. A positive clone hybridoma cell line 7E10 was screened out.
  • Positive clone hybridoma 7E10 cell line 5000-10000 cells, total RNA was isolated using Trizol (Invitrogen), and reverse transcriptase (product of Invitrogen) was used to obtain cDNA. The above operations are carried out in accordance with the manufacturer's instructions. PCR was carried out using the following primers and conditions, and the amplification enzyme used was KOD plus (TOYOBO) to ensure that possible mutations were reduced during amplification.
  • the amplification primers for the Fab region of the antibody were 4 groups, mouse IgG-5 ' / mouse IgGl-3 ', mouse IgG-5 ' / mouse IgG2a-3, , mouse IgG-5 ' / mouse IgG3_3, , (LC 1 +LC2+LC3+LC4+LC5+LC6+LC7 ) / mouse kappa-3 ' , extension time 1 min.
  • the PCR product was electrophoretically recovered and cloned into the vector pMD19_T for sequencing. according to
  • Table 1 Chimeric antibody related primer design Amplified mouse heavy mouse IgG - 5' AGGTCCARCTKCTCGAGTCWGG (R: A/G, K: G/T, W: A/T) (SEQ ID NO: 6) Chain Fab region mouse IgGl - 3' AGGCTTACTAGTACAATCCCTGGGCACAAT (SEQ ID NO: 7)
  • LC7 mouse kappa-3 GCGCCGTCTAGAATTAACACTCATTCCTGTTGAA (SEQ ID NO: 17) light chain murine letter 7E10LP1: at
  • Zone splicing 7E10L P4 GCCACCGTACGGGTGCAGAAATAAATTCCCAGAT (SEQ ID NO: 21)
  • Heavy chain mouse letter 7E10LHP1 at
  • variable 7E10LH P2 TGACTCGAGCAGCTGGACCTCACACTGCACACCCTTCAGGAT (SEQ ID NO: 25) Region and human constant 7E10LH P3: ATCCTGAAGGGTGTGCAGTGTGAGGTCCAGCTGCTCGAGTCA (SEQ ID NO: 26) Site splicing 7E10LH P4: CCTTGGTGGAGGCACTTGCACAGTAATAGACTGCA (SEQ ID NO: 27)
  • Hybridoma cell signal peptide region amplification Primers were designed based on the results of sequence analysis of heavy and light chain Fab regions, and the signal peptide region of hybridoma cells was amplified by cRACE method. The PCR product was electrophoresed and cloned into the vector pMD19-T to obtain pMD19-7E10L and pMD19-7E10H, and sequenced.
  • Human IgG1 heavy and light chain constant regions were amplified by human IgG1 heavy and light chain constant region genes for PCR amplification, and cloned into vector pMD19-T sequencing c- splicing to construct chimeric heavy chain and chimeric light chain genes using 7E10LP1-P6, respectively.
  • the primers and the primers of 7E10LHP1-P6 were used as templates in 7E10 reverse transcription, and the signal peptide region, variable region, heavy and light chain of human IgG1 of 7E10 were detected by splicing PCR (SOE-PCR).
  • SOE-PCR splicing PCR
  • the constant regions were spliced to obtain chimeric heavy chain and chimeric light chain genes.
  • the PCR products of both were electrophoretically recovered and cloned into vector PMD19-T sequencing.
  • SEQ ID NO: 1 chimeric light chain sequence:
  • Nhel locus Nhel locus +Kozak sequence +signal peptide +FRl+
  • SEQ ID NO: 2 chimeric heavy chain:
  • Amino acid sequence (SEQ ID NO: 2):
  • chimeric heavy chain and chimeric light chain genes containing sequences of heavy and light chain variable regions can be obtained by total synthesis or splicing PCR. It is well known to those skilled in the art. Construction of eukaryotic expression vectors pCI-7E10H, pCI-7E10L, pCI_7E10LH of chimeric antibody
  • the chimeric light chain cloning vector PMD-7E10L and the expression vector pCI-neo were digested with Nhel and Mlul, respectively, and electrophoresed and recovered, T4 ligase 16 °C is connected for 2 h.
  • the ligation product transforms TOP10 competent bacteria. After the recombinant plasmid was identified by PCR and restriction enzyme digestion, the positive clone pCI-7E10L was screened.
  • the chimeric heavy chain cloning vector pMD-7E10H and the expression vector pCI-neo were digested with EcoRI and Notl, respectively. After electrophoresis recovery, T4 ligase was ligated at 16 °C for 2 h. The ligation product transforms T0P10 competent bacteria. After the recombinant plasmid was identified by PCR and restriction enzyme digestion, the positive clone pCI-7E10H was selected.
  • a chimeric expression vector was constructed, polyA and CMV were cloned into pMD-19T vector, Mlul and Notl restriction sites were added at both ends, and EcoRI sites were added after CMV sequence and before Notl restriction site, using EcoRI and Notl.
  • the chimeric heavy chain cloning vector PMD-7E10H was digested, the heavy chain fragment was excised, and the target fragment was recovered by electrophoresis.
  • EcoRI and Notl were digested with pMD-polyA-CMV, and T4 ligase was ligated at 16 °C for 2 h. The ligation product transforms T0P10 competent bacteria.
  • the positive clone pMD-polyA-CMV-7E10H was screened.
  • pCI_neo, pMD-polyA-CMV-7E10H were digested with Mlul and Notl, and pCI-neo and polyA-CMV_7E10H fragments were recovered by electrophoresis, and T4 ligase was ligated at 16 °C for 2 h. The ligation product transforms T0P10 competent bacteria.
  • the positive clone pCI-polyA-CMV-7E10H was screened out.
  • pMD_7E10L and pCI-polyA-CMV-7E10H were digested with Nhel and Mlul, and the digested product was recovered by electrophoresis.
  • T4 ligase was ligated at 16 °C for 2 h. The ligation product transforms T0P10 competent bacteria. After the recombinant plasmid was identified by PCR and restriction enzyme digestion, the positive clone pCI-7E10LH was screened.
  • Cell transfection was performed according to the Lip O f ec tamine TM 2000 protocol. Take 1 well in a 6-well plate as an example. The brief procedure is as follows: Inoculate 4 ⁇ 10 5 cells, 37 ° C. 5 % C0 2 was cultured for 18 h and then changed for 1 time. After 12 h, the transfection was started. The ideal confluence of adherent cells at transfection was 90-95%. 4 ⁇ ⁇ plasmid DNA and 10 ⁇ Lip O f ec tami ne TM 2000 were diluted to 250 ⁇ l with anti-antibiotic-free and serum-free medium, respectively, and gently mixed, and allowed to stand at room temperature for 15 min to form a liposome complex.
  • the cells were washed once with PBS, and 2 ml of F12 was added as a medium, and then the liposome complex was dropped, and the plate was shaken back and forth, and gently mixed. After incubating at 37 ° C, 5 % C0 2 for 6 h, replace 5 ml of F12 complete medium containing 10% FBS for 48-72 h, then culture with serum-free medium EX-CELL 302 containing no H, T, Gly. Cell clones of the dhfr + phenotype were screened.
  • EX-CELL 302 The cells cultured in EX-CELL 302 were digested, counted, and subcultured in a 96-well culture plate at 1.5 cells/well for sandwich ELISA using goat anti-human IgG (H+L) polyclonal antibody. Clones with high expression of chimeric antibodies were screened. The cell clone with the highest expression of chimeric antibody was selected and passaged for 1:5 and then cultured with EX-CELL 302 selection medium containing 30 nmol/L methotrexate (MTX). After the cells were acclimated to 30 nmol/L MTX, subcloning was continued as described above.
  • MTX methotrexate
  • the cell clone with the highest antibody expression was sequentially cultured in the same manner as the EX-CELL 302 selection medium containing 100 nmol/L MTX and 1 Mmol/L MTX, and subcloned. After the 96-well plate cells were substantially overgrown, The medium was further cultured for 2 days, and the supernatant was assayed for the chimeric antibody content. The cell clone with the highest expression of the chimeric antibody was screened by this method. The cell clone was transferred to a 25 cm 2 flask to expand the culture. After the cell was full, the medium was further cultured for 4 days, and the supernatant was assayed for the chimeric antibody content, and the antibody content measured this time was used. The cell clone El with the highest expression of chimeric antibody was selected, and the amino acid sequence of the chimeric antibody expressed by sequencing was confirmed to be in accordance with expectations.
  • the human mouse chimeric antibody gene against angiostatin was constructed and successfully expressed in CH0-DG44 cells, and the product was abbreviated as 7E10.
  • the expression product retained the antigen-binding ability of the parental mouse, while replacing the murine source with a human source in the constant region, and the expression level in the secreted clone supernatant was 40 ng/ml.
  • Indirect ELISA was used to detect the antigen binding and humanity of 7E10. Each sample was tested with goat anti-human IgG (H+L) _HRP enzyme secondary antibody and goat anti-mouse IgG (H+L)-HRP.
  • Double antibody sandwich ELISA but coated with 10 ⁇ ⁇ / ml of goat anti-human IgG (H+L) polyclonal antibody overnight, with goat anti-human IgG (H+L)-HRP enzyme secondary antibody.
  • Indirect ELISA was performed with rhATP5B coated plate.
  • 7E10 in transfected cell clone E1 culture supernatant could be combined with coated rhATP5B. It was recognized by goat anti-human IgG (H+L) and showed a positive reaction.
  • goat anti-mouse IgG (H+L)-HRP was used as the secondary antibody, the parental m7E10 was positive, while the chimeric antibody 7E10 was negative (the results are shown in Table 2. This demonstrates that 7E10 contains a constant region fragment of human antibody, and m7E10 can be combined with rhATP5B.
  • Double-antibody sandwich ELISA was used to coat the enzyme plate with goat anti-human IgG (H+L) polyclonal antibody, and goat anti-human IgG (H+L)-HRP was used as the secondary antibody.
  • Human IgG1 was used as the standard curve to identify clone E1.
  • the expression level of 7E10 in the culture supernatant was 40. 05 ng/ml (see Table 3 for the results).
  • mice (ICR, 16-21 g, male), purchased for 5 days before the experiment, and were cultured with the animal model of the Experimental Animal Center of Ncapturing University: The lung cancer cells A549 were expanded and cultured, and each 3 million was inoculated subcutaneously in nude mice. Set high
  • 7E10 injection was prepared by a conventional method. 5 ⁇ The 7E10 injection was low dose group: 7E10 concentration was 0. 05mg/ml
  • 7E10 injection medium dose group 7E10 concentration is 0. lmg/ml
  • 7E10injection high dose group 7E10 concentration is 0. 4mg/ml
  • the experiment was divided into 6 groups, 10 mice in each group, of which 11 were positive and negative control groups.
  • the 6 groups were: 1. 5% glucose negative control group; 2. conventional paclitaxel positive control group; 3. 7E10 low dose group; 4. 7E10 medium dose group; 5. 7E10 high dose group; 6. 7E10+anti VEGF Antibody group.
  • the experiment was performed by double-blind method, and the experimenter only got 6 codes.
  • Table 2 list of tumor weight of each group (unit: g) Group 5% grape conventional yew 7E10 injection 7E10 injection 7E10 injection high 7E10 + anti number sugar negative pair alcohol positive injection low dose medium dose group dose group VEGF antibody group (2), according to group group
  • Table 3 Average tumor weight, tumor inhibition rate, and statistical P value list for each group
  • the positive control of group B was generally small, and there were 6 tumor-bearing mice with clear tumor edges and surrounding tissues, which were easy to separate.
  • the tumors were generally small, and there were 6 tumor-bearing mice with clear tumor edges and surrounding tissues, which were easy to separate.
  • the 7E10+antiVEGF antibody group most of the tumors were relatively small, and the tumor tumors of the 8 tumor-bearing mice were clear and easy to separate from the surrounding tissues.
  • Experimental tumor cells human lung adenocarcinoma cell line (A549), human high metastatic lung cancer cells (95-D)
  • A549 cells were seeded in a 24-well plate at a concentration of 2 ⁇ 10 5 /ml, 1 ml/well, and 7E10 antibody was added to culture in 1640 medium (including 10%). After fetal bovine serum), cultured at 37 °C, 5 % C02, and saturated humidity for 24 and 48 h, the cells were collected by centrifugation, washed with PBS, and the Annexin-V kit was used to detect early apoptosis (refer to the kit instructions for specific procedures). The results showed that the antibody acted on A549 cells, causing 24 and 48 hours later. The percentage of apoptosis was 22.3% and 47.5%, respectively.
  • the supernatant of the 96-well plate was aspirated, and 50 L / well of 0.05 mM ADP solution was added and reacted for 2 minutes.
  • the ATP detection kit (Molecular Probes, A22066) instructions, prepare a standard reaction solution of 90 L / hole into the test white plate in advance, and check the background signal on the machine. Take 10 L of the reaction supernatant and add it to the white plate to which the standard reaction solution has been added. The reaction was allowed to stand at room temperature for 10 minutes.
  • AnalystTM HT (Molecular Devices) detects and calculates inhibition at 562 nm. Four replicate wells were set for each concentration and the experiment was repeated three times. The results are shown in Figures 6 and 7.

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Abstract

L'invention concerne un anticorps monoclonal chimère de l'homme et de la souris contre le récepteur de l'angiostatine, ainsi que des molécules d'ADN codant l'anticorps, un procédé de préparation de l'anticorps et l'utilisation de l'anticorps dans la fabricaiton de médicaments antitumoraux.
PCT/CN2011/071723 2010-11-12 2011-03-11 Anticorps monoclonal chimère de l'homme et de la souris contre le récepteur de l'angiostatine et son utilisation WO2012062072A1 (fr)

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