WO2021212785A1 - 一种全人源单克隆抗体及其应用 - Google Patents

一种全人源单克隆抗体及其应用 Download PDF

Info

Publication number
WO2021212785A1
WO2021212785A1 PCT/CN2020/122793 CN2020122793W WO2021212785A1 WO 2021212785 A1 WO2021212785 A1 WO 2021212785A1 CN 2020122793 W CN2020122793 W CN 2020122793W WO 2021212785 A1 WO2021212785 A1 WO 2021212785A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
seq
cov
sars
variable region
Prior art date
Application number
PCT/CN2020/122793
Other languages
English (en)
French (fr)
Inventor
桂勋
王双
王荣娟
焦莎莎
蒋雯
陈奔
顾春英
Original Assignee
迈威(上海)生物科技股份有限公司
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 迈威(上海)生物科技股份有限公司 filed Critical 迈威(上海)生物科技股份有限公司
Publication of WO2021212785A1 publication Critical patent/WO2021212785A1/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
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • 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
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • 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]

Definitions

  • the present invention belongs to the field of antibody engineering, and specifically relates to a monoclonal antibody against coronavirus and its application, in particular to a human monoclonal antibody that binds to coronavirus RBD, a preparation method and application.
  • the 2019 Novel Coronavirus was discovered due to a 2019 viral pneumonia case and was named by the World Health Organization on January 12, 2020.
  • Coronavirus is a large virus family, known to cause colds and more serious diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS).
  • MERS Middle East Respiratory Syndrome
  • SARS Severe Acute Respiratory Syndrome
  • the new coronavirus is a new strain of coronavirus that has never been found in the human body before.
  • the main inflammatory indicators in the laboratory test significantly decreased, the ratio of lymphocytes increased, the key indicators such as blood oxygen saturation and viral load were all improved, and the clinical signs and symptoms improved significantly.
  • Recovered patients' plasma is mainly used for patients with rapid disease progression, severe, and critically ill patients with new coronary pneumonia. In principle, the course of the disease does not exceed 3 weeks; if the new coronavirus nucleic acid test is positive or the clinical expert determines that the patient has viremia, it should be used as soon as possible in the acutely advanced stage of the disease.
  • the SARS spike protein RBD and the new coronavirus spike protein RBD have cross-neutralizing epitope peptides.
  • the anti-SARS monoclonal antibody CR3022 can bind to the new coronavirus spike protein RBD (Emerging Microbes & Infections, 9(1): 382-385, 20200217).
  • the homology modeling method was used to clarify the hot spots and key residues of the protein-protein interaction interface of the new coronavirus CTD1/human ACE2 complex, and to screen candidate inhibitors that target the CTD1 region and the ACE2 binding surface to block the virus and Recognition and binding of human ACE2 protein.
  • ADE antibody-dependent enhancement
  • Some viruses have a significantly enhanced ability to replicate or infect with the assistance of specific antibodies, which can cause more serious pathological damage during the infection process.
  • the popular explanation is that antibodies cannot neutralize the virus, but act as a "Trojan horse", making the virus more capable of infecting immune cells, producing more offspring viruses, and causing severe symptoms.
  • the ADE (antibody-dependent enhancement) effect that is, the antibody-dependent enhancement effect, was first discovered in the process of dengue virus infection.
  • Dengue virus that binds to non-neutralizing antibodies can enter macrophages through the bypass to proliferate, and then When the second infection is caused, especially when the virus strain of the second infection is different from the first infection, the symptoms of the second infection are more severe and the ADE effect occurs.
  • feline infectious peritonitis virus (FIPV) vaccines in the early 1980s found that low-titer neutralizing antibodies against the S protein can aggravate symptoms and lead to more serious deaths.
  • studies on SARS virus and MERS virus have found that low-affinity antibodies against S protein produced by vaccination can mediate the virus's entry into immune cells.
  • the present invention isolates coronavirus RBD-specific memory B cells from the PBMCs of recovered personnel after the new coronavirus infection, and amplifies the light and heavy chain variable region sequences of the obtained antibodies, which are transiently expressed, and at least 20 antibodies are detected. It can specifically bind to the coronavirus RBD with high affinity, and at least 7 antibodies can block or inhibit the binding of the new coronavirus RBD to the host receptor ACE2.
  • the seven antibody molecules that can block or inhibit the binding of the new coronavirus RBD to the host receptor ACE2 did not produce ADE effects during the SARS-CoV-2 infection of THP-1 cells and K562 cells, and two of them
  • the two antibodies Corn-01 and Corn-05 can promote SARS-CoV-2 to enter Raji host cells, and there is an ADE effect.
  • the ADE effect occurs in the process of infecting host cells. in particular:
  • the present invention provides a SARS-CoV-2 S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof, characterized in that: the monoclonal antibody is obtained from a blood sample during the recovery period of a patient infected with a novel coronavirus A fully human antibody that specifically binds to the RBD region of the SARS-CoV-2 S protein obtained from a single B-cell clone isolated in, has no ADE effect on SARS-CoV-2 infected THP-1 cells and K562 cells.
  • the SARS-CoV-2 S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of the present invention is characterized in that the monoclonal antibody does not produce ADE effect on SARS-CoV-2 infection of Raji cells .
  • the SARS-CoV-2 S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of the present invention is characterized in that the monoclonal antibody has a partial concentration range of 10-10000ng against SARS-CoV -2 Infection of Raji cells produces ADE effect.
  • the SARS-CoV-2 S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of the present invention is characterized in that the monoclonal antibody has a partial concentration range of 50-3000ng against SARS-CoV -2 Infection of Raji cells produces ADE effect.
  • the SARS-CoV-2 S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of the present invention is characterized in that the monoclonal antibody has high affinity with SARS-CoV-2 S protein RBD
  • the KD value is 5.0 ⁇ 10 ⁇ 9 M or less.
  • the SARS-CoV-2 S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of the present invention is characterized in that the monoclonal antibody blocks the binding of ACEII and SARS-CoV-2 S protein RBD
  • the IC50 value of is less than 50nM, preferably less than 30nM, 25nM, 20nM, 15nM or 10nM.
  • the SARS-CoV-2 S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of the present invention is characterized in that the point mutation in the Fc section of the monoclonal antibody changes its binding to the receptor .
  • the SARS-CoV-2 S protein-ACEII receptor binding blocking monoclonal antibody or fragments thereof of the present invention is characterized in that the point mutation in the Fc segment of the monoclonal antibody reduces or eliminates its interaction with Fc ⁇ Rs. Combine.
  • the SARS-CoV-2 S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of the present invention is characterized in that the point mutations in the Fc segment of the monoclonal antibody include Amino acid substitutions, deletions or insertion mutations are carried out at any one or two positions in the group consisting of 235 positions.
  • the SARS-CoV-2 S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of the present invention is characterized in that the point mutations in the Fc segment of the monoclonal antibody include L234A and L235A mutations.
  • the SARS-CoV-2 S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of the present invention is characterized in that the heavy chain constant region of the monoclonal antibody has SEQ ID NO: 16 the sequence of.
  • the SARS-CoV-2 S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of the present invention is SARS-CoV-2 S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of the present invention
  • Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 An amino acid sequence obtained by substitution, deletion, addition, or insertion of one or more amino acid residues on the basis of the amino acid sequence;
  • Its light chain variable region has the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 Based on the amino acid sequence, one or more amino acid residues are substituted, deleted, added, or inserted into the obtained amino acid sequence.
  • the SARS-CoV-2 S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of the present invention is characterized in that:
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence obtained by substitution, deletion, addition, or insertion of one or more amino acid residues on the basis of the amino acid sequence shown in SEQ ID NO:1 ; And, the light chain variable region has the amino acid sequence shown in SEQ ID NO: 2, or one or more amino acid residues are substituted, deleted, added, or inserted on the basis of the amino acid sequence shown in SEQ ID NO: 2 Amino acid sequence of
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 3, or the amino acid sequence obtained by substitution, deletion, addition, or insertion of one or more amino acid residues on the basis of the amino acid sequence shown in SEQ ID NO: 3 ; And, the light chain variable region has the amino acid sequence shown in SEQ ID NO: 4, or one or more amino acid residues are substituted, deleted, added, or inserted on the basis of the amino acid sequence shown in SEQ ID NO: 4 Amino acid sequence of
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence obtained by substitution, deletion, addition, or insertion of one or more amino acid residues on the basis of the amino acid sequence shown in SEQ ID NO: 5 ; And, the light chain variable region has the amino acid sequence shown in SEQ ID NO: 6, or one or more amino acid residues are substituted, deleted, added, or inserted on the basis of the amino acid sequence shown in SEQ ID NO: 6 Amino acid sequence of
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence obtained by substitution, deletion, addition, or insertion of one or more amino acid residues on the basis of the amino acid sequence shown in SEQ ID NO: 7 ; And, the light chain variable region has the amino acid sequence shown in SEQ ID NO: 8, or one or more amino acid residues are substituted, deleted, added, or inserted on the basis of the amino acid sequence shown in SEQ ID NO: 8 Amino acid sequence of
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence obtained by substitution, deletion, addition, or insertion of one or more amino acid residues on the basis of the amino acid sequence shown in SEQ ID NO: 9 ; And, the light chain variable region has the amino acid sequence shown in SEQ ID NO: 10, or one or more amino acid residues are substituted, deleted, added, or inserted on the basis of the amino acid sequence shown in SEQ ID NO: 10 Amino acid sequence of
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence obtained by substitution, deletion, addition, or insertion of one or more amino acid residues on the basis of the amino acid sequence shown in SEQ ID NO: 11 ; And, the light chain variable region has the amino acid sequence shown in SEQ ID NO: 12, or one or more amino acid residues are substituted, deleted, added, or inserted on the basis of the amino acid sequence shown in SEQ ID NO: 12 Amino acid sequence of
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 13, or an amino acid sequence obtained by substitution, deletion, addition, or insertion of one or more amino acid residues on the basis of the amino acid sequence shown in SEQ ID NO: 13 ; And, the light chain variable region has the amino acid sequence shown in SEQ ID NO: 14, or one or more amino acid residues are substituted, deleted, added, or inserted on the basis of the amino acid sequence shown in SEQ ID NO: 14 The amino acid sequence.
  • the SARS-CoV-2 S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of any one of the present invention is characterized in that:
  • the heavy chain variable region has the HCDR1, HCDR2 and HCDR3 of the antibody heavy chain variable region shown in any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 13; or is the same as SEQ ID NO: 1, 3 Any one of, 5, 7, 9, 11, 13 has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity HCDR1, HCDR2 and HCDR3 of the variable region of the heavy chain of the antibody;
  • the light chain variable region has the LCDR1-3, LCDR2, and LCDR3 of the light chain variable region of the antibody shown in any of SEQ ID NO: 2, 4, 6, 8, 10, 12, and 14; or the same as SEQ ID NO: 2 , 4, 6, 8, 10, 12, 14 have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%
  • the sequence of identity is the HCDR1, HCDR2 and HCDR3 of the variable region of the antibody light chain.
  • the SARS-CoV-2 S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of the invention is characterized in that:
  • the heavy chain variable region has the HCDR1, HCDR2 and HCDR3 of the antibody heavy chain variable region shown in SEQ ID NO: 1; and the light chain variable region has the LCDR1 of the antibody light chain variable region shown in SEQ ID NO: 2 LCDR2 and LCDR3;
  • the heavy chain variable region has the HCDR1, HCDR2 and HCDR3 of the antibody heavy chain variable region shown in SEQ ID NO: 3; and the light chain variable region has the LCDR1 of the antibody light chain variable region shown in SEQ ID NO: 4 LCDR2 and LCDR3;
  • the heavy chain variable region has the HCDR1, HCDR2 and HCDR3 of the antibody heavy chain variable region shown in SEQ ID NO: 5; and the light chain variable region has the LCDR1 of the antibody light chain variable region shown in SEQ ID NO: 6 LCDR2 and LCDR3;
  • the heavy chain variable region has the HCDR1, HCDR2 and HCDR3 of the antibody heavy chain variable region shown in SEQ ID NO: 7; and the light chain variable region has the LCDR1 of the antibody light chain variable region shown in SEQ ID NO: 8 LCDR2 and LCDR3;
  • the heavy chain variable region has the HCDR1, HCDR2 and HCDR3 of the antibody heavy chain variable region shown in SEQ ID NO: 9; and the light chain variable region has the LCDR1 of the antibody light chain variable region shown in SEQ ID NO: 10 LCDR2 and LCDR3;
  • the heavy chain variable region has the HCDR1, HCDR2 and HCDR3 of the antibody heavy chain variable region shown in SEQ ID NO: 11; and the light chain variable region has the LCDR1 of the antibody light chain variable region shown in SEQ ID NO: 12 LCDR2 and LCDR3; or
  • the heavy chain variable region has the HCDR1, HCDR2 and HCDR3 of the antibody heavy chain variable region shown in SEQ ID NO: 13; and the light chain variable region has the LCDR1 of the antibody light chain variable region shown in SEQ ID NO: 14 LCDR2 and LCDR3.
  • the present invention provides a polynucleotide encoding the aforementioned monoclonal antibody or fragment thereof of the present invention.
  • the present invention provides a nucleic acid construct comprising the aforementioned polynucleotide of the present invention.
  • the nucleic acid construct of the present invention is used to express the aforementioned monoclonal antibody or fragment thereof of the present invention.
  • the present invention provides a host cell comprising the aforementioned polynucleotide of the present invention or the aforementioned nucleic acid construct of the present invention.
  • the present invention provides a composition comprising one or more monoclonal antibodies or fragments thereof selected from the group consisting of the aforementioned monoclonal antibodies or fragments thereof of the present invention, and optionally pharmaceutically acceptable a.
  • the composition of the present invention comprises any two, three, four, five, selected from the group consisting of the monoclonal antibody or fragments thereof according to any one of claims 1 to 13, Six, or seven monoclonal antibodies or fragments thereof.
  • the present invention provides the monoclonal antibody or fragments thereof according to any one of the foregoing of the present invention, the foregoing polynucleotide of the present invention, the foregoing nucleic acid construct of the present invention, the foregoing host cell of the present invention, and the foregoing combination of the present invention.
  • the application of the present invention is characterized in that the prevention or treatment of SARS-CoV-2 infection includes reducing or reducing the risk of SARS-CoV-2 infection, alleviating SARS-CoV-2 infection-related diseases (such as COVID- 19), shorten the course of SARS-CoV-2 infection-related diseases (such as COVID-19), promote the recovery of SARS-CoV-2 infection-related diseases (such as COVID-19), and reduce SARS-CoV-2 infections. die.
  • SARS-CoV-2 infection-related diseases such as COVID- 19
  • shorten the course of SARS-CoV-2 infection-related diseases such as COVID-19
  • promote the recovery of SARS-CoV-2 infection-related diseases such as COVID-19
  • reduce SARS-CoV-2 infections die.
  • the present invention provides a method for preventing or treating SARS-CoV-2 infection, which is characterized by administering to a subject in need an effective amount of the monoclonal antibody or fragments thereof according to any one of the foregoing of the present invention.
  • the aforementioned polynucleotide of the present invention, the aforementioned nucleic acid construct of the present invention, the aforementioned host cell of the present invention, and the aforementioned composition of the present invention are characterized by administering to a subject in need an effective amount of the monoclonal antibody or fragments thereof according to any one of the foregoing of the present invention.
  • the method of the present invention is characterized in that the subject is at risk of SARS-CoV-2 infection, has SARS-CoV-2 infection-related diseases (such as COVID-19), or has SARS-CoV -2 The possibility of death due to infection.
  • SARS-CoV-2 infection-related diseases such as COVID-19
  • SARS-CoV -2 The possibility of death due to infection.
  • the method of the present invention is characterized in that the method enables the subject to reduce or reduce the risk of SARS-CoV-2 infection, alleviate the symptoms of SARS-CoV-2 infection-related diseases (such as COVID-19), Shorten the course of SARS-CoV-2 infection-related diseases (such as COVID-19), promote the recovery of SARS-CoV-2 infection-related diseases (such as COVID-19), and reduce deaths caused by SARS-CoV-2 infection.
  • SARS-CoV-2 infection-related diseases such as COVID-19
  • Shorten the course of SARS-CoV-2 infection-related diseases such as COVID-19
  • promote the recovery of SARS-CoV-2 infection-related diseases such as COVID-19
  • the present invention provides a method for eliminating the ADE effect of antibodies when SARS-CoV-2 infects host cells, wherein the antibody is a neutralizing antibody against SARS-CoV-2 S protein.
  • Point mutations in the Fc segment changed the binding of the anti-SARS-CoV-2 S protein antibody to its receptor.
  • the method of the present invention for eliminating the ADE effect of the antibody when SARS-CoV-2 infects host cells wherein the point mutation in the Fc segment reduces or eliminates the binding of the anti-SARS-CoV-2 S protein antibody to Fc ⁇ Rs.
  • the method of the present invention for eliminating the ADE effect of the antibody when SARS-CoV-2 infects host cells wherein the Fc segment point mutation is included in any one or two of the group consisting of position 234 and position 235
  • the site undergoes amino acid substitution, deletion or insertion mutation.
  • the method for eliminating the ADE effect of the antibody of the present invention when SARS-CoV-2 infects host cells is characterized in that the neutralizing antibody against SARS-CoV-2 S protein is selected from any one of the foregoing of the present invention The monoclonal antibody or fragments thereof.
  • the present invention provides an antibody that specifically binds to SARS-CoV-2 S protein, wherein the antibody is based on natural human anti-SARS-CoV-2 S protein antibody. Segment point mutation avoids the use of natural human anti-SARS-CoV-2 S protein antibodies to prevent the ADE effect (antibody-dependent enhancement effect) when SARS-CoV-2 infects host cells;
  • the antibody that specifically binds to the SARS-CoV-2 S protein does not produce an ADE effect on SARS-CoV-2 infected THP-1 cells, Raji cells, and K562 cells within a concentration range of 10-10000ng/mL. .
  • the antibody of the present invention wherein the natural human anti-SARS-CoV-2 S protein antibody can neutralize the infection of Vero E host cells by SARS-CoV-2.
  • the antibody of the present invention wherein the natural human anti-SARS-CoV-2 S protein antibody has an ADE effect on SARS-CoV-2 infection of Raji cells at least in a partial concentration range of 10-10000ng/mL.
  • the antibody of the present invention wherein the human anti-SARS-CoV-2 S protein antibody has an ADE effect on SARS-CoV-2 infected Raji cells at least in a partial concentration range of 50-3000 ng/mL.
  • the antibody of the present invention wherein the natural human anti-SARS-CoV-2 S protein antibody
  • Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 An amino acid sequence obtained by substitution, deletion, addition, or insertion of one or more amino acid residues on the basis of the amino acid sequence;
  • Its light chain variable region has the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 Based on the amino acid sequence, one or more amino acid residues are substituted, deleted, added, or inserted into the obtained amino acid sequence.
  • the antibody of the present invention wherein the natural human anti-SARS-CoV-2 S protein antibody,
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence obtained by substitution, deletion, addition, or insertion of one or more amino acid residues on the basis of the amino acid sequence shown in SEQ ID NO:1 ; And, the light chain variable region has the amino acid sequence shown in SEQ ID NO: 2, or one or more amino acid residues are substituted, deleted, added, or inserted on the basis of the amino acid sequence shown in SEQ ID NO: 2 Amino acid sequence of
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 3, or the amino acid sequence obtained by substitution, deletion, addition, or insertion of one or more amino acid residues on the basis of the amino acid sequence shown in SEQ ID NO: 3 ; And, the light chain variable region has the amino acid sequence shown in SEQ ID NO: 4, or one or more amino acid residues are substituted, deleted, added, or inserted based on the amino acid sequence shown in SEQ ID NO: 4 Amino acid sequence of
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence obtained by substitution, deletion, addition, or insertion of one or more amino acid residues on the basis of the amino acid sequence shown in SEQ ID NO: 5 ; And, the light chain variable region has the amino acid sequence shown in SEQ ID NO: 6, or one or more amino acid residues are substituted, deleted, added, or inserted on the basis of the amino acid sequence shown in SEQ ID NO: 6 Amino acid sequence of
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence obtained by substitution, deletion, addition, or insertion of one or more amino acid residues on the basis of the amino acid sequence shown in SEQ ID NO: 7 ; And, the light chain variable region has the amino acid sequence shown in SEQ ID NO: 8, or one or more amino acid residues are substituted, deleted, added, or inserted on the basis of the amino acid sequence shown in SEQ ID NO: 8 Amino acid sequence of
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence obtained by substitution, deletion, addition, or insertion of one or more amino acid residues on the basis of the amino acid sequence shown in SEQ ID NO: 9 ; And, the light chain variable region has the amino acid sequence shown in SEQ ID NO: 10, or one or more amino acid residues are substituted, deleted, added, or inserted on the basis of the amino acid sequence shown in SEQ ID NO: 10 Amino acid sequence of
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence obtained by substitution, deletion, addition, or insertion of one or more amino acid residues on the basis of the amino acid sequence shown in SEQ ID NO: 11 ; And, the light chain variable region has the amino acid sequence shown in SEQ ID NO: 12, or one or more amino acid residues are substituted, deleted, added, or inserted on the basis of the amino acid sequence shown in SEQ ID NO: 12 Amino acid sequence of
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 13, or an amino acid sequence obtained by substitution, deletion, addition, or insertion of one or more amino acid residues on the basis of the amino acid sequence shown in SEQ ID NO: 13 ; And, the light chain variable region has the amino acid sequence shown in SEQ ID NO: 14, or one or more amino acid residues are substituted, deleted, added, or inserted on the basis of the amino acid sequence shown in SEQ ID NO: 14 The amino acid sequence.
  • the SARS-CoV-2 S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of any one of the present invention is characterized in that:
  • the heavy chain variable region has the HCDR1, HCDR2 and HCDR3 of the antibody heavy chain variable region shown in any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 13; or is the same as SEQ ID NO: 1, 3 Any one of, 5, 7, 9, 11, 13 has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity HCDR1, HCDR2 and HCDR3 of the variable region of the heavy chain of the antibody;
  • the light chain variable region has the LCDR1-3, LCDR2 and LCDR3 of the light chain variable region of the antibody shown in any of SEQ ID NO: 2, 4, 6, 8, 10, 12, and 14; or the same as SEQ ID NO: 2 , 4, 6, 8, 10, 12, 14 have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%
  • the sequence of identity is the HCDR1, HCDR2 and HCDR3 of the variable region of the antibody light chain.
  • the SARS-CoV-2 S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of the invention is characterized in that:
  • the heavy chain variable region has the HCDR1, HCDR2 and HCDR3 of the antibody heavy chain variable region shown in SEQ ID NO: 1; and the light chain variable region has the LCDR1 of the antibody heavy chain variable region shown in SEQ ID NO: 2 LCDR2 and LCDR3;
  • the heavy chain variable region has the HCDR1, HCDR2 and HCDR3 of the antibody heavy chain variable region shown in SEQ ID NO: 3; and the light chain variable region has the LCDR1 of the antibody heavy chain variable region shown in SEQ ID NO: 4 LCDR2 and LCDR3;
  • the heavy chain variable region has the HCDR1, HCDR2 and HCDR3 of the antibody heavy chain variable region shown in SEQ ID NO: 5; and the light chain variable region has the LCDR1 of the antibody heavy chain variable region shown in SEQ ID NO: 6 LCDR2 and LCDR3;
  • the heavy chain variable region has the HCDR1, HCDR2 and HCDR3 of the antibody heavy chain variable region shown in SEQ ID NO: 7; and the light chain variable region has the LCDR1 of the antibody heavy chain variable region shown in SEQ ID NO: 8 LCDR2 and LCDR3;
  • the heavy chain variable region has the HCDR1, HCDR2 and HCDR3 of the antibody heavy chain variable region shown in SEQ ID NO: 9; and the light chain variable region has the LCDR1 of the antibody heavy chain variable region shown in SEQ ID NO: 10 LCDR2 and LCDR3;
  • the heavy chain variable region has the HCDR1, HCDR2 and HCDR3 of the antibody heavy chain variable region shown in SEQ ID NO: 11; and the light chain variable region has the LCDR1 of the antibody heavy chain variable region shown in SEQ ID NO: 12 LCDR2 and LCDR3; or
  • the heavy chain variable region has the HCDR1, HCDR2, and HCDR3 of the antibody heavy chain variable region shown in SEQ ID NO: 13; and the light chain variable region has the LCDR1 of the antibody heavy chain variable region shown in SEQ ID NO: 14 LCDR2 and LCDR3.
  • the point mutation in the Fc segment changes the binding of the anti-SARS-CoV-2 S protein antibody to its receptor.
  • the antibody of the present invention wherein the point mutation of the Fc segment reduces or eliminates the binding of the anti-SARS-CoV-2 S protein antibody to Fc ⁇ Rs.
  • the antibody of the present invention wherein the antibody does not produce an ADE effect on SARS-CoV-2 infected THP-1 cells, Raji cells, and K562 cells within a concentration range of 10-10000 ng/mL.
  • the Fc segment point mutation includes amino acid substitution, deletion or insertion mutation at any one or two positions in the group consisting of the 234th position and the 235th position.
  • the Fc segment point mutation includes substitution, deletion or insertion mutation of a single amino acid at any one or two positions in the group consisting of position 234 and position 235.
  • the antibody of the present invention wherein the Fc segment point mutations include L234A and L235A mutations.
  • the present invention provides the application of an antibody in the preparation of a drug for treating SARS-CoV-2 infection, wherein the antibody is as described in the first aspect of the present invention.
  • the present invention provides the application of antibodies in the preparation of drugs for the treatment of diseases caused by SARS-CoV-2 infection, wherein the antibodies are as described in the first aspect of the present invention, and the diseases include COVID-19. .
  • the present invention provides a polynucleotide encoding the antibody according to the first aspect of the present invention.
  • the present invention provides a vector comprising the polynucleotide according to the fourth aspect of the present invention.
  • the present invention provides a host cell comprising the polynucleotide according to the fourth aspect of the present invention or the vector according to the fourth aspect of the present invention.
  • the present invention provides a pharmaceutical composition comprising one or more antibodies selected from the group consisting of the antibodies of the first aspect of the present invention, and optionally pharmaceutically acceptable a.
  • the present invention provides a method for eliminating the ADE effect of an antibody when SARS-CoV-2 infects host cells, wherein the antibody is anti-SARS-CoV-2 S protein neutralizing
  • the antibody changes the binding of the anti-SARS-CoV-2 S protein antibody to its receptor by performing point mutations on the Fc segment of the antibody.
  • the method of the present invention for eliminating the ADE effect of antibodies when SARS-CoV-2 infects host cells wherein the point mutation in the Fc segment reduces or eliminates the binding of the anti-SARS-CoV-2 S protein antibody to Fc ⁇ Rs.
  • the Fc segment point mutation is included in any one or two of the group consisting of the 234th and 235th positions Make amino acid substitutions, deletions, or insertion mutations.
  • coronavirus refers to members of the order Nidovirales, Coronaviridae, and Coronavirus.
  • the coronavirus of the present invention mainly relates to coronaviruses that infect humans, including HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2 (2019-nCov),
  • the coronavirus of the present invention particularly relates to SARS-CoV, MERS-CoV, and SARS-CoV-2 (2019-nCov).
  • the term "specificity" refers to determining whether the protein is present in a protein and/or other biological heterogeneous population, for example, the binding reaction of the monoclonal antibody of the present invention with the SARS-CoV-2 RBD protein. Therefore, under the specified conditions, a specific ligand/antigen binds to a specific receptor/antibody, and does not bind to other proteins present in the sample in a significant amount.
  • antibody herein is intended to include full-length antibodies and any antigen-binding fragments thereof (abbreviated as antibody fragments) or single chains.
  • Full-length antibodies are glycoproteins containing at least two heavy (H) chains and two light (L) chains, the heavy and light chains are connected by disulfide bonds.
  • Each heavy chain is composed of a heavy chain variable region (VH for short) and a heavy chain constant region.
  • the heavy chain constant region is composed of three domains, namely CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (abbreviated as VL) and a light chain constant region.
  • the constant region of the light chain consists of a domain CL.
  • the VH and VL regions can also be divided into hypervariable regions called complementarity determining regions (CDR), which are separated by more conservative framework regions (FR) regions.
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, FR3, FR4 from the amino terminal to the carboxy terminal.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • the constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including a variety of immune system cells (for example, effector cells) and the first component (C1q) of the traditional complement system.
  • variable region refers to the amino acid residues of an antibody that are responsible for antigen binding.
  • CDR region sequence can be defined by IMGT, Kabat, Chothia and AbM methods or any CDR region sequence determination method well known in the art to identify amino acid residues in the variable region.
  • Antibody CDRs can be identified as hypervariable regions originally defined by Kabat et al., for example, residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) of the light chain variable domain and heavy Residues 31-35 (H1), 50-65 (H2) and 95-102 (H3) of the chain variable domain, see Kabat EA et al., 1991, Sequences of Proteins of Immunological Interest (protein sequence of immune target) , 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md.; CDR positions can also be identified as defined by the "hypervariable loop" (HVL) structure originally described by Chothia et al.
  • HVL hypervariable loop
  • IMGT immunoglobulin variable regions
  • CDR regions are defined according to IMGT numbering, for example, 27-32 (L1), 50-52 (L2) and For residues 89-97 (L3) and residues 26-35 (H1), 51-57 (H2) and 93-102 (H3) of the heavy chain variable domain, please refer to Dev.Comp such as Lefranc MP etc. Immunol., 2003, 27:55-77, which is incorporated herein by reference.
  • CDR identification includes "AbM definition”, which is a compromise between Kabat and Chothia and obtained using Oxford Molecular's AbM antibody model software; or "contact definition” of CDR, which is based on the observed antigen contact and Elaborated in MacCallum RM et al., 1996, J. Mol Biol., 262:732-745.
  • configuration definition the position of CDR can be identified as residues that contribute to antigen binding, see, for example, Makabe K et al., 2008, J. Biol Chem., 283:1156-1166.
  • the CDR contained in the antibody or antigen-binding fragment thereof of the present invention can be determined according to various numbering systems known in the art.
  • the CDR contained in the antibody or antigen-binding fragment thereof of the present invention is preferably determined by the Kabat, Chothia or IMGT numbering system, especially by the Kabat numbering system.
  • monoclonal antibody or “monoclonal antibody” or “monoclonal antibody composition” refers to an antibody molecule product of single molecular composition.
  • the monoclonal antibody composition exhibits a single binding specificity and affinity for a specific epitope.
  • antigen-binding fragment of an antibody (or antibody fragment for short) refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been confirmed that the antigen-binding function of antibodies can be implemented by fragments of full-length antibodies.
  • binding fragments included in the "antigen-binding portion" of the antibody include (i) Fab fragments, monovalent fragments composed of VL, VH, CL, and CH1; (ii) F(ab')2 fragments, including hinge region two A bivalent fragment of two Fab fragments connected by a sulfur bridge; (iii) an Fd fragment composed of VH and CH1; (iv) an Fv fragment composed of antibody single-arm VL and VH; (v) a dAb fragment composed of VH ( Ward et al., (1989) Nature 341: 544-546); (vi) Isolated complementarity determining regions (CDR); and (vii) Nanobodies, which contain a single variable domain and two constant domains Heavy chain variable region.
  • Fab fragments monovalent fragments composed of VL, VH, CL, and CH1
  • F(ab')2 fragments including hinge region two A bivalent fragment of two Fab fragments connected by a sulfur bridge
  • the two domains VL and VH of the Fv fragment are encoded by different genes, they can be connected by recombination through a synthetic linker that makes the two into a single protein chain, where the VL and VH regions pair to form a monovalent molecule (called Single-chain Fc (scFv); see, for example, Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883).
  • scFv Single-chain Fc
  • These single chain antibodies are also intended to be included in the meaning of the term.
  • These antibody fragments can be obtained by common techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.
  • the antigen-binding fragments of the present invention include those capable of specifically binding to coronavirus RBD.
  • antibody binding fragments include, for example, but not limited to, Fab, Fab', F(ab') 2 , Fv fragments, single chain Fv (scFv) fragments, and single domain fragments.
  • the Fab fragment contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • CH1 first constant domain
  • the difference between Fab' fragments and Fab fragments lies in the addition of a few residues at the carboxy terminus of the CH1 domain of the heavy chain, including one or more cysteines from the hinge region of an antibody.
  • Fab' fragments are generated by cleaving the disulfide bond at the hinge cysteine of the F(ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art.
  • Fab and F(ab')2 fragments lack the crystallizable (Fc) region of intact antibody fragments, are cleared more rapidly from the animal’s circulation, and may have less non-specific tissue binding than intact antibodies (see, for example, Wahl et al. Human, 1983, J. Nucl. Med. 24:316).
  • the "Fc” region is a crystallizable constant region of an antibody fragment that does not contain an antigen-specific binding region.
  • the Fc region is composed of two identical protein fragments, derived from the second and third constant domains (CH2 and CH3 domains, respectively) of the two heavy chains of the antibody.
  • IgM and IgE Fc regions contain three heavy chain constant domains (CH2, CH3 and CH4 domains) in each polypeptide chain.
  • the "Fv” fragment is the smallest fragment of an antibody that contains a complete target recognition and binding site.
  • This region is composed of a dimer (VH-VL dimer) of one heavy chain and one light chain variable domain in tight non-covalent bonding.
  • VH-VL dimer dimer
  • the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer.
  • the six CDRs confer target binding specificity to the antibody.
  • even a single variable domain (or half of an Fv containing only three CDRs specific for the target) may have the ability to recognize and bind to the target, although its affinity is lower than the entire binding site.
  • Single chain Fv or “scFv” antibody binding fragments comprise the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form a structure that facilitates target binding.
  • a "single domain fragment” consists of a single VH or VL domain that shows sufficient affinity for the coronavirus RBD.
  • the single domain fragments are camelized (see, for example, Riechmann, 1999, Journal of Immunological Methods 231:25-38).
  • the anti-coronavirus RBD antibody of the present invention includes a derivatized antibody.
  • derivatized antibodies are usually glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting/blocking groups, proteolytic cleavage, and linked to cell ligands or other proteins.
  • protecting/blocking groups proteolytic cleavage, and linked to cell ligands or other proteins.
  • Any of numerous chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.
  • the derivative may contain one or more unnatural amino acids, for example, using ambrx technology (see, for example, Wolfson, 2006, Chem. Biol. 13(10): 1011-2).
  • Human antibodies include antibodies having the amino acid sequence of human immunoglobulin, and include antibodies isolated from human immunoglobulin libraries or animals that are transgenic for one or more human immunoglobulins, and Does not express endogenous immunoglobulins.
  • Human antibodies can be prepared by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT Publications WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO 96/33735; and WO 91/10741.
  • transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes to produce human antibodies. See, for example, PCT Publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598.
  • companies such as LakePharma, Inc.
  • antibody that recognizes an antigen and "antibody specific for an antigen” are used interchangeably with the term “antibody that specifically binds to an antigen” herein.
  • high affinity for IgG antibodies means that the KD for the antigen is 1.0 ⁇ 10 -6 M or less, preferably 5.0 ⁇ 10 -8 M or less, more preferably 1.0 ⁇ 10 -8 M or less, 5.0 ⁇ 10 -9 M or less, more preferably 1.0 ⁇ 10 -9 M or less.
  • "high affinity" binding may vary.
  • “high affinity” binding of IgM subtypes means that the KD is 10 -6 M or less, preferably 10 -7 M or less, and more preferably 10 -8 M or less.
  • Kassoc or “Ka” refers to the rate of association of a specific antibody-antigen interaction
  • Kdis or “Kd” refers to the rate of dissociation of a specific antibody-antigen interaction
  • KD refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (Kd/Ka) and is expressed in molar concentration (M).
  • M molar concentration
  • the KD value of the antibody can be determined by a method known in the art.
  • a preferred way to determine the KD of an antibody is to use a surface plasmon resonance (SPR) measurement, preferably to use a biosensing system such as the BiacoreTM system.
  • SPR surface plasmon resonance
  • EC50 also called half-maximal effect concentration, refers to the concentration of antibody that causes 50% of the maximum effect.
  • IC50 refers to the half inhibitory concentration of the antagonist being measured. It can indicate that a certain drug or substance (inhibitor) is inhibiting half of certain biological procedures (or certain substances included in this procedure, such as enzymes, cell receptors or microorganisms).
  • epitope refers to the site on an antigen where B and/or T cells respond.
  • B cell epitopes can all be formed by continuous amino acids or discrete amino acids juxtaposed by the tertiary folding of proteins. Epitopes formed by consecutive amino acids are typically retained when exposed to a denaturing solvent, while epitopes formed by tertiary folding are typically lost when treated with a denaturing solvent.
  • An epitope typically includes at least 3, and more usually at least 5 or 8-10 amino acids in a unique spatial conformation.
  • composition provides a composition comprising an antibody or antigen-binding fragment thereof as described herein.
  • the composition according to the present invention can be administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerance, and similar properties.
  • suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerance, and similar properties.
  • suitable formulations can be found in the formula books known to all medical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
  • formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as LIPOFECTINTM), DNA conjugates, anhydrous absorption pastes, oil-in-water And water-in-oil emulsion, emulsion carbowax (polyethylene glycol of various molecular weight), semi-solid gel and semi-solid mixture containing carbowax. See also Powell et al. "Compendium of Excipients for Parenteral Formulations” PDA (1998) “Journal of Pharmaceutical Science and Technology (J Pharm Sci Technol)” 52: 238-311.
  • the composition of the present invention is preferably an injectable preparation, and the injectable preparation may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injection, drip infusion, and the like.
  • injectable preparations can be prepared by publicly known methods.
  • injection preparations can be prepared, for example, by dissolving, suspending or emulsifying the above-described antibody or salt thereof in a sterile aqueous or oily medium conventionally used for injection.
  • aqueous medium for injection there are, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, etc., which can be used in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, Polyethylene glycol), non-ionic surfactants [e.g. polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)] and the like.
  • solubilizers such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, Polyethylene glycol), non-ionic surfactants [e.g. polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)] and the like.
  • solubilizers such as sesame oil, soybean oil, etc., which can be used in combination with solubilizers such as
  • amino acid mutation means herein an amino acid substitution, insertion, and/or deletion in a polypeptide sequence, or a change to a part that is chemically linked to a protein.
  • the mutation can be a change in the carbohydrate or PEG structure attached to the protein.
  • amino acid mutation means herein an amino acid substitution, insertion and/or deletion in a polypeptide sequence.
  • amino acid mutations are generally for amino acids encoded by DNA, such as the 20 amino acids that have codons in DNA and RNA.
  • amino acid substitution or “substitution” means herein that an amino acid at a specific position in the parent polypeptide sequence is replaced by a different amino acid. Specifically, in some embodiments, the substitution is for a non-naturally occurring amino acid at a specific position, which is not naturally occurring in the organism or in any organism.
  • substitution E272Y refers to a variant polypeptide in which the glutamic acid at position 272 is replaced by tyrosine, in this case an Fc variant.
  • the protein is engineered to change the nucleic acid coding sequence without changing the starting amino acid (e.g.
  • CGG encoding arginine
  • CGA still encoding arginine to increase the expression level of the host organism
  • Amino acid substitution that is to say, although a new gene encoding the same protein is produced, if the protein has the same amino acid at its starting specific position, it is not an amino acid substitution.
  • amino acid insertion or “insertion” means the addition of an amino acid sequence at a specific position in the parent polypeptide sequence.
  • -233E or 233E indicates that glutamic acid is inserted after position 233 and before position 234.
  • -233ADE or A233ADE indicates that AlaAspGlu is inserted after position 233 and before position 234.
  • amino acid deletion or “deletion” means the removal of the amino acid sequence at a specific position in the parent polypeptide sequence.
  • G236- or G236# or G236del indicates the deletion of glycine at position 236.
  • EDA233- or EDA233# indicates that the sequence GluAspAla is deleted from position 233.
  • identity is used to refer to the degree of sequence similarity between two polypeptides or between two nucleic acids.
  • a certain position in the two sequences to be compared is occupied by the same base or amino acid monomer subunit (for example, a certain position in each of the two DNA molecules is occupied by adenine, or two A certain position in each of the polypeptides is occupied by lysine)
  • the molecules are the same at that position.
  • the "percent identity” between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared ⁇ 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match).
  • the comparison is made when two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol., 48:443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). You can also use the algorithms of E. Meyers and W. Miller (Comput. Appl.
  • Biosci., 4:11-17, 1988 that have been integrated into the ALIGN program (version 2.0), and use the PAM120 weight residue table (weight residue table)
  • a gap length penalty of 12 and a gap penalty of 4 are used to determine the percent identity between two amino acid sequences.
  • the Needleman and Wunsch (J.MoI.Biol., 48:444-453, 1970) algorithm in the GAP program integrated into the GCG software package can be used, and Blossum 62 Matrix or PAM250 matrix and gap weight of 16, 14, 12, 10, 8, 6 or 4 and length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences sex.
  • residue means a position in a protein and the identity of its related amino acid.
  • Asparagine 297 also known as Asn297 or N297
  • residue 297 in the human antibody IgG1 is residue 297 in the human antibody IgG1.
  • nucleic acid molecule and “polynucleotide” are intended to include DNA molecules and RNA molecules.
  • the nucleic acid molecule may be single-stranded or double-stranded, and may be cDNA.
  • a "host cell” is a cell in which a vector can proliferate and its DNA can be expressed, and the cell may be a prokaryotic cell or a eukaryotic cell. The term also includes any progeny of the subject host cell. It should be understood that not all offspring are the same as the parent cell, because mutations may occur during the replication process, and such offspring are included.
  • vector and “nucleic acid construct” refer to a nucleic acid molecule capable of transporting another nucleic acid linked to it.
  • plasmid refers to a circular double-stranded DNA loop in which additional DNA segments can be connected.
  • viral vector in which additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in the host cell into which they are introduced (for example, bacterial vectors with bacterial origins of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • vectors can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicate with the host genome.
  • certain vectors can direct the expression of genes to which they are effectively linked.
  • Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").
  • expression vectors useful in recombinant DNA technology usually exist in the form of plasmids.
  • viral vectors for example, replication defective retroviruses, adenoviruses, and adeno-associated viruses
  • an effective amount refers to an amount sufficient to obtain or at least partially obtain the desired effect.
  • an effective amount for preventing a disease e.g., infection
  • an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent a disease that has already been suffered.
  • the patient s disease and the amount of its complications. It is completely within the abilities of those skilled in the art to determine such an effective amount.
  • the effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient’s own immune system, the patient’s general conditions such as age, weight and sex, the way the drug is administered, and other treatments that are administered at the same time and many more.
  • prevention refers to a method performed in order to prevent or delay the occurrence of a disease or condition or symptom (for example, an infection) in a subject or minimize its effect if it occurs.
  • treatment refers to methods performed in order to obtain beneficial or desired clinical results.
  • beneficial or desired clinical outcomes include, but are not limited to, reduction in the rate of disease progression, improvement or reduction of disease state, and regression or improvement of prognosis, whether detectable or undetectable.
  • the amount of therapeutic agent effective to alleviate the symptoms of any particular disease can vary depending on factors such as the patient's disease state, age and weight, and the ability of the drug to cause a desired response in the subject. Whether the symptoms of the disease are alleviated can be assessed by any clinical measurement, which is usually used by doctors or other skilled healthcare providers to assess the severity or progression of the symptoms.
  • the present invention uses single lymphocyte cloning technology to expand and obtain multiple specific antibodies from a large number of single B cells in patients with SARS-CoV-2 infection during the convalescent stage.
  • SARS-CoV-2 CoV-2 S protein-ACEII binding blocking activity analysis obtained 7 strains that can specifically bind to SARS-CoV-2 S protein RBD with high affinity and can block SARS-CoV-2 from binding to the host cell receptor ACEII
  • the cloned antibody provides the amino acid sequence of the CDRs of its light and heavy chains.
  • the monoclonal antibody of the present invention has achieved good neutralization and sealing technical effects in both the pseudovirus neutralization test and the SARS-CoV-2 virus particle neutralization test.
  • the present invention performs ADE analysis on the 7 monoclonal antibodies with SARS-CoV-2 S protein-ACEII binding blocking activity obtained by screening, and it is used in the process of SARS-CoV-2 infection of THP-1 cells and K562 cells. None of them produce ADE effect; it is suggested that the 7 monoclonal antibodies screened by the present invention have lower ADE effect in clinical application.
  • the 7 monoclonal antibodies of the present invention are not only fully human antibodies that naturally exist in the serum of recovered patients, but also target multiple different natural epitopes of the SARS-CoV-2 S protein RBD, so they are particularly suitable for "cocktail antibody therapy". .
  • the research of the present invention found that the antibodies Corn-01 and Corn-05 in a specific concentration range can produce ADE effects when the SARS-CoV-2 virus infects Raji host cells. Combined with research reports in the prior art, the mechanism by which neutralizing antibodies against SARS-CoV-2 produces ADE effects was explored. According to the difference in the types of Fc receptors on Raji host cells and other host cells that do not produce ADE effects, the anti-SARS-CoV-2 neutralizing antibody was analyzed.
  • the mechanism of the SARS-CoV-2 neutralizing antibody producing ADE effect may be caused by the binding of the antibody to the Fc ⁇ RI receptor on the host cell membrane; thus enabling those skilled in the art to block the anti-SARS-CoV-2 neutralizing antibody Binding with the host cell Fc ⁇ RIIb receptor reduces or eliminates the ADE effect, that is, the virus blocking activity of the antibody is retained, and the possible ADE effect is eliminated, which improves the safety of human clinical application.
  • FIG. 1 Fusion expression of SARS-CoV-2 S1 protein and His.
  • Figure 2 Fusion expression of SARS-CoV-2 S1 protein and mFc.
  • FIG. 3 Fusion expression of SARS-CoV-2 S1 protein RBD and His.
  • FIG. 4 Fusion expression of SARS-CoV-2 S1 protein RBD and mFc.
  • Figure 5 Fusion expression of human ACE2 and human Fc.
  • Figure 6 The ability of recombinant antibodies Corn-02, Corn-10 and Corn-01 to block the binding of S1RBD-mFc to ACE2.
  • CR3022 is a monoclonal antibody against SARS-Cov RBD disclosed in US2010172917A1;
  • Corn-10 is a monoclonal antibody that specifically binds to S1RBD-mFc prepared in Example 4 of the present invention.
  • Figure 7 The ability of recombinant antibodies Corn-02, Corn-10 and Corn-01 to block the binding of S1-mFc to ACE2.
  • Figure 8 The blocking effect of recombinant antibodies on the binding of S1RBD-mFc and ACEII-hFc.
  • Figure 9 Curves of the blocking effect of Corn-01 and Corn-07 on the binding of S1RBD-mFc and ACEII-hFc (competitive ELISA method).
  • Figure 10 The blocking effect of Corn-01 and Corn-07 on the combination of S1RBD-His and ACEII-his (false virus infection fluorescence detection method).
  • Figure 11 The ADE effect of Corn-01 in the process of SARS-CoV-2 virus particles infecting different host cells
  • Figure 12 ADE effects of Corn-01, Corn-04, Corn-05, and Corn-06 on SARS-CoV-2 virus particles infecting Raji cells
  • Figure 13 The ADE effect of Corn-01, Corn-05, and Corn-07 on the SARS-CoV-2 virus particles infecting Raji cells
  • Figure 14 The ADE effect of Corn-01 and Corn-01-LALA on SARS-CoV-2 virus particles infecting Raji cells
  • Figure 15 The ADE effect of Corn-05 and Corn-05-LALA on SARS-CoV-2 virus particles infecting Raji cells
  • FIG. 16 Neutralizing activity of Corn-01 and Corn-05 against SARS-CoV-2 virus particles
  • FIG. 17 Analysis of the neutralization activity of Corn-01 and Corn-01-LALA on SARS-CoV-2 virus particles
  • Figure 18 Analysis of the neutralization activity of Corn-05 and Corn-05-LALA on SARS-CoV-2 virus particles.
  • Example 1 Recombinant expression of SARS-CoV-2 antigen and host receptor
  • the fully synthetic gene S1RBD (Accession: QHD43416.1, 319-541aa) was cloned into the eukaryotic transient expression vector with His tag or mFc tag at the C-terminus by restriction digestion method, and the obtained expression plasmid was transferred into Escherichia coli was amplified, and S1RBD-his and S1RBD-mFc expression plasmids were isolated and obtained. According to the operating instructions of the transfection reagent 293fectin (Cat:12347019, Gibco), the plasmids were transferred into HEK293 cells for recombinant expression.
  • S1RBD-mFc is purified by ProA affinity chromatography column to obtain S1RBD-mFc protein (amino acid sequence is shown in SEQ ID NO: 18).
  • S1RBD-his (amino acid sequence shown in SEQ ID NO: 17) was purified by HisTrap HP affinity chromatography column to obtain S1RBD-his protein. The purity of the obtained recombinant protein was checked by SDS-PAGE ( Figure 3-4).
  • the S1 gene (Accession: QHD43416.1, 1-685aa) was cloned from the purchased full-length SARS-CoV-2 expression vector (Cat: VG40589-UT, Beijing Yiqiao Shenzhou) by PCR, and digested by restriction enzymes Respectively clone into the eukaryotic transient expression vector with mFc tag or His tag at the C-terminal, transfer the obtained expression plasmid into Escherichia coli for amplification, and separate and obtain S1-mFc and S1-His expression plasmids, and according to the transfection reagent 293fectin (Cat:12347019, Gibco) instructions, transfer the plasmid into HEK293 cells for recombinant expression.
  • ACEII extracellular region gene ACEII (1-615) (Accession: NP_068576.1, 1-615aa) was cloned from the purchased full-length human ACEII expression vector (Cat: HG10108-ACR, Beijing Yiqiao Shenzhou) by PCR.
  • Example 2 Isolation of SARS-CoV-2 S1 protein RBD-specific memory B cells
  • the specific memory B cells of patients recovered from the new coronavirus infection were detected and sorted.
  • the new crown antibody detection kit was used to detect the IgG and IgM antibodies in the patient's serum, and the serum samples that were positive for the new crown antibody IgG were selected.
  • the RosetteSep kit (Cat: 15064, STEMCELL) was used to enrich the B cells, and on this basis, FITC-labeled S1-RBD-his was used to capture the memory B cells specifically bound by the new crown RBD and perform flow cytometry single cells sorting.
  • Example 3 Amplification of human anti-SARS-CoV-2RBD antibody sequence
  • RNA magnetic beads (Nanjing Novena) to extract RNA from single B cells and reverse transcribed into cDNA.
  • the specific method is as follows:
  • Two-step PCR was used to amplify the antibody heavy chain and light chain (Kappa) variable region genes.
  • the primer sequence is derived from pages 114 to 117 of the Human Monoclonal Antibodies book. The specific method is as follows:
  • the PCR products were separated and purified by agarose gel electrophoresis, and the antibody light and heavy chain variable regions were sequenced.
  • Example 4 Expression of human anti-SARS-CoV-2RBD antibody and preliminary screening identification of specific binding
  • the 122 pairs of sequences after sequencing were analyzed, 49 pairs of antibody light and heavy chain variable region genes were further synthesized, and they were cloned into the transient expression vector of the whole antibody for recombinant expression and specific identification.
  • the fully synthesized antibody heavy chain variable region was cloned into the eukaryotic transient expression vector pKN041 upstream of the heavy chain constant region encoding gene of human IgG1 by restriction enzyme digestion, and the fully synthesized antibody light chain variable region was cloned into the real body by restriction enzyme digestion.
  • the supernatant was taken and used with Fortebio's Octet QKe system instrument, using anti-human antibody Fc capture antibody (AHC) bioprobe to capture the antibody Fc segment to determine the binding of the antibody to S1RBD.
  • AHC anti-human antibody Fc capture antibody
  • the antibody supernatant and the antibody flow through the surface of the AHC probe (Cat:18-0015, PALL) for 240s.
  • S1RBD-mFc KN expression, lot: 20200217A
  • the concentration of S1RBD-mFc recombinant protein was 100 nM.
  • the binding time is 300s
  • the dissociation time is 300s.
  • the software was used to perform 1:1 Langmuir binding mode fitting to calculate the kinetic constant of antigen-antibody binding. A total of 39 antibodies were detected, of which 20 antibodies specifically bind to S1RBD-mFc (Table 1).
  • the clones with high affinity for the supernatant Fortebio detection were subjected to further ELISA blocking activity detection.
  • the specific method is as follows:
  • Coating plate Coating human ACE2-hFc (1-615) (KN expression, lot: 20200213C), the concentration is 0.75ug/ml; each well is 100ul; 4°CO/N;
  • Blocking 5% BSA in PBS, 37°C, 120min, wash the plate 4 times with PBST;
  • HRP-anti-mouse IgG (Cat:115-035-071, Jackson ImmunoResearch) (1:5000) 37°C, 45min, wash the plate 4 times with PBST;
  • TMB (Cat: ME142, Beijing Taitianhe Biology) color development, 37°C, 10min;
  • Reading Read and record the absorbance value of the orifice plate at a wavelength of 450nm.
  • ELISA was performed using the gradient dilution method.
  • the above-mentioned antibody to be tested in the step of adding the primary antibody, the above-mentioned antibody to be tested (initial concentration 40 ⁇ g/mL, 1.5-fold serial dilution, 12 gradients) and S1-RBD- mFc 70ng/ml, each take 100uL equal volume and mix well.
  • Figures 8-9 The results are shown in Figures 8-9.
  • Example 7 ADE effect of Corn-01 antibody on SARS-CoV-2 infected cells
  • Infect lymphocyte cells THP-1, Raji and K562 with the new coronavirus particles from the National Inspection Institute for ADE detection of antibodies. After neutralizing the neocorona antibody to be evaluated with different concentrations of 750TCID50/well pseudoviral particles (transfected with luciferase reporter gene) at 37°C, inoculate 1x105/well host cells respectively, and place them at 37°C, 5% CO2 Cultivate for 20-28h in the incubator. After 28-28h, take out the cell plate and add 100ul luciferase detection reagent (G7940, Promega) to each well, and react for 2 minutes in the dark. The fluorescence detector reads and evaluates the strength of ADE according to the intensity of fluorescence signal.
  • Example 8 Detection of ADE effect of neutralizing antibody during virus infection of sensitive host cells
  • Figure 12 and Figure 13 show that different anti-SARS-CoV-2 S1 protein RBD region neutralizing antibodies have differences in their ability to cause ADE effects during virus infection of sensitive host cells.
  • the three antibodies Corn-04, Corn-06 and Corn-07 in the form of IgG1 have no obvious ADE effect in the concentration range of 1-1000ng/ml, while Corn-01 and Corn-05 are in a certain concentration range (50ng/ml). /ml-3000ng/ml) ADE phenomenon occurs.
  • Raji cells were infected with the new coronavirus pseudovirus particles from the China Inspection Institute for ADE detection of antibodies. After neutralizing the neocorona antibody to be evaluated with 750TCID50/well pseudoviral particles (transfected with luciferase reporter gene) at 37°C, inoculate 1x105/well Raji cells respectively, and place them in a CO2 incubator at 37°C Cultivate within 20-28h. After 20-28h, take out the cell plate and add 100 ⁇ l of luciferase detection reagent to each well, and react for 2min in the dark. The fluorescence detector reads and evaluates the strength of ADE according to the intensity of fluorescence signal.
  • Fc receptors on Raji host cells and other host cells that do not produce ADE effects determine the location of the Fc point mutation, and detect the affinity of the antibody to the Fc receptor on the host cell after the mutation.
  • the Fc of Corn-01 and Corn-05 were subjected to LALA mutation (Fc-L234A, L235A) to construct Corn-01-LALA and Corn-05-LALA.
  • LALA mutation Fc-L234A, L235A
  • Octet QKe system Fortebio protein interaction system Octet QKe system was used to determine Corn-05 (wtIgG1) and Corn-05 LALA and Fc ⁇ RI (CD64) recombinant protein (10256-H08H, Yiqiao Shenzhou) and Fc ⁇ RIIa (CD32A) recombinant protein (10374-H08H1) , Yiqiao Shenzhou) affinity.
  • the capture antibody (AHC) bio-probe of anti-human antibody Fc segment is used to capture the antibodies Corn-05 and Corn-05-LALA. 15 ⁇ g/ml antibody flows through the surface of the AHC probe (Cat:18-5060, PALL) for 120s. Recombinant antigen at a concentration of 100 nM was used as the mobile phase.
  • the binding time is 300s, and the dissociation time is 300s.
  • the blank control response value was subtracted, and the software was used to perform 1:1 Langmuir binding mode fitting to calculate the kinetic constant of antigen-antibody binding. The results are shown in Table 4.
  • Example 10 The point mutation of the antibody Fc eliminates the ADE effect of the antibody on the host cell infected by the new coronavirus
  • Example 7 In order to verify whether reducing the affinity of neutralizing antibody Fc and host cell receptor Fc ⁇ RI through point mutations can reduce or eliminate its ADE effect on host cells infected by the new coronavirus, the mutated Corn-01-LALA and Corn-05-LALA Compared with Corn-01 and Corn-05 before mutation, the method of Example 7 was used to detect the ADE effect on the host cells infected by the new coronavirus. The results are shown in Figure 14 and Figure 15.
  • Figures 14 and 15 show that Corn-01-LALA and Corn-05-LALA have introduced L234A and L235A mutations in the Fc segment, which eliminates the ADE phenomenon.
  • the fully human neutralizing antibody against the RBD region of the SARS-CoV-2 S1 protein may cause the ADE effect during the SARS-CoV-2 infection of the host.
  • the ADE effect may be caused by the antibody Fc segment and the host cell.
  • the FcrRI receptor is mediated, so by introducing L234A and L235A mutations in the Fc segment, the binding of the antibody to the FcrRI receptor is greatly reduced, thereby avoiding the occurrence of ADE.
  • Example 11 The point mutation of the antibody Fc does not affect the neutralizing activity of the antibody against the new coronavirus

Abstract

一种全人源单克隆抗体或其片段，所述单克隆抗体是从COVID‐19患者康复期血液样本中分离单个B细胞克隆获得的特异性结合SARS‐CoV‐2 S蛋白RBD区的全人源抗体，其对SARS‐CoV‐2感染THP‐1细胞和K562细胞均不产生ADE效应。所述单克隆抗体与SARS‐CoV‐2 S蛋白RBD具有高亲和性，KD值为5.0×10 ‐9M以下；并且所述单克隆抗体对SARS‐CoV‐2 S蛋白RBD与ACEII的结合具有阻断活性，IC50值小于50nM。

Description

一种全人源单克隆抗体及其应用 技术领域
本发明属于抗体工程领域，具体涉及一种针对冠状病毒的单抗及其应用，特别是涉及一种结合冠状病毒RBD的人源单抗、制备方法及应用。
背景技术
2019新型冠状病毒(2019-nCoV)，因2019年病毒性肺炎病例而被发现，2020年1月12日被世界卫生组织命名。冠状病毒是一个大型病毒家族，已知可引起感冒以及中东呼吸综合征(MERS)和严重急性呼吸综合征(SARS)等较严重疾病。新型冠状病毒是以前从未在人体中发现的冠状病毒新毒株。
人感染了冠状病毒后常见体征有呼吸道症状、发热、咳嗽、气促和呼吸困难等。在较严重病例中，感染可导致肺炎、严重急性呼吸综合征、肾衰竭，甚至死亡。目前对于新型冠状病毒所致疾病没有特异治疗方法，需根据患者临床情况进行治疗。《新型冠状病毒感染的肺炎诊疗方案(试行第五版)》公布，在重型、危重型病人治疗的其他治疗措施中，可采用恢复期血浆治疗。2月8日，首期在江夏区第一人民医院开展了3名危重患者的新冠特免血浆治疗，目前连同后续医院治疗的危重病人超过了10人。经临床反映，患者接受治疗12至24小时后，实验室检测主要炎症指标明显下降，淋巴细胞比例上升，血氧饱和度、病毒载量等重点指标全面向好，临床体征和症状明显好转。围绕康复者血浆中和病毒的治疗目的，细化了临床使用的适应证、禁忌症和不宜使用的情况。康复者血浆主要用于病情进展较快、重症、危重症新冠肺炎患者。原则上病程不超过3周；新冠病毒核酸检测阳性或临床专家判定患者存在病毒血症，在病情急性进展期应当尽早使用。尽管康复患者血浆疗法在临床上取得了一定的成效，然而由于抗原患者血浆来源有限、纯化抗体安全隐患高、特异性抗体效价不稳定。效价高、性能稳定、安全性好的单克隆抗体对于控制新冠状病毒疫情具有良好的应用前景。目前现有文献已经公开或教导了针对新冠病毒RBD制备保护性中和单抗的报道。利用新冠病毒刺突蛋白RBD产生抗新冠病毒的保护性中和抗体(如：bioRxiv，“SARS-CoV-2 and SARS-CoV Spike-RBD Structure and Receptor Binding Comparison and Potential Implications on Neutralizing Antibody  and Vaccine Development”，20200220)。SARS刺突蛋白RBD和新冠病毒刺突蛋白RBD存在交叉中和表位肽，抗SARS的单克隆抗体CR3022能够结合新冠病毒刺突蛋白RBD(Emerging Microbes & Infections,9(1):382-385,20200217)。采用同源建模的方法明确了新冠病毒病毒CTD1/人ACE2复合物的蛋白-蛋白相互作用界面的热点和关键残基，筛选靶向CTD1区域与ACE2结合表面的候选抑制剂，阻断病毒与人体ACE2蛋白的识别与结合。
随着抗SARS-CoV-2疫苗和治疗性抗体研究的持续推进，最近有报道表明部分治疗性抗体用于SARS-CoV-2治疗时可产生ADE现象。所谓的ADE，是指抗体依赖的增强作用，某些病毒在特异性抗体协助下复制或感染能力显著增强，在感染过程中会引发更严重病理损伤。通俗的解释就是，抗体不能中和病毒，反而充当了“特洛伊木马”，让病毒感染免疫细胞的能力更强，产生更多的子代病毒，造成严重症状。ADE(antibody-dependent enhancement)效应，即抗体依赖的增强效应，最早是在登革热病毒感染过程中被发现的，结合非中和性抗体的登革热病毒可以通过旁路进入巨噬细胞里面进行增殖，然后造成第二次感染，尤其是第二次感染和第一次感染的病毒株不一样的时候，第二次感染的症状更重，发生ADE效应。对于冠状病毒，上世纪八十年代初对猫传染性腹膜炎病毒(FIPV)的疫苗研究中发现针对S蛋白的低效价中和抗体反而会加剧症状、导致更严重的死亡。在对人造成感染的冠状病毒中，对SARS病毒和MERS病毒的研究发现疫苗接种产生的针对S蛋白的低亲和力抗体，可以介导病毒进入免疫细胞。基于冠状病毒建立感染的分子机理，如果抗新冠病毒SARS-CoV-2抗体也会产生ADE效应，则将严重影响抗新冠病毒SARS-CoV-2疫苗和治疗性抗体的临床应用。然而，对于具体的抗新冠病毒SARS-CoV-2 S蛋白的中和抗体是否产生ADE效应、产生ADE效应的分子机制、以及应对措施，目前还未见报道。
发明内容
为解决上述问题，本发明从新冠病毒感染后康复人员PBMC中分离冠状病毒RBD特异性记忆B细胞、扩增获得抗体的轻重链可变区序列，进行了瞬时表达，经检测其中至少20个抗体能够以高亲和力特异性结合冠状病毒RBD，并且至少7个抗体能够阻断或抑制新型冠状病毒RBD与宿主受体ACE2的结合。进 一步研究发现所述能够阻断或抑制新型冠状病毒RBD与宿主受体ACE2结合的7个抗体分子在SARS‐CoV‐2感染THP‐1细胞和K562细胞的过程中均不产生ADE效应，其中两个抗体Corn‐01、Corn‐05则能够促进SARS‐CoV‐2进入Raji宿主细胞，存在ADE效应。通过Raji宿主细胞的膜结合型Fc受体种类分析抗体产生ADE的机理，并进而通过对抗体Fc段突变降低或消除了抗体通过Fc与宿主细胞FcγRs受体的结合，从而避免了在SARS‐CoV‐2感染宿主细胞过程中产生ADE效应。具体而言：
一方面，本发明提供一种SARS‐CoV‐2 S蛋白‐ACEII受体结合阻断性单克隆抗体或其片段，其特征在于：所述单克隆抗体是从新型冠状病毒感染患者康复期血液样本中分离单个B细胞克隆获得的特异性结合SARS‐CoV‐2 S蛋白RBD区的全人源抗体，其对SARS‐CoV‐2感染THP‐1细胞和K562细胞均不产生ADE效应。
优选的，本发明所述SARS‐CoV‐2 S蛋白‐ACEII受体结合阻断性单克隆抗体或其片段，其特征在于所述单克隆抗体对SARS‐CoV‐2感染Raji细胞不产生ADE效应。
优选的，本发明所述SARS‐CoV‐2 S蛋白‐ACEII受体结合阻断性单克隆抗体或其片段，其特征在于所述单克隆抗体在10‐10000ng的部分浓度范围内对SARS‐CoV‐2感染Raji细胞产生ADE效应。
优选的，本发明所述SARS‐CoV‐2 S蛋白‐ACEII受体结合阻断性单克隆抗体或其片段，其特征在于所述单克隆抗体在50‐3000ng的部分浓度范围内对SARS‐CoV‐2感染Raji细胞产生ADE效应。
优选的，本发明所述SARS‐CoV‐2 S蛋白‐ACEII受体结合阻断性单克隆抗体或其片段，其特征在于所述单克隆抗体与SARS‐CoV‐2 S蛋白RBD具有高亲和性，KD值为5.0×10 ‐9M以下。
优选的，本发明所述SARS‐CoV‐2 S蛋白‐ACEII受体结合阻断性单克隆抗体或其片段，其特征在于所述单克隆抗体阻断ACEII与SARS‐CoV‐2 S蛋白RBD结合的IC50值小于50nM，优选小于30nM、25nM、20nM、15nM或10nM。
优选的，本发明所述SARS‐CoV‐2 S蛋白‐ACEII受体结合阻断性单克隆抗体或其片段，其特征在于所述单克隆抗体Fc段的点突变改变了其与受体的结合。
优选的，本发明所述SARS‐CoV‐2 S蛋白‐ACEII受体结合阻断性单克隆抗体或其片段，其特征在于所述单克隆抗体Fc段的点突变降低或消除了其与FcγRs的结合。
优选的，本发明所述SARS‐CoV‐2 S蛋白‐ACEII受体结合阻断性单克隆抗体或其片段，其特征在于所述单克隆抗体Fc段的点突变包括在由第234位、第235位组成的组中任意一个或两个位点进行氨基酸的替换、缺失或插入突变。
优选的，本发明所述SARS‐CoV‐2 S蛋白‐ACEII受体结合阻断性单克隆抗体或其片段，其特征在于所述单克隆抗体Fc段的点突变包括L234A、L235A突变。
优选的，本发明所述SARS‐CoV‐2 S蛋白‐ACEII受体结合阻断性单克隆抗体或其片段，其特征在于所述单克隆抗体的重链恒定区具有SEQ ID NO:16所示的序列。
在一个具体的实施例中，本发明所述SARS‐CoV‐2 S蛋白‐ACEII受体结合阻断性单克隆抗体或其片段，
其重链可变区具有SEQ ID NO:1、3、5、7、9、11、13所示氨基酸序列，或在SEQ ID NO:1、3、5、7、9、11、13所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；
其轻链可变区具有SEQ ID NO:2、4、6、8、10、12、14所示氨基酸序列，或在SEQ ID NO:2、4、6、8、10、12、14所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列。
优选的，本发明所述SARS-CoV-2 S蛋白-ACEII受体结合阻断性单克隆抗体或其片段，其特征在于：
重链可变区具有SEQ ID NO:1所示氨基酸序列，或在SEQ ID NO:1所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；并且，轻链可变区具有SEQ ID NO:2所示氨基酸序列，或在SEQ ID NO:2所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；
重链可变区具有SEQ ID NO:3所示氨基酸序列，或在SEQ ID NO:3所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；并且，轻链可变区具有SEQ ID NO:4所示氨基酸序列，或在SEQ  ID NO:4所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；
重链可变区具有SEQ ID NO:5所示氨基酸序列，或在SEQ ID NO:5所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；并且，轻链可变区具有SEQ ID NO:6所示氨基酸序列，或在SEQ ID NO:6所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；
重链可变区具有SEQ ID NO:7所示氨基酸序列，或在SEQ ID NO:7所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；并且，轻链可变区具有SEQ ID NO:8所示氨基酸序列，或在SEQ ID NO:8所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；
重链可变区具有SEQ ID NO:9所示氨基酸序列，或在SEQ ID NO:9所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；并且，轻链可变区具有SEQ ID NO:10所示氨基酸序列，或在SEQ ID NO:10所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；
重链可变区具有SEQ ID NO:11所示氨基酸序列，或在SEQ ID NO:11所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；并且，轻链可变区具有SEQ ID NO:12所示氨基酸序列，或在SEQ ID NO:12所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；
或者，
重链可变区具有SEQ ID NO:13所示氨基酸序列，或在SEQ ID NO:13所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；并且，轻链可变区具有SEQ ID NO:14所示氨基酸序列，或在SEQ ID NO:14所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列。
在另一个具体的实施例中，本发明任一项所述SARS-CoV-2 S蛋白-ACEII 受体结合阻断性单克隆抗体或其片段，其特征在于：
重链可变区具有SEQ ID NO:1、3、5、7、9、11、13中任一所示抗体重链可变区的HCDR1、HCDR2和HCDR3；或者与SEQ ID NO:1、3、5、7、9、11、13中任一具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％、99％、或100％同一性的序列所述抗体重链可变区的HCDR1、HCDR2和HCDR3；
并且，
轻链可变区具有SEQ ID NO:2、4、6、8、10、12、14中任一所示抗体轻链可变区的LCDR1-3、LCDR2和LCDR3；或者与SEQ ID NO:2、4、6、8、10、12、14中任一具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％、99％、或100％同一性的序列所述抗体轻链可变区的HCDR1、HCDR2和HCDR3。
优选的，发明所述SARS-CoV-2 S蛋白-ACEII受体结合阻断性单克隆抗体或其片段，其特征在于：
重链可变区具有SEQ ID NO:1所示抗体重链可变区的HCDR1、HCDR2和HCDR3；并且，轻链可变区具有SEQ ID NO:2所示抗体轻链可变区的LCDR1、LCDR2和LCDR3；
重链可变区具有SEQ ID NO:3所示抗体重链可变区的HCDR1、HCDR2和HCDR3；并且，轻链可变区具有SEQ ID NO:4所示抗体轻链可变区的LCDR1、LCDR2和LCDR3；
重链可变区具有SEQ ID NO:5所示抗体重链可变区的HCDR1、HCDR2和HCDR3；并且，轻链可变区具有SEQ ID NO:6所示抗体轻链可变区的LCDR1、LCDR2和LCDR3；
重链可变区具有SEQ ID NO:7所示抗体重链可变区的HCDR1、HCDR2和HCDR3；并且，轻链可变区具有SEQ ID NO:8所示抗体轻链可变区的LCDR1、LCDR2和LCDR3；
重链可变区具有SEQ ID NO:9所示抗体重链可变区的HCDR1、HCDR2和HCDR3；并且，轻链可变区具有SEQ ID NO:10所示抗体轻链可变区的LCDR1、LCDR2和LCDR3；
重链可变区具有SEQ ID NO:11所示抗体重链可变区的HCDR1、HCDR2 和HCDR3；并且，轻链可变区具有SEQ ID NO:12所示抗体轻链可变区的LCDR1、LCDR2和LCDR3；或
重链可变区具有SEQ ID NO:13所示抗体重链可变区的HCDR1、HCDR2和HCDR3；并且，轻链可变区具有SEQ ID NO:14所示抗体轻链可变区的LCDR1、LCDR2和LCDR3。
第二方面，本发明提供一种多核苷酸，其编码本发明前述的单克隆抗体或其片段。
第三方面，本发明提供一种核酸构建体，其包含本发明前述的多核苷酸。
优选的，本发明所述核酸构建体用于表达本发明前述单克隆抗体或其片段。
第四方面，本发明提供一种宿主细胞，其包含本发明前述的多核苷酸或本发明前述的核酸构建体。
第五方面，本发明提供一种组合物，其包含一种或多种选自由本发明前述单克隆抗体或其片段组成的组中的单克隆抗体或其片段，以及任选的药学上可接受的载体。
优选的，本发明所述的组合物，其包含选自由权利要求1至13中任一项所述单克隆抗体或其片段组成的组中的任意两种、三种、四种、五种、六种、或七种单克隆抗体或其片段。
第五方面，本发明提供本发明前述任一项所述单克隆抗体或其片段、本发明前述的多核苷酸、本发明前述的核酸构建体、本发明前述的宿主细胞、本发明前述的组合物在制备预防或治疗SARS‐CoV‐2感染药物中的应用。
优选的，本发明所述的应用，其特征在于所述预防或治疗SARS‐CoV‐2感染包括减少或降低SARS‐CoV‐2感染的风险、缓解SARS‐CoV‐2感染相关疾病(例如COVID‐19)的症状、缩短SARS‐CoV‐2感染相关疾病(例如COVID‐19)的病程、促进SARS‐CoV‐2感染相关疾病(例如COVID‐19)的康复、减少SARS‐CoV‐2感染导致的死亡。
第六方面，本发明提供一种预防或治疗SARS‐CoV‐2感染的方法，其特征在于对有需要的受试者施用有效量的本发明前述任一项所述单克隆抗体或其片段、本发明前述的多核苷酸、本发明前述的核酸构建体、本发明前述的宿主细胞、本发明前述的组合物。
优选的，本发明所述的方法，其特征在于所述受试者遭受SARS‐CoV‐2感染的风险、具有SARS‐CoV‐2感染相关疾病(例如COVID‐19)、或具有因SARS‐CoV‐2感染而导致死亡的可能。
优选的，本发明所述的方法，其特征在于所述方法使受试者减少或降低SARS‐CoV‐2感染的风险、缓解SARS‐CoV‐2感染相关疾病(例如COVID‐19)的症状、缩短SARS‐CoV‐2感染相关疾病(例如COVID‐19)的病程、促进SARS‐CoV‐2感染相关疾病(例如COVID‐19)的康复、减少SARS‐CoV‐2感染导致的死亡。
第七方面，本发明提供一种消除抗体在SARS‐CoV‐2感染宿主细胞时ADE效应的方法，其中，所述的抗体为抗SARS‐CoV‐2 S蛋白的中和性抗体，通过对抗体Fc段进行点突变改变了抗SARS‐CoV‐2 S蛋白抗体与其受体的结合。
优选的，本发明所述消除抗体在SARS‐CoV‐2感染宿主细胞时ADE效应的方法，其中所述Fc段的点突变降低或消除了抗SARS‐CoV‐2 S蛋白抗体与FcγRs的结合。
优选的，本发明所述消除抗体在SARS‐CoV‐2感染宿主细胞时ADE效应的方法，其中所述Fc段点突变包括在由第234位、第235位组成的组中任意一个或两个位点进行氨基酸的替换、缺失或插入突变。
优选的，本发明所述消除抗体在SARS‐CoV‐2感染宿主细胞时ADE效应的方法，其特征在于所述抗SARS‐CoV‐2 S蛋白的中和性抗体选自本发明前述任一项所述单克隆抗体或其片段。
在一个具体的实施例中，本发明提供一种特异性结合SARS‐CoV‐2 S蛋白的抗体，其中，所述抗体是在天然人源抗SARS‐CoV‐2 S蛋白抗体的基础上进行Fc段点突变，避免了天然人源抗SARS‐CoV‐2 S蛋白抗体用于阻止SARS‐CoV‐2感染宿主细胞时的ADE效应(抗体依赖的增强效应)；
所述特异性结合SARS‐CoV‐2 S蛋白的抗体在10‐10000ng/mL的浓度范围内对SARS‐CoV‐2感染THP‐1细胞、Raji细胞、K562细胞都不产生ADE效应。。
进一步，本发明所述的抗体，其中，所述天然人源抗SARS‐CoV‐2 S蛋白抗体能够中和SARS‐CoV‐2对Vero E宿主细胞的感染。
进一步，本发明所述的抗体，其中，所述天然人源抗SARS‐CoV‐2 S蛋白抗体至少在10‐10000ng/mL的部分浓度范围内对SARS‐CoV‐2感染Raji细胞具有 ADE效应。
进一步，本发明所述的抗体，其中，所述人源抗SARS‐CoV‐2 S蛋白抗体至少在50‐3000ng/mL的部分浓度范围内对SARS‐CoV‐2感染Raji细胞具有ADE效应。
进一步，本发明所述的抗体，其中，所述天然人源抗SARS‐CoV‐2 S蛋白抗体
其重链可变区具有SEQ ID NO:1、3、5、7、9、11、13所示氨基酸序列，或在SEQ ID NO:1、3、5、7、9、11、13所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；
其轻链可变区具有SEQ ID NO:2、4、6、8、10、12、14所示氨基酸序列，或在SEQ ID NO:2、4、6、8、10、12、14所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列。
优选的，本发明所述的抗体，其中，所述天然人源抗SARS‐CoV‐2 S蛋白抗体，
重链可变区具有SEQ ID NO:1所示氨基酸序列，或在SEQ ID NO:1所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；并且，轻链可变区具有SEQ ID NO:2所示氨基酸序列，或在SEQ ID NO:2所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；
重链可变区具有SEQ ID NO:3所示氨基酸序列，或在SEQ ID NO:3所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；并且，轻链可变区具有SEQ ID NO:4所示氨基酸序列，或在SEQ ID NO:4所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；
重链可变区具有SEQ ID NO:5所示氨基酸序列，或在SEQ ID NO:5所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；并且，轻链可变区具有SEQ ID NO:6所示氨基酸序列，或在SEQ ID NO:6所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；
重链可变区具有SEQ ID NO:7所示氨基酸序列，或在SEQ ID NO:7所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；并且，轻链可变区具有SEQ ID NO:8所示氨基酸序列，或在SEQ ID NO:8所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；
重链可变区具有SEQ ID NO:9所示氨基酸序列，或在SEQ ID NO:9所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；并且，轻链可变区具有SEQ ID NO:10所示氨基酸序列，或在SEQ ID NO:10所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；
重链可变区具有SEQ ID NO:11所示氨基酸序列，或在SEQ ID NO:11所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；并且，轻链可变区具有SEQ ID NO:12所示氨基酸序列，或在SEQ ID NO:12所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；
或者，
重链可变区具有SEQ ID NO:13所示氨基酸序列，或在SEQ ID NO:13所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；并且，轻链可变区具有SEQ ID NO:14所示氨基酸序列，或在SEQ ID NO:14所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列。
在另一个具体的实施例中，本发明任一项所述SARS‐CoV‐2 S蛋白‐ACEII受体结合阻断性单克隆抗体或其片段，其特征在于：
重链可变区具有SEQ ID NO:1、3、5、7、9、11、13中任一所示抗体重链可变区的HCDR1、HCDR2和HCDR3；或者与SEQ ID NO:1、3、5、7、9、11、13中任一具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％、99％、或100％同一性的序列所述抗体重链可变区的HCDR1、HCDR2和HCDR3；
并且，
轻链可变区具有SEQ ID NO:2、4、6、8、10、12、14中任一所示抗体轻链 可变区的LCDR1‐3、LCDR2和LCDR3；或者与SEQ ID NO:2、4、6、8、10、12、14中任一具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％、99％、或100％同一性的序列所述抗体轻链可变区的HCDR1、HCDR2和HCDR3。
优选的，发明所述SARS‐CoV‐2 S蛋白‐ACEII受体结合阻断性单克隆抗体或其片段，其特征在于：
重链可变区具有SEQ ID NO:1所示抗体重链可变区的HCDR1、HCDR2和HCDR3；并且，轻链可变区具有SEQ ID NO:2所示抗体重链可变区的LCDR1、LCDR2和LCDR3；
重链可变区具有SEQ ID NO:3所示抗体重链可变区的HCDR1、HCDR2和HCDR3；并且，轻链可变区具有SEQ ID NO:4所示抗体重链可变区的LCDR1、LCDR2和LCDR3；
重链可变区具有SEQ ID NO:5所示抗体重链可变区的HCDR1、HCDR2和HCDR3；并且，轻链可变区具有SEQ ID NO:6所示抗体重链可变区的LCDR1、LCDR2和LCDR3；
重链可变区具有SEQ ID NO:7所示抗体重链可变区的HCDR1、HCDR2和HCDR3；并且，轻链可变区具有SEQ ID NO:8所示抗体重链可变区的LCDR1、LCDR2和LCDR3；
重链可变区具有SEQ ID NO:9所示抗体重链可变区的HCDR1、HCDR2和HCDR3；并且，轻链可变区具有SEQ ID NO:10所示抗体重链可变区的LCDR1、LCDR2和LCDR3；
重链可变区具有SEQ ID NO:11所示抗体重链可变区的HCDR1、HCDR2和HCDR3；并且，轻链可变区具有SEQ ID NO:12所示抗体重链可变区的LCDR1、LCDR2和LCDR3；或
重链可变区具有SEQ ID NO:13所示抗体重链可变区的HCDR1、HCDR2和HCDR3；并且，轻链可变区具有SEQ ID NO:14所示抗体重链可变区的LCDR1、LCDR2和LCDR3。
进一步，本发明所述的抗体，其中所述Fc段的点突变改变了抗SARS‐CoV‐2 S蛋白抗体与其受体的结合。
进一步，本发明所述的抗体，其中，所述Fc段的点突变降低或消除了抗 SARS‐CoV‐2 S蛋白抗体与FcγRs的结合。
进一步，本发明所述的抗体，其中，所述抗体在10‐10000ng/mL的浓度范围内对SARS‐CoV‐2感染THP‐1细胞、Raji细胞、K562细胞都不产生ADE效应。
进一步，本发明所述的抗体，其中，Fc段点突变包括在由第234位、第235位组成的组中任意一个或两个位点进行氨基酸的替换、缺失或插入突变。
进一步，本发明所述的抗体，其中，Fc段点突变包括在由第234位、第235位组成的组中任意一个或两个位点进行单个氨基酸的替换、缺失或插入突变。
进一步，本发明所述的抗体，其中，所述Fc段点突变包括L234A、L235A突变。
在另一个具体的实施例中，本发明提供抗体在制备治疗SARS‐CoV‐2感染药物中的应用，其中所述抗体如本发明第一方面所述。
在另一个具体的实施例中，本发明提供抗体在制备治疗SARS‐CoV‐2感染导致的疾病药物中的应用，其中所述抗体如本发明第一方面所述，所述疾病包括COVID‐19。
在另一个具体的实施例中，本发明提供一种多核苷酸，其编码本发明第一方面所述的抗体。
在另一个具体的实施例中，本发明提供一种载体，其包含本发明第四方面所述的多核苷酸。
在另一个具体的实施例中，本发明提供一种宿主细胞，其包含本发明第四方面所述的多核苷酸或本发明第四方面所述的载体。
在另一个具体的实施例中，本发明提供一种药物组合物，其包含一种或多种选自由本发明第一方面所述抗体组成的组中的抗体，以及任选的药学上可接受的载体。
在另一个具体的实施例中，本发明提供一种消除抗体在SARS-CoV-2感染宿主细胞时ADE效应的方法，其中，所述的抗体为抗SARS-CoV-2 S蛋白的中和性抗体，通过对抗体Fc段进行点突变改变了抗SARS-CoV-2 S蛋白抗体与其受体的结合。
进一步，本发明所述消除抗体在SARS-CoV-2感染宿主细胞时ADE效应的方法，其中，所述Fc段的点突变降低或消除了抗SARS-CoV-2 S蛋白抗体与 FcγRs的结合。
进一步，本发明所述消除抗体在SARS-CoV-2感染宿主细胞时ADE效应的方法，所述Fc段点突变包括在由第234位、第235位组成的组中任意一个或两个位点进行氨基酸的替换、缺失或插入突变。
为更好理解本发明，首先定义一些术语。其他定义则贯穿具体实施方式部分而列出。
术语“冠状病毒”是指套式病毒目(Nidovirales)、冠状病毒科(Coronaviridae)、冠状病毒属(Coronavirus)的成员。本发明所述冠状病毒主要涉及感染人的冠状病毒，包括HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、MERS-CoV、SARS-CoV-2(2019-nCov)，本发明所述冠状病毒特别涉及SARS-CoV、MERS-CoV、SARS-CoV-2(2019-nCov)。
术语“特异性”是指在蛋白和/或其他生物异质群体中确定是否存在所述蛋白，例如本发明所述单抗与SARS-CoV-2RBD蛋白的结合反应。因此，在所指定的条件下，特定的配体/抗原与特定的受体/抗体结合，并且并不以显著的量与样本中存在的其它蛋白结合。
本文中的术语“抗体”意在包括全长抗体及其任何抗原结合片段(简称抗体片段)或单链。全长抗体是包含至少两条重(H)链和两条轻(L)链的糖蛋白，重链和轻链由二硫键连接。各重链由重链可变区(简称VH)和重链恒定区构成。重链恒定区由三个结构域构成，即CH1、CH2和CH3。各轻链由轻链可变区(简称VL)和轻链恒定区构成。轻链恒定区由一个结构域CL构成。VH和VL区还可以划分为称作互补决定区(CDR)的高变区，其由较为保守的框架区(FR)区分隔开。各VH和VL由三个CDR以及四个FR构成，从氨基端到羧基端以FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4的顺序排布。重链和轻链的可变区包含与抗原相互作用的结合域。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子的结合，包括多种免疫系统细胞(例如，效应细胞)和传统补体系统的第一组分(C1q)。
术语“超变区”或“CDR区”或“互补决定区”是指负责抗原结合的抗体氨基酸残基。CDR区序列可以由IMGT、Kabat、Chothia和AbM方法来定义或本领域熟知的任何CDR区序列确定方法而鉴定的可变区内的氨基酸残基。抗体CDR可鉴定为最初由Kabat等人定义的高变区，例如，轻链可变结构域的 24-34(L1)、50-56(L2)和89-97(L3)位残基和重链可变结构域的31-35(H1)、50-65(H2)和95-102(H3)位残基，参见Kabat EA等,1991,Sequences ofProteins of Immunological Interest(免疫目的物的蛋白质序列),第5版,PublicHealth Service,National Institutes of Health,Bethesda,Md.；CDR的位置亦可鉴定为最初由Chothia等人描述的“超变环”(HVL)结构来界定的。IMGT(ImMunoGeneTics)也提供了包括CDR的免疫球蛋白可变区的编号系统，根据IMGT编号定义CDR区，例如，轻链可变结构域的27-32(L1)、50-52(L2)和89-97(L3)位残基和重链可变结构域的26-35(H1)、51-57(H2)和93-102(H3)位残基，参见如Lefranc MP等的Dev.Comp.Immunol.,2003,27:55-77，其通过引用并入本文。用于CDR鉴定的其它方法包括“AbM定义”，其为Kabat与Chothia之间的折衷且使用Oxford Molecular′s AbM抗体模型软件得到；或CDR的“接触定义”，其基于所观察的抗原接触且阐述于MacCallum RM等人,1996,J.Mol Biol.,262:732-745中。CDR的“构型定义”方法中，CDR的位置可鉴定为对抗原结合作出贡献的残基，参见例如Makabe K等人,2008,J.Biol Chem.,283:1156-1166。本发明的抗体或其抗原结合片段含有的CDR可根据本领域已知的各种编号系统确定。在某些实施方案中，本发明的抗体或其抗原结合片段含有的CDR优选地通过Kabat、Chothia或IMGT编号系统确定，特别是通过Kabat编号系统确定。
术语“单克隆抗体”或“单抗”或“单克隆抗体组成”是指单一分子组成的抗体分子制品。单克隆抗体组成呈现出对于特定表位的单一结合特异性和亲和力。
本文中的术语，抗体的“抗原结合片段”(或简称为抗体片段)，是指抗体的保持有特异结合抗原能力的一个或多个片段。已证实，抗体的抗原结合功能可以通过全长抗体的片段来实施。包含在抗体的“抗原结合部分”中的结合片段的例子包括(i)Fab片段，由VL、VH、CL和CH1构成的单价片段；(ii)F(ab′)2片段，包含铰链区二硫桥连接的两个Fab片段的二价片段；(iii)由VH和CH1构成的Fd片段；(iv)由抗体单臂VL和VH构成的Fv片段；(v)由VH构成的dAb片段(Ward et al.，(1989)Nature 341：544-546)；(vi)分离的互补决定区(CDR)；以及(vii)纳米抗体，一种包含单可变结构域和两个恒定结构域的重链可变区。此外，尽管Fv片段的两个结构域VL和VH由不同的基因编码，它们可以通过重组法经由使两者成为单蛋白链的合成接头而连接，其中VL和VH区配对形成单价分 子(称为单链Fc(scFv)；参见例如Bird et al.，(1988)Science 242：423-426；and Huston et al.，(1988)Proc.Natl.Acad.Sci.USA 85：5879-5883)。这些单链抗体也意在包括在术语涵义中。这些抗体片段可以通过本领域技术人员已知的常用技术而得到，且片段可以通过与完整抗体相同的方式进行功能筛选。
本发明的抗原结合片段包括能够特异性结合冠状病毒RBD的那些。抗体结合片段的实例包括例如但不限于Fab、Fab'、F(ab') 2、Fv片段、单链Fv(scFv)片段和单结构域片段。
Fab片段含有轻链的恒定结构域和重链的第一恒定结构域(CH1)。Fab'片段与Fab片段的不同之处在于在重链CH1结构域的羧基末端处的少数残基的添加，包括来自抗体铰链区的一个或多个半胱氨酸。通过切割在F(ab')2胃蛋白酶消化产物的铰链半胱氨酸处的二硫键产生Fab'片段。抗体片段的另外化学偶联是本领域普通技术人员已知的。Fab和F(ab')2片段缺乏完整抗体的片段可结晶(Fc)区，从动物的循环中更快速地清除，并且可能具有比完整抗体更少的非特异性组织结合(参见例如，Wahl等人，1983，J.Nucl.Med.24:316)。
如本领域通常理解的，“Fc”区是不包含抗原特异性结合区的抗体的片段可结晶恒定区。在IgG、IgA和IgD抗体同种型中，Fc区由两个相同的蛋白质片段组成，衍生自抗体的两条重链的第二和第三恒定结构域(分别为CH2和CH3结构域)。IgM和IgE Fc区在每条多肽链中含有三个重链恒定结构域(CH2、CH3和CH4结构域)。
“Fv”片段是含有完整靶识别和结合位点的抗体的最小片段。该区域由以紧密的非共价结合的一个重链和一个轻链可变结构域的二聚体(VH-VL二聚体)组成。在该构型中，每个可变结构域的三个CDR相互作用，以限定在VH-VL二聚体的表面上的靶结合位点。通常，六个CDR对抗体赋予靶结合特异性。然而，在一些情况下，甚至单个可变结构域(或仅包含对于靶特异性的三个CDR的Fv的一半)可以具有识别且结合靶的能力，尽管其亲和力低于整个结合位点。
“单链Fv”或“scFv”抗体结合片段包含抗体的VH和VL结构域，其中这些结构域存在于单条多肽链中。一般地，Fv多肽进一步包含在VH和VL结构域之间的多肽接头，其致使scFv能够形成有利于靶结合的结构。
“单结构域片段”由对冠状病毒RBD显示出足够亲和力的单个VH或VL结构域组成。在一个具体实施方案中，单结构域片段是骆驼化的(参见例如，Riechmann，1999，Journal ofImmunological Methods 231:25–38)。
本发明的抗冠状病毒RBD的抗体包括衍生化抗体。例如，衍生化抗体通常通过糖基化、乙酰化、聚乙二醇化、磷酸化、酰胺化、通过已知保护/封闭基团的衍生化、蛋白酶解切割、与细胞配体或其它蛋白质的连接来修饰。可以通过已知技术进行众多化学修饰中的任一种，所述技术包括但不限于特定的化学切割、乙酰化、甲酰化、衣霉素的代谢合成等。另外，衍生物可以含有一种或多种非天然氨基酸，例如，使用ambrx技术(参见例如，Wolfson，2006，Chem.Biol.13(10):1011-2)。
“人源抗体”包括具有人免疫球蛋白的氨基酸序列的抗体，并且包括从人免疫球蛋白文库或动物中分离的抗体，所述动物对于一种或多种人免疫球蛋白是转基因的，并且不表达内源免疫球蛋白。人抗体可以通过本领域已知的各种方法制备，所述方法包括使用衍生自人免疫球蛋白序列的抗体文库的噬菌体展示方法。参见美国专利号4,444,887和4,716,111；以及PCT公开WO 98/46645；WO 98/50433；WO 98/24893；WO 98/16654；WO 96/34096；WO96/33735；和WO 91/10741。还可以使用不能表达功能性内源免疫球蛋白，但可以表达人免疫球蛋白基因的转基因小鼠来产生人抗体。参见例如，PCT公开WO 98/24893；WO 92/01047；WO 96/34096；WO 96/33735；美国专利号5,413,923；5,625,126；5,633,425；5,569,825；5,661,016；5,545,806；5,814,318；5,885,793；5,916,771；和5,939,598。另外，使用与上述类似的技术，公司例如LakePharma，Inc.(Belmont，CA)或Creative BioLabs(Shirley，NY)可以从事于提供针对所选抗原的人抗体。可以使用被称为“引导选择”的技术生成识别所选表位的全人抗体。在该方法中，选择的非人单克隆抗体，例如小鼠抗体，用于引导识别相同表位的完全人抗体的选择(参见，Jespers等人，1988，Biotechnology 12:899-903)。
术语“识别抗原的抗体”以及“对抗原特异的抗体”在本文中与术语“特异结合抗原的抗体”交替使用。
术语“高亲和性”对于IgG抗体而言，是指对于抗原的KD为1.0×10 -6M以下，优选5.0×10 -8M以下，更优选1.0×10 -8M以下、5.0×10 -9M以下，更优选1.0×10 -9M以下。对于其他抗体亚型，“高亲和性”结合可能会变化。例如，IgM亚型的“高亲和性”结合是指KD为10 -6M以下，优选10 -7M以下，更优选10 -8M以下。
术语“Kassoc”或“Ka”是指特定抗体-抗原相互作用的结合速率，而术语“Kdis”或“Kd”是指特定抗体-抗原相互作用的离解速率。术语“KD”是指解离常数，由Kd与Ka比(Kd/Ka)得到，并以摩尔浓度(M)表示。抗体的KD值可以通过领域内已知的方法确定。优选的确定抗体KD的方式是使用表面等离子共振仪(SPR)测得的，优选使用生物传感系统例如BiacoreTM系统测得。
术语“EC50”，又叫半最大效应浓度，是指引起50％最大效应的抗体浓度。
术语“IC50”，是指被测量的拮抗剂的半抑制浓度。它能指示某一药物或者物质(抑制剂)在抑制某些生物程序(或者是包含在此程序中的某些物质，比如酶，细胞受体或是微生物)的半量。
如本文所使用，“表位”是指抗原上B和/或T细胞作出反应的位点。B细胞表位可均由连续氨基酸或因蛋白质的三级折叠而并列的不连续氨基酸形成。由连续氨基酸形成的表位在暴露于变性溶剂时典型地被保留，而通过三级折叠而形成的表位在用变性溶剂处理时典型地丢失。表位典型地包括呈独特空间构象的至少3个，且更通常是至少5或8-10个氨基酸。
如本文所使用，“组合物”，本发明提供包含如本文所述抗体或其抗原结合片段的组合物。根据本发明的组合物可以与适合的载剂、赋形剂和并入调配物中以提供改良的转移、递送、耐受性和类似性质的其它药剂一起给予。在所有医药化学工作者已知的配方书中可以发现许多适当的调配物：Remington′s Pharmaceutical Sciences，MackPublishing Company，Easton，PA。这些调配物包括例如粉剂、糊剂、膏剂、果冻、蜡、油、脂质、含脂质(阳离子或阴离子)的囊泡(如LIPOFECTINTM)、DNA结合物、无水吸收糊剂、水包油和油包水乳液、乳液碳蜡(各种分子量的聚乙二醇)、半固体凝胶和含有碳蜡的半固体混合物。还参见Powell等人“Compendium of excipients for parenteral formulations”PDA(1998)《医药科学技术杂志(J Pharm Sci Technol)》52：238-311。
本发明所述组合物优选为可注射制剂，可注射制剂可以包括用于静脉内、皮下、皮内和肌肉内注射、点滴输注等的剂型。这些可注射制剂可以通过公众已知的方法来制备。举例来说，可以例如通过将上文所描述的抗体或其盐溶解、悬浮或乳化在常规用于注射的无菌水性介质或油性介质中来制备注射制剂。作为用于注射的水性介质，存在例如生理盐水、含有葡萄糖和其它助剂的等张溶液等，其可以与如以下的适当的增溶剂组合使用：醇(例如乙醇)、多元醇(例如丙二醇、聚乙二醇)、非离子表面活性剂[例如聚山梨醇酯80、HCO-50(氢化蓖麻油的聚氧乙烯(50mol)加合物)]等。作为油性介质，存在已采用的例如芝麻油、大豆油等，其可与如苯甲酸苯甲酯、苯甲醇等增溶剂组合使用。因此制备的注射优选填充于适当的安瓿中。
如本文所使用，“氨基酸突变”在本文中意思指多肽序列中的氨基酸取代、插入和/或缺失，或针对以化学方式连接至蛋白质的部分的改变。举例来说，突变可以是附接至蛋白质的碳水化合物或PEG结构的改变。“氨基酸突变”在本文中意思指多肽序列中的氨基酸取代、插入和/或缺失。为清楚起见，除非另外说明，否则氨基酸突变通常是针对DNA编码的氨基酸，例如在DNA和RNA中具有密码子的20种氨基酸。
如本文所使用，“氨基酸取代”或“取代”在本文中意思指亲本多肽序列中特定位置处的氨基酸被不同氨基酸置换。确切地说，在一些实施方案中，取代是针对特定位置处非天然存在的氨基酸，这些氨基酸不是生物体内天然存在的或是任何生物体中的。举例来说，取代E272Y是指272位的谷氨酸被酪氨酸置换的变体多肽，在此情形中是Fc变体。为清楚起见，蛋白质被工程改造成改变核酸编码序列但不改变起始氨基酸(例如CGG(编码精氨酸)变为CGA(仍编码精氨酸)，用以增加宿主生物体表达水平)不是“氨基酸取代”；也就是说，尽管产生了编码同一蛋白质的新基因，但如果该蛋白质在其起始的特定位置处具有相同氨基酸，就不是氨基酸取代。
如本文所使用，“氨基酸插入”或“插入”意思指在亲本多肽序列中的特定位置处添加氨基酸序列。举例来说，-233E或233E指示在233位后并且在234位前插入谷氨酸。另外，-233ADE或A233ADE指示在233位后并且在234位前插入AlaAspGlu。
如本文所使用，“氨基酸缺失”或“缺失”意思指去除亲本多肽序列中特定位置处的氨基酸序列。举例来说，G236-或G236#或G236del指示在236位处的甘氨酸缺失。另外，EDA233-或EDA233#表示自233位开始缺失序列GluAspAla。
如本文所使用，“同一性”用于指两个多肽之间或两个核酸之间的序列相似程度。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.,48:443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl.Biosci.，4:11-17,1988)的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J.MoI.Biol.,48:444-453,1970)算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gapweight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文所使用，“残基”意思指蛋白质中的位置及其相关氨基酸的身份。举例来说，天冬酰胺297(又称为Asn297或N297)是在人类抗体IgG1中297位的残基。
如本文所使用，“核酸分子”、“多核苷酸”旨在包括DNA分子和RNA分子。核酸分子可以是单链或双链的，并且可以是cDNA。
如本文所使用，“宿主细胞”在其中载体可以增殖并且其DNA可以表达的细胞，所述细胞可以是原核细胞或者真核细胞。该术语还包括受试宿主细胞的 任何后代。应理解，并不是所有的后代都与亲本细胞相同，因为在复制过程中可能会发生突变，这类后代被包括在内。
如本文所使用，“载体”、“核酸构建体”是指能够运输与其连接的另一种核酸的核酸分子。一种类型的载体是“质粒”，其是指其中可以连接另外的DNA区段的环状双链DNA环。另一种类型的载体是病毒载体，其中额外的DNA区段可以连接到病毒基因组中。某些载体能够在它们被导入的宿主细胞中自主复制(例如，具有细菌复制起点和游离型哺乳动物载体的细菌载体)。其他载体(例如非附加型哺乳动物载体)可以在导入宿主细胞后整合到宿主细胞的基因组中，并由此与宿主基因组一起复制。此外，某些载体能够指导它们有效连接的基因的表达。这种载体在本文中被称为“重组表达载体”(或简称为“表达载体”)。通常，在重组DNA技术中有用的表达载体通常以质粒的形式存在。然而，也包括其他形式的表达载体，如病毒载体(例如，复制缺陷型逆转录病毒，腺病毒和腺伴随病毒)，其起到等同的功能。
如本文所使用，“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如，感染)有效量是指，足以预防，阻止，或延迟疾病(例如，感染)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
术语“预防”是指，为了阻止或延迟疾病或病症或症状(例如，感染)在受试者体内的发生或如果它发生作用减到最小而实施的方法。
术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。有益或所需的临床结果包括但不限于，降低疾病进展速率，改善或减轻疾病状态，和消退或改善的预后，无论是可检测或是不可检测的。有效缓解任何特定疾病症状的治疗剂的量可以根据诸如患者的疾病状态，年龄和体重以及药物在受试者中引起期望的反应的能力等因素而变化。疾病症状是否得到缓解可以通过任何临床测量来评估，这些测量通常由医生或其他熟练的医疗保健提供者用于评估该症状的严重程度或进展状态。
与现有技术相比，本发明的技术方案具有以下优点：
第一，本发明采用单个淋巴细胞克隆技术，从SARS-CoV-2感染康复期患者的大量单个B细胞中扩增获得了多个特异性抗体，经过重组表达、特异性结合能力筛选、SARS-CoV-2 S蛋白‐ACEII结合阻断活性分析获得了能以高亲和力、特异性结合SARS-CoV-2 S蛋白RBD，且能够阻断SARS-CoV-2结合宿主细胞受体ACEII的7株单克隆抗体，提供了其轻重链CDRs区的氨基酸序列。筛选过程中发现，抗体阻断SARS-CoV-2结合宿主细胞受体ACEII的活性与其特异性结合SARS-CoV-2 S蛋白RBD的亲和力并无正相关性。本发明的单抗在假病毒中和试验、SARS-CoV-2病毒颗粒中和试验中都取得了良好的中和封闭的技术效果。
第二，本发明对筛选获得的具有SARS‐CoV‐2 S蛋白‐ACEII结合阻断活性的7株单克隆抗体进行ADE分析，其在SARS-CoV-2感染THP-1细胞和K562细胞的过程均不产生ADE效应；提示本发明筛选的7株单抗在临床应用中具有较低的ADE效应。另外，本发明的7株单克隆抗体不仅是康复患者血清中天然存在的全人源抗体，而且针对SARS‐CoV‐2 S蛋白RBD多个不同的天然表位，因此特别适合“鸡尾酒抗体疗法”。
第三，本发明研究发现特定浓度范围的抗体Corn‐01、Corn‐05在SARS‐CoV‐2病毒感染Raji宿主细胞时会产生ADE效应。结合现有技术的研究报道，探索了抗SARS‐CoV‐2的中和性抗体产生ADE效应的机理，根据Raji宿主细胞与其它不产生ADE效应的宿主细胞上Fc受体种类的不同，分析抗SARS‐CoV‐2中和性抗体产生ADE效应的机理可能是由于抗体与宿主细胞膜上FcγRI受体的结合导致的；从而使得本领域技术人员能够通过阻断抗SARS‐CoV‐2中和性抗体与宿主细胞FcγRIIb受体的结合而降低或消除ADE效应，即保留了抗体的病毒阻断活性，又消除了可能的ADE效应，提高了人体临床应用的安全性。
附图说明
通过阅读下文优选实施方式的详细描述，各种其他的优点和益处对于本领域普通技术人员将变得清楚明了。附图仅用于示出优选实施方式的目的，而并不认为是对本发明的限制。而且在整个附图中，用相同的参考符号表示相同的部件。在附图中：
图1：SARS-CoV-2 S1蛋白与His的融合表达。
图2：SARS-CoV-2 S1蛋白与mFc的融合表达。
图3：SARS-CoV-2 S1蛋白RBD与His的融合表达。
图4：SARS-CoV-2 S1蛋白RBD与mFc的融合表达。
图5：人ACE2与人Fc的融合表达。
图6：重组抗体体Corn‐02、Corn‐10、Corn‐01阻断S1RBD-mFc与ACE2结合的能力。CR3022是US2010172917A1公开的一株抗SARS-Cov RBD的单抗；Corn‐10是本发明实施例4中制备的与S1RBD-mFc有特异性结合的单抗。
图7：重组抗体Corn‐02、Corn‐10、Corn‐01阻断S1-mFc与ACE2结合的能力。
图8：重组抗体对S1RBD-mFc与ACEII-hFc结合的阻断作用曲线图。
图9：Corn‐01、Corn‐07对S1RBD-mFc与ACEII-hFc结合阻断作用曲线图(竞争ELISA法)。
图10：Corn‐01、Corn‐07对S1RBD-His与ACEII-his结合的阻断作用曲线图(假病毒感染荧光检测法)。
图11：Corn‐01在SARS-CoV-2病毒颗粒感染不同宿主细胞过程中的ADE效应
图12：Corn‐01、Corn‐04、Corn‐05、Corn‐06对SARS-CoV-2病毒颗粒感染Raji细胞的ADE效应
图13：Corn‐01、Corn‐05、Corn‐07对SARS-CoV-2病毒颗粒感染Raji细胞的ADE效应
图14：Corn‐01、Corn‐01‐LALA对SARS-CoV-2病毒颗粒感染Raji细胞的ADE效应
图15：Corn‐05、Corn‐05‐LALA对SARS-CoV-2病毒颗粒感染Raji细胞的ADE效应
图16：Corn‐01、Corn‐05对SARS-CoV-2病毒颗粒的中和活性
图17：Corn‐01、Corn‐01‐LALA对SARS-CoV-2病毒颗粒的中和活性分析
图18：Corn‐05、Corn‐05‐LALA对SARS-CoV-2病毒颗粒的中和活性分析。
具体实施方式
下面将参照附图更详细地描述本公开的示例性实施方式。虽然附图中显示了本公开的示例性实施方式，然而应当理解，可以以各种形式实现本公开而不应被这里阐述的实施方式所限制。相反，提供这些实施方式是为了能够更透彻地理解本公开，并且能够将本公开的范围完整的传达给本领域的技术人员。
实施例1：SARS-CoV-2抗原及宿主受体的重组表达
将全合成的基因S1RBD(Accession：QHD43416.1，319-541aa)，通过酶切方法分别克隆入C端带有His标签或mFc标签的真核瞬时表达载体中，将获得的表达质粒，转入大肠杆菌扩增，分离获得S1RBD-his和S1RBD-mFc表达质粒，并根据转染试剂293fectin(Cat:12347019，Gibco)的操作说明，将质粒转入HEK293细胞中重组表达。细胞转染后5-6天，取培养上清，S1RBD-mFc利用ProA亲和层析柱对表达上清进行纯化，获得S1RBD-mFc蛋白(氨基酸序列如SEQ ID NO:18所示)。S1RBD-his(氨基酸序列如SEQ ID NO:17所示)利用HisTrap HP柱亲和层析柱对表达上清进行纯化，获得S1RBD-his蛋白。并将获得的重组蛋白通过SDS-PAGE检测纯度(图3-4)。
通过PCR的方法从购买的全长SARS‐CoV‐2表达载体(Cat:VG40589‐UT，北京义翘神州)中克隆出S1基因(Accession：QHD43416.1，1‐685aa)，并通过酶切方法分别克隆入C端带有mFc标签或His标签的真核瞬时表达载体中，将获得的表达质粒，转入大肠杆菌扩增，分离获得S1‐mFc、S1‐His表达质粒，并根据转染试剂293fectin(Cat:12347019，Gibco)的操作说明，将质粒转入HEK293细胞中重组表达。细胞转染后5‐6天，取培养上清，纯化，获得S1–mFc(氨基酸序列如SEQ ID NO:20所示)、S1‐His蛋白(氨基酸序列如SEQ ID NO:19所示)。并将获得的重组蛋白通过SDS‐PAGE检测纯度(图1‐2)。
通过PCR的方法从购买的全长人ACEII表达载体(Cat:HG10108‐ACR，北京义翘神州)中克隆出ACEII胞外区基因ACEII(1‐615)(Accession：NP_068576.1，1‐615aa)，并通过酶切方法分别克隆入C端带有hFc标签的真核瞬时表达载体中，将获得的表达质粒，转入大肠杆菌扩增，分离获得，ACEII(1‐615)‐hFc表达质粒，并根据转染试剂293fectin(Cat:12347019，Gibco)的操作说明，将质粒转入HEK293细胞中重组表达。细胞转染后5‐6天，取培养上清，利用ProA 亲和层析柱对表达上清进行纯化，获得ACEII(1‐615)‐hFc蛋白(氨基酸序列如SEQ ID NO:21所示)，并将获得的重组蛋白通过SDS‐PAGE检测纯度(图5)。
实施例2：SARS‐CoV‐2 S1蛋白RBD特异性记忆B细胞的分离
对新型冠状病毒感染康复患者的特异性记忆B细胞进行检测和分选，采用新冠抗体检测试剂盒对患者血清中IgG及IgM抗体进行了检测，选择新冠抗体IgG阳性的血清样本。利用RosetteSep试剂盒(Cat:15064,STEMCELL)对B细胞进行了富集，在此基础上采用FITC标记的S1‐RBD‐his去捕获新冠RBD特异性结合的记忆性B细胞并进行流式单细胞分选。
实施例3：人源抗SARS‐CoV‐2RBD抗体序列的扩增
采用RNA磁珠(南京诺唯赞)提取单B细胞的RNA，反转录成cDNA，具体方法如下：
1.每孔分装5μl Catch Buffer B(TCL+1％β‐ME)，分选单个记忆性B细胞。
2.贴膜，2000rpm离心1min。
3.每孔加入10μl H2O和33μl Beads，吹吸混匀，室温作用10min。
4.置磁力架，室温5min，弃上清。
5.用200μl无核酸酶水新鲜配置的80％乙醇漂洗磁珠，室温30s，弃上清。
6.重复漂洗一次，弃上清，风干3min。
7.移下磁力架，每孔加12μl Mix 1，吹吸5次，室温作用5min。
8.置磁力架，室温2min，转移10μl至新板，300g离心30s，运行程序1。
9.每孔加10μl Mix 2，混匀、离心，运行程序2。
10.合成好的cDNA尽快进行PCR。
Mix 1：310μl H2O+50μl dNTP+20μl Random 6+20μl Oligo_dT
Mix 2：170μl H2O+160μl Buffer+40μl DTT+20μl RNase I+10μl RTase IV(Cat:EN0601 and 18090010,ThermoFisher)
程序1：65℃5min→4℃∞
程序2：23℃10min→50℃30min→80℃10min→4℃∞
采用两步PCR扩增抗体重链以及轻链(Kappa)可变区基因。引物序列源自于Human Monoclonal Antibodies书中第114至117页。具体方法如下：
第一轮PCR(Ig‐VH1、Ig‐VK1)，反应体系(20μl)：
Figure PCTCN2020122793-appb-000001
运行程序：
94℃5min→(94℃30s→51℃30s→72℃55s)×15Cycles
→(94℃30s→56℃30s→72℃55s)×30Cycles
→72℃8min
→4℃∞
第二轮PCR(Ig‐VH2、Ig‐VK2)，反应体系(20μl)：
Figure PCTCN2020122793-appb-000002
运行程序：
94℃5min→(94℃30s→57℃30s→72℃45s)×50Cycles
→72℃10min
→4℃∞
琼脂糖凝胶电泳分离纯化PCR产物并进行抗体轻、重链可变区测序。
实施例4：人源抗SARS‐CoV‐2RBD抗体的表达及特异性结合初筛鉴定
对测序后的122对序列进行分析、并进一步合成其中的49对抗体轻重链可变区基因，将其克隆入全抗体瞬时表达载体中，进行重组表达和特异性鉴定。 将全合成的抗体重链可变区，通过酶切克隆入真核瞬时表达载体pKN041的人IgG1的重链恒定区编码基因的上游，全合成的抗体轻链可变区通过酶切克隆入真核瞬时表达载体pKN019的人轻链Cκ的编码基因的上游，构建轻、重链表达载体，获得轻链和重链表达质粒，转入大肠杆菌扩增，分离获得抗体轻链和重链质粒，并根据转染试剂293fectin(Cat:12347019，Gibco)的操作说明，将抗体的轻、重链质粒转入HEK293细胞中重组表达。
细胞转染后24小时，取上清利用Fortebio公司的Octet QKe system仪器，采用抗人抗体Fc段的捕获抗体(AHC)生物探针捕获抗体Fc段的方法测定抗体与S1RBD的结合。测定时将抗体上清及抗体，流经AHC探针(Cat:18‐0015，PALL)表面，时间为240s。S1RBD‐mFc(KN表达，lot:20200217A)作为流动相，S1RBD‐mFc重组蛋白浓度为100nM。结合时间为300s，解离时间为300s。实验完毕，用软件进行1：1Langmuir结合模式拟合，计算抗原抗体结合的动力学常数。共检测了39个抗体，其中与S1RBD‐mFc有特异性结合的抗体为20个(表1)。
表1 候选抗体与S1RBD‐mFc的特异性结合动力学参数。
名称 KD(M) Kon(1/Ms) Kdis(1/s)
1(Corn-07) 4.24E-10 1.73E+05 7.35E-05
2 2.98E-09 1.04E+05 3.09E-04
3 5.59E-07 2.79E+04 1.56E-02
4 1.99E-05 1.60E+03 3.18E-02
5(Corn-01) 4.02E-10 2.01E+05 8.09E-05
6(Corn-8) 2.88E-09 1.55E+06 4.46E-03
7 1.16E+14 1.97E+03 2.29E+17
8 7.06E-06 4.68E+04 3.30E-01
9 2.18E+16 5.34E+03 1.17E+20
10 6.75E+08 2.14E+04 1.45E+13
11(Corn-03) 1.55E-09 1.47E+05 2.27E-04
12 1.37E+22 9.34E+04 1.28E+27
13 1.67E+09 3.75E+03 6.26E+12
14 7.44E+19 3.18E+03 2.36E+23
15 5.11E-09 1.97E+06 1.00E-02
16(Corn-09) 1.45E-09 6.65E+05 9.66E-04
17(Corn-02) 2.60E-10 3.85E+05 1.00E-04
18 3.18E-09 2.12E+05 6.76E-04
19 6.69E-06 5.76E+04 3.86E-01
20 2.07E-08 1.10E+06 2.29E-02
21 2.43E+09 1.39E+04 3.36E+13
22(Corn-10) 1.43E-09 6.17E+05 8.83E-04
23 4.56E-06 2.89E+05 1.32E+00
24(Corn-04) 7.43E-10 2.49E+05 1.85E-04
25 1.26E+19 8.62E+03 1.09E+23
26 3.35E+07 3.00E+04 1.01E+12
27 1.36E-08 1.01E+06 1.37E-02
28 4.93E-09 2.72E+06 1.34E-02
29 2.50E-09 1.66E+05 4.15E-04
30 8.10E-09 2.59E+05 2.10E-03
31 3.19E-10 5.10E+05 1.63E-04
32(Corn-05) 1.39E-09 1.94E+05 2.69E-04
33 3.91E-09 6.85E+05 2.68E-04
34 1.65E+23 1.81E+04 2.97E+27
35 6.35E-10 1.65E+05 1.05E-04
36 1.30E-09 5.65E+05 7.32E-04
37 1.03E+08 3.71E+05 3.82E+13
38(Corn-06) 3.70E-09 1.09E+05 4.02E-04
39 3.74E-06 1.50E+05 5.61E-01
根据表1可知，39株成功表达的抗体分子中经过初筛对S1RBD‐mFc具有特异性结合能力的20株抗体分别为克隆编号1、2、29、5、6、31、15、22、16、17、18、36、35、33、27、28、38、32、24、11。从初筛亲和力高的抗体克隆选择1、17、22、5、24、32、38、11进一步研究。所述抗体克隆5、17、11、24、32、38、1在下文中也被称为Corn‐01、Corn‐02、Corn‐03、Corn‐04、Corn‐05、Corn‐06、Corn‐07。
抗体Corn-01
重链可变区(SEQ ID NO:1)：
Figure PCTCN2020122793-appb-000003
轻链可变区(SEQ ID NO:2)：
Figure PCTCN2020122793-appb-000004
抗体Corn-02
重链可变区(SEQ ID NO:3)：
Figure PCTCN2020122793-appb-000005
轻链可变区(SEQ ID NO:4)：
Figure PCTCN2020122793-appb-000006
抗体Corn-03
重链可变区(SEQ ID NO:5)：
Figure PCTCN2020122793-appb-000007
轻链可变区(SEQ ID NO:6)：
Figure PCTCN2020122793-appb-000008
抗体Corn-04
重链可变区(SEQ ID NO:7)：
Figure PCTCN2020122793-appb-000009
轻链可变区(SEQ ID NO:8)：
Figure PCTCN2020122793-appb-000010
抗体Corn-05
重链可变区(SEQ ID NO:9)：
Figure PCTCN2020122793-appb-000011
轻链可变区(SEQ ID NO:10)：
Figure PCTCN2020122793-appb-000012
抗体Corn-06
重链可变区(SEQ ID NO:11)：
Figure PCTCN2020122793-appb-000013
轻链可变区(SEQ ID NO:12)：
Figure PCTCN2020122793-appb-000014
抗体Corn-07
重链可变区(SEQ ID NO:13)：
Figure PCTCN2020122793-appb-000015
轻链可变区(SEQ ID NO:14)：
Figure PCTCN2020122793-appb-000016
注：Corn‐01至Corn‐07抗体轻重链可变区序列中下划线加粗的部分为抗体的CDRs区，所述CDRs区是以Kabat法分析确定的。
实施例5：抗体的ELISA阻断活性
将上清Fortebio检测亲和力高的克隆进行进一步的ELISA阻断活性检测。具体方法如下：
1.包板：包被human ACE2‐hFc(1‐615)(KN表达，lot:20200213C)，浓度为0.75ug/ml；每孔100ul；4℃O/N；
2.封闭：5％BSA in PBS，37℃,120min,PBST洗板4次；
3.加一抗：120ul 30ng/ml S1‐RBD‐mFc(KN表达，lot:20200217A)，加入10ug/ml浓度的Corn‐09、CR3022、Corn‐08、Corn‐02、Corn‐10、Corn‐01各120ul和5+17(1:1混合，5ug/ml)，略震荡混匀放置50min，每孔取2个100ul平行加入human ACE2‐hFc(1‐615)包被孔；
4.加二抗：HRP‐anti‐mouse IgG(Cat:115‐035‐071,Jackson Immuno Research)(1:5000)37℃，45min，PBST洗板4次；
5.显色：TMB(Cat:ME142，北京泰天河生物)显色，37℃，10min；
6.终止：2M HCL终止反应；
7.读数：读取并记录波长450nm下孔板的吸光度值。
结果如图6～7所示，对于S1RBD与ACEII，S1与ACEII的结合，重组抗体Corn‐01和Corn‐02有明显的阻断活性，且具有协同效应。重组抗体Corn‐10不能阻断S1RBD与ACE2的结合，但能够抑制S1与ACE2的结合。上述结果表明抗体Corn‐01和Corn‐02二者与ACE2，在SARS-CoV-2 S1蛋白上具有相同的结合位点，因而能够直接阻断S1RBD与ACE2的结合。
进一步对于抗体Corn‐01、Corn‐03、Corn‐04、Corn‐05、Corn‐06、Corn‐07，采用梯度稀释法进行ELISA。简言之，根据上述方法包被、封闭之后，在添加一抗的步骤中，将上述待测抗体(起始浓度为40μg/mL，1.5倍连续稀释，12个梯度)和与S1-RBD-mFc 70ng/ml，各取100uL等体积混匀，37℃放置50min后，每个样本取两个100μl平行加入到ACE2-hFc包被孔；然后加二抗、显色、终止、读数。结果如图8～9所示。
6株抗体均能明显抑制ACEII‐hFc与S1RBD‐mFc的结合，其半数抑制浓度IC50如表2所示：
表2：抗体对ACEII‐hFc与S1RBD‐mFc的结合的半数抑制浓度
  IC50(nM)
Corn‐01 2.588
Corn‐03 0.678
Corn‐04 0.432
Corn‐05 0.505
Corn‐06 0.822
Corn‐07 0.340
实施例6.抗体对SARS-CoV-2的病毒中和试验方法
6.1假病毒感染试验——荧光法
对于Corn‐01和Corn‐07中和SARS‐CoV‐2的活性进行预估，采用中检院的新冠假病毒感染Huh7细胞中和活性实验观察。将不同浓度的待评估抗体与750TCID50/孔的假病毒颗粒(转染荧光素酶报告基因)在37℃进行中和反应后，分别接种2×10 4/孔huh7细胞，置于37℃的CO2培养箱内培养20‐28h。20‐28h后取出细胞板每孔加入100μl荧光素酶检测试剂，避光反应2min，荧光检测仪读数，计算中和抑制率，并根据中和抑制率结果，利用Reed‐Muench法计算IC50。
结果如图10所示，Corn‐07能够剂量依赖地抑制假病毒颗粒进入宿主细胞，经过Reed‐Muench法计算，IC50值为62ng/mL，高于Corn‐01(IC50,281ng/mL)的中和活性。
6.2 SARS‐CoV‐2病毒感染试验——细胞病变中和滴定法
1)取生长状态良好的Vero‐E6细胞消化，调整细胞密度为1×10 5/mL接种于96孔板，100μL/孔(即每孔10 4个细胞)，放置于37℃、5％CO 2培养箱培养12‐16h；
2)12‐16h后，弃去孔中培养基，首先分别加入终浓度为不同稀释度(最高浓度100μg/ml，3倍稀释8次)的样品50μl，然后每孔加入10 2CCID50的SARS‐CoV‐2病毒50μl。同时设置细胞对照，病毒对照。
3)于加样后72h观察并测定细胞病变情况，实验结果如表3所示。
表3 抗体中和SARS‐CoV‐2病毒感染宿主细胞的活性检测结果‐EC50
Figure PCTCN2020122793-appb-000017
实施例7：Corn‐01抗体对SARS‐CoV‐2感染细胞的ADE作用
采用中检院的新冠病毒颗粒感染淋巴细胞细胞(THP‐1、Raji和K562)进行抗体的ADE检测。将不同浓度的待评估新冠抗体与750TCID50/孔的假病毒颗粒(转染荧光素酶报告基因)在37℃进行中和反应后，分别接种1x105/孔宿主细胞，置于37℃，5％CO2培养箱内培养20‐28h。28‐28h后取出细胞板每孔加入100ul荧光素酶检测试剂(G7940，Promega)，避光反应2min，荧光检测仪读数，根据荧光信号强度评价ADE的强弱。
结果表明(图11)，IgG1形式的Corn‐01对Raji细胞在一定浓度范围内(50ng/ml‐3000ng/ml)表现出了明显的ADE作用；而对于THP‐1和K562在1‐1000ng/ml的浓度范围内均未产生ADE效应。
实施例8：中和性抗体在病毒感染敏感宿主细胞过程中ADE效应的检测
利用实施例7的方法，采用Raji细胞对前期筛选鉴定的5种抗SARS-CoV-2S1蛋白RBD区的全人源中和性抗体检测ADE效应，结果如图12、图13所示。
图12、图13表明，不同的抗SARS-CoV-2 S1蛋白RBD区中和性抗体在病毒感染敏感宿主细胞过程中引起ADE效应的能力存在差异。IgG1形式的Corn‐04、Corn‐06和Corn‐07三个抗体在1-1000ng/ml的浓度范围内并未观察到明显的ADE效应，而Corn‐01和Corn‐05在一定浓度范围(50ng/ml-3000ng/ml)内产生了ADE现象。
采用中检院的新冠假病毒颗粒感染Raji细胞进行抗体的ADE检测。将不同浓度的待评估新冠抗体与750TCID50/孔的假病毒颗粒(转染荧光素酶报告基因)在37℃进行中和反应后，分别接种1x105/孔Raji细胞，置于37℃的CO2培养箱内培养20‐28h。20‐28h后取出细胞板每孔加入100μl荧光素酶检测试剂，避光反应2min，荧光检测仪读数，根据荧光信号强度评价ADE的强弱。
实施例9：抗体Fc点突变改变了对宿主细胞受体亲和力的影响
根据Raji宿主细胞与其它不产生ADE效应的宿主细胞上Fc受体种类，确定Fc点突变的位置，并检测突变后抗体对宿主细胞上Fc受体的亲和力。将Corn‐01和Corn‐05的Fc进行LALA突变(Fc-L234A,L235A)，构建Corn‐01‐LALA 和Corn‐05‐LALA。突变前的野生型重链恒定区的氨基酸序列如SEQ ID NO:15所示，突变后的突变型重链恒定区的氨基酸序列如SEQ ID NO:16所示。
应用Fortebio蛋白相互作用系统Octet QKe system测定测定Corn‐05(wtIgG1)和Corn‐05‐LALA与FcγRI(CD64)重组蛋白(10256‐H08H,义翘神州)和FcγRIIa(CD32A)重组蛋白(10374‐H08H1,义翘神州)的亲和力。采用抗人抗体Fc段的捕获抗体(AHC)生物探针捕获抗体Corn‐05和Corn‐05‐LALA。15μg/ml抗体流经AHC探针(Cat:18‐5060，PALL)表面，时间为120s。浓度100nM重组抗原作为流动相。结合时间为300s，解离时间为300s。实验完毕，扣除空白对照响应值，用软件进行1：1Langmuir结合模式拟合，计算抗原抗体结合的动力学常数，结果如表4所示。
表4.Corn‐05和Corn‐05‐LALA对CD64和CD32A亲和力常数
Sample ID Loading Sample ID Response KD(M) kon(1/Ms) kdis(1/s)
CD64-His Corn‐05 0.1174 1.45E-08 1.20E+05 1.73E-03
CD64-His Corn‐05-LALA 0.0344 3.66E-05 7.89E+02 2.89E-02
CD32A-His Corn‐05 0.2302 5.60E-08 1.89E+06 1.06E-01
CD32A-His Corn‐05-LALA 0.2254 6.62E-08 1.94E+06 1.29E-01
表4的结果表明：与wtIgG1型的Corn‐05相比，LALA突变后其与各FcγRs的亲和力发生了改变。
实施例10：抗体Fc的点突变消除了抗体对新冠病毒感染宿主细胞的ADE效应
为了验证通过点突变降低中和性抗体Fc与宿主细胞受体FcγRI的亲和力是否能够降低或消除其对新冠病毒感染宿主细胞的ADE效应，将突变后的Corn‐01‐LALA、Corn‐05‐LALA分别与突变前的Corn‐01、Corn‐05进行对比，采用实施例7的方法检测对新冠病毒感染宿主细胞的ADE效应。结果如图14、图15所示。
图14、图15表明：Corn‐01‐LALA、Corn‐05‐LALA由于在Fc段引入了L234A、L235A突变，消除了ADE现象。结合实施例9的实验结果可以推断抗SARS-CoV-2 S1蛋白RBD区的全人源中和性抗体在SARS-CoV-2感染宿主过程中引起ADE效应可能是通过抗体Fc段与宿主细胞上FcrRI受体介导的，因此通过在Fc段引入L234A、L235A突变后极大降低了抗体与FcrRI受体的结合，从 而避免了ADE的发生。
实施例11：抗体Fc的点突变不影响抗体对新冠病毒的中和活性
采用中检院的新冠病毒颗粒感染Huh-7细胞进行抗体的中和活性检测。将不同浓度的待评估新冠抗体与750TCID50/孔的假病毒颗粒(转染荧光素酶报告基因)在37℃进行中和反应后，分别接种2x10 4/孔Huh-7细胞，置于37℃，5％CO 2培养箱内培养20-28h。28-28h后取出细胞板每孔加入100ul荧光素酶检测试剂(G7940，Promega)，避光反应2min，荧光检测仪读数，根据荧光信号强度评价抗体对病毒的中和活性，结果如图16所示。图16的结果表明，wtIgG1形式的Corn‐01、Corn‐05呈现了良好的剂量依赖的病毒中和活性。
将消除了ADE效应的Corn‐01和Corn‐05的LALA突变体(Fc-L234A，L235A)的中和活性进行检测，评估wtIgG1与LALA突变体(L234A，L235A)的中和活性的差异，结果如图18和图19所示。
图17和图18的结果表明与Corn‐01、Corn‐05相比，Fc段LALA突变体Corn‐01‐LALA、Corn‐05‐LALA的中和活性并未发生明显改变。由此可知，对抗SARS-CoV-2中和性抗体的Fc段进行上述点突变改造能够保留其病毒中和活性。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。

Claims (29)

  1. 一种SARS-CoV-2 S蛋白-ACEII受体结合阻断性单克隆抗体或其片段，其特征在于：所述单克隆抗体是从新型冠状病毒感染患者康复期血液样本中分离单个B细胞克隆获得的特异性结合SARS-CoV-2 S蛋白RBD区的全人源抗体，其对SARS-CoV-2感染THP-1细胞和K562细胞均不产生ADE效应。
  2. 如权利要求1所述SARS-CoV-2 S蛋白-ACEII受体结合阻断性单克隆抗体或其片段，其特征在于所述单克隆抗体对SARS-CoV-2感染Raji细胞不产生ADE效应。
  3. 如权利要求1所述SARS-CoV-2 S蛋白-ACEII受体结合阻断性单克隆抗体或其片段，其特征在于所述单克隆抗体在10-10000ng的部分浓度范围内对SARS-CoV-2感染Raji细胞产生ADE效应。
  4. 如权利要求3所述SARS-CoV-2 S蛋白-ACEII受体结合阻断性单克隆抗体或其片段，其特征在于所述单克隆抗体在50-3000ng的部分浓度范围内对SARS-CoV-2感染Raji细胞产生ADE效应。
  5. 如权利要求1所述SARS-CoV-2 S蛋白-ACEII受体结合阻断性单克隆抗体或其片段，其特征在于所述单克隆抗体与SARS-CoV-2 S蛋白RBD具有高亲和性，KD值为5.0×10 -9M以下。
  6. 如权利要求1所述SARS-CoV-2 S蛋白-ACEII受体结合阻断性单克隆抗体或其片段，其特征在于所述单克隆抗体阻断SARS-CoV-2 S蛋白RBD与ACEII结合的IC50值小于50nM，优选小于30nM、25nM、20nM、15nM或10nM。
  7. 如权利要求1所述SARS-CoV-2 S蛋白-ACEII受体结合阻断性单克隆抗体或其片段，其特征在于所述单克隆抗体Fc段的点突变改变了其与受体的结合。
  8. 如权利要求7所述SARS-CoV-2 S蛋白-ACEII受体结合阻断性单克隆抗体或其片段，其特征在于所述单克隆抗体Fc段的点突变降低或消除了其与FcγRs的结合。
  9. 如权利要求7或8所述SARS-CoV-2 S蛋白-ACEII受体结合阻断性单克隆抗体或其片段，其特征在于所述单克隆抗体Fc段的点突变包括在由第234位、第235位组成的组中任意一个或两个位点进行氨基酸的替换、缺失或插入突变。
  10. 如权利要求9所述SARS-CoV-2 S蛋白-ACEII受体结合阻断性单克隆 抗体或其片段，其特征在于所述单克隆抗体Fc段的点突变包括L234A、L235A突变。
  11. 如权利要求7至10中任一项所述SARS-CoV-2 S蛋白-ACEII受体结合阻断性单克隆抗体或其片段，其特征在于所述单克隆抗体的重链恒定区具有SEQ ID NO:16所示的序列。
  12. 如权利要求1至11中任一项所述SARS-CoV-2 S蛋白-ACEII受体结合阻断性单克隆抗体或其片段，
    其重链可变区具有SEQ ID NO:1、3、5、7、9、11、13所示氨基酸序列，或在SEQ ID NO:1、3、5、7、9、11、13所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；
    其轻链可变区具有SEQ ID NO:2、4、6、8、10、12、14所示氨基酸序列，或在SEQ ID NO:2、4、6、8、10、12、14所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列。
  13. 如权利要求12所述SARS-CoV-2 S蛋白-ACEII受体结合阻断性单克隆抗体或其片段，其特征在于：
    重链可变区具有SEQ ID NO:1所示氨基酸序列，或在SEQ ID NO:1所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；并且，轻链可变区具有SEQ ID NO:2所示氨基酸序列，或在SEQ ID NO:2所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；
    重链可变区具有SEQ ID NO:3所示氨基酸序列，或在SEQ ID NO:3所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；并且，轻链可变区具有SEQ ID NO:4所示氨基酸序列，或在SEQ ID NO:4所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；
    重链可变区具有SEQ ID NO:5所示氨基酸序列，或在SEQ ID NO:5所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；并且，轻链可变区具有SEQ ID NO:6所示氨基酸序列，或在SEQ ID NO:6所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、 添加、插入获得的氨基酸序列；
    重链可变区具有SEQ ID NO:7所示氨基酸序列，或在SEQ ID NO:7所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；并且，轻链可变区具有SEQ ID NO:8所示氨基酸序列，或在SEQ ID NO:8所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；
    重链可变区具有SEQ ID NO:9所示氨基酸序列，或在SEQ ID NO:9所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；并且，轻链可变区具有SEQ ID NO:10所示氨基酸序列，或在SEQ ID NO:10所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；
    重链可变区具有SEQ ID NO:11所示氨基酸序列，或在SEQ ID NO:11所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；并且，轻链可变区具有SEQ ID NO:12所示氨基酸序列，或在SEQ ID NO:12所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；
    或者，
    重链可变区具有SEQ ID NO:13所示氨基酸序列，或在SEQ ID NO:13所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列；并且，轻链可变区具有SEQ ID NO:14所示氨基酸序列，或在SEQ ID NO:14所示氨基酸序列的基础上进行一个或多个氨基酸残基的取代、缺失、添加、插入获得的氨基酸序列。
  14. 如权利要求1至11中任一项所述SARS-CoV-2 S蛋白-ACEII受体结合阻断性单克隆抗体或其片段，其特征在于：
    重链可变区具有SEQ ID NO:1、3、5、7、9、11、13中任一所示抗体重链可变区的HCDR1、HCDR2和HCDR3；或者与SEQ ID NO:1、3、5、7、9、11、13中任一具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％、99％、或100％同一性的序列所述抗体重链可变区的HCDR1、HCDR2和HCDR3；
    并且，
    轻链可变区具有SEQ ID NO:2、4、6、8、10、12、14中任一所示抗体轻链可变区的LCDR1-3、LCDR2和LCDR3；或者与SEQ ID NO:2、4、6、8、10、12、14中任一具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％、99％、或100％同一性的序列所述抗体轻链可变区的HCDR1、HCDR2和HCDR3。
  15. 如权利要求14所述SARS-CoV-2 S蛋白-ACEII受体结合阻断性单克隆抗体或其片段，其特征在于：
    重链可变区具有SEQ ID NO:1所示抗体重链可变区的HCDR1、HCDR2和HCDR3；并且，轻链可变区具有SEQ ID NO:2所示抗体轻链可变区的LCDR1、LCDR2和LCDR3；
    重链可变区具有SEQ ID NO:3所示抗体重链可变区的HCDR1、HCDR2和HCDR3；并且，轻链可变区具有SEQ ID NO:4所示抗体轻链可变区的LCDR1、LCDR2和LCDR3；
    重链可变区具有SEQ ID NO:5所示抗体重链可变区的HCDR1、HCDR2和HCDR3；并且，轻链可变区具有SEQ ID NO:6所示抗体轻链可变区的LCDR1、LCDR2和LCDR3；
    重链可变区具有SEQ ID NO:7所示抗体重链可变区的HCDR1、HCDR2和HCDR3；并且，轻链可变区具有SEQ ID NO:8所示抗体轻链可变区的LCDR1、LCDR2和LCDR3；
    重链可变区具有SEQ ID NO:9所示抗体重链可变区的HCDR1、HCDR2和HCDR3；并且，轻链可变区具有SEQ ID NO:10所示抗体轻链可变区的LCDR1、LCDR2和LCDR3；
    重链可变区具有SEQ ID NO:11所示抗体重链可变区的HCDR1、HCDR2和HCDR3；并且，轻链可变区具有SEQ ID NO:12所示抗体轻链可变区的LCDR1、LCDR2和LCDR3；
    或者，
    重链可变区具有SEQ ID NO:13所示抗体重链可变区的HCDR1、HCDR2和HCDR3；并且，轻链可变区具有SEQ ID NO:14所示抗体轻链可变区的LCDR1、LCDR2和LCDR3。
  16. 一种多核苷酸，其编码权利要求1至15中任一项所述单克隆抗体或其片段。
  17. 核酸构建体，其包含权利要求16所述的多核苷酸。
  18. 宿主细胞，其包含权利要求16所述的多核苷酸或权利要求17所述的核酸构建体。
  19. 组合物，其包含一种或多种选自由权利要求1至15中任一项所述单克隆抗体或其片段组成的组中的单克隆抗体或其片段，以及任选的药学上可接受的载体。
  20. 如权利要求19所述的组合物，其包含选自由权利要求1至15中任一项所述单克隆抗体或其片段组成的组中的任意两种、三种、四种、五种、六种、或七种单克隆抗体或其片段。
  21. 权利要求1至15中任一项所述单克隆抗体或其片段、权利要求16所述的多核苷酸、权利要求17所述的核酸构建体、权利要求18所述的宿主细胞、权利要求19或20所述的组合物在制备预防或治疗SARS‐CoV‐2感染药物中的应用。
  22. 如权利要求21所述的应用，其特征在于所述预防或治疗SARS‐CoV‐2感染包括减少或降低SARS‐CoV‐2感染的风险、缓解SARS‐CoV‐2感染相关疾病(例如COVID‐19)的症状、缩短SARS‐CoV‐2感染相关疾病(例如COVID‐19)的病程、促进SARS‐CoV‐2感染相关疾病(例如COVID‐19)的康复、减少SARS‐CoV‐2感染导致的死亡。
  23. 一种预防或治疗SARS‐CoV‐2感染的方法，其特征在于对有需要的受试者施用有效量的权利要求1至15中任一项所述单克隆抗体或其片段、权利要求16所述的多核苷酸、权利要求17所述的核酸构建体、权利要求18所述的宿主细胞、和/或权利要求19或20所述的组合物。
  24. 如权利要求23所述的方法，其特征在于所述受试者遭受SARS‐CoV‐2感染的风险、具有SARS‐CoV‐2感染相关疾病(例如COVID‐19)、或具有因SARS‐CoV‐2感染而导致死亡的可能。
  25. 如权利要求23或24所述的方法，其特征在于所述方法使受试者减少或降低SARS‐CoV‐2感染的风险、缓解SARS‐CoV‐2感染相关疾病(例如COVID‐19) 的症状、缩短SARS‐CoV‐2感染相关疾病(例如COVID‐19)的病程、促进SARS‐CoV‐2感染相关疾病(例如COVID‐19)的康复、减少SARS‐CoV‐2感染导致的死亡。
  26. 一种降低或消除抗体在SARS‐CoV‐2感染宿主细胞时ADE效应的方法，其中，所述的抗体为抗SARS‐CoV‐2 S蛋白的中和性抗体，通过对抗体Fc段进行点突变改变了抗SARS‐CoV‐2 S蛋白抗体与其受体的结合。
  27. 如权利要求26所述消除抗体在SARS‐CoV‐2感染宿主细胞时ADE效应的方法，其中Fc段的点突变降低或消除了抗SARS‐CoV‐2 S蛋白抗体与FcγRs的结合。
  28. 如权利要求26或27所述消除抗体在SARS‐CoV‐2感染宿主细胞时ADE效应的方法，其中Fc段的点突变包括在由第234位、第235位组成的组中任意一个或两个位点进行氨基酸的替换、缺失或插入突变。
  29. 如权利要求26‐28中任一项所述消除抗体在SARS‐CoV‐2感染宿主细胞时ADE效应的方法，其特征在于所述抗SARS‐CoV‐2 S蛋白的中和性抗体选自权利要求1至15中任一项所述单克隆抗体或其片段。
PCT/CN2020/122793 2020-04-20 2020-10-22 一种全人源单克隆抗体及其应用 WO2021212785A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010315901 2020-04-20
CN202010315901.5 2020-04-20

Publications (1)

Publication Number Publication Date
WO2021212785A1 true WO2021212785A1 (zh) 2021-10-28

Family

ID=78094469

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/122793 WO2021212785A1 (zh) 2020-04-20 2020-10-22 一种全人源单克隆抗体及其应用

Country Status (2)

Country Link
CN (1) CN113527473A (zh)
WO (1) WO2021212785A1 (zh)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114044821A (zh) * 2022-01-10 2022-02-15 中国人民解放军军事科学院军事医学研究院 一种抗新冠病毒全人源广谱中和抗体zwc12及应用
CN114989291A (zh) * 2021-11-25 2022-09-02 浙江安维珞诊断技术有限公司 一种靶向RBD的抗SARS-CoV-2全人源化单克隆抗体及应用
WO2023102692A1 (zh) * 2021-12-06 2023-06-15 广州医科大学附属市八医院 协同中和新型冠状病毒的人源抗体和抗体组合及其应用
WO2023148641A1 (en) * 2022-02-02 2023-08-10 Translational Health Science And Technology Institute Monoclonal antibodies specific to receptor binding domain of sars-cov2 and uses thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110974950A (zh) * 2020-03-05 2020-04-10 广州恩宝生物医药科技有限公司 一种用于预防SARS-CoV-2感染的腺病毒载体疫苗

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110974950A (zh) * 2020-03-05 2020-04-10 广州恩宝生物医药科技有限公司 一种用于预防SARS-CoV-2感染的腺病毒载体疫苗

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHONGQING MEDICAL UNIVERSITY: "Our School Successfully Built an Antibody Rapid Screening Technology Platform and Obtained a New Coronavirus Fully Human Monoclonal Antibody", JOURNAL OF CHONGQING MEDICAL UNIVERSITY, no. 3, 28 March 2020 (2020-03-28), pages 292,429, XP055855516 *
SUN CHUNYUN, CHEN LONG, YANG JI, LUO CHUNXIA, ZHANG YANJING, LI JING, YANG JIAHUI, ZHANG JIE, XIE LIANGZHI: "SARS-CoV-2 and SARS-CoV Spike-RBD Structure and Receptor Binding Comparison and Potential Implications on Neutralizing Antibody and Vaccine Development", BIORXIV, 20 February 2020 (2020-02-20), pages 1 - 18, XP055812679, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2020.02.16.951723v1.full.pdf> [retrieved on 20210610], DOI: 10.1101/2020.02.16.951723 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114989291A (zh) * 2021-11-25 2022-09-02 浙江安维珞诊断技术有限公司 一种靶向RBD的抗SARS-CoV-2全人源化单克隆抗体及应用
CN114989291B (zh) * 2021-11-25 2023-01-06 浙江安维珞诊断技术有限公司 一种靶向RBD的抗SARS-CoV-2全人源化单克隆抗体及应用
WO2023102692A1 (zh) * 2021-12-06 2023-06-15 广州医科大学附属市八医院 协同中和新型冠状病毒的人源抗体和抗体组合及其应用
CN114044821A (zh) * 2022-01-10 2022-02-15 中国人民解放军军事科学院军事医学研究院 一种抗新冠病毒全人源广谱中和抗体zwc12及应用
WO2023148641A1 (en) * 2022-02-02 2023-08-10 Translational Health Science And Technology Institute Monoclonal antibodies specific to receptor binding domain of sars-cov2 and uses thereof

Also Published As

Publication number Publication date
CN113527473A (zh) 2021-10-22

Similar Documents

Publication Publication Date Title
WO2021212785A1 (zh) 一种全人源单克隆抗体及其应用
CN112390879B (zh) 靶向SARS-CoV-2的抗体及其制备方法和应用
CN110016079B (zh) 抗呼吸道合胞病毒的中和抗体及其应用
EP1476468B1 (en) Human monoclonal antibody fab fragments directed against hcv e2 glycoprotein and endowed with in vitro neutralizing activity
CN111333723B (zh) 针对狂犬病病毒g蛋白的单克隆抗体及其用途
WO2021218879A1 (zh) Sars-cov-2中和抗体及其制备和应用
Duan et al. A novel disulfide-stabilized single-chain variable antibody fragment against rabies virus G protein with enhanced in vivo neutralizing potency
JP2023528826A (ja) Ｓａｒｓ関連コロナウイルスに対する中和抗体
WO2014043509A2 (en) Antigen binding molecule with terminal modifications
CN106565840B (zh) 抗乙肝表面抗原的抗体及其用途
CN112409488A (zh) 针对多种冠状病毒的单克隆抗体及应用
TW202334429A (zh) Sars-cov-2棘蛋白特異性抗體及其用途
EP4206224A1 (en) Human antibody or antigen-binding fragment thereof against coronavirus spike protein
WO2022216223A1 (en) Vaccine and/or antibody for viral infection
US20230065377A1 (en) Human antibodies to alphaviruses
CN115315442B (zh) Sars-cov-2抗体及其应用
CN115087667B (zh) 特异性结合SARS-CoV-2的抗原结合蛋白
JP2024509933A (ja) 抗水痘帯状疱疹ウイルスの抗体およびその使用
Roh et al. Generation of a chickenized catalytic anti-nucleic acid antibody by complementarity-determining region grafting
US20220251173A1 (en) Anti-hepatitis b virus antibodies and use thereof
WO2023131262A1 (zh) 特异性结合SARS-CoV-2的抗原结合蛋白
RU2817696C1 (ru) Моноклональное антитело iC1 и его антигенсвязывающий фрагмент, селективно связывающие рецептор-связывающий домен Spike-белка вируса SARS-CoV-2, обладающие вируснейтрализующей активностью
RU2803082C2 (ru) Антитела против вируса гепатита в и их применение
WO2024053719A1 (ja) コロナウイルス変異株に対するヒト抗体またはその抗原結合断片
WO2023071953A1 (zh) 抗cd26抗体及其应用

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: 20932361

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: 20932361

Country of ref document: EP

Kind code of ref document: A1