WO2021180110A1 - Utilisation d'anticorps humanisé anti-basigine pour la préparation d'un médicament pour le traitement de la nouvelle pneumonie à coronavirus - Google Patents

Utilisation d'anticorps humanisé anti-basigine pour la préparation d'un médicament pour le traitement de la nouvelle pneumonie à coronavirus Download PDF

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WO2021180110A1
WO2021180110A1 PCT/CN2021/079907 CN2021079907W WO2021180110A1 WO 2021180110 A1 WO2021180110 A1 WO 2021180110A1 CN 2021079907 W CN2021079907 W CN 2021079907W WO 2021180110 A1 WO2021180110 A1 WO 2021180110A1
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humanized antibody
basigin
seq
antibody
cypa
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PCT/CN2021/079907
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Chinese (zh)
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陈志南
朱平
边惠洁
张征
尉丁
杨向民
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中国人民解放军第四军医大学
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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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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 belongs to the technical field of novel antibody drugs in the treatment of viral pneumonia. Specifically, the present invention relates to the application of an anti-BASIGIN humanized antibody for preparing drugs for the treatment of new coronavirus (COVID-19) pneumonia.
  • Coronavirus is a type of single-stranded positive-stranded RNA virus that can infect humans and a variety of vertebrates. It is an important pathogen that causes common colds and upper respiratory tract infections, and seriously threatens human health.
  • the novel coronavirus (COVID-19) that was circulating at the end of 2019 has the characteristics of rapid spread, heavy epidemic, and rapid progress. In some patients, it causes progressive impairment of respiratory function and causes death.
  • the treatment of 2019-nCoV mainly focuses on supporting symptomatic treatment and antiviral treatment. There is a lack of specific drugs, and there is an urgent need to develop new prevention and treatment drugs.
  • Coronaviruses belong to the Coronavirus family (Coronaviridae), which includes 4 genera: Alpha Coronavirus ( ⁇ -CoV), Beta Coronavirus ( ⁇ -CoV), Gamma Coronavirus ( ⁇ -CoV), Delta Coronavirus ( ⁇ - CoV). So far, there are six known human coronaviruses. Two of the coronaviruses have a high transmission rate and a high case fatality rate. They are the pathogens severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East Respiratory Syndrome that caused the outbreak of SARS in Guangdongzhou, China in 2003 and MERS in Saudi Arabia in 2012. Coronavirus (MERS-CoV), they all belong to ⁇ -coronavirus. MERS symptoms usually include fever, cough, and shortness of breath, and even develop into pneumonia. The case fatality rate is about 34.4%. The symptoms of SARS usually include fever, chills and body pain, and even develop into pneumonia. The fatality rate is about 9.6%.
  • SARS-CoV pathogens severe acute respiratory syndrome coronavirus
  • New type of coronavirus pneumonia is pneumonia caused by a new type of coronavirus (COVID-19) infection.
  • COVID-19 coronavirus
  • Coronavirus contains at least four structural proteins: spike protein S protein (spike), E protein (Envelope), M protein (membrane) and N protein (nucleocapsid).
  • S protein spike protein S protein
  • E protein envelope
  • M protein membrane
  • N protein nucleocapsid
  • the S protein promotes host attachment and the fusion of virus and cell membrane during virus infection.
  • the N protein is mainly involved in maintaining the stability of the genome and the formation of the nucleocapsid.
  • CyPA plays an important role in the process of virus replication, assembly, release and infection.
  • CyPA not only directly participates in the budding and release of progeny viruses, but also appears on the surface of the virus membrane after the virus is released.
  • S protein In cooperation with the S protein, it participates in the identification and binding process of the coronavirus and target cells.
  • CD147 molecule is one of the receptor molecules of CyPA. It binds to CyPA through the extracellular Pro180 and Gly181 sites to mediate the invasion of coronavirus and is a co-receptor for coronavirus to invade host cells.
  • ELISA results show that the half maximum effect concentration (EC 50 ) between the two is 135 ⁇ g/mL; SPR results show that the affinity constant (K D ) between the two is 8.7 ⁇ 10 -6 M.
  • K D affinity constant
  • Our previous studies have shown that by blocking the binding site of the CD147 molecule of CyPA, it can effectively block the SARS-CoV infection of target cells and realize the treatment of viral infections.
  • CD147 is a highly glycosylated transmembrane glycoprotein with a molecular weight of 50-60kD. It is a member of the immunoglobulin superfamily (IgSF). This molecule is named differently in different tissues, such as EMMPRIN, Neurothelin, M6antigen, BASIGIN, etc. In humans, CD147 consists of a total of 269 amino acids, which can be divided into extracellular, transmembrane and intracellular regions. The first 21 residues after the initial translation of the N-terminus are signal peptides, 22-205 constitute the extracellular region, 206-229 are the transmembrane region, with a typical leucine zipper structure, and the C-terminus 230-269 is the intracellular region.
  • IgSF immunoglobulin superfamily
  • CD147 is a multifunctional membrane protein target molecule, which not only participates in the tumor development process, but also plays an important role in the facilitating process of virus invasion of host cells.
  • the specific possible mechanism of action is that CyPA binds to the N protein of SARS-CoV, and then the exposed CyPA binds to the host cell membrane molecule CD147 to cause the virus to invade.
  • the CD147 antagonistic peptide HAb18G-AP-9 prevents the virus from invading by blocking CD147 to prevent its circulating life cycle. Or it is possible that the CD147 antagonist peptide can enter the infected cell by blocking the SARS-CoV N protein binding to CyPA and then binding with CD147 on the ER to inhibit the budding and formation process of the virus, thereby achieving the process of blocking the virus invasion.
  • Related research content Published in J Infect Dis. 2005,191(5):755-760.
  • CD147 is a receptor molecule for coronaviruses including new coronaviruses to invade cells.
  • the BASIGIN molecule that targets cells is expected to become an important target for new anti-coronavirus drugs.
  • McAb Monoclonal antibodies
  • HAMA human anti-mouse antibody
  • the present invention verifies through experiments that the anti-BASIGIN human antibody inhibits the biological activity of the neocoronavirus infecting host cells.
  • the purpose of the present invention is to provide an anti-BASIGIN humanized antibody to inhibit the infection of host cells by the new coronavirus, and to inhibit the application of new coronary pneumonia.
  • One of the objectives of the present invention is to provide an anti-human BASIGIN humanized antibody (hereinafter referred to as "Basigin-hAb”) for the preparation of drugs for the treatment of novel coronavirus pneumonia.
  • Basigin-hAb an anti-human BASIGIN humanized antibody
  • the anti-human BASIGIN humanized antibody of the present invention is based on the hybridoma cell line HAb18Gedomab2 (preservation number CCTCC-C200636, patent number: ZL200710007452.2) that secretes the murine antibody 6H8 (also known as HAb18GC2) against human BASIGIN molecule ,
  • HAb18Gedomab2 preservation number CCTCC-C200636, patent number: ZL200710007452.2
  • the present invention also provides the amino acid sequence of the light chain variable region of the anti-human BASIGIN humanized antibody is shown in SEQ ID NO: 1, and the heavy chain amino acid sequence is shown in SEQ ID NO: 3; the light chain variable region nucleotides The sequence is the sequence shown in SEQ ID NO: 2, and the nucleotide sequence of the heavy chain variable region is the sequence shown in SEQ ID NO: 4; both of them include the immunoglobulin light chain variable region and the immunoglobulin heavy chain Variable region
  • the immunoglobulin light chain variable region includes CDR1: as shown in SEQ ID NO: 5 in the sequence list; CDR2: as shown in SEQ ID NO: 6 in the sequence list; CDR3: as shown in SEQ ID NO: in the sequence list 7 shown;
  • the immunoglobulin heavy chain variable region contains CDR1: as shown in SEQ ID NO: 8 in the sequence list; CDR2: as shown in SEQ ID NO: 9 in the sequence list; CDR3: as shown in SEQ ID NO: in the sequence list 10 shown.
  • the monoclonal antibody is a humanized IgG2 antibody recombinantly expressed by CHO cells (CHO DG44, a mutated Chinese hamster ovary cell).
  • the molecule consists of two heavy chains containing 442 amino acids and two heavy chains containing 214 amino acids. The light chains of amino acids are linked by disulfide bonds.
  • anti-human BASIGIN humanized antibody for the preparation of drugs for the treatment of novel coronavirus pneumonia.
  • Anti-human BASIGIN humanized antibody blocks the interaction between CD147 molecule and the new coronavirus S protein; anti-human BASIGIN humanized antibody blocks the interaction between CD147 molecule and cyclophilin A (CyPA).
  • amino acid sequence of the light chain variable region (VL) of the anti-human BASIGIN humanized antibody is shown in SEQ ID NO: 1
  • amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO: 3. Show.
  • nucleotide sequence of the light chain variable region (VL) of the anti-human BASIGIN humanized antibody is shown in SEQ ID NO: 2
  • nucleotide sequence of the heavy chain variable region (VH) is shown in SEQ ID NO: 4 is shown.
  • the light chain (VL) constant region of the anti-human BASIGIN humanized antibody is ⁇
  • the heavy chain (VH) constant region is IgG2.
  • the anti-human BASIGIN humanized antibody comprises a heavy chain variable region (VH), wherein the VH comprises: the amino acid sequence shown in SEQ ID NO: 3.
  • the anti-human BASIGIN humanized antibody wherein the VH comprises an amino acid sequence as shown in SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.
  • the anti-human BASIGIN humanized antibody comprises a light chain variable region (VL), wherein the VL comprises as shown in SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7. CDR shown.
  • VL light chain variable region
  • the anti-human BASIGIN humanized antibody wherein the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO:1.
  • the anti-human BASIGIN humanized antibody wherein the light chain variable region (VL) comprises the nucleotide sequence shown in SEQ ID NO: 2.
  • the CD147 molecule can interact with the new coronavirus S protein and the virus-binding protein CyPA to mediate the invasion of the new coronavirus into host cells; blocking CD147 and the new coronavirus S protein and the virus-binding protein CyPA by anti-human BASIGIN humanized antibodies
  • the interaction can effectively inhibit virus infection of target cells, and inhibit CyPA-mediated chemotaxis, realizing the treatment of virus-infected host cells. Therefore, the present invention provides for the first time the application of anti-human BASIGIN humanized antibody in the treatment of novel coronavirus pneumonia.
  • Figure 1 The results of the SPR method of the present invention to determine the affinity of CD147 and the new coronavirus S protein COVID-19 Spike-RBD.
  • the curves in the figure represent the binding of 0.4, 0.6, 0.8, 1.0 and 1.2 ⁇ M COVID-19 Spike-RBD protein to CD147, respectively Power IP curve;
  • Figure 2 The results of the SPR method of the present invention to determine the affinity between the new coronavirus N protein and CyPA, the curves in the figure represent the binding kinetic curves of 200, 400 and 800nM SARS-CoV-2N protein and CyPA, respectively;
  • Figure 3 The results of the SPR method of the present invention to determine the affinity between CD147 and CyPA.
  • the curves in the figure represent the binding kinetic curves of 1, and 30 ⁇ MCyPA protein and CD147 respectively;
  • Figure 4 The ELISA method of the present invention determines the results of the interaction between CD147 and the new coronavirus S protein COVID-19 Spike-RBD; where the abscissa is the logarithmic value of the CD147-his fusion protein concentration at different concentrations, and the ordinate is the optical density at 450 nm ( OD) value;
  • Figure 5 Competitive ELISA method of the embodiment of the present invention to determine the anti-BASIGIN humanized antibody inhibits the interaction between CD147 and the new coronavirus S protein (COVID-19 Spike-RBD), where the abscissa is the pair of anti-BASIGIN humanized antibody concentration Value, the ordinate is the optical density (OD) value measured at 450nm);
  • Fig. 6 Competitive ELISA method in the embodiment of the present invention determines the result of anti-BASIGIN humanized antibody inhibiting the interaction between CD147 and CyPA, where the abscissa is the logarithmic value of the anti-BASIGIN humanized antibody concentration, and the ordinate is the optical density measured at 450 nm ( OD) value);
  • Figure 7 The anti-BASIGIN humanized antibody of the present invention inhibits CyPA-mediated cell chemotaxis
  • Figure 8 The anti-BASIGIN humanized antibody of the present invention inhibits COVID-19 infection cell dose-effect curve
  • Figure 9 The anti-BASIGIN humanized antibody of the present invention inhibits the number-effect curve of COVID-19 virus nucleic acid copy.
  • Immunoglobulin refers to a class of structurally related glycoproteins.
  • the glycoprotein consists of two pairs of polypeptide chains, a pair of low molecular weight light chains (L) and a pair of high molecular weight heavy chains (H). All four chains pass The disulfide bonds connect to each other.
  • Each heavy chain typically consists of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region (abbreviated as CH herein).
  • Each light chain typically consists of a light chain variable region (abbreviated as VL herein) and a light chain constant region (abbreviated as CL herein).
  • the light chain constant region typically consists of one domain CL.
  • VH and VL can be further subdivided into hypervariable regions (or hypervariable hypervariable regions in sequence and/or the form of structurally defined loops), also called complementarity determining regions (CDR), interspersed with more conservative regions
  • the area is called the frame area (FR).
  • Each VH and VL typically consists of three CDRs and four FRs, arranged in the following order from the amino terminal to the carboxy terminal, FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see Chothia and Lesk, 1987 ).
  • the numbering of amino acid residues in this region can be performed by the method described by Kabat et al.
  • Humanized antibody refers to an antibody derived from a non-human antibody, typically a murine animal, which retains or substantially retains the antigen-binding properties of the parent antibody, but it The immunity in humans is low. Since the antibody of the present invention is defined by structural and functional characteristics, “humanized antibody” can be used interchangeably with “antibody”.
  • CDR Complementarity determining region
  • the framework region (FR) refers to the amino acid sequence inserted between the CDRs. These parts of the antibody are used to hold the CDRs in place (allowing the CDRs to bind to the antigen). Both the light chain variable region and the heavy chain variable region comprise a framework region (FR) and typically three CDRs.
  • the constant region refers to the part of an antibody molecule that confers effector functions.
  • the constant region of the subject humanized antibody is derived from human immunoglobulin.
  • the heavy chain constant region can be selected from five isotypes: ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ . Further, various subclasses of heavy chains (for example, IgG subclasses of heavy chains) can cause different effector functions. Therefore, by selecting a desired heavy chain constant region, antibodies with desired effector functions can be produced.
  • Preferred heavy chain constant regions are ⁇ 1 (IgG1), ⁇ 2 (IgG2), ⁇ 3 (IgG3) and ⁇ 4 (IgG4), more preferably ⁇ 2 (IgG2).
  • the light chain constant region may be of ⁇ or ⁇ type, preferably ⁇ type.
  • variable or constant regions of an immunoglobulin heavy or light chain can be linked as described by using standard recombinant DNA techniques to create a suitable host that can be expressed (thus producing the immunoglobulin Chain (one or more)), or variable and constant regions that can be linked by chemical synthesis using peptides.
  • the parent antibody of the humanized antibody of the present invention is the murine antibody 6H8 called anti-human BASIGIN molecule, which is produced by the hybridoma cell line HAb18Gedomab2, and the hybridoma cell line HAb18Gedomab2 was deposited in China on December 13, 2006. Culture Collection (CCTCC-C200636, patent number: ZL200710007452.2).
  • the anti-human BASIGIN humanized antibody of the present invention retains the ability to specifically bind to the parent antibody to recognize the antigen, and the parent antibody is used to produce this humanized antibody HP6H8-1.
  • An anti-BASIGIN humanized antibody And its application application number: 201610285139.4).
  • Example 1 SPR method to determine the interaction between the new coronavirus S protein (COVID-19 Spike-RBD) and CD147
  • the BIAcore 3000 system and the CM5 sensor chip are used to determine the molecular binding dynamics parameters between the new coronavirus S protein (COVID-19 Spike-RBD) and CD147.
  • the detection buffer system is PBS (pH7.4), and the detection temperature is 25°C; 35 ⁇ g CD147 is fixed to the surface of the CM5 chip by amino coupling; the interaction is detected in Kinetic Analysis/Concentration Series/Direct Binding mode, and the flow rate is set to 10 ⁇ L/min , The binding time is 10min, and the dissociation time is 10min; 0.4, 0.8, 1.0 and 1.2 ⁇ M of the new coronavirus S protein (COVID-19 Spike-RBD) of the tested samples are injected into different channels respectively. Results BIAevaluation software was used for fitting analysis to determine the kinetic constants.
  • Example 2 SPR method to determine the interaction between the new coronavirus N protein and cyclophilin A (CyPA)
  • the BIAcore 3000 system and CM5 sensor chip are used to measure the molecular binding dynamics parameters between the new coronavirus N protein and cyclophilin A (CyPA).
  • the detection buffer system is PBS (pH7.4), and the detection temperature is 25°C; CyPA is fixed to the surface of the CM5 chip by amino coupling; the chip is inactivated with 1M ethanolamine-HCl (Bio-Rad); the chip is used after rotating 90 degrees Rinse with buffer (PBS/0.005% Tween 20) until the baseline is stable.
  • Example 3 SPR method to determine the interaction between CD147 and cyclophilin A (CyPA)
  • the SPR was used to monitor the binding of CD147 to cyclophilin A (CyPA) in real time, and the affinity constant (KD) was measured to reflect the affinity of CD147 to CyPA.
  • the ProteOn XPR36 (Bio-Rad, XPR36) instrument was used to determine the affinity of CD147 with cyclophilin A (CyPA).
  • the GLC chip Bio-Rad, 1765011 was activated with 0.04M EDC+0.01Msulfo-NHS (Bio-Rad). Dilute CD147 to 10mM with 10mM NaAc (pH 4.5), and inject it onto the chip at a rate of 30ul/min, so that the antigen is coupled to the activated chip through amino groups.
  • the chip was inactivated with 1M ethanolamine-HCl (Bio-Rad); the chip was rotated 90 degrees and washed with buffer (PBS/0.005% Tween 20) until the baseline was stable.
  • buffer PBS/0.005% Tween 20
  • 1 and 30 ⁇ M cyclophilin A (CyPA) samples were injected respectively.
  • the sampling speed is 30ul/min.
  • the sample binding time is 60s, and the dissociation time is 900s; the Kinetic-Langmuir model is used for data analysis.
  • Example 4 ELISA method to determine the interaction capacity between CD147 and the new coronavirus S protein (COVID-19 Spike-RBD)
  • the ELISA method was used to detect the interaction between CD147 and the new coronavirus S protein (COVID-19 Spike-RBD). 5 ⁇ g/mL COVID-19 Spike-RBD-his fusion protein was coated on a microwell plate, and then incubated with different concentrations of CD147-his fusion protein (2-fold dilution, 400-0.195 ⁇ g/mL) at 37°C for 1 hour. After washing three times with PBST, incubate with HAb18 murine anti-CD147 antibody for 1 hour; then incubate with HRP-labeled goat anti-mouse antibody for 1 hour. Add 100 ⁇ L TMB, develop color for 4 minutes, then add 50 ⁇ L 2M H2SO4 to stop the reaction. A full-wavelength microplate reader (Epoch, BioTek Instruments, Inc.) was used to measure the optical density (OD) value at 450 nm, and a four-parameter fitting was used to calculate the half maximum effect concentration (EC 50 ).
  • OD optical density
  • Example 5 Competitive inhibition experiment of anti-BASIGIN humanized antibody inhibiting the interaction between CD147 and the new coronavirus S protein (COVID-19 Spike-RBD)
  • the competitive inhibition ELISA method was used to detect the ability of the anti-BASIGIN humanized antibody to compete with the new coronavirus S protein (COVID-19 Spike-RBD) to bind to CD147.
  • 100 ⁇ L of CD147-his fusion protein (5 ⁇ g/mL) was coated on the microtiter plate. Then incubate with 200 ⁇ g/mL COVID-19 Spike-RBD and different concentrations of anti-BASIGIN humanized antibody (2-fold dilution, from 200-0.781 ⁇ g/mL) for 1 hour. Rinse with PBST three times, incubate with anti-CD147 murine antibody HAb18, and incubate with HRP-labeled goat anti-mouse antibody after washing.
  • Example 6 Competitive inhibition experiment of anti-BASIGIN humanized antibody inhibiting the interaction between CD147 and its ligand CyPA
  • the competitive inhibition ELISA method was used to test the ability of anti-BASIGIN humanized antibody to compete with CyPA for CD147 binding.
  • the prepared CyPA-his fusion protein was coated on a microplate. Then, it was incubated with 1 mg/mL CD147-Tag fusion protein and different concentrations of anti-BASIGIN humanized antibody (2-fold dilution, from 200-0.049 ⁇ g/mL) for 1 hour. Rinse with PBST three times, incubate with anti-CD147 murine antibody HAb18, and incubate with HRP-labeled goat anti-mouse antibody after washing. After adding 100 ⁇ l TMB, the color was developed for 4 minutes, and then 1M H 2 SO 4 was added to stop the reaction. The optical density (OD) value at 450 nm was measured with a microplate spectrophotometer (Epoch, BioTek Instruments, Inc.).
  • the ability of anti-BASIGIN humanized antibody to inhibit CyPA-mediated chemotaxis was evaluated.
  • the human peripheral blood mononuclear cells were separated, and the peripheral blood mononuclear cells were suspended in RPMI1640 culture medium containing 20% FBS.
  • the experiment was divided into serum-free control group, anti-BASIGIN humanized antibody group (10 ⁇ g/mL, upper chamber), CyPA group (200 ⁇ g/L, lower chamber), and Mepal beads (10 ⁇ g/mL, upper chamber) + CyPA (200 ⁇ g/ L, lower room) group.
  • CI chemotactic index
  • Example 8 Experiment of anti-BASIGIN humanized antibody inhibiting new coronavirus (COVID-19) infection in host cells
  • a 96-well plate virus micro-virus inhibition experiment method was used to evaluate the efficacy of anti-BASIGIN humanized antibodies in inhibiting the infection of host epithelial cells by the new coronavirus (COVID-19).
  • 1 ⁇ 10 4 /well of Vero E6 cells were plated on a 96-well plate at 37°C and cultured overnight. Aspirate and discard the supernatant, add 100 ⁇ l culture medium (containing 3.125, 6.25, 12.5, 25, 50 , 100, 150, 200 ⁇ g/mL Mebelbead antibody concentration), incubate at 37°C for 1h, and then add 100 ⁇ l virus solution (containing 100TCID 50 /Well) Incubate for 1 h, remove the above liquid, and wash 5 times with PBS. Supplement 200 ⁇ l of cell culture medium (containing 3.125, 6.25, 12.5, 25, 50, 100, 150, 200 ⁇ g/mL Mebelbead antibody concentration group), continue to culture for 2 days, observe cell pathology.
  • 100 ⁇ l culture medium containing 3.125, 6.25, 12.5, 25, 50, 100, 150, 200 ⁇ g/mL Mebelbead antibody concentration group
  • Example 9 Anti-BASIGIN humanized antibody inhibits nucleic acid copy number of new coronavirus (COVID-19) virus; pharmacodynamic experiment
  • 1 ⁇ 10 4 /well of Vero E6 cells were plated on a 96-well plate at 37°C and cultured overnight. Aspirate and discard the supernatant, add 100 ⁇ l culture medium (containing 3.125, 6.25, 12.5, 25, 50 , 100, 150, 200 ⁇ g/mL Mebelbead antibody concentration), incubate at 37°C for 1h, and then add 100 ⁇ l virus solution (containing 100TCID 50 /Well) Incubate for 1 h, remove the above liquid, and wash 5 times with PBS. Supplement 200 ⁇ l of cell culture medium (containing 3.125, 6.25, 12.5, 25, 50, 100, 150, 200 ⁇ g/mL Mebelbead antibody concentration group), continue to culture for 2 days, observe cell pathology.
  • 100 ⁇ l culture medium containing 3.125, 6.25, 12.5, 25, 50, 100, 150, 200 ⁇ g/mL Mebelbead antibody concentration group
  • Aspirate the supernatant use the fluorescent quantitative PCR method, use the standard plasmid as the standard, serially dilute, establish a standard curve, detect the copy number of viral nucleic acid in the culture solution, and calculate the inhibition rate. Drawing an amount of using Graphpad prism - effect curve, EC 50. At the same time, a virus control group and a blank control group are set up.
  • Mepelizumab can effectively reduce the number of viral nucleic acid copies and has a dose-dependent relationship.

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Abstract

L'invention concerne l'utilisation d'un anticorps humanisé anti-BASIGINE humaine pour la préparation d'un médicament pour le traitement de la nouvelle pneumonie à coronavirus. L'anticorps humanisé anti-BASIGINE humaine a une séquence d'acides aminés d'une région variable à chaîne légère telle que représentée dans la SEQ ID NO : 1 et une séquence d'acides aminés d'une chaîne lourde telle que représentée dans la SEQ ID NO : 3. La séquence nucléotidique de la région variable à chaîne légère est la séquence telle que représentée dans la SEQ ID NO : 2, et la séquence nucléotidique de la région variable à chaîne lourde est la séquence telle que représentée dans la SEQ ID NO : 4. L'anticorps peut spécifiquement reconnaître et se lier au co-récepteur CD147 de cellules épithéliales hôtes que le virus envahit, bloquer l'interaction de CD147 avec la protéine S de coronavirus et de CD147 avec la cyclophiline A (CyPA), ce qui permet d'empêcher le coronavirus d'infecter les cellules hôtes.
PCT/CN2021/079907 2020-03-11 2021-03-10 Utilisation d'anticorps humanisé anti-basigine pour la préparation d'un médicament pour le traitement de la nouvelle pneumonie à coronavirus WO2021180110A1 (fr)

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CN202010166717.9 2020-03-11
CN202010166717.9A CN111420048B (zh) 2020-03-11 2020-03-11 抗basigin人源化抗体用于制备治疗新型冠状病毒肺炎药物的应用

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