WO2021258625A1 - 抗sar-cov-2(covid-19)全人源单克隆抗体及其制法与应用 - Google Patents

抗sar-cov-2(covid-19)全人源单克隆抗体及其制法与应用 Download PDF

Info

Publication number
WO2021258625A1
WO2021258625A1 PCT/CN2020/129881 CN2020129881W WO2021258625A1 WO 2021258625 A1 WO2021258625 A1 WO 2021258625A1 CN 2020129881 W CN2020129881 W CN 2020129881W WO 2021258625 A1 WO2021258625 A1 WO 2021258625A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
seq
antigen
cov
binding fragment
Prior art date
Application number
PCT/CN2020/129881
Other languages
English (en)
French (fr)
Inventor
万晓春
李俊鑫
Original Assignee
中国科学院深圳先进技术研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院深圳先进技术研究院 filed Critical 中国科学院深圳先进技术研究院
Publication of WO2021258625A1 publication Critical patent/WO2021258625A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • 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
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/04—Drugs for disorders of the respiratory system for throat disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/02—Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the invention belongs to the field of immunology, and specifically relates to an anti-SAR-COV-2 (COVID-19) fully human monoclonal antibody and its preparation method and application.
  • HAMA anti-mouse antibody response
  • Human-derived monoclonal antibodies have high specificity and significant curative effects in the treatment of inflammation, cancer, influenza, especially coronaviruses.
  • COVID-19 is an acute respiratory infectious disease caused by a SAR-COV-2 coronavirus, and there is still a lack of effective drugs and vaccines.
  • the human antibodies that neutralize the virus are some specific antibodies produced by human B lymphocytes, which can bind to the antigen on the surface of the virus, thereby preventing the virus from adhering to the target cell receptor, preventing the virus from invading the cell, and effectively preventing SAR-COV-2 influenza.
  • the present invention provides an anti-SAR-COV-2 antibody or antigen-binding fragment thereof, which specifically neutralizes SAR-COV-2.
  • the present invention provides an anti-SAR-COV-2 antibody or antigen-binding fragment thereof, which specifically neutralizes SAR-COV-2.
  • One aspect of the present invention provides an isolated anti-SAR-COV-2 antibody or antigen-binding fragment thereof; it has any of the following groups of three complementarity determining regions (HCDR) of the heavy chain variable region and three light chain variables The complementary determining region of the variable region (LCDR):
  • HCDR1 GTFFNNYV SEQ ID No: 1;
  • HCDR2 ISYDGSVK SEQ ID No: 2;
  • HCDR3 AKPHVLFWLGEGKGPFDY SEQ ID No: 3;
  • LCDR1 QGISSY SEQ ID No: 4;
  • LCDR2 AAS SEQ ID No: 5;
  • LCDR3 QQLNSYPLT SEQ ID No: 6.
  • Another aspect of the present invention provides an isolated anti-SAR-COV-2 antibody or antigen-binding fragment thereof, which has a heavy chain variable region and a light chain variable region as shown below;
  • the antibody or antigen-binding fragment thereof is a humanized antibody, more preferably a fully humanized antibody.
  • the antibody or antigen-binding fragment thereof specifically binds to the S protein on the surface of SAR-COV-2.
  • the antibody is a monoclonal antibody or a polyclonal antibody, preferably a monoclonal antibody.
  • the antibody or antigen-binding fragment thereof specifically binds to the S protein on the surface of SAR-COV-2.
  • Another aspect of the present invention provides a nucleotide sequence that encodes the aforementioned antibody or antigen-binding fragment thereof.
  • Another aspect of the present invention provides a vector comprising the aforementioned nucleotide sequence.
  • Another aspect of the present invention provides a host cell comprising the aforementioned vector or vector group.
  • the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells or suitable for preparing antibodies Or other cells of antigen-binding fragments thereof.
  • kits comprising the aforementioned antibody or antigen-binding fragment thereof.
  • Another aspect of the present invention provides a detection reagent, which comprises the aforementioned antibody or antigen-binding fragment thereof.
  • Another aspect of the present invention provides the use of the above-mentioned antibody or antigen-binding fragment thereof as a detection reagent, and the reagent is used for the following purposes: enzyme-linked immunosorbent assay (ELISA), western blot (Western Blot), flow cytometry (FACS), immunohistochemistry (IHC) detection or immuno-PCR.
  • ELISA enzyme-linked immunosorbent assay
  • FACS flow cytometry
  • IHC immunohistochemistry
  • the antibody or its antigen-binding fragment can be coupled alone or with a chemical bond, electrostatic adsorption or hydrophobic adsorption, and the linking conjugate includes horseradish peroxidase (HRP), alkaline phosphatase (AP), Biotin (Biotin), Fluorescein isothiocyanate (FITC), Cy3, Cy5, magnetic beads and agarose and other conjugates are connected.
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • Biotin Biotin
  • FITC Fluorescein isothiocyanate
  • Cy3, Cy5 magnetic beads and agarose and other conjugates are connected.
  • the detection reagent can be used for non-diagnostic or therapeutic purposes.
  • Another aspect of the present invention provides a pharmaceutical composition, the antibody or antigen-binding fragment thereof isolated as described above, and pharmaceutically acceptable excipients.
  • the antibody or antigen-binding fragment thereof blocks or reduces the binding of the S protein of SAR-COV-2 to the cell surface receptor of the subject
  • the cell surface receptor is preferably a cell blood vessel Tentensin converting enzyme-related carboxypeptidase (ACE2).
  • Another aspect of the present invention provides the use of an anti-SAR-COV-2 antibody or antigen-binding fragment thereof in the preparation of a medicament for preventing, treating or alleviating at least one symptom or indication of SAR-COV-2 infection.
  • the drug is an oral or injection preparation.
  • Another aspect of the present invention provides a method for preventing, treating or alleviating at least one symptom or indication of SAR-COV-2 infection, the method comprising combining the antibody or antigen-binding fragment of any one of the foregoing or the foregoing drug The composition is administered to the subject.
  • the at least one symptom or indication is selected from the group consisting of lung inflammation, alveolar injury, fever, cough, dyspnea, hypoxemia, acute respiratory distress syndrome, sepsis Symptomatic shock, coagulopathy, metabolic acidosis, nasal congestion, runny nose, sore throat, diarrhea, organ failure, septic shock and death.
  • the pharmaceutical composition or the antibody or antigen-binding fragment thereof is administered in combination with a second therapeutic agent.
  • the second therapeutic agent is selected from the following group: anti-inflammatory drugs (such as corticosteroids and non-steroidal anti-inflammatory drugs), antiviral drugs, different antibodies against the S protein of SAR-COV-2, and for SAR-COV -2 vaccines, antibiotics, dietary supplements such as antioxidants, and any other palliative therapy for the treatment of SAR-COV-2 infection, drugs to relieve the above symptoms or indications.
  • the pharmaceutical composition or the antibody or antigen-binding fragment thereof is administered subcutaneously, intravenously, intracutaneously, intraperitoneally, orally, intramuscularly or intracranially.
  • the anti-SAR-COV-2 antibody of the present invention can target to bind to the S protein of SAR-COV-2 virus, with high specificity, and can effectively block the surface S protein of SAR-COV-2 virus from the subject cells Surface receptor binding.
  • the genes of the fully human antibodies of the present invention are completely derived from human genes and have no components of other species. They do not have toxic and side effects such as anti-mouse and anti-antibodies in the human body, and have better biological characteristics. Capacitive, more suitable and more potential to become a macromolecular drug for the treatment of influenza virus.
  • the single B cell used in the present invention has simple operations to develop antibodies against SAR-COV-2 virus. It is fast, and the human antibody produced has the advantages of high affinity and specificity.
  • Figure 1 is a flow cytometric test result of NTH-3T3 expressing CD40L in Example 1.
  • Fig. 2 is a diagram showing the result of sorting memory B cells by flow cytometry in Example 1.
  • Figure 3 is a graph of the results of the ELISA experiment in Example 1.
  • Figure 4 is a schematic diagram of the amino acid and nucleotide sequences of the heavy chain variable region and the light chain variable region.
  • the term “antibody” refers to a molecule comprising at least one antigen binding site that immunospecifically binds to a specific target antigen target. Therefore, the term “antibody” includes, but is not limited to, full-length antibodies and/or variants thereof, fragments thereof, peptide bodies and variants thereof, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and at least two Multispecific antibodies (such as bispecific antibodies), human antibodies, humanized antibodies, and antibody mimics that mimic the structure and/or function of antibodies or designated fragments or parts thereof, including single-chain antibodies and Fragment.
  • the binding of the antibody to the target can cause a variety of effects, such as but not limited to such binding in vitro, in situ and/or in vivo regulation, reduction, increase, antagonism, agonism, reduction, slowing, blocking, inhibition, elimination and/or Interference with at least one target activity or binding, or receptor activity or binding.
  • the antibodies of the present disclosure encompass antibody fragments capable of binding to biomolecules (such as antigens or receptors) or parts thereof, including but not limited to Fab, Fab' and F(ab')2, pFc', Fd, single domain antibodies (sdAb), variable fragment (Fv), single-chain variable fragment (scFv) or disulfide-linked Fv (sdFv); bifunctional antibody or bivalent bifunctional antibody; linear antibody; single-chain antibody molecule; by antibody Multispecific antibodies formed by fragments.
  • biomolecules such as antigens or receptors
  • Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass.
  • type e.g., IgG, IgE, IgM, IgD, IgA, and IgY
  • class e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2
  • subclass e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2
  • the term "monoclonal antibody” refers to an antibody derived from a group of antibodies of the same species, that is, except for the possibility of a small number of mutations that may occur naturally, each antibody constituting the population is the same. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations containing different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to its specificity, the advantage of monoclonal antibodies is that they can be synthesized without contaminating other antibodies. The modifier "monoclonal" should not be interpreted as requiring the production of antibodies by any specific method.
  • HCDR has the same meaning as the heavy chain complementarity determining region
  • LCDR has the same meaning as the light chain complementarity determining region
  • monoclonal antibodies include "chimeric" antibodies in which a portion of the heavy chain and/or light chain is the same or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific anti-class or subclass, and The rest of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and fragments of these antibodies exhibit the desired biological activity.
  • SAR-COV-2 is also referred to as "new coronavirus”, which refers to a newly-occurring virus that causes new coronavirus pneumonia (COVID-19).
  • the S protein refers to the spike protein on the coronavirus.
  • SARS-CoV-2 recognizes the ACE2 on the cell surface of the human body through the spike protein on the surface of the virus and infects host cells. By blocking the S protein on the surface of the coronavirus SARS-CoV-2, it can effectively inhibit the virus from adhering to the target cell receptor and prevent the virus from invading the cell.
  • humanized antibody as used herein includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human humanized antibody of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or in vitro site-specific mutagenesis or by somatic mutation in vivo).
  • anti-"antigen-binding fragment and the like as used herein includes any naturally-occurring, enzymatically obtainable, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex.
  • antigen-binding fragment of an antibody has one or more fragments that have the ability to bind to the S protein of SAR-COV-2.
  • the present invention provides an anti-SAR-COV-2 fully human monoclonal antibody or a biologically active fragment derived from the monoclonal antibody capable of specifically binding to SAR-COV-2, wherein the heavy and light chain CDR1,
  • the amino acid sequences of CDR2 and CDR3 regions are as follows:
  • Heavy chain CDR1 region GTFFNNYV SEQ ID No: 1;
  • Heavy chain CDR2 region ISYDGSVK SEQ ID No: 2;
  • Heavy chain CDR3 region AKPHVLFWLGEGKGPFDY SEQ ID No: 3;
  • Light chain CDR2 region AAS SEQ ID No: 5;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 7, or an amino acid sequence with equivalent functions is formed by substitution, deletion or addition of one or several amino acids in this sequence;
  • amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 8, or an amino acid sequence with equivalent functions formed by substitution, deletion or addition of one or several amino acids in this sequence
  • the heavy chain amino acid sequence of the antibody is as shown in SEQ ID NO: 6, or the sequence is an amino acid sequence with equivalent functions formed by substitution, deletion or addition of one or several amino acids; and/or
  • the light chain amino acid sequence of the antibody is shown in SEQ ID NO: 8, or an amino acid sequence with equivalent functions formed by substitution, deletion or addition of one or several amino acids in this sequence.
  • the ELISA experiment verified that the anti-SAR-COV-2 fully human monoclonal antibody of the present invention can target and bind the S protein of the SAR-COV-2 virus.
  • the antibody of the present invention is a fully human monoclonal antibody. Compared with murine antibodies, the genes of fully human antibodies are completely derived from human genes, without components of other species, and no anti-mouse anti-antibody occurs in the human body. Toxic and side effects, with better biocompatibility, more suitable and more potential to become a macromolecular drug for the treatment of influenza virus.
  • the present invention provides a gene encoding the anti-SAR-COV-2 fully human monoclonal antibody of the present invention.
  • the gene includes a nucleotide sequence encoding the amino acid shown above.
  • the nucleotide sequence is as follows (the following sequence is only exemplary, and those skilled in the art can design other nucleotide sequences that can be translated into the desired amino acid sequence according to the specific amino acid sequence) :
  • the nucleotide sequence encoding the variable region of the heavy chain is:
  • the nucleotide sequence encoding the variable region of the light chain is:
  • the present invention provides a vector containing the gene as described above.
  • the present invention provides a cell containing the gene as described above or the vector as described above.
  • the present invention provides a method for producing the anti-SAR-COV-2 fully human monoclonal antibody or a biologically active fragment derived from the monoclonal antibody capable of specifically binding to SAR-COV-2, the method Including culturing the above-mentioned gene or genetically engineered cell containing the above-mentioned vector containing the heavy and light chain encoding the anti-SAR-COV-2 fully human monoclonal antibody or directly culturing the above-mentioned cells, collecting and purifying to obtain the anti-SAR-COV-2 fully human Source monoclonal antibodies.
  • the technology is simple and quick to operate, and the human antibodies produced have high affinity and specificity.
  • the improved technology for isolating the monoclonal antibodies of the present invention with functions of neutralizing viruses or killing tumors from memory B cells can be further used. It greatly reduces the tedious operation and cost.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-SAR-COV-2 fully human monoclonal antibody of the present invention or a monoclonal antibody derived from the monoclonal antibody capable of specifically binding to SAR-COV-2 Biologically active fragments.
  • the present invention provides the anti-SAR-COV-2 fully human monoclonal antibody or the biologically active fragment derived from the monoclonal antibody capable of specifically binding to SAR-COV-2 or the pharmaceutical composition Application in the preparation of medicines for treating diseases caused by SAR-COV-2 virus.
  • the present invention provides a kit for detecting the level of SAR-COV-2 virus, which contains the anti-SAR-COV-2 fully human monoclonal antibody of the present invention or a specific monoclonal antibody derived from the monoclonal antibody.
  • the kit further contains a second antibody and an enzyme or fluorescent or radiolabel for detection, and a buffer; the second antibody For example, it is an anti-antibody against the monoclonal antibody of the present invention.
  • lentivirus to establish 3T3-CD40L feeder cells.
  • the lentiviral expression vector pLVX-CD40L was constructed and transfected into 293T cells. The viral supernatant was collected on the fourth day of transfection.
  • Activated NIH-3T3 cells cultured for 3 generations, infected with lentivirus, continued to culture and passage 3 times.
  • Figure 1 is to express CD40L 3T3 cells and 3T3 cells transfected with empty vector pLVX (with ZxGreen) were stained with anti-CD40L with APC, and then analyzed by flow cytometry. It was found that all 3T3-CD40L feeder cells express CD40L. When the cells grow to 80%-90%, digest and collect the cells at a concentration of 1 ⁇ 10 7 cells per milliliter.
  • cryopreservation solution Place it in a radiometer for 5000rads radiation, and resuspend the cells in the cryopreservation solution at a concentration of 3.5 ⁇ 10 7 cells per milliliter. Aliquot 1ml into cryovials and freeze in liquid nitrogen (can be stored for 2 years).
  • the memory B cells were added to the mixed medium, mixed and then diluted in a 384-well plate with 1 cell per well with a volume of 50 ⁇ l, and placed in a 37°C, 5% CO 2 incubator for static culture. After 13 days, the supernatant was taken for ELISA to obtain human monoclonal antibodies.
  • the surface antigen S protein was purchased from Yiqiao Shenzhou Company, which is immunogenic, and the anti-S protein antibody can be SAR-COV-2 influenza virus. Perform ELISA experiments on the human monoclonal antibodies in the supernatant obtained above, specifically:
  • the B cells that can secrete antibodies that bind to the SAR-COV-2 virus obtained in Example 1 were lysed, and the lysate was taken to perform reverse transcription of RNA to obtain the PCR template cDNA of the human antibody gene.
  • Design and synthesize primers for cloning antibody genes use cDNA as template to clone antibody heavy and light chain genes, and send them to Jinweizhi Company for sequencing. specifically:
  • Reverse transcription system 150ng random primer (invitrogen, 48190-011), 0.5 ⁇ l 10mM dNTP (Invitrogen, 18427-088), 1 ⁇ l 0.1M DTT (Invitrogen, 18080-044), 0.5%v/v Igepal CA -630 (Sigma, I3021-50ML), 4U RNAsin (Promega), 6U Prime RNAse Inhibitor (Eppendorf) and 50U III reverse transcriptase (Invitrogen, 18080-044), supplement with DEPC water to 14 ⁇ l/well.
  • KOD-Plus-Neo (TOYOBO, KOD401) kit PCR to amplify the heavy and light chains of antibody genes, 40 ⁇ L system: 3.5 ⁇ L cDNA, 20nM mixed primers, 4 ⁇ L buffer, 4 ⁇ L 2mM dNTPs , 2.4 ⁇ L MgSO 4 , 1 ⁇ L KOD.
  • amino acid sequence of the variable region of the antibody gene heavy chain is shown in SEQ ID No: 7
  • the sequencing result of the PCR product of the variable region of the antibody gene light chain is as shown in SEQ ID No: 10.
  • amino acid sequence of the variable region of the light chain of the antibody gene is shown in SEQ ID No: 8;
  • HCDR1 GTFFNNYV SEQ ID No: 1;
  • HCDR2 ISYDGSVK SEQ ID No: 2;
  • HCDR3 AKPHVLFWLGEGKGPFDY SEQ ID No: 3;
  • LCDR1 QGISSY SEQ ID No: 4;

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Virology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Genetics & Genomics (AREA)
  • Pulmonology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Cardiology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Heart & Thoracic Surgery (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Epidemiology (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Otolaryngology (AREA)
  • Obesity (AREA)
  • Diabetes (AREA)

Abstract

本发明提供一种抗SAR-COV-2抗体或其抗原结合片段及其应用，其具有SEQ ID No：1-3所示的重链互补决定区，以及SEQ ID No：4-6所示的轻链互补决定区。本发明的抗体为人源化抗体，副作用低，亲和力和特异性高。

Description

抗SAR-COV-2(COVID-19)全人源单克隆抗体及其制法与应用 技术领域
本发明属于免疫学领域，具体地涉及抗SAR-COV-2(COVID-19)全人源单克隆抗体及其制法与应用。
背景技术
在2018年全球十大畅销药中，有8个是全人源或人源化单克隆抗体药物。排名第一的是艾伯维公司治疗关节炎的抗TNFa单克隆抗体Humira，这是一个全人源单克隆抗体，已经是连续6年销售额100亿以上的药王。从1986年第一个单克隆抗体药物上市开始，单抗药物经历了鼠源单抗药物(如Orthoclone OKT3)、嵌合单抗药物(Rituximab)、人源化单抗药物(Herceptin)和全人源单抗药物(Humira)等阶段。由于人体出现抗鼠抗体反应(HAMA)，鼠源单抗药物、嵌合单抗药物已经逐渐被淘汰，目前占据市场的单克隆抗体药物全都是人源化单克隆抗体药物。与国际先进的人源抗体生产技术相比，深圳乃至全中国都有很大差距，主要表现在人源抗体药物领域的创新能力薄弱，自主研发的品种少，目前还没有原创人源化单克隆抗体药物上市的报道，庞大的抗体药物市场被国外药企占领。我国要改变落后局面，争夺消费潜力巨大的国内外抗体药物市场，亟需攻克全人源单克隆抗体技术。
人源单克隆抗体在治疗炎症、癌症、流行性感冒特别是冠状病毒等方面具有高特异性的显著疗效。COVID-19是由一种SAR-COV-2冠状病毒引起的急性呼吸道传染病，至今仍然缺乏有效的药物和疫苗。新冠状病毒在入侵细胞时需要依赖病毒自身表达的特定分子与人细胞上的受体结合，才能感染细胞，并进一步扩增。中和病毒的人源抗体是人B淋巴细胞产生的某些特异抗体，能够与病毒表面的抗原结合，从而阻止该病毒黏附靶细胞受体，防止病毒侵入细胞，能够高效防治SAR-COV-2流行性感冒。
发明内容
为解决上述问题，本发明提供一种抗SAR-COV-2的抗体或其抗原结合片段，其特异性中和SAR-COV-2。
为解决上述问题，本发明提供一种抗SAR-COV-2的抗体或其抗原结合片段，其特异性中和SAR-COV-2。
本发明一方面提供一种分离的抗SAR-COV-2的抗体或其抗原结合片段；其具有如下任一组的三个重链可变区的互补决定区(HCDR)和三个轻链可变区的互补决定区(LCDR)：
HCDR1：GFTFNNYV SEQ ID No：1；
HCDR2：ISYDGSVK SEQ ID No：2；
HCDR3：AKPHVLFWLGEGKGPFDY SEQ ID No：3；
LCDR1：QGISSY SEQ ID No：4；
LCDR2：AAS SEQ ID No：5；
LCDR3：QQLNSYPLT SEQ ID No：6。
本发明另一个方面提供了一种分离的抗SAR-COV-2的抗体或其抗原结合片段，其具有如下所示的重链可变区和轻链可变区；
重链可变区：
Figure PCTCN2020129881-appb-000001
轻链可变区：
Figure PCTCN2020129881-appb-000002
在本发明的技术方案中，所述的抗体或其抗原结合片段为人源化抗体，更优选为全人源化抗体。
在本发明的技术方案中，所述的抗体或其抗原结合片段特异性结合SAR-COV-2表面S蛋白。
在本发明的技术方案中，所述的抗体为单克隆抗体或多克隆抗体，优选为单克隆抗体。
在本发明的技术方案中，所述的抗体或其抗原结合片段特异性结合SAR-COV-2表面S蛋白。
本发明再一个方面提供了一种核苷酸序列，其编码如前述的抗体或其抗原结合片段。
本发明再一个方面提供了一种载体，包含前述的核苷酸序列。
本发明再一个方面提供了一种宿主细胞，包含前述载体或载体组，优选地，所述宿主细胞是原核的或真核的，更优选的选自酵母细胞、哺乳动物细胞或适用于制备抗体或其抗原结合片段的其它细胞。
本发明再一个方面提供了一种试剂盒，所述试剂盒包含如前述的抗体或其抗原结合片段。
本发明再一个方面提供了一种检测试剂，所述检测试剂包含如前述的抗体或其抗原结合片段。
本发明再一个方面提供了上述抗体或其抗原结合片段作为检测试剂的用途，所述试剂用 于以下用途的试剂：酶联免疫吸附检测(ELISA)、免疫印迹(Western Blot)、流式细胞术(FACS)、免疫组织化学(IHC)检测或者免疫PCR。
在上述在免疫学检测中，抗体或其抗原结合片段可单独或与通过化学键偶联，静电吸附或者亲疏水性吸附，而连接缀合物包括辣根过氧化物酶(HRP)，碱性磷酸酶(AP)，生物素(Biotin)，异硫氰酸荧光素(FITC)，Cy3、Cy5、磁珠和琼脂糖等缀合物连接。
在本发明的技术方案中，检测试剂可用于非诊断或治疗目的检测。
本发明再一个方面提供了一种药物组合物，其如前所述的分离的抗体或其抗原结合片段和药物上可接受辅料。
在本发明的技术方案中，其中所述抗体或其抗原结合片段阻断或降低SAR-COV-2的S蛋白与受试者的细胞表面受体结合，所述细胞表面受体优选为细胞血管紧张素转化酶相关性羧肽酶(ACE2)。
本发明再一个方面提供了一种抗SAR-COV-2的抗体或其抗原结合片段在制备预防、治疗或缓解SAR-COV-2感染的至少一种症状或适应症的药物中的用途。
在本发明的技术方案中，所述的药物为口服或注射制剂。
本发明再一个方面提供了一种预防、治疗或缓解SAR-COV-2感染的至少一种症状或适应症的方法，所述方法包括将前述任一项的抗体或其抗原结合片段或前述药物组合物施用于受试者。
在本发明的技术方案中，其中所述至少一种症状或适应症选自下组：肺部炎症、肺泡损伤、发热、咳嗽、呼吸困难、低血氧症、急性呼吸窘迫综合征、脓毒症休克、凝血功能障碍、代谢性酸中毒、鼻塞、流涕、咽痛、腹泻、器官衰竭、败血性休克和死亡。
在本发明的技术方案中，其中所述药物组合物或所述抗体或其抗原结合片段与第二治疗剂组合施用。其中所述第二治疗剂选自下组：抗炎症药物(如皮质类固醇和非甾体抗炎症药物)、抗病毒药物、针对SAR-COV-2的S蛋白的不同的抗体、对于SAR-COV-2的疫苗、抗生素、膳食补充剂如抗氧化剂和治疗SAR-COV-2感染的任何其他姑息疗法、缓解上述症状或适应症的药物。
在本发明的技术方案中，其中所述药物组合物或所述抗体或其抗原结合片段通过皮下、静脉内、皮内、腹膜内、口服、肌内或颅内施用。
有益效果
(1)本发明所述抗SAR-COV-2抗体可以靶向结合SAR-COV-2病毒的S蛋白，特异性高，能有效阻断SAR-COV-2病毒表面S蛋白与受试者细胞表面受体的结合。
(2)相比鼠源抗体，本发明全人源抗体的基因完全来源于人的基因，没有其他种属的成分，在人体内不发生抗鼠抗抗体等毒副作用，具有更好的生物相容性，更适合和更有潜力成为治疗流感病毒的大分子药物。
(3)相较于现有技术提供的噬菌体展示技术制备抗SAR-COV-2病毒人源单克隆抗体的方法，本发明采用的单个B细胞开发抗SAR-COV-2病毒的抗体具有操作简单快捷，生产的人源抗体具有高亲和力和特异性等优点。
附图说明
图1为实施例1 NTH-3T3表达CD40L的流式检测结果图。
图2为实施例1流式细胞仪分选记忆B细胞结果图。
图3为实施例1 ELISA实验结果图。
图4为重链可变区与轻链可变区的氨基酸和核苷酸序列示意图。
具体实施方式
为了使本发明的上述目的、特征和优点能够更加明显易懂，下面对本发明的具体实施方式做详细的说明，但不能理解为对本发明的可实施范围的限定。
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现结合具体实施例对本发明的技术方案进行以下详细说明，应理解这些实例仅用于说明本发明而不用于限制本发明的范围。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
如本文所用，术语“抗体”是指包含至少一个抗原结合位点的分子，所述抗原结合位点免疫特异性地结合至特定的目标抗原靶标。因此，术语“抗体”包括但不限于全长抗体和/或其变体，其片段，肽体及其变体，单克隆抗体(包括全长单克隆抗体)、多克隆抗体、由至少两个完整的抗体形成的多特异性抗体(例如双特异性抗体)、人抗体、人源化抗体和模拟抗体的结构和/或功能的抗体模拟物或其指定片段或部分，包括单链抗体及其片段。抗体与靶标的结合可引起多种作用，例如但不限于这种结合在体外、原位和/或体内调节、减少、增加、拮抗、激动、减轻、减缓、阻断、抑制、消除和/或干扰至少一种靶标活性或结合，或受体活性或结合。因此，本公开的抗体涵盖能够结合生物分子(例如抗原或受体)或其部分的抗体片段，包括但不限于Fab、Fab'和F(ab')2、pFc'、Fd、单结构域抗体(sdAb)、可变片段(Fv)、单链可变片段(scFv)或二硫化物连接的Fv(sdFv)；双功能抗体或二价双功能抗体；线性抗体；单链抗 体分子；由抗体片段形成的多特异性抗体。抗体可以是任何类型(例如IgG、IgE、IgM、IgD、IgA和IgY)，类别(例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)或亚类。
如本文所用，术语“单克隆抗体”是指得自一群基本上同种的抗体的抗体，即除了可能存在少量可能天然发生的突变之外，构成群体的各个抗体是相同的。单克隆抗体是高度特异性的，针对单个抗原位点。此外，与包含针对不同决定簇(表位)的不同抗体的多克隆抗体制备物相反，每个单克隆抗体针对抗原上的单一决定簇。除了其特异性之外，单克隆抗体的优点还在于其可以未污染其它抗体而合成。修饰语“单克隆”不应解释为需要通过任何特定方法产生抗体。
如本文所用，术语HCDR与重链互补决定区含义相同，LCDR与轻链互补决定区含义相同。
如本文所用，单克隆抗体包括“嵌合”抗体，其中重链和/或轻链的一部分与衍生自特定物种或属于特定抗类别或亚类的抗体中的相应序列相同或同源，而所述链剩余的与衍生自另一物种或属于另一抗体类别或亚类的抗体中的相应序列相同或同源，且这些抗体的片段，表现出所需的生物活性。
如本文所用，术语“SAR-COV-2”也称作“新型冠状病毒”，指新发生的引起新型冠状病毒肺炎(COVID-19)的病毒。
如本文所用，S蛋白指冠状病毒上的刺突蛋白(Spike protein)，SARS-CoV-2通过病毒表面的刺突蛋白识别人体内细胞表面的ACE2并侵染宿主细胞。通过阻断冠状病毒SARS-CoV-2表面的S蛋白可以有效抑制病毒黏附靶细胞受体，防止病毒侵入细胞。
如本文使用的术语"人源化抗体"包括具有源自人种系免疫球蛋白序列的可变和恒定区的抗体。本发明的人人源化抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(例如由随机或体外位点特异性诱变或通过体内体细胞突变引入的突变)。
如本文使用的术语抗"抗原结合片段"等包括任何天然存在的、酶学可获得的、合成的或基因工程的特异性结合抗原以形成复合物的多肽或糖蛋白。如本文使用的术语抗体的"抗原结合片段"与SAR-COV-2的S蛋白具有结合能力的一个或多个片段。
一方面，本发明提供抗SAR-COV-2全人源单克隆抗体或来源于该单克隆抗体的能够特异性结合SAR-COV-2的生物活性片段，其中，该抗体的重轻链CDR1、CDR2及CDR3区的氨基酸序列分别如下所示：
重链CDR1区：GFTFNNYV SEQ ID No：1；
重链CDR2区：ISYDGSVK SEQ ID No：2；
重链CDR3区：AKPHVLFWLGEGKGPFDY SEQ ID No：3；
轻链CDR1区：QGISSY SEQ ID No：4；
轻链CDR2区：AAS SEQ ID No：5；
轻链CDR3区：QQLNSYPLT SEQ ID No：6。
在一些实施方式中，该抗体的重链可变区氨基酸序列如SEQ ID NO:7所示，或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列；
Figure PCTCN2020129881-appb-000003
和/或
该抗体的轻链可变区氨基酸序列如SEQ ID NO:8所示，或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列
Figure PCTCN2020129881-appb-000004
在一些实施方式中，该抗体的重链氨基酸序列如SEQ ID NO:6所示，或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列；和/或
该抗体的轻链氨基酸序列如SEQ ID NO:8所示，或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列。
经ELISA实验验证，本发明所述抗SAR-COV-2全人源单克隆抗体可以靶向结合SAR-COV-2病毒的S蛋白。本发明所述抗体为全人源性单克隆抗体，相比鼠源抗体，全人源抗体的基因完全来源于人的基因，没有其他种属的成分，在人体内不发生抗鼠抗抗体等毒副作用，具有更好的生物相容性，更适合和更有潜力成为治疗流感病毒的大分子药物。
另一方面，本发明提供编码本发明所述的抗SAR-COV-2全人源单克隆抗体的基因。在一些实施方式中，所述基因包含编码具有上述所示的氨基酸的核苷酸序列。
在一些具体实施方式中，，该核苷酸序列如下所示(以下序列仅为示例性，本领域技术人员根据具体的氨基酸序列，可以设计其他可以翻译为所需氨基酸序列的核苷酸序列)：
编码重链可变区的核苷酸序列为：
Figure PCTCN2020129881-appb-000005
编码轻链可变区的核苷酸序列为：
Figure PCTCN2020129881-appb-000006
本发明抗体的重链可变区和轻链可变区序列示意图见图4，其中灰色框中的显示的是CDR区。
另一方面，本发明提供含如上所述基因的载体。
再一方面，本发明提供含如上所述基因或如上所述载体的细胞。
再一方面，本发明提供一种产生所述抗SAR-COV-2全人源单克隆抗体或来源于该单克隆抗体的能够特异性结合SAR-COV-2的生物活性片段的方法，该方法包括培养含编码抗SAR-COV-2全人源单克隆抗体的重轻链的上述基因或上述载体的基因工程细胞或直接培养上述细胞，收集，纯化得所述抗SAR-COV-2全人源单克隆抗体。
现有技术中存在采用噬菌体展示技术制备抗SAR-COV-2病毒人源单克隆抗体的方法，尽管该方法具有生产成本低、不经过免疫和细胞融合等繁琐工作的优点，但是其缺点也比较明显，从非免疫抗体库中获得的抗体往往亲和力不足、受外源基因转化率的限制、抗体库的库容量不足以涵盖动物的抗体多样性等。本发明从病人的血液中分离分泌功能抗体的B细胞，然后提取RNA和合成cDNA，从中克隆分泌目的抗体的基因，最后重组和表达全人源单克隆抗体。该技术操作简单快捷，生产的人源抗体具有高亲和力和特异性，此外，可进一步采用改进的从记忆B细胞中分离具有中和病毒功能或杀伤肿瘤功能的本发明所述单克隆抗体技术，更是大大减少了繁琐操作和成本。
另一方面，本发明提供一种药物组合物，其包含本发明所述的抗SAR-COV-2全人源单克隆抗体或来源于该单克隆抗体的能够特异性结合SAR-COV-2的生物活性片段。
另一方面，本发明提供所述的抗SAR-COV-2全人源单克隆抗体或来源于该单克隆抗体的能够特异性结合SAR-COV-2的生物活性片段或所述的药物组合物在制备用于治疗由SAR-COV-2病毒引起的疾病的药物中的应用。
另一方面，本发明提供一种检测SAR-COV-2病毒水平的试剂盒，其含有本发明所述的抗SAR-COV-2全人源单克隆抗体或来源于该单克隆抗体的能够特异性结合SAR-COV-2的生物活性片段；在一些实施方式中，所述的试剂盒还含有第二抗体和用于检测的酶或荧光或放射标记物，以及缓冲液；所述第二抗体例如为抗本发明所述单克隆抗体的抗抗体。
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现结合具体实施例对本 发明的技术方案进行以下详细说明，应理解这些实例仅用于说明本发明而不用于限制本发明的范围。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
实施例1
(1)构建稳定表达CD40L的NTH-3T3细胞系(3T3-CD40L)
利用慢病毒建立3T3-CD40L饲养细胞。构建慢病毒表达载体pLVX-CD40L，转染293T细胞，转染第四天收集病毒上清液。活化NIH-3T3细胞，培养3代后用慢病毒感染，继续培养并传代3次。利用流式细胞仪进行分选FITC荧光强度在MFI附近的细胞，重新加入至培养瓶中，37℃，5％CO 2培养箱中培养和检测，检测结果如图1所示，其是将表达CD40L的3T3细胞和空载体pLVX(带有ZxGreen)转染的3T3细胞分别用带有APC的抗CD40L染色，然后上流式细胞仪分析。结果发现，所有3T3-CD40L饲养细胞都表达CD40L。当细胞长到80％～90％时，消化收集细胞，浓度为每毫升1×10 7细胞。置于辐射仪中进行5000rads辐射，冻存液重悬细胞，浓度为每毫升3.5×10 7细胞，分装1ml在冷冻小管，液氮冻存(可以保存2年)。
(2)记忆B细胞的分选和活化
用淋巴分离液分离和冻存曾经感染SAR-COV-2病毒的康复病人的PBMC，每管10～50×10 6细胞，冻存在液氮罐中。配制PBMC流式染色液，其成分如下表1所示
表1 PBMC流式染色液
抗体 体积(μL)
CD19-PE-Cy7 0.5
IgM-PE 1.0
IgA-APC 2.5
IgD-FITC 2.5
PBS-1％(wt/vol)BSA 43.5
解冻PBMC，加入上述PBMC流式染色液并在流式细胞仪上分选，结果如图2所示，分选出CD19 +IgM –IgA –IgD –的记忆B细胞，细胞纯度需在90％以上，若低于90％，重复分选过程。配制激活B细胞的混合培养基，如下表2所示：
表2
组分 体积
完全IMDM培养基 336mL
IL-2(10,000U mL -1) 3.5mL
IL-21(100μg mL -1) 175μL
步骤(1)中得到的3T3-CD40L 10mL
将记忆B细胞加入到混合培养基中，混匀后有限稀释在384孔板，每孔1个细胞，体积为50μl，置于37℃，5％CO 2培养箱中静置培养。13天后，取上清液进行ELISA，获得人源单克隆抗体。
(3)人源单克隆抗体结合SAR-COV-2病毒的表面抗原S蛋白实验
表面抗原S蛋白购自义翘神州公司，具有免疫原性，抗S蛋白抗体可以SAR-COV-2流感病毒。对上述获得的上清液人源单克隆抗体进行ELISA实验，具体地：
(1)将100ng/100μl的SAR-COV-2病毒的HA蛋白包被在96孔酶标板中，每孔100μl；
(2)放置4℃冰箱过夜；
(3)用PBST溶液洗涤三遍，每孔加5％的脱脂奶粉溶液200μl，37℃孵育1小时；
(4)用PBST溶液洗涤三遍，加100μl没有感染病毒的正常人血清(阴性对照)或上清液，各三个重复；
(5)37℃孵育1小时后用PBST溶液洗涤三遍；
(6)以1:5000稀释带HRP的抗人IgG抗体(abcam)，加入酶标版中，每孔100μl；
(7)37℃孵育1小时后用PBST溶液洗涤三遍；
(8)每孔加100μl TMB底物溶液(Thermo Scientific)，37℃5分钟；
(9)每孔加终止溶液2M硫酸100μl，立刻在酶标仪中450nm波长检测吸光值。其结果如图3所示，ELISA实验表明本发明获得的人源单克隆抗体可以靶向结合SAR-COV-2病毒的S蛋白。
实施例2人源化单克隆抗体基因的克隆
将实施例1获得的能够分泌结合SAR-COV-2病毒的抗体的B细胞进行裂解，取裂解液进行RNA的反转录，获得人源抗体基因的PCR模板cDNA。设计和合成克隆抗体基因的引物，以cDNA为模板克隆抗体的重链和轻链的基因，并且送金唯智公司测序。具体地：
(1)将裂解后的B细胞液转移至96孔板(Eppendorf，030133366)。
(2)反转录体系：150ng随机引物(invitrogen,48190-011),0.5μl 10mM dNTP(Invitrogen,18427-088),1μl 0.1M DTT(Invitrogen,18080-044),0.5％v/v Igepal CA-630(Sigma,I3021-50ML),4U RNAsin(Promega),6U Prime RNAse Inhibitor(Eppendorf)and 50U
Figure PCTCN2020129881-appb-000007
III reverse transcriptase(Invitrogen,18080-044)，补DEPC水至14μl/well。
(3)反转录反应程序：42℃，10min；25℃，10min；50℃，60min；94℃，5min。
(4)cDNA保存在-20℃。
(5)引物的设计和合成:
正向引物5′-3′序列(Forward Primer 5′-3′sequence)
重链可变区PCR引物:
5′VH1 CTGCAACCGGTGTACATTCCCAGGTGCAGCTGGTGCAG(SEQ ID NO:11)
5′VH1/5 CTGCAACCGGTGTACATTCCGAGGTGCAGCTGGTGCAG(SEQ ID NO:12)
5′VH3 CTGCAACCGGTGTACATTCTGAGGTGCAGCTGGTGGAG(SEQ ID NO:13)
5′VH3-23 CTGCAACCGGTGTACATTCTGAGGTGCAGCTGTTGGAG(SEQ ID NO:14)
5′VH4 CTGCAACCGGTGTACATTCCCAGGTGCAGCTGCAGGAG(SEQ ID NO:15)
5′VH 4-34 CTGCAACCGGTGTACATTCCCAGGTGCAGCTACAGCAGTG(SEQ ID NO:16)
5′VH 1-18 CTGCAACCGGTGTACATTCCCAGGTTCAGCTGGTGCAG(SEQ ID NO:17)
5′VH 1-24 CTGCAACCGGTGTACATTCCCAGGTCCAGCTGGTACAG(SEQ ID NO:18)
5′VH3-33 CTGCAACCGGTGTACATTCTCAGGTGCAGCTGGTGGAG(SEQ ID NO:19)
5′VH 3-9 CTGCAACCGGTGTACATTCTGAAGTGCAGCTGGTGGAG(SEQ ID NO:20)
5′VH4-39 CTGCAACCGGTGTACATTCCCAGCTGCAGCTGCAGGAG(SEQ ID NO:21)
5′VH 6-1 CTGCAACCGGTGTACATTCCCAGGTACAGCTGCAGCAG(SEQ ID NO:22)
3′JH 1/2/4/5 TGCGAAGTCGACGCTGAGGAGACGGTGACCAG(SEQ ID NO:23)
3′JH 3 TGCGAAGTCGACGCTGAAGAGACGGTGACCATTG(SEQ ID NO:24)
3′JH 6 TGCGAAGTCGACGCTGAGGAGACGGTGACCGTG(SEQ ID NO:25)κ轻链可变区PCR产物
5′Vκ1-5 CTGCAACCGGTGTACATTCTGACATCCAGATGACCCAGTC(SEQ ID NO:26)
5′Vκ1-9 TTGTGCTGCAACCGGTGTACATTCAGACATCCAGTTGACCC AGTCT(SEQ ID NO:27)
5′Vκ1D-43 CTGCAACCGGTGTACATTGTGCCATCCGGATGACCCAGTC(SEQ ID NO:28)
5′Vκ2-24 CTGCAACCGGTGTACATGGGGATATTGTGATGACCCAGAC(SEQ ID NO:29)
5′Vκ2-28 CTGCAACCGGTGTACATGGGGATATTGTGATGACTCAGTC(SEQ ID  NO:30)
5′Vκ2-30 CTGCAACCGGTGTACATGGGGATGTTGTGATGACTCAGTC(SEQ ID NO:31)
5′Vκ3-11 TTGTGCTGCAACCGGTGTACATTCAGAAATTGTGTTGACAC AGTC(SEQ ID NO:32)
5′Vκ3-15 CTGCAACCGGTGTACATTCAGAAATAGTGATGACGCAGTC(SEQ ID NO:33)
5′Vκ3-20 TTGTGCTGCAACCGGTGTACATTCAGAAATTGTGTTGACG CAGTCT(SEQ ID NO:34)
5′Vκ4-1 CTGCAACCGGTGTACATTCGGACATCGTGATGACCCAGTC(SEQ ID NO:35)
3′Jκ1/4 GCCACCGTACGTTTGATYTCCACCTTGGTC(SEQ ID NO:36)
3′Jκ2 GCCACCGTACGTTTGATCTCCAGCTTGGTC(SEQ ID NO:37)
3′Jκ3 GCCACCGTACGTTTGATATCCACTTTGGTC(SEQ ID NO:38)
3′Jκ5 GCCACCGTACGTTTAATCTCCAGTCGTGTC(SEQ ID NO:39)
(6)用KOD-Plus-Neo(TOYOBO，KOD401)试剂盒PCR分别扩增抗体基因的重链和轻链，40μL体系：3.5μL cDNA，20nM混合引物，4μL缓冲液(buffer)，4μL 2mM dNTPs，2.4μL MgSO 4，1μL KOD。
(7)反应程序：94℃，2min；45个循环：98℃,10s；58℃,30s；68℃,28s。
(8)对扩增产物进行琼脂糖凝胶，结果发现，抗体轻链为，大小为，重链大小是。
(9)抗体基因重链可变区PCR产物测序结果如SEQ ID No:9所示序列：
Figure PCTCN2020129881-appb-000008
抗体基因重链可变区氨基酸序列如SEQ ID No:7所示序列
Figure PCTCN2020129881-appb-000009
抗体基因轻链可变区PCR产物测序结果如SEQ ID No:10所示序列，
Figure PCTCN2020129881-appb-000010
抗体基因轻链可变区的氨基酸序列如SEQ ID No:8所示序列；
Figure PCTCN2020129881-appb-000011
对应地，其CDR区序列如下所示：
HCDR1：GFTFNNYV SEQ ID No：1；
HCDR2：ISYDGSVK SEQ ID No：2；
HCDR3：AKPHVLFWLGEGKGPFDY SEQ ID No：3；
LCDR1：QGISSY SEQ ID No：4；
LCDR:2：AAS SEQ ID No：5；
LCDR3：QQLNSYPLT SEQ ID No：6
上述结果显示上清液中含有能够结合SAR-COV-2病毒的抗体。
最后说明的是：以上实施例仅用于说明本发明的实施过程和特点，而非限制本发明的技术方案，尽管参照上述实施例对本发明进行了详细说明，本领域的普通技术人员应当理解：依然可以对本发明进行修改或者等同替换，而不脱离本发明的精神和范围的任何修改或局部替换，均应涵盖在本发明的保护范围当中。

Claims (11)

  1. 一种分离的抗SAR-COV-2的抗体或其抗原结合片段，其具有如下任一组的三个重链互补决定区(HCDR)和三个轻链互补决定区(LCDR)：
    HCDR1：GFTFNNYV SEQ ID No：1；
    HCDR2：ISYDGSVK SEQ ID No：2；
    HCDR3：AKPHVLFWLGEGKGPFDY SEQ ID No：3；
    LCDR1：QGISSY SEQ ID No：4；
    LCDR2：AAS SEQ ID No：5；
    LCDR3：QQLNSYPLT SEQ ID No：6。
  2. 根据权利要求1所述的分离的抗SAR-COV-2的抗体或其抗原结合片段，其具有如下所示的重链可变区和轻链可变区；
    重链可变区：
    QVQLVESGGGVVQPGTSLRLSCAASGFTFNNYVMHWVRQAPGKGLEWVAVISYDGSVKYYADSVKGRFTISRDHSKNTLYLHMNSLRPEDTAVYYCAKPHVLFWLGEGKGPFDYWGQGTLVTVSS SEQ ID No：7
    轻链可变区：
    DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYPLTFGPGTRVDIKR SEQ ID No：8。
  3. 根据权利要求1或权利要求2任一项所述的抗体或其抗原结合片段，所述的抗体或其抗原结合片段为人源化的抗体或其抗原结合片段。
  4. 一种核苷酸序列，其特征在于：其编码如权利要求1-3任一项所述的抗体或其抗原结合片段。
  5. 一种载体，其特征在于：包含权利要求4所述的核苷酸序列。
  6. 一种宿主细胞，其特征在于：包含权利要求5所述载体。
  7. 一种试剂盒，所述试剂盒包含如权利要求1-3任一项所述的抗体或其抗原结合片段。
  8. 一种检测试剂，所述检测试剂包含如权利要求1-3任一项所述的抗体或其抗原结合片 段。
  9. 如权利要求1-3任一项所述的抗体或其抗原结合片段作为检测试剂的用途，所述试剂用于以下用途的试剂：酶联免疫吸附检测、免疫印迹、流式细胞术、免疫组织化学检测或者免疫PCR。
  10. 一种药物组合物，其如权利要求1-3任一项所述的分离的抗体或其抗原结合片段和药物上可接受辅料。
  11. 权利要求1-3任一项所述的抗SAR-COV-2的抗体或其抗原结合片段，或权利要求10所述的药物组合物在制备预防、治疗或缓解SAR-COV-2感染的至少一种症状或适应症的药物中的用途；
    优选地，所述至少一种症状或适应症选自下组：新型冠状病毒肺炎、肺部炎症、肺泡损伤、发热、咳嗽、呼吸困难、低血氧症、急性呼吸窘迫综合征、脓毒症休克、凝血功能障碍、代谢性酸中毒、鼻塞、流涕、咽痛、腹泻、器官衰竭、败血性休克和死亡。
PCT/CN2020/129881 2020-06-22 2020-11-18 抗sar-cov-2(covid-19)全人源单克隆抗体及其制法与应用 WO2021258625A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010574813.7 2020-06-22
CN202010574813.7A CN113896788B (zh) 2020-06-22 2020-06-22 抗sar-cov-2（covid-19）全人源单克隆抗体及其制法与应用

Publications (1)

Publication Number Publication Date
WO2021258625A1 true WO2021258625A1 (zh) 2021-12-30

Family

ID=79186506

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/129881 WO2021258625A1 (zh) 2020-06-22 2020-11-18 抗sar-cov-2(covid-19)全人源单克隆抗体及其制法与应用

Country Status (2)

Country Link
CN (1) CN113896788B (zh)
WO (1) WO2021258625A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114397453B (zh) * 2022-03-25 2022-06-07 江苏美克医学技术有限公司 新型冠状病毒突变株的检测试剂盒及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111153991A (zh) * 2020-02-26 2020-05-15 北京博奥森生物技术有限公司 一种人SARS-CoV-2单克隆抗体及其制备方法和应用
CN111303279A (zh) * 2020-03-17 2020-06-19 中国医学科学院病原生物学研究所 一种针对新型冠状病毒的单域抗体及其应用
CN111303280A (zh) * 2020-03-22 2020-06-19 中国人民解放军军事科学院军事医学研究院 高中和活性抗SARS-CoV-2全人源单克隆抗体及应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109957012B (zh) * 2017-12-14 2021-04-09 中国科学院深圳先进技术研究院 抗h7n9全人源单克隆抗体8e17及其制法与应用
GB202003632D0 (en) * 2020-03-12 2020-04-29 Harbour Antibodies Bv SARS-Cov-2 (SARS2, COVID-19) antibodies

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111153991A (zh) * 2020-02-26 2020-05-15 北京博奥森生物技术有限公司 一种人SARS-CoV-2单克隆抗体及其制备方法和应用
CN111303279A (zh) * 2020-03-17 2020-06-19 中国医学科学院病原生物学研究所 一种针对新型冠状病毒的单域抗体及其应用
CN111303280A (zh) * 2020-03-22 2020-06-19 中国人民解放军军事科学院军事医学研究院 高中和活性抗SARS-CoV-2全人源单克隆抗体及应用

Also Published As

Publication number Publication date
CN113896788B (zh) 2023-05-23
CN113896788A (zh) 2022-01-07

Similar Documents

Publication Publication Date Title
CN111620945B (zh) 一种抗新型冠状病毒的单克隆抗体或其衍生体
CN101374944B (zh) 用于获得永生化抗体分泌细胞的方法
JP7235256B2 (ja) 重症熱性血小板減少症候群ウイルスの外膜糖タンパク質に結合する抗体及びその用途
JP5814790B2 (ja) デングウイルス中和抗体およびその使用
TWI733695B (zh) 治療發炎性疾病之方法
CN112574300B (zh) 抗sar-cov-2全人源单克隆抗体及其制法与应用
CN113853387A (zh) 能结合胸腺基质淋巴细胞生成素的抗体及其应用
WO2022143816A1 (zh) 针对SARS-CoV-2和SARS-CoV的全人广谱交叉中和抗体及其应用
WO2022228183A1 (zh) 抗siglec15抗体及其制备方法和用途
WO2020119663A1 (zh) 抗h7n9全人源单克隆抗体4e18及其制法与应用
WO2020019222A2 (zh) 抗H7N9全人源单克隆抗体hIg311及其制法与应用
WO2021258625A1 (zh) 抗sar-cov-2(covid-19)全人源单克隆抗体及其制法与应用
WO2021248276A1 (zh) 一种抗sar-cov-2抗体或其抗原结合片段及其应用
WO2021088904A1 (zh) 抗人程序性死亡配体-1(pd-l1)的抗体及其用途
WO2020119664A1 (zh) 抗h7n9全人源单克隆抗体2g3及其制法与应用
WO2020119666A1 (zh) 抗h7n9全人源单克隆抗体3f12及其制法与应用
WO2023092739A1 (zh) 抗sar-cov-2全人源单克隆抗体6g18及其制法与应用
WO2023070310A1 (zh) 抗sar-cov-2全人源单克隆抗体及其制法与应用
WO2019011167A1 (zh) 双特异性重组蛋白
CN109957012B (zh) 抗h7n9全人源单克隆抗体8e17及其制法与应用
CN114573690B (zh) 抗sar-cov-2全人源单克隆抗体及其制法与应用
CN113831409B (zh) 一种抗sar-cov-2抗体或其抗原结合片段及其应用
CN116023475A (zh) 抗sar-cov-2全人源单克隆抗体及其制法与应用
CN107459575B (zh) 具有抑制病原体的免疫抑制功能的单克隆抗体，其抗原结合片段，以及产生该抗体的杂交瘤
CN115724954A (zh) 抗SARS-CoV-2病毒的单克隆抗体6G21H及其制备方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20942344

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20942344

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC