CN113683686A - Preparation and application of neutralizing monoclonal antibody against new coronavirus - Google Patents

Preparation and application of neutralizing monoclonal antibody against new coronavirus Download PDF

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CN113683686A
CN113683686A CN202010421833.0A CN202010421833A CN113683686A CN 113683686 A CN113683686 A CN 113683686A CN 202010421833 A CN202010421833 A CN 202010421833A CN 113683686 A CN113683686 A CN 113683686A
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antibody
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variable region
light chain
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黄忠
张超
王亚磊
徐诗奇
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Institut Pasteur of Shanghai of CAS
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    • C07—ORGANIC CHEMISTRY
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    • 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
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    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

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Abstract

The invention provides a preparation and application of a neutralizing monoclonal antibody for resisting a new coronavirus. Specifically, the invention provides a monoclonal antibody of an anti-SARS-CoV 2Spike protein RBD structural domain. The antibody of the present invention can identify SARS-CoV-2 with high affinity, has strong neutralizing capacity on SARS-CoV-2 pseudovirus infection and strong receptor competitive capacity. The antibody provided by the invention has a good clinical application prospect.

Description

Preparation and application of neutralizing monoclonal antibody against new coronavirus
Technical Field
The invention belongs to the field of biotechnology or medicine, and particularly relates to preparation and application of a neutralizing monoclonal antibody against a new coronavirus.
Background
In acute infectious diseases, most of the infectious diseases are viral infectious diseases, the incidence rate of the viral infectious diseases is high, and the death rate is high. Because the detection and diagnosis means are limited, the outbreak of new epidemic caused by new viruses often has the characteristics of paroxysmal, random, unpredictable and the like, once the outbreak occurs, if no effective prevention and treatment means exists, the large-scale epidemic is very easy to cause, and the health and life safety of people is seriously threatened.
Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) is the causative agent of novel coronavirus pneumonia (COVID-19). SARS-CoV-2 is an enveloped virus, and like SARS-CoV, belongs to the genus Beta-coronavirus within the family Coronaviridae. The trimeric spike (spike) glycoprotein on the surface of SARS-CoV-2 virus mediates entry of the virus into the host cell. The S protein has two functional subunits: the S1 subunit mediates cell adsorption (there are four domains NTD, RBD, SD1, SD2), and the S2 subunit is responsible for fusion of the viral envelope and cell membrane. The virus binds to the receptor human angiotensin converting enzyme 2(ACE2) protein via the RBD (receptor binding domain) in the S1 subunit, and thus adheres to the cell surface.
So far, the transmission route of 2019-nCoV virus is not completely mastered, and is known to be transmitted by droplets and contact, and human-borne and medical staff infection, certain community transmission risks and the possibility of virus variation exist. There is currently no specific preventive or therapeutic approach for diseases caused by the novel coronavirus.
At present, no specific vaccine or antiviral drug exists for severe pneumonia diseases caused by SARS-CoV-2 (same as 2019-nCoV) coronavirus. These infectious diseases seriously affect the human life health, and the development of antiviral drugs with good efficacy is imminent. Aiming at SARS-CoV-2 coronavirus, a low-toxicity and high-efficiency antiviral drug is developed to meet the clinical requirements of SARS-CoV-2 coronavirus infected patients at home and abroad, and has great social significance.
In view of the foregoing, there is an urgent need in the art to develop effective diagnostic and therapeutic methods against SARS-CoV-2 coronavirus for the diagnosis and treatment of pneumonia caused by infection with the novel coronavirus.
Disclosure of Invention
The invention aims to provide an effective diagnosis and treatment method for SARS-CoV-2 coronavirus.
Specifically, the invention aims to provide preparation and application of a neutralizing monoclonal antibody against SARS-CoV-2 virus.
In a first aspect of the invention, there is provided a heavy chain variable region of an antibody, said heavy chain variable region having complementarity determining regions CDRs selected from the group consisting of:
VH-CDR1 shown in SEQ ID NO. 4 or 16, VH-CDR2 shown in SEQ ID NO. 5 or 17, and VH-CDR3 shown in SEQ ID NO. 6 or 18;
wherein, any one of the amino acid sequences also comprises a derivative sequence which is optionally added, deleted, modified and/or substituted by at least one amino acid and can retain the binding affinity with the RBD structural domain of the SARS-CoV-2S protein.
In another preferred embodiment, the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO 3 or 15.
In a second aspect of the invention, there is provided a heavy chain of an antibody, said heavy chain having a heavy chain variable region as described in the first aspect of the invention.
In another preferred embodiment, the heavy chain further comprises a heavy chain constant region.
In another preferred embodiment, the heavy chain constant region is of human or murine origin.
In another preferred embodiment, the heavy chain constant region is a human antibody heavy chain IgG1 constant region.
In another preferred embodiment, the amino acid sequence of the heavy chain is as shown in SEQ ID NO 2 or 14.
In a third aspect of the present invention, there is provided a light chain variable region of an antibody, said light chain variable region having complementarity determining regions CDRs selected from the group consisting of:
VL-CDR1 shown in SEQ ID NO. 10 or 22, VL-CDR2 shown in SEQ ID NO. 11 or 23, and VL-CDR3 shown in SEQ ID NO. 12 or 24;
wherein, any one of the amino acid sequences also comprises a derivative sequence which is optionally added, deleted, modified and/or substituted by at least one amino acid and can retain the binding affinity with the RBD structural domain of the SARS-CoV-2S protein.
In another preferred embodiment, the variable region of the light chain has the amino acid sequence shown in SEQ ID NO 9 or 21.
In a fourth aspect of the invention, there is provided a light chain of an antibody, said light chain having a light chain variable region as described in the third aspect of the invention.
In another preferred embodiment, the light chain further comprises a light chain constant region.
In another preferred embodiment, the light chain constant region is of human or murine origin.
In another preferred embodiment, the light chain constant region is a human antibody light chain kappa constant region.
In another preferred embodiment, the amino acid sequence of the light chain is shown in SEQ ID NO 8 or 20.
In a fifth aspect of the invention, there is provided an antibody having a heavy chain variable region as described in the first aspect of the invention, and/or a light chain variable region as described in the third aspect of the invention;
alternatively, the antibody has a heavy chain as described in the second aspect of the invention, and/or a light chain as described in the fourth aspect of the invention;
wherein, any one of the amino acid sequences also comprises a derivative sequence which is optionally added, deleted, modified and/or substituted by at least one amino acid and can retain the binding affinity with the RBD structural domain of the SARS-CoV-2S protein.
In another preferred embodiment, the number of the amino acids to be added, deleted, modified and/or substituted is 1 to 5 (e.g., 1 to 3, preferably 1 to 2, and more preferably 1).
In another preferred embodiment, the derivative sequence which is added, deleted, modified and/or substituted with at least one amino acid and which retains the binding affinity of the RBD domain of the SARS-CoV-2S protein is an amino acid sequence having a homology or sequence identity of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.
In another preferred embodiment, the antibody further comprises a heavy chain constant region and/or a light chain constant region.
In another preferred embodiment, said heavy chain constant region is of human origin and/or said light chain constant region is of human origin.
In another preferred embodiment, the heavy chain constant region is a human antibody heavy chain IgG1 constant region and the light chain constant region is a human antibody light chain kappa constant region.
In another preferred embodiment, the heavy chain variable region of the antibody further comprises a framework region of human origin, and/or the light chain variable region of the antibody further comprises a framework region of human origin.
In another preferred embodiment, the heavy chain variable region of the antibody further comprises a murine framework region, and/or the light chain variable region of the antibody further comprises a murine framework region.
In another preferred embodiment, the antibody is selected from the group consisting of: an antibody of animal origin, a chimeric antibody, a humanized antibody, a fully human antibody, or a combination thereof.
In another preferred embodiment, the antibody is a partially or fully humanized, or fully human monoclonal antibody.
In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.
In another preferred embodiment, the antibody is a full-length protein, or an antigen-binding fragment of an antibody.
In another preferred embodiment, the antibody is a bispecific antibody, or a multispecific antibody.
In another preferred embodiment, the antibody is in the form of a drug conjugate.
In another preferred embodiment, the antibody has one or more properties selected from the group consisting of:
(a) specifically binds to the RBD domain of SARS-CoV-2S protein;
(b) blocking the combination of SARS-CoV-2 virus and human angiotensin converting enzyme 2(ACE 2); and
(c) effectively neutralize SARS-CoV-2 virus infection.
In another preferred embodiment, the antibody has a heavy chain variable region according to the first aspect of the invention and a light chain variable region according to the third aspect of the invention;
wherein said heavy chain variable region and said light chain variable region comprise CDRs selected from the group consisting of:
(1) VH-CDR1 shown in SEQ ID NO. 4, VH-CDR2 shown in SEQ ID NO. 5, VH-CDR3 shown in SEQ ID NO. 6, VL-CDR1 shown in SEQ ID NO. 10, VL-CDR2 shown in SEQ ID NO. 11, and VL-CDR3 shown in SEQ ID NO. 12; or
(2) VH-CDR1 shown in SEQ ID NO:16, VH-CDR2 shown in SEQ ID NO:17, VH-CDR3 shown in SEQ ID NO:18, VL-CDR1 shown in SEQ ID NO:22, VL-CDR2 shown in SEQ ID NO:23, and VL-CDR3 shown in SEQ ID NO: 24.
In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 9.
In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO. 15, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO. 21.
In another preferred embodiment, the amino acid sequence of the heavy chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity to the amino acid sequence set forth in SEQ ID NO 3 or 15.
In another preferred embodiment, the amino acid sequence of the light chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity to the amino acid sequence set forth in SEQ ID NO 9 or 21.
In a sixth aspect of the present invention, there is provided a recombinant protein comprising:
(i) a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; and
(ii) optionally a tag sequence to facilitate expression and/or purification.
In another preferred embodiment, the tag sequence comprises a 6His tag.
In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.
In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.
In a seventh aspect of the invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of: the heavy chain variable region according to the first aspect of the invention, the heavy chain according to the second aspect of the invention, the light chain variable region according to the third aspect of the invention, the light chain according to the fourth aspect of the invention, the antibody according to the fifth aspect of the invention, or the recombinant protein according to the sixth aspect of the invention.
In another preferred embodiment, the polynucleotide encoding the heavy chain is as shown in SEQ ID NO 1 or 13; and/or, the polynucleotide encoding the light chain is shown as SEQ ID NO. 7 or 19.
In an eighth aspect of the invention, there is provided a vector comprising a polynucleotide according to the seventh aspect of the invention.
In another preferred embodiment, the carrier comprises: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.
In a ninth aspect of the invention there is provided a genetically engineered host cell comprising a vector according to the eighth aspect of the invention or having integrated into its genome a polynucleotide according to the seventh aspect of the invention.
In a tenth aspect of the present invention, there is provided an antibody conjugate comprising:
(a) an antibody moiety selected from the group consisting of: a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, or a combination thereof; and
(b) a coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle/nanorod, a nanomagnet, a viral coat protein, or a VLP, or a combination thereof.
In another preferred embodiment, said antibody moiety is coupled to said coupling moiety by a chemical bond or a linker.
In another preferred embodiment, the radionuclide includes:
(i) a diagnostic isotope selected from the group consisting of: tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or combinations thereof; and/or
(ii) A therapeutic isotope selected from the group consisting of: lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133Yb-169, Yb-177, or a combination thereof.
In another preferred embodiment, the coupling moiety is a drug or toxin.
In another preferred embodiment, the drug is a cytotoxic drug.
In another preferred embodiment, the cytotoxic agent is selected from the group consisting of: an anti-tubulin drug, a DNA minor groove binding agent, a DNA replication inhibitor, an alkylating agent, an antibiotic, a folate antagonist, an anti-metabolite drug, a chemotherapeutic sensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.
Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs include, for example, auristatins (auristatins), camptothecins (camptothecins), duocarmycins/duocarmycins (duocarmycins), etoposides (etoposides), maytansinoids (maytansinoids) and maytansinoids (e.g., DM1 and DM4), taxanes (taxanes), benzodiazepines (benzodiazepines), or benzodiazepine-containing drugs (e.g., pyrrolo [1,4] benzodiazepines (PBDs), indobenzodiazepines (indoxazepines) and benzodiazepines (oxyphenoxazepines)), or combinations thereof.
In another preferred embodiment, the toxin is selected from the group consisting of:
auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), aureomycin, maytansinoid, ricin A-chain, combretastatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthrax toxin dione, actinomycin, diphtheria toxin, Pseudomonas Exotoxin (PE) A, PE40, abrin a chain, modeccin a chain, alpha-sarcina, gelonin, mitogelonin (mitogellin), restrictocin (rettstricon), phenomycin, enomycin, curcin (curcin), crotin, calicheamicin, soapwort (Sapaonaria officinalis) inhibitor, glucocorticoid, or a combination thereof.
In another preferred embodiment, the conjugated moiety is a detectable label.
In another preferred embodiment, the conjugate is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (e.g., IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin).
In another preferred embodiment, the immunoconjugate comprises: multivalent (e.g., divalent) of (a).
In another preferred embodiment, the multivalent is (a) comprising multiple repeats in the amino acid sequence of the immunoconjugate.
In an eleventh aspect of the invention there is provided an immune cell which expresses or has exposed outside the cell membrane an antibody according to the fifth aspect of the invention.
In another preferred embodiment, the immune cells comprise NK cells and T cells.
In another preferred embodiment, the immune cell is from a human or non-human mammal (e.g., a mouse).
In a twelfth aspect of the present invention, there is provided a pharmaceutical composition comprising:
(i) an active ingredient selected from the group consisting of: a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, an antibody conjugate according to the tenth aspect of the invention, an immune cell according to the eleventh aspect of the invention, or a combination thereof; and
(ii) a pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition is a liquid preparation.
In another preferred embodiment, the pharmaceutical composition is an injection.
In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of the antibody according to the fifth aspect of the present invention, the recombinant protein according to the sixth aspect of the present invention, the antibody conjugate according to the tenth aspect of the present invention, the immune cell according to the eleventh aspect of the present invention, or the combination thereof, and 0.01 to 99.99% of the pharmaceutically acceptable carrier, wherein the percentages are mass percentages of the pharmaceutical composition.
In a thirteenth aspect of the invention there is provided the use of an active ingredient selected from the group consisting of: a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, an antibody conjugate according to the tenth aspect of the invention, an immune cell according to the eleventh aspect of the invention, or a combination thereof, wherein the active ingredients are used (a) in the preparation of a diagnostic reagent or kit for SARS-CoV-2 viral infection; and/or (b) preparing a medicament for preventing and/or treating SARS-CoV-2 virus infection.
In another preferred embodiment, the diagnostic reagent is a test strip or test plate.
In another preferred embodiment, the diagnostic reagent or kit is used for: detecting SARS-CoV-2S protein or a fragment thereof in the sample.
In another preferred embodiment, the antibody is in the form of A Drug Conjugate (ADC).
In a fourteenth aspect of the present invention, there is provided a method for in vitro detection of SARS-CoV-2 virus or SARS-CoV-2S protein or a fragment thereof in a sample, the method comprising the steps of:
(1) contacting said sample in vitro with an antibody according to the fifth aspect of the invention;
(2) detecting the formation of an antigen-antibody complex, wherein the formation of the complex indicates the presence of SARS-CoV-2 virus or SARS-CoV-2S protein or a fragment thereof in the sample.
In another preferred embodiment, said detection comprises diagnostic or non-diagnostic.
In a fifteenth aspect of the present invention, there is provided a kit comprising:
(1) a first container comprising an antibody according to the fifth aspect of the invention; and/or
(2) A second container comprising a secondary antibody directed against the antibody of the fifth aspect of the invention;
alternatively, the first and second electrodes may be,
the kit contains a detection plate, and the detection plate comprises: a substrate (support plate) and a test strip comprising an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, an antibody conjugate according to the tenth aspect of the invention, an immune cell according to the eleventh aspect of the invention, or a combination thereof.
In a sixteenth aspect of the present invention, there is provided a method of producing a recombinant polypeptide which is an antibody according to the fifth aspect of the present invention or a recombinant protein according to the sixth aspect of the present invention, the method comprising:
(a) culturing a host cell according to the ninth aspect of the invention under conditions suitable for expression; and
(b) isolating said recombinant polypeptide from the culture.
In a seventeenth aspect of the present invention, there is provided a pharmaceutical combination comprising:
(i) a first active ingredient comprising an antibody according to the fifth aspect of the invention, or a recombinant protein according to the sixth aspect of the invention, or an antibody conjugate according to the tenth aspect of the invention, or an immune cell according to the eleventh aspect of the invention, or a pharmaceutical composition according to the twelfth aspect of the invention, or a combination thereof;
(ii) a second active ingredient comprising an additional drug for the treatment of SAR-CoV-2 viral infection.
In another preferred example, the other drugs for treating SAR-CoV-2 virus infection include: other protective monoclonal antibodies or small molecular chemical drugs such as Reidesvir or other Chinese patent drugs.
In an eighteenth aspect of the present invention, there is provided a method for diagnosing SAR-CoV-2 virus infection, comprising the steps of:
(i) obtaining a sample from a subject, contacting said sample with an antibody according to the fifth aspect of the invention; and
(ii) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates that the subject is a confirmed patient of SAR-CoV-2 virus.
In another preferred embodiment, the sample is a blood sample or a pharyngeal swab sample, or a sample from another tissue or organ.
In a nineteenth aspect of the present invention, there is provided a method for treating a disease infected with SARS-CoV-2 virus, comprising the steps of: administering to a subject in need thereof an effective amount of an antibody according to the fifth aspect of the invention, or a recombinant protein according to the sixth aspect of the invention, or an antibody conjugate according to the tenth aspect of the invention, or an immune cell according to the eleventh aspect of the invention, a pharmaceutical composition according to the twelfth aspect of the invention, or a combination thereof.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the binding activity of monoclonal antibodies to RBD proteins.
Among them, (A-B) shows the ELISA assay monoclonal antibody (2H2 and 3C1) response to SARS-CoV-2RBD (A) and SARS-CoV RBD (B). Zika virus monoclonal antibody 5F8 was used as an isotype control (ctr).
FIG. 2 shows the affinity of mAbs for RBD proteins.
Wherein (A-B) shows the binding affinity of the BLI-measured monoclonal antibody to SARS-CoV-2 RBD. Binding affinity at steady state is expressed as the equilibrium dissociation constant (KD).
FIG. 3 shows the receptor competition activity of the mAbs.
Among them, monoclonal antibodies (2H2 and 3C1) were assayed for their ability to compete with the receptor ACE2 (with Fc fragment tag of human antibody) for binding to RBD protein by ELISA, followed by detection with a secondary antibody against human Fc. Zika virus monoclonal antibody 5F8 was used as an isotype control (ctr).
FIG. 4 neutralizing activity of mAbs.
Pseudoviruses were incubated with four-fold serial dilutions of purified antibodies 2H2 and 3C1 for 1 hour, respectively, and then added to VeroE6 cells. Intracellular luciferase activity was measured approximately 48 hours post infection and the half maximal inhibitory concentration (IC50) of each antibody was calculated using GraphPad Prism software. Data are presented as mean ± standard error of mean.
FIG. 5 shows the expression and identification of chimeric mabs.
Wherein, the chimeric antibodies (A-C) of 2H2 and 3C1 are expressed in 293T cells in small quantity, and supernatant is taken at 48 hours to detect the expression and activity of the antibodies. (A) Western blot detection of the expressed chimeric antibody in the non-reduced state is shown. The secondary antibody is horseradish peroxidase (HRP) conjugated anti-human IgG. c2H2, chimeric 2H2 antibody. C3C1, chimeric 3C1 antibody. (B) ELISA was shown to detect the response of the expressed chimeric antibody to SARS-CoV-2 RBD. For each antibody, the activity of the expression products of the individual heavy chain, the individual light chain, the whole antibody (heavy chain plus light chain) was verified separately. (C) ELISA was shown to detect the competitive activity of the expressed chimeric antibody on the binding of ACE2 receptor (with human antibody Fc-tag and biotinylated) to SARS-CoV-2 RBD. The secondary antibody is HRP-coupled streptavidin, and can detect biotinylated receptor signals.
Detailed Description
The inventor of the invention develops an antibody which is highly effective and specific to 2019 novel coronavirus for the first time through extensive and intensive research and a large amount of screening. The results show that two SARS-CoV-2 specific monoclonal antibodies (named 2H2 and 3C1), especially 2H2, prepared from RBD immunized mice in the invention can effectively inhibit pseudovirus infection at cellular level, and can play a role in neutralization by inhibiting the binding of ACE2 receptor and RBD protein. The present invention has been completed based on this finding.
Antibodies
As used herein, the term "antibody" or "immunoglobulin" is an heterotetrameric glycan protein of about 150000 daltons with the same structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable region (VH) followed by a plurality of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Particular amino acid residues form the interface between the variable regions of the light and heavy chains.
As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, which are in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held close together by the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIH Publ. No.91-3242, Vol I, 647-669 (1991)). The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.
The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two distinct classes (termed kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be assigned to different classes based on the amino acid sequence of their heavy chain constant regions. There are mainly 5 classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA 2. The heavy chain constant regions corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
In general, the antigen binding properties of an antibody can be described by 3 specific regions in the heavy and light chain variable regions, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, and the β -sheets formed by the FRs between them are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type.
In the present invention, "VH-CDR 1" and "CDR-H1" are used interchangeably and refer to CDR1 of the heavy chain variable region; "VH-CDR 2" and "CDR-H2" are used interchangeably and refer to CDR2 of the heavy chain variable region; "VH-CDR 3" and "CDR-H3" are used interchangeably and refer to CDR3 of the heavy chain variable region. "VL-CDR 1" and "CDR-L1" are used interchangeably and refer to CDR1 of the light chain variable region; "VL-CDR 2" and "CDR-L2" are used interchangeably and refer to CDR2 of the light chain variable region; "VL-CDR 3" and "CDR-L3" are used interchangeably and refer to CDR3 of the light chain variable region.
The invention includes not only intact antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.
In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be obtained by standard DNA recombination techniques, and are useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as chimeric antibodies having a variable region derived from a murine monoclonal antibody, and a constant region derived from a human immunoglobulin (see, e.g., U.S. Pat. No. 4,816,567 and U.S. Pat. No. 4,816,397, which are hereby incorporated by reference in their entirety). Humanized antibodies refer to antibody molecules derived from non-human species having one or more Complementarity Determining Regions (CDRs) derived from the non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Pat. No. 5,585,089, herein incorporated by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using recombinant DNA techniques well known in the art.
In the present invention, the antibody may be monospecific, bispecific, trispecific, or more multispecific.
In the present invention, the antibody of the present invention also includes conservative variants thereof, which means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids having similar or similar properties as compared with the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variants are preferably produced by amino acid substitutions according to Table A.
TABLE A
Initial residue(s) Representative substitutions Preferred substitutions
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
In the present invention, the antibody is an antibody that specifically binds to the RBD domain of SARS-CoV-2S protein. The invention provides a high specificity and high affinity antibody directed against the RBD domain of SARS-CoV-2S protein, comprising a heavy chain variable region (VH) amino acid sequence and a light chain comprising a light chain variable region (VL) amino acid sequence.
Preferably, the first and second electrodes are formed of a metal,
the heavy chain variable region (VH) has complementarity determining regions CDRs selected from the group consisting of:
VH-CDR1 shown in SEQ ID NO. 4 or 16,
VH-CDR2 shown in SEQ ID NO 5 or 17, and
VH-CDR3 shown in SEQ ID NO 6 or 18;
the light chain variable region (VL) has Complementarity Determining Regions (CDRs) selected from the group consisting of:
VL-CDR1 shown in SEQ ID NO 10 or 22,
VL-CDR2 shown in SEQ ID NO. 11 or 23, and
VL-CDR3 shown in SEQ ID NO 12 or 24;
wherein, any one of the amino acid sequences also comprises a derivative sequence which is optionally added, deleted, modified and/or substituted by at least one amino acid and can retain the binding affinity with the RBD structural domain of the SARS-CoV-2S protein.
In another preferred embodiment, the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence is preferably an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% homology or sequence identity.
Methods for determining sequence homology or identity known to those of ordinary skill in the art include, but are not limited to: computer Molecular Biology (computerized Molecular Biology), Lesk, a.m. ed, oxford university press, new york, 1988; biological calculation: informatics and genomic Projects (Biocomputing: information and Genome Projects), Smith, d.w. eds, academic press, new york, 1993; computer Analysis of Sequence Data (Computer Analysis of Sequence Data), first part, Griffin, a.m. and Griffin, h.g. eds, Humana Press, new jersey, 1994; sequence Analysis in Molecular Biology (Sequence Analysis in Molecular Biology), von Heinje, g., academic Press, 1987 and Sequence Analysis primers (Sequence Analysis Primer), Gribskov, m. and Devereux, j. eds M Stockton Press, New York, 1991 and Carllo, h. and Lipman, d.s., SIAM j.applied Math., 48:1073 (1988). The preferred method of determining identity is to obtain the greatest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to: the GCG program package (Devereux, J. et al, 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al, 1990). BLASTX programs are publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al, NCBI NLM NIH Bethesda, Md.20894; Altschul, S. et al, 1990). The well-known Smith Waterman algorithm can also be used to determine identity.
Preferably, the antibody described herein is one or more of a full-length antibody protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody fragment (scFv), a single domain antibody (sdAb), and a single-domain antibody (sign-domain antibody), and a monoclonal antibody or a polyclonal antibody produced from the above antibodies. The monoclonal antibody can be developed by various means and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc., and the monoclonal antibody is prepared from wild-type or transgenic mice by the hybridoma technology in the mainstream.
The antibody full-length protein is conventional in the art and comprises a heavy chain variable region, a light chain variable region, a heavy chain constant region and a light chain constant region. The heavy chain variable region and the light chain variable region of the protein, the human heavy chain constant region and the human light chain constant region form a full-length protein of a fully human antibody. Preferably, the antibody full-length protein is IgG1, IgG2, IgG3 or IgG 4; more preferably IgG 1.
The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from an animal-derived antibody, a chimeric antibody, a humanized antibody, more preferably a humanized antibody, a human-animal chimeric antibody, and still more preferably a fully humanized antibody.
The antibody derivatives of the present invention may be single chain antibodies, and/or antibody fragments, such as: fab, Fab ', (Fab') 2 or other antibody derivatives known in the art, and the like, as well as any one or more of IgA, IgD, IgE, IgG, and IgM antibodies or antibodies of other subtypes.
The single-chain antibody is a conventional single-chain antibody in the field and comprises a heavy chain variable region, a light chain variable region and a short peptide of 15-20 amino acids.
Among them, the animal is preferably a mammal such as a mouse.
The antibodies of the invention may be chimeric, humanized, CDR grafted and/or modified antibodies targeting the SARS-CoV-2Spike protein.
In the above-mentioned aspect of the present invention, the number of amino acids to be added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, and more preferably 15 to 20% of the total number of amino acids in the original amino acid sequence.
In the above-mentioned aspect of the present invention, the number of the amino acids to be added, deleted, modified and/or substituted may be 1 to 7, more preferably 1 to 5, still more preferably 1 to 3, and still more preferably 1 to 2.
In another preferred embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO 2 or 14.
In another preferred embodiment, the variable region of the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO 8 or 20.
In an embodiment of the invention, the antibody targeting the RBD domain of SARS-CoV-2S protein is 2H2 or 3C 1. Preferably, the antibody targeting the RBD domain of the SARS-CoV-2S protein is 2H 2.
In a more preferred embodiment, each antibody of the invention specifically comprises each of the following VL and VH sequences, as well as Fc, CL and CH1 sequences.
Table B summary of antibody sequences
Figure BDA0002497240540000161
Figure BDA0002497240540000171
Figure BDA0002497240540000181
Encoding polynucleotides
The present invention also provides a polynucleotide encoding the above antibody or a recombinant protein comprising the same or a heavy chain variable region or a light chain variable region thereof.
Preferably, the nucleotide sequence of the nucleic acid for encoding the heavy chain is shown as the sequence table SEQ ID NO 1 or 13; and/or the nucleotide sequence of the nucleic acid for encoding the light chain is shown as the sequence table SEQ ID NO 7 or 19.
More preferably, the nucleotide sequence of the nucleic acid for encoding the heavy chain variable region is shown as the sequence table SEQ ID NO 1; and the nucleotide sequence of the nucleic acid for coding the light chain variable region is shown as a sequence table SEQ ID NO. 7.
The preparation method of the nucleic acid is a preparation method which is conventional in the field, and preferably comprises the following steps: obtaining the nucleic acid molecule coding the protein by gene cloning technology, or obtaining the nucleic acid molecule coding the protein by artificial complete sequence synthesis method.
Those skilled in the art know that the base sequence of the amino acid sequence encoding the above protein may be appropriately introduced with substitutions, deletions, alterations, insertions or additions to provide a polynucleotide homolog. The homologue of the polynucleotide of the present invention may be prepared by substituting, deleting or adding one or more bases of a gene encoding the protein sequence within a range in which the activity of the antibody is maintained.
Carrier
The invention also provides a recombinant expression vector comprising the nucleic acid.
Wherein said recombinant expression vector is obtainable by methods conventional in the art, i.e.: the nucleic acid molecule is connected to various expression vectors to construct the nucleic acid molecule. The expression vector is any vector conventionally used in the art so long as it can carry the aforementioned nucleic acid molecule. The carrier preferably comprises: various plasmids, cosmids, bacteriophages or viral vectors, etc.
The invention also provides a recombinant expression transformant containing the recombinant expression vector.
Wherein, the preparation method of the recombinant expression transformant is a preparation method which is conventional in the field, and preferably comprises the following steps: transforming the recombinant expression vector into a host cell. The host cell is any host cell conventionally used in the art, so long as it is sufficient that the recombinant expression vector is stably self-replicating and the nucleic acid carried thereby can be efficiently expressed. Preferably, the host cell is an e.coli TG1 or e.coli BL21 cell (expressing a single chain antibody or Fab antibody), or an HEK293 or CHO cell (expressing a full length IgG antibody). The recombinant expression plasmid is transformed into a host cell to obtain a recombinant expression transformant preferred in the present invention. Wherein the transformation method is a transformation method conventional in the art, preferably a chemical transformation method, a thermal shock method or an electric transformation method.
Preparation of antibodies
The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique, for example, by PCR amplification or genomic library screening. Alternatively, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.
Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.
At present, the DNA sequence encoding the antibody of the invention (or a fragment thereof, or a derivative thereof) has been obtained entirely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Preferred animal cells include (but are not limited to): CHO-S, HEK-293 cells.
Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the invention. The antibody of the invention is then purified by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, using conventional separation and purification means well known to those skilled in the art.
The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis by Munson et al, anal. biochem.,107:220 (1980).
The antibody of the present invention may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.
Antibody-drug conjugates (ADC)
The invention also provides an antibody-conjugated drug (ADC) based on the antibody of the invention.
Typically, the antibody-conjugated drug comprises the antibody, and an effector molecule to which the antibody is conjugated, and preferably chemically conjugated. Wherein the effector molecule is preferably a therapeutically active drug. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug or a radionuclide.
The antibody of the invention may be conjugated to the effector molecule by a coupling agent. Examples of the coupling agent may be any one or more of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond. The non-selective coupling agent is a compound which enables effector molecules and antibodies to form covalent bonds, such as glutaraldehyde and the like. The coupling agent using carboxyl can be any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (coupling site is acylhydrazone).
Certain residues on the antibody (e.g., Cys or Lys, etc.) are used to attach to a variety of functional groups, including imaging agents (e.g., chromophores and fluorophores), diagnostic agents (e.g., MRI contrast agents and radioisotopes), stabilizing agents (e.g., ethylene glycol polymers) and therapeutic agents. The antibody may be conjugated to a functional agent to form an antibody-functional agent conjugate. Functional agents (e.g., drugs, detection reagents, stabilizers) are coupled (covalently linked) to the antibody. The functional agent may be attached to the antibody directly, or indirectly through a linker.
Antibodies may be conjugated to drugs to form Antibody Drug Conjugates (ADCs). Typically, the ADC comprises a linker between the drug and the antibody. The linker may be degradable or non-degradable. Degradable linkers are typically susceptible to degradation in the intracellular environment, e.g., the linker degrades at the site of interest, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers such as glucuronide-containing linkers that can be degraded by glucuronidase. The peptidyl linker may comprise, for example, a dipeptide such as valine-citrulline, phenylalanine-lysine or valine-alanine. Other suitable degradable linkers include, for example, pH sensitive linkers (e.g., linkers that hydrolyze at a pH of less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions in which the antibody is hydrolyzed by a protease.
Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and attachment is achieved by the reactive group. Thiol-specific reactive groups are preferred and include: for example maleimide compounds, haloamides (for example iodine, bromine or chlorine); halogenated esters (e.g., iodo, bromo, or chloro); halomethyl ketones (e.g., iodo, bromo, or chloro), benzyl halides (e.g., iodo, bromo, or chloro); vinyl sulfone, pyridyl disulfide; mercury derivatives such as 3, 6-bis- (mercuric methyl) dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiolsulfonate. The linker may comprise, for example, a maleimide linked to the antibody via a thiosuccinimide.
The drug may be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, the linker links the antibody and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or keto group that may form a bond with the linker. In the case of a drug directly attached to a linker, the drug has a reactive group prior to attachment to the antibody.
In the present invention, a drug-linker can be used to form an ADC in a single step. In other embodiments, bifunctional linker compounds may be used to form ADCs in a two-step or multi-step process. For example, a cysteine residue is reacted with a reactive moiety of a linker in a first step, and in a subsequent step, a functional group on the linker is reacted with a drug, thereby forming an ADC.
Generally, the functional group on the linker is selected to facilitate specific reaction with a suitable reactive group on the drug moiety. As a non-limiting example, azide-based moieties may be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker by 1, 3-dipolar cycloaddition between the azide and the alkynyl group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other attachment strategies, such as those described in bioconjugation technology, second edition (Elsevier), are well known to those skilled in the art. It will be appreciated by those skilled in the art that for selective reaction of a drug moiety and a linker, each member of a complementary pair may be used for both the linker and the drug when the reactive functional group of the complementary pair is selected.
The present invention also provides a method of preparing an ADC, which may further comprise: the antibody is conjugated to a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).
In certain embodiments, the methods of the invention comprise: the antibody is conjugated to the bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the method of the present invention further comprises: the antibody linker conjugate is bound to the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody through the linker.
In some embodiments, the antibody drug conjugate ADC has the formula:
Figure BDA0002497240540000221
wherein:
ab is an antibody, and Ab is an antibody,
LU is a joint;
d is a drug;
and subscript p is a value selected from 1 to 8.
Applications of
The invention also provides the use of the antibodies, antibody conjugates ADC, recombinant proteins, and/or immune cells of the invention, for example for the preparation of a diagnostic formulation or for the preparation of a medicament.
Preferably, the medicament is a medicament for preventing and/or treating SARS-CoV-2 virus infection.
Detection use and kit
The antibodies of the invention or ADCs thereof may be used in detection applications, for example for the detection of samples, to provide diagnostic information.
In the present invention, the specimen (sample) used includes cells, tissue samples and biopsy specimens.
Preferably, the sample is a blood sample or a pharyngeal swab sample from a subject.
The term "biopsy" as used herein shall include all kinds of biopsies known to the person skilled in the art. Thus biopsies as used in the present invention may comprise e.g. resection samples of tumours, tissue samples prepared by endoscopic methods or needle biopsy of organs.
Samples for use in the present invention include fixed or preserved cell or tissue samples.
The invention also provides a kit containing the antibody (or fragment thereof) of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.
Pharmaceutical composition
The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the above antibody or active fragment thereof or fusion protein thereof or ADC thereof or corresponding immune cell, and a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated.
The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration. Typically, the route of administration of the pharmaceutical composition of the present invention is preferably injection administration or oral administration. The injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid or liquid form, and may be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, a capsule, a granule, an injection or an infusion, etc.
The antibody of the present invention may also be used for cell therapy by intracellular expression of a nucleotide sequence, for example, for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.
The pharmaceutical composition of the invention is a pharmaceutical composition for preventing and/or treating SARS-CoV-2 virus infection diseases.
The pharmaceutical composition of the present invention can be directly used for binding SARS-CoV-2S protein molecules or fragments thereof, and thus can be used for preventing and treating diseases caused by viral infection.
The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the monoclonal antibody (or conjugate thereof) of the present invention as described above and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.
In the present invention, preferably, the pharmaceutical composition of the present invention further comprises one or more pharmaceutically acceptable carriers. The medicinal carrier is a conventional medicinal carrier in the field, and can be any suitable physiologically or pharmaceutically acceptable medicinal auxiliary material. The pharmaceutical adjuvant is conventional in the field, and preferably comprises pharmaceutically acceptable excipient, filler or diluent and the like. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the protein and 0.01-99.99% of a pharmaceutical carrier, wherein the percentage is the mass percentage of the pharmaceutical composition.
In the present invention, preferably, the pharmaceutical composition is administered in an effective amount, which is an amount that alleviates or delays the progression of the disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be based in part on the consideration of the condition to be treated and the result sought. One skilled in the art can determine an effective amount by using such factors as an individual basis and using no more than routine experimentation.
In the case of pharmaceutical compositions, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 20 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
The invention provides the application of the pharmaceutical composition in preparing a medicament for preventing and/or treating SARS-CoV-2 virus infection diseases. Preferably, the SARS-CoV-2 virus infection is pneumonia.
Coronavirus (coronavirus)
As used herein, the terms "novel coronavirus", "2019-nCov" or "SARS-CoV-2" are used interchangeably, the 2019 novel coronavirus being the 7 th coronavirus known to infect humans and causing new coronary pneumonia (COVID-19), one of the serious infectious diseases threatening global human health.
Coronaviruses (CoV) belong to the family of the Nidovirales (Nidovirales) Coronaviridae (Coronaviridae), a enveloped positive-strand RNA virus, a subfamily of which contains four genera, alpha, beta, delta and gamma.
Among the coronaviruses currently known to infect humans, HCoV-229E and HCoV-NL63 belong to the genus alpha coronavirus, and HCoV-OC43, SARS-CoV, HCoV-HKU1, MERS-CoV and SARS-CoV-2 are all the genus beta coronavirus. SARS-CoV-2 is also known as 2019-nCov.
Highly pathogenic coronaviruses SARS-CoV and MERS-CoV, which outbreak in 2003 and 2012, respectively, both belong to the genus beta coronavirus. The novel coronavirus (SARS-CoV-2) which is outbreak in 2019 and has 80% similarity with SARS-CoV and 40% similarity with MERS-CoV, and also belongs to the beta genus coronavirus.
The genome of the virus is a single-strand positive-strand RNA, is one of RNA viruses with the largest genome, and codes comprise replicase, spike protein, envelope protein, nucleocapsid protein and the like. In the initial stage of viral replication, the genome is translated into two peptide chains of up to several thousand amino acids, the precursor Polyprotein (Polyprotein), which is subsequently cleaved by proteases to yield nonstructural proteins (e.g., RNA polymerase and helicase) and structural proteins (e.g., spike protein) and accessory proteins.
The main advantages of the invention include:
1) antibody 2H2 specifically recognizes SARS-COV-2 with high affinity.
2) Antibody 3C1 can cross-recognize SARS-COV and SARS-COV-2 with high affinity.
3) Antibodies 2H2 and 3C1 have strong neutralizing capacity against SARS-COV-2 pseudovirus infection.
4) The antibodies 2H2 and 3C1 have strong receptor competition capability
5) Two murine antibodies can be easily humanized without affecting their biological activity.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
Materials and methods
1. Cells
The mouse myeloma cell line SP2/0 was cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% Fetal Bovine Serum (FBS). Human embryonic kidney cell line 293F in serum-free medium FreeStyleTM293 expression medium (Gibco, USA).
2. Protein
Expressing the recombinant SARS-CoV-2RBD protein. The gene segment encoding the 320-550 residue of the S protein is synthesized after being optimized by a preferred codon and cloned to pcDNA3.4 plasmid, the upstream of the gene is the signal peptide of interleukin 10, and the downstream is a histidine tag.
Expressing the recombinant ACE2 receptor protein. RNA was extracted from the human intestinal cell line Caco2 cells and inverted to cDNA. A DNA fragment encoding the extracellular domain (residues 18-740) of ACE2 was amplified by PCR and cloned into the modified pcDNA3.4 plasmid. The upstream of the gene is the signal peptide of interleukin 10, and the downstream is the hinge region (hinge) and crystallizable fragment (Fc segment) of human IgG1 and a histidine tag.
The above two recombinant expression plasmids were each transiently transfected into 293F cells using Polyethyleneimine (PEI). After about 4 days of culture, the supernatant was collected and then purified by Ni column. ACE2-Fc was biotinylated using the EZ-Link TM Sulfo-NHS-LC-LC-Biotin kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions, followed by the use of ZebaTMThe desalting column (Thermo Fisher Scientific) was centrifuged to remove excess unreacted biotin.
3. Preparation of monoclonal antibody
Animal studies were approved by the animal welfare and use committee of the shanghai pasteur institute. All mice were purchased from Shanghai laboratory animal center (SLAC, China).
Mice were immunized as follows:
on day 0, RBD-Fc fusion protein (Iceland, Proteus; 100. mu.g/dose) was mixed with aluminum hydroxide adjuvant (500. mu.g/dose; Invivogen, USA) and CpG (25. mu.g/dose), and 6-8 weeks old female BALB/c mice were injected intraperitoneally.
At 8 days, 50. mu.g/dose of RBD-Fc fusion protein was mixed and emulsified with an equal volume of Freund's complete adjuvant (sigma, USA) and then injected subcutaneously into mice.
On day 13, 50. mu.g/dose of RBD-Fc fusion protein was mixed and emulsified with an equal volume of TiterMax adjuvant (Sigma) and then injected subcutaneously into mice.
At 22 days, the boost was performed by tail vein injection of 75 μ g RBD protein. Four days after the boost, splenocytes were harvested and fused with SP2/0 myeloma cells under the action of polyethylene glycol (PEG)1450 (Sigma).
The fused cells were cultured for 8 days in hypoxanthine, aminopterin and thymidine (HAT; Sigma) selective growth medium.
The antigen binding and receptor competition ability of the antibody in the hybridoma supernatant was examined by ELISA as described below. Positive hybridoma cells were cloned 2 to 4 times by limiting dilution to obtain a monoclonal cell line. Selected hybridoma clones were amplified and subsequently injected intraperitoneally into liquid paraffin-induced BALB/c mice. Ascites fluid was then collected and the monoclonal antibody was purified using a HiTrap Protein G HP affinity chromatography column (GE Healthcare, USA).
4. Enzyme-linked immunosorbent assay (ELISA)
And detecting the antigen binding capacity of the antibody. 100 ng/well of RBD protein was coated in a microtiter ELISA plate (Nunc, USA) overnight at 4 ℃. Blocking was then performed with 5% skim milk powder in PBS-Tween20 (PBST). After washing with PBST, 50. mu.L/well of hybridoma supernatant or two-fold serial dilutions of purified mAb were added followed by incubation at 37 ℃ for 2 hours. After washing, horseradish peroxidase (HRP) -conjugated anti-mouse IgG (1: 10,000 dilution; Sigma-Aldrich, USA) was added and incubated at 37 ℃ for 1 hour. After development, the absorbance at 450nm was monitored using a microplate reader. Note: cytospecific monoclonal antibody 5F8, a Zika virus (ZIKV) E protein, was used as an isotype control.
And detecting the receptor competitive power of the antibody. 40 ng/well of RBD protein was coated in ELISA plates overnight at 4 ℃. Blocking was then performed with 5% skim milk powder in PBS-Tween20 (PBST). After washing with PBST, 25. mu.L/well of hybridoma supernatant or two-fold serial dilutions of purified mAb and 25. mu.L/well (20ng) of ACE2-Fc receptor protein or biotinylated ACE2-Fc receptor protein were added, followed by incubation at 37 ℃ for 2 hours. After washing, horseradish peroxidase (HRP) conjugated anti-human IgG (Abcam, usa) or horseradish peroxidase (HRP) conjugated streptavidin (proteintech, usa) was added and incubated at 37 ℃ for 1 hour. After development, the absorbance at 450nm was monitored using a microplate reader.
The light and heavy chain types of the mabs were identified by ELISA using the SBA cloning System-HRP kit (Southern Biotech, USA) according to the manufacturer's instructions.
5. Preparation and neutralization assay of pseudoviruses
A brief preparation of SARS-CoV-2 pseudovirus based on Murine Leukemia Virus (MLV) is as follows:
the S protein encoding plasmid, MLV Gag-Pol packaging plasmid and MLV transfer plasmid encoding the luciferase reporter gene were mixed with Lipofectamine 2000(Life Technologies) and co-transfected into HEK293T cells. Cells were incubated with transfection medium for 18 hours at 37 ℃. Then, the transfection medium was removed, DMEM containing 10% FBS was added, and incubation was performed at 37 ℃ for another 30 hours. The supernatant was then collected and filtered through a 0.45 μm membrane.
The pseudovirus neutralization assay was as follows:
VeroE6 cells were cultured and plated into 48-well plates. 50 μ L of the antibody sample diluted in a 4-fold gradient was incubated with 150 μ L of pseudovirus at 37 ℃ for 1 hour, added to the cells, and incubated at 37 ℃ for 12 hours. After removal of the medium, fresh medium was added and incubated at 37 ℃ for a further 48 hours. Intracellular Luciferase signals were then detected using the Luciferase Assay System (Promega) kit according to the manufacturer's instructions. The percent neutralization was calculated according to the following formula: 100 × (fluorescence of given sample-fluorescence of cell simplex control sample)/(fluorescence of pseudovirus simplex control sample-fluorescence of cell simplex control sample). The median inhibitory concentration (IC50) for each mab was calculated by non-linear regression using GraphPad Prism software. IC50 is defined as the concentration of antibody required to inhibit 50% of viral infection compared to infection with a control sample of pseudovirus only.
6. Biofilm interferometry (BLI) assay
The binding affinity of the antibody to the antigen is determined. The BLI assay was performed on an Octet RED96 machine (PallFort Bio, USA) according to the manufacturer's instructions. Briefly, histidine-tagged RBD protein was dialyzed into 0.01M PBS, diluted to 30 ng/. mu.L with kinetic solution (0.01M PBS supplemented with 0.1% bovine serum albumin and 0.02% Tween 20), and then immobilized on a biosensor (PallForte Bio) with Ni-NTA until saturation. The antigen-bound biosensor was placed in a well containing a series of diluted monoclonal antibody samples for 500s to allow antigen-antibody binding, and then immersed in a kinetic solution for 500s to dissociate. The equilibrium dissociation constant (KD) was calculated using Octet data analysis software (pallfortebio).
7. Determination and analysis of monoclonal antibody sequences
Total RNA was isolated from hybridoma cells using TRIzol reagent (Invitrogen, usa). First strand cDNA was then synthesized using antibody type specific primers and M-MLV reverse transcriptase (Promega, USA). PCR amplification was performed using Ex Taq enzyme (Takara, Japan) and degenerate primers, followed by sequencing, to obtain the variable region sequences of the heavy and light chains of the mAbs. The position of the Complementarity Determining Regions (CDRs) was determined using IgBLAST tools.
8. Expression and preliminary identification of anti-SARS-CoV-2 chimeric monoclonal antibody
The variable region genes of the heavy chain and the light chain of the murine monoclonal antibody are cloned into a modified pcDNA3.4 vector respectively, and the vector comprises an interleukin 10(IL-10) signal sequence and a constant region gene (gamma1, kappa) of human immunoglobulin. The resulting light and heavy chain expression plasmids were co-transfected into HEK293T cells by Lipofectamine 2000(Life Technologies), 6 hours later replaced with serum-free medium, and 42 hours later the supernatant was harvested. Detecting the expression and the characteristics of the chimeric antibody.
Example 1: preparation and biochemical identification of anti-SARS-CoV-2 monoclonal antibody
1.1 obtaining of antibodies
To prepare monoclonal antibodies against SARS-CoV-2, spleen cells of mice immunized with SARS-CoV-2RBD-Fc fusion protein were fused with SP2/0 myeloma cells to obtain hybridomas. Screening of hybridoma cells was performed by detecting the antigen binding and receptor competition ability of the antibody in the supernatant of hybridoma cells by ELISA. Positive hybridoma cells were subcloned by limiting dilution.
Finally, 2 stable clones (2H2 and 3C1) were obtained. mAbs 2H2 and 3C1 belong to the IgG1 and kappa subtypes and are characterized as shown in Table 1.
TABLE 1 characterization of anti-SARS-CoV-2 monoclonal antibodies
Figure BDA0002497240540000291
Among them, the KD (equilibrium) value of the antibody interacting with SARS-CoV-2RBD was determined by BLI (see FIG. 2).
1.2 detection of antigen recognition ability
The ability of the monoclonal antibodies to recognize different antigens including the RBD of SARS-CoV-2 and the RBD of SARS-CoV was examined by ELISA.
The results are shown in FIG. 1. The results showed that both mAbs 2H2 and 3C1 reacted with the RBD of SARS-CoV-2, whereas the corresponding isotype control antibody 5F8 was completely non-reactive. mAb 2H2 was unable to bind to the RBD of SARS-CoV, whereas 3C1 was able to bind efficiently. These results indicate that mab 2H2 recognizes a sequence position unique to SARS-CoV-2, while mab 3C1 is directed to a conserved region position common to both coronaviruses.
1.3 detection of binding affinity
Subsequently, the binding affinity of the two mabs was determined quantitatively. Biofilm interference assay (BLI) measurements were performed. In this experiment, RBDs of SARS-CoV-2 were immobilized on a sensor and then allowed to interact with different concentrations of monoclonal antibody.
The results are shown in FIG. 2. The results showed that the equilibrium dissociation constant (KD) of 2H2 was 7.5 nM; the KD value of 3C1 was 13 nM. This demonstrates that both mabs have high affinity for the RBD antigen.
1.4 detection of antibody blocking Capacity
Since the receptor ACE2 can bind to RBD protein with high affinity, in this example, monoclonal antibody was tested by ELISA for its ability to compete with the receptor ACE2 (with human antibody Fc fragment tag) for binding to RBD protein, and for human Fc signal, ACE2 signal.
The results are shown in FIG. 3. Both mab 2H2 and 3C1 were able to inhibit the binding of RBD protein and the receptor ACE2 dose-dependently, whereas the control antibody 5F8 had no inhibitory effect at all. These results indicate that both mAbs have receptor competitive activity.
1.5 detection of neutralizing Effect on SARS-CoV-2
The neutralizing effect of the two monoclonal antibodies on SARS-CoV-2 was determined by a pseudovirus neutralization assay.
The results are shown in FIG. 4. Both mAbs 2H2 and 3C1 were effective in neutralizing infection with the SARS-CoV-2 pseudovirus. The results in FIG. 1 show that the half maximal inhibitory concentration (IC50) of 2H2 and 3C1 was determined to be 0.005, 1.911 μ g/mL, respectively. These results indicate that mAb 2H2 neutralizes SARS-CoV-2 more efficiently than mAb 3C 1. In contrast, Zika virus (ZIKV) specific mAb 5F8(IgG1 isotype control) did not show any neutralization at the maximum tested concentration (100. mu.g/mL) (see Table 1).
Example 2: anti SARS-CoV-2 monoclonal antibody sequence
In this example, the antibody sequence was determined. RNA is extracted from hybridoma cells, and then variable region sequences are obtained by a degenerate primer method.
Sequencing results showed that the sequences of the variable regions of monoclonal antibodies 2H2 and 3C1 were significantly different, and gene fragments of completely different germline gene families were used (see table 2 and appendix).
These results indicate that clones 2H2 and 3C1 were from different hybridoma cell progenitors. Sequence comparison analysis showed that the heavy chain variable regions (V) of mAb 2H2 and mAb 3C1H) Has 49.6% similarity with the light chain variable region (V) of monoclonal antibody 2H2 and monoclonal antibody 3C1L) Has a sequence similarity of 57.7%.
Example 3: expression identification of chimeric monoclonal antibodies
The variable regions of murine 2H2 and 3C1 were ligated to the constant regions of human IgG1 heavy chain and human kappa light chain, respectively, to construct human-murine chimeric mabs and expressed in HEK293T cells.
The humanized properties of chimeric mabs 2H2(C2H2) and 3C1(C3C1) were confirmed by reaction with anti-human IgG secondary antibodies in western blot analysis (fig. 5A). Both expressed C2H2 and C3C1 intact antibodies strongly bound to the RBD protein of SARS-CoV-2, whereas neither the expressed light chain nor the expressed heavy chain was effective in binding to the RBD protein (FIG. 5B). Receptor competition experiments show that the expressed complete antibodies of C2H2 and C3C1 can strongly compete with the receptor ACE2 for binding to RBD protein (FIG. 5C).
Taken together, these data indicate that humanized antibodies C2H2 and C3C1 retain high binding affinity and biological activity.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Shanghai Pasteur institute of Chinese academy of sciences
<120> preparation and application of neutralizing monoclonal antibody against novel coronavirus
<130> P2020-0888
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Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln
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Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Asn Gly
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Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met
35 40 45
Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Ser Pro Ser Leu Lys
50 55 60
Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln His Tyr Leu
65 70 75 80
Gln Leu Asn Ser Val Thr Ser Glu Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Ser Asp Tyr His Gly Ser Lys Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr
115 120 125
Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu
130 135 140
Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp
145 150 155 160
Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser
180 185 190
Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser
195 200 205
Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys
210 215 220
Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro
225 230 235 240
Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr
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Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser
260 265 270
Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg
275 280 285
Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile
290 295 300
Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn
305 310 315 320
Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys
325 330 335
Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu
340 345 350
Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe
355 360 365
Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala
370 375 380
Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr
385 390 395 400
Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly
405 410 415
Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His
420 425 430
Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys
435 440
<210> 15
<211> 119
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 15
Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Asn Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met
35 40 45
Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Ser Pro Ser Leu Lys
50 55 60
Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln His Tyr Leu
65 70 75 80
Gln Leu Asn Ser Val Thr Ser Glu Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Ser Asp Tyr His Gly Ser Lys Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser
115
<210> 16
<211> 8
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 16
Gly Asp Ser Ile Thr Asn Gly Tyr
1 5
<210> 17
<211> 7
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 17
Ile Ser Tyr Ser Gly Ser Thr
1 5
<210> 18
<211> 13
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 18
Ala Ser Asp Tyr His Gly Ser Lys Tyr Tyr Phe Asp Tyr
1 5 10
<210> 19
<211> 645
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 19
gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaca cagggtcagc 60
atcacctgca aggccagtca ggatgtgggt aatgatgtag cctggtatca acagaaacca 120
gggcaatctc ctaaactact gatttattgg gcatccaccc ggcacactgg agtccctgat 180
cgcttcacag gcagtggatc tgggacagat ttcactctca ccattagcaa tgtgcagtct 240
gaagacttgg cagattattt ctgtcagcaa tataacagat atccgtacac gttcggaggg 300
gggaccaagc tggaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca 360
tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac 420
cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg 480
aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg 540
ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca 600
tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag 645
<210> 20
<211> 214
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 20
Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
His Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Asn Asp
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser
65 70 75 80
Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Arg Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala
100 105 110
Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly
115 120 125
Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile
130 135 140
Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu
145 150 155 160
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
165 170 175
Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
180 185 190
Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser
195 200 205
Phe Asn Arg Asn Glu Cys
210
<210> 21
<211> 107
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 21
Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
His Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Asn Asp
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser
65 70 75 80
Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Arg Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 22
<211> 6
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 22
Gln Asp Val Gly Asn Asp
1 5
<210> 23
<211> 3
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 23
Trp Ala Ser
1
<210> 24
<211> 9
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 24
Gln Gln Tyr Asn Arg Tyr Pro Tyr Thr
1 5

Claims (10)

1. An antibody heavy chain variable region having Complementarity Determining Regions (CDRs) selected from the group consisting of:
VH-CDR1 shown in SEQ ID NO. 4 or 16, VH-CDR2 shown in SEQ ID NO. 5 or 17, and VH-CDR3 shown in SEQ ID NO. 6 or 18;
wherein, any one of the amino acid sequences also comprises a derivative sequence which is optionally added, deleted, modified and/or substituted by at least one amino acid and can retain the binding affinity with the RBD structural domain of the SARS-CoV-2S protein.
2. An antibody heavy chain having the heavy chain variable region of claim 1.
3. An antibody light chain variable region having Complementarity Determining Regions (CDRs) selected from the group consisting of:
VL-CDR1 shown in SEQ ID NO. 10 or 22, VL-CDR2 shown in SEQ ID NO. 11 or 23, and VL-CDR3 shown in SEQ ID NO. 12 or 24;
wherein, any one of the amino acid sequences also comprises a derivative sequence which is optionally added, deleted, modified and/or substituted by at least one amino acid and can retain the binding affinity with the RBD structural domain of the SARS-CoV-2S protein.
4. A light chain of an antibody, wherein said light chain has the variable region of the light chain of claim 3.
5. An antibody having the heavy chain variable region of claim 1, and/or the light chain variable region of claim 3;
alternatively, the antibody has a heavy chain according to claim 2, and/or a light chain according to claim 4;
wherein, any one of the amino acid sequences also comprises a derivative sequence which is optionally added, deleted, modified and/or substituted by at least one amino acid and can retain the binding affinity with the RBD structural domain of the SARS-CoV-2S protein.
6. A recombinant protein, said recombinant protein comprising:
(i) the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, or the antibody of claim 5; and
(ii) optionally a tag sequence to facilitate expression and/or purification.
7. A polynucleotide encoding a polypeptide selected from the group consisting of: the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, the antibody of claim 5, or the recombinant protein of claim 6.
8. A vector comprising the polynucleotide of claim 7.
9. A genetically engineered host cell comprising the vector or genome of claim 8 having the polynucleotide of claim 7 integrated therein.
10. An antibody conjugate, comprising:
(a) an antibody moiety selected from the group consisting of: the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, the antibody of claim 5, the recombinant protein of claim 6, or a combination thereof; and
(b) a coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle/nanorod, a nanomagnet, a viral coat protein, or a VLP, or a combination thereof.
CN202010421833.0A 2020-05-18 2020-05-18 Preparation and application of neutralizing monoclonal antibody against new coronavirus Pending CN113683686A (en)

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Application Number Priority Date Filing Date Title
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Publications (1)

Publication Number Publication Date
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