CN117088970A - Preparation and application of broad-spectrum anti-coronavirus monoclonal antibody - Google Patents

Preparation and application of broad-spectrum anti-coronavirus monoclonal antibody Download PDF

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Publication number
CN117088970A
CN117088970A CN202310607960.3A CN202310607960A CN117088970A CN 117088970 A CN117088970 A CN 117088970A CN 202310607960 A CN202310607960 A CN 202310607960A CN 117088970 A CN117088970 A CN 117088970A
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antibody
variable region
chain variable
heavy chain
light chain
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Inventor
王鹏飞
曹志伟
赵晓宇
裘天颐
乔芮
李嘉言
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Fudan University
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Fudan University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6839—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting material from viruses
    • A61K47/6841—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting material from viruses the antibody targeting a RNA virus
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2510/00—Genetically modified cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Abstract

The invention provides preparation and application of a broad-spectrum anti-coronavirus monoclonal antibody, and in particular discloses 3 monoclonal antibodies aiming at novel coronavirus SARS-CoV and/or SARS-CoV-2 Receptor Binding Domain (RBD) proteins, nucleic acid sequences for encoding the antibodies and antibody fragments and a preparation method thereof. In vitro experiments prove that the antibody of the invention has strong neutralization effect on various tested SARS-CoV, SARS-CoV-2 and mutant pseudoviruses thereof, and has broad-spectrum neutralization capability.

Description

Preparation and application of broad-spectrum anti-coronavirus monoclonal antibody
Technical Field
The invention relates to the field of biological medicine, in particular to preparation and application of a broad-spectrum anti-coronavirus monoclonal antibody.
Background
Various vaccines against SARS-CoV-2, including inactivated vaccines, mRNA vaccines, and recombinant protein vaccines. The popularization of the vaccines effectively improves the immunity resistance of the inoculator to SARS-CoV-2 and reduces the serious disease rate caused by virus infection. However, because SARS-CoV-2 has the characteristic that RNA virus is easy to mutate, the existing vaccine and monoclonal antibody approved for use have more or less immune escape effect on the mutant strain which is continuously appeared, and the expectation of effectively suppressing the pandemic cannot be achieved. Worse still, coronaviruses derived from other animal hosts (such as bats, dogs, pangolins, etc.) have the potential to spread across species to humans, which would accelerate the rate of variation of SARS-CoV-2.
Therefore, research and development of a broad-spectrum neutralizing antibody capable of simultaneously aiming at a plurality of coronaviruses are of great significance for coping with the present or future possible occurrence of coronavirus epidemic.
Disclosure of Invention
The invention aims to provide a broad-spectrum neutralizing antibody capable of simultaneously aiming at a plurality of coronaviruses.
In a first aspect, the present invention provides a heavy chain variable region of an antibody, said heavy chain variable region comprising the following three complementarity determining region CDRs:
CDR1 as shown in SEQ ID NO 1 or 2 or 3,
CDR2 as shown in SEQ ID NO 4 or 5 or 6, and
CDR3 as shown in SEQ ID NO 7 or 8 or 9.
In another preferred embodiment, the CDRs of the heavy chain variable region comprise SEQ ID NO: N H ,N H +3, and N H +6, where N H 1 or 2 or 3, respectively.
In another preferred embodiment, any of the above amino acid sequences further comprises a derivative sequence that is optionally added, deleted, modified and/or substituted with at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and is capable of retaining the Receptor Binding Domain (RBD) protein binding affinity of the novel coronaviruses SARS-CoV and/or SARS-CoV-2.
In another preferred embodiment, the heavy chain variable region further comprises an FR region of human origin or an FR region of murine origin.
In another preferred embodiment, the heavy chain variable region has the amino acid sequence shown in any one of SEQ ID NOs 10 to 12.
In a second aspect the invention provides a heavy chain of an antibody, said heavy chain having a heavy chain variable region according to the first aspect of the invention.
In another preferred embodiment, the heavy chain of the antibody further comprises a heavy chain constant region.
In another preferred embodiment, the heavy chain constant region is of human, murine or rabbit origin.
In a third aspect, the invention provides a light chain variable region of an antibody, said light chain variable region comprising the following three complementarity determining region CDRs:
CDR1' shown in SEQ ID NO 13 or 14 or 15,
CDR2' as shown in SEQ ID NO 16 or 17 or 18, and
CDR3' shown in SEQ ID NO 19 or 20 or 21.
In another preferred embodiment, the CDRs of the light chain variable region comprise SEQ ID NO: N L ,N L +3, and N L +6, where N L 13 or 14 or 15 respectively.
In another preferred embodiment, any of the above amino acid sequences further comprises a derivative sequence that is optionally added, deleted, modified and/or substituted with at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and is capable of retaining the RBD protein binding affinity of the novel coronaviruses SARS-CoV and/or SARS-CoV-2.
In another preferred embodiment, the light chain variable region further comprises an FR region of human origin or an FR region of murine origin.
In another preferred embodiment, the light chain variable region has the amino acid sequence set forth in any one of SEQ ID NOS.22-24.
In a fourth aspect the invention provides a light chain of an antibody, said light chain having a light chain variable region according to the third aspect of the invention.
In another preferred embodiment, the light chain of the antibody further comprises a light chain constant region.
In another preferred embodiment, the light chain constant region is of human, murine or rabbit origin.
In a fifth aspect, the invention provides an antibody having:
(1) A heavy chain variable region according to the first aspect of the invention; and/or
(2) The light chain variable region according to the third aspect of the invention.
In another preferred embodiment, the antibody has: a heavy chain according to the second aspect of the invention; and/or a light chain according to the fourth aspect of the invention.
In another preferred embodiment, the antibody is a specific antibody against SARS-CoV and/or SARS-CoV-2, preferably a specific antibody against SARS-CoV and/or SARS-CoV-2RBD protein.
In another preferred embodiment, the antibody has affinity (EC) 50 ) Is less than or equal to 100. Mu.g/ml (e.g., 1-100. Mu.g/ml), preferably less than or equal to 1. Mu.g/ml, more preferably less than or equal to 0.01. Mu.g/ml.
In another preferred embodiment, the antibody has affinity (EC) for the RBD protein of the novel coronavirus SARS-CoV-2 50 ) Is less than or equal to 100. Mu.g/ml (e.g., 1-100. Mu.g/ml), preferably less than or equal to 1. Mu.g/ml, more preferably less than or equal to 0.01. Mu.g/ml.
In another preferred embodiment, the antibody is selected from the group consisting of: an animal-derived antibody, a chimeric antibody, a humanized antibody, or a combination thereof.
In another preferred embodiment, the CDR regions of the humanized antibody comprise 1, 2, or 3 amino acid changes.
In another preferred embodiment, the animal is a non-human mammal, preferably a mouse, sheep, rabbit.
In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.
In another preferred embodiment, the antibody is an antibody full-length protein, or an antigen-binding fragment.
In another preferred embodiment, the antibody is a bispecific antibody, or a multispecific antibody.
In another preferred embodiment, the antibody is a monoclonal antibody.
In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.
In another preferred embodiment, the antibody further comprises a heavy chain constant region and/or a light chain constant region.
In another preferred embodiment, the heavy chain constant region is of human origin and/or the light chain constant region is of human origin.
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 framework region of murine origin, and/or the light chain variable region of the antibody further comprises a framework region of murine origin.
In another preferred embodiment, the heavy chain variable region sequence of the antibody is as shown in any one of SEQ ID NOs 10 to 12; and/or
The light chain variable region sequence of the antibody is shown in any one of SEQ ID NOs 22-24.
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 any one of SEQ ID NOS.10-12.
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 any one of SEQ ID NOS.22-24.
In another preferred embodiment, the number of amino acids added, deleted, modified and/or substituted does not exceed 40%, preferably 20%, more preferably 10% of the total number of amino acids of the initial amino acid sequence.
In another preferred embodiment, the at least one amino acid sequence that has been added, deleted, modified and/or substituted is an amino acid sequence having a homology of at least 80%.
In another preferred embodiment, the derivative sequence with at least one amino acid added, deleted, modified and/or substituted has the activity of binding to the RBD protein of SARS-CoV and/or SARS-CoV-2.
In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO. 10 and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO. 22.
In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO. 11 and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO. 23.
In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO. 12 and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO. 24.
In another preferred embodiment, the antibody is of the IgG type.
In another preferred embodiment, the antibody is in the form of a drug conjugate.
In a sixth aspect, the present invention provides a recombinant protein having:
(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 assist 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 another preferred embodiment, the recombinant protein specifically binds SARS-CoV and/or SARS-CoV-2RBD protein.
In a seventh aspect the invention provides a CAR construct, the antigen binding region of which is an scFv that specifically binds to SARS-CoV and/or SARS-CoV-2RBD proteins, and which scFv 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.
In an eighth aspect, the invention provides a recombinant immune cell expressing an exogenous CAR construct according to the seventh aspect of the invention; or the immune cells express or are exposed outside the cell membrane with an antibody according to the fifth aspect of the invention.
In another preferred embodiment, the immune cells are selected from the group consisting of: NK cells, T cells.
In another preferred embodiment, the immune cells are derived from a human or non-human mammal (e.g., a mouse).
In a ninth aspect, the present invention provides an antibody drug 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, or an antibody according to the fifth 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, drug, toxin, cytokine, radionuclide, enzyme, gold nanoparticle/nanorod, nanomagnetic particle, viral coat protein or VLP, or a combination thereof.
In another preferred embodiment, the antibody moiety is coupled to the coupling moiety via a chemical bond or linker.
In another preferred embodiment, the radionuclide comprises:
(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-133 Yb-169, yb-177, or combinations thereof.
In another preferred embodiment, the coupling moiety is a drug or a 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 folic acid antagonist, an antimetabolite, a chemosensitizer, 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 including, for example, auristatins (auristatins), camptothecins (camptothecins), duocarmycin/duocarmycin (duocarmycins), etoposides (etoposides), maytansinoids (maytansines) and maytansinoids (maytansinoids) (e.g., DM1 and DM 4), taxanes (taxanes), benzodiazepines (benzodiazepines), or benzodiazepine-containing drugs (benzodiazepine containing drugs) (e.g., pyrrolo [1,4] benzodiazepines (PBDs), indoline benzodiazepines (indoxazepines) and oxazolobenzodiazepines (oxybenzodiazepines)), vinca alkaloids (vilos), 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, mestaneol, ricin a-chain, combretastatin, docamicin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide (tenoposide), vincristine, vinblastine, colchicine, dihydroxyanthrax, diketo, actinomycin, diphtheria toxin, pseudomonas Exotoxin (PE) A, PE, abrin a chain, a-chain of jezosin, α -octacocin, gelonin, mitogellin, restrictocin (retproctrocin), phenol, enomycin, curcin, crotonin, calicheamicin, saporin (Sapaonaria officinalis), glucocorticoids, or combinations thereof.
In another preferred embodiment, the coupling 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 (computerized tomography) contrast agents, or enzymes capable of producing a detectable product, 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 proteins (BPHL)), chemotherapeutic agents (e.g., cisplatin).
In another preferred embodiment, the immunoconjugate comprises: multivalent (e.g., divalent).
In another preferred embodiment, the multivalent means that multiple repeats (a) are included in the amino acid sequence of the immunoconjugate.
In a tenth aspect the present invention provides the use of an active ingredient 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, or the antibody according to the fifth aspect of the invention, the recombinant protein according to the sixth aspect of the invention, or a combination thereof, for use in (a) preparing a diagnostic reagent or kit for a novel coronavirus infection; and/or (b) preparing a medicament for preventing and/or treating a novel coronavirus infection.
In another preferred embodiment, the diagnostic reagent is a test strip or a test plate.
In another preferred embodiment, the diagnostic reagent or kit is for: detecting SARS-CoV and/or SARS-CoV-2RBD protein or fragments thereof in the sample.
In another preferred embodiment, the antibody is in the form of A Drug Conjugate (ADC).
In another preferred embodiment, the novel coronavirus includes wild-type novel coronaviruses and mutant novel coronaviruses.
In another preferred embodiment, the wild-type novel coronavirus comprises SARS-CoV, SARS-CoV-2.
In another preferred embodiment, the mutant novel coronavirus comprises: novel coronavirus mutants Alpha, beta, delta, gamma, delta, omicron ba.1, omicron BA.2, omicronBA.5, omicron xbb.1 and Omicron bq.1.1.
In another preferred embodiment, the reagent comprises a chip, an immune microparticle coated with an antibody.
In an eleventh aspect, the present invention provides 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 immune cell according to the eighth aspect of the invention, an antibody drug conjugate according to the ninth aspect of the invention, or a combination thereof; and
(ii) A pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition is a liquid formulation.
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 heavy chain variable region according to the first aspect of the invention, the heavy chain variable region 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, or the antibody according to the fifth aspect of the invention, the recombinant protein according to the sixth aspect of the invention, the immune cell according to the eighth aspect of the invention, the antibody drug conjugate according to the ninth aspect of the invention, or a combination thereof, and 0.01 to 99.99% of the pharmaceutically acceptable carrier, said percentages being mass percentages of the pharmaceutical composition.
In another preferred embodiment, the pharmaceutical composition is used for the prevention and/or treatment of novel coronavirus infections.
In a twelfth aspect, the invention provides a polynucleotide encoding a polypeptide selected from the group consisting of:
(1) 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; or (b)
(2) A recombinant protein according to the sixth aspect of the invention;
(3) A CAR construct according to the seventh aspect of the invention.
According to a thirteenth aspect of the present invention there is provided a vector comprising a polynucleotide according to the twelfth aspect of the present invention.
In another preferred embodiment, the carrier comprises: bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.
In a fourteenth aspect the present invention provides a genetically engineered host cell comprising a vector according to the thirteenth aspect of the invention or a polynucleotide according to the twelfth aspect of the invention integrated in the genome.
In a fifteenth aspect the present invention provides a method for in vitro detection of a novel coronavirus in a sample, said method comprising the steps of:
(1) Contacting the sample with an antibody according to the fifth aspect of the invention;
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of SARS-CoV and/or SARS-CoV-2 virus RBD protein or fragment thereof in the sample.
In another preferred embodiment, the detection comprises diagnostic or non-diagnostic.
In a sixteenth aspect, the present invention provides a method for detecting SARS-CoV and/or SARS-CoV-2RBD protein or fragment thereof in a sample in vitro, said method comprising the steps of:
(1) Contacting the sample with an antibody according to the fifth aspect of the invention;
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of SARS-CoV and/or SARS-CoV-2RBD protein or fragment thereof in the sample.
In another preferred embodiment, the detection comprises diagnostic or non-diagnostic.
A seventeenth aspect of the present invention provides a detection plate, the detection plate comprising: a substrate (support) and a test strip comprising an antibody according to the fifth aspect of the invention or an antibody drug conjugate according to the ninth aspect of the invention.
In an eighteenth aspect, the present invention provides 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 second antibody against an antibody according to the fifth aspect of the invention;
alternatively, the kit contains a detection plate according to the seventeenth aspect of the invention.
In a nineteenth aspect, the present invention provides a method for producing a recombinant polypeptide, the method comprising:
(a) Culturing a host cell according to the fourteenth aspect of the invention under conditions suitable for expression;
(b) Isolating the recombinant polypeptide from the culture, said recombinant polypeptide being an antibody according to the fifth aspect of the invention or a recombinant protein according to the sixth aspect of the invention.
In a twentieth aspect the present invention provides a pharmaceutical combination comprising:
(i) A first 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 immune cell according to the eighth aspect of the invention, an antibody drug conjugate according to the ninth aspect of the invention, or a combination thereof;
(ii) A second active ingredient comprising other agents for treating novel coronavirus infections.
In a twenty-first aspect, the present invention provides a novel diagnostic method for coronavirus infection comprising:
(i) Obtaining a sample from a subject, and 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 a complex indicates that the subject is a novel coronavirus diagnostic patient.
In another preferred embodiment, the sample is a blood sample or a pharyngeal swab sample, or a sample in another tissue organ.
In a twenty-second aspect, the invention provides a method of treating a novel coronavirus infection, the method comprising: administering to a subject in need thereof an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, a CAR construct according to the seventh aspect of the invention, an immune cell according to the eighth aspect of the invention, an antibody drug conjugate according to the ninth aspect of the invention, a pharmaceutical composition according to the eleventh aspect of the invention, a pharmaceutical combination according to the twentieth aspect of the invention, or a combination thereof.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1. Binding capacity of antibodies to different coronavirus spike proteins is different.
FIG. 2. Antibodies have broad-spectrum neutralization against different highly pathogenic coronavirus pseudoviruses.
FIG. 3 shows that the antibodies have a certain capacity of broad-spectrum anti-SARS-CoV-2 five mutant strains.
FIG. 4. Antibodies still maintain neutralizing capacity against part of the SARS-CoV-2Omicron sub-line mutant pseudovirus.
FIG. 5 binding ability of antibodies to the cellular receptor binding Region (RBD) and N-terminal domain (NTD) proteins of the S protein domain of SARS-CoV-2.
FIG. 6 competitive binding of antibodies to related known epitopes within RBD.
FIG. 7 shows the relationship of antibodies competing with the receptor ACE2 for SARS-CoV-2 spike protein trimer.
Detailed Description
Through extensive and intensive studies, the present inventors have unexpectedly developed, for the first time, a class of antibodies having high specificity and high affinity for novel coronavirus SARS-CoV and/or SARS-CoV-2RBD proteins, and chimeric antigen receptor immune cells based on the antibodies with high specificity, through extensive screening. In particular, the present invention unexpectedly results in monoclonal antibodies against novel coronaviruses with extremely excellent affinity and specificity. The results of the invention show that 3 monoclonal antibodies (named PW5-4, PW5-5 and PW 5-535) prepared by the invention have strong neutralization effect on various tested SARS-CoV, SARS-CoV-2 and mutant pseudoviruses thereof, and the broad-spectrum neutralization capability is proved. On this basis, the present invention has been completed.
Terminology
In order that the present disclosure may be more readily understood, certain terms are first defined. As used in the present application, each of the following terms shall have the meanings given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.
The term "about" may refer to a value or composition that is within an acceptable error of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or measured.
As used herein, a "Chimeric Antigen Receptor (CAR)" is a fusion protein comprising an extracellular domain capable of binding an antigen, a transmembrane domain from which the extracellular domain is derived from a different polypeptide, and at least one intracellular domain. "Chimeric Antigen Receptor (CAR)" is also referred to as "chimeric receptor", "T-body" or "Chimeric Immune Receptor (CIR)". By "extracellular domain capable of binding an antigen" is meant any oligopeptide or polypeptide capable of binding an antigen. An "intracellular domain" refers to any oligopeptide or polypeptide known as a domain that transmits a signal to activate or inhibit an intracellular biological process.
As used herein, "domain" refers to a region in a polypeptide that is independent of other regions and that folds into a specific structure.
As used herein, "single chain variable region fragment (ScFv)" refers to a single chain polypeptide derived from an antibody that retains the ability to bind an antigen. Examples of ScFv include antibody polypeptides formed by recombinant DNA techniques, and wherein Fv regions of immunoglobulin heavy (H chain) and light (L chain) chain fragments are linked via a spacer sequence. Various methods of engineering ScFv are known to those skilled in the art.
As used herein, the term "treatment" refers to the administration of an internally or externally applied therapeutic agent comprising any of the antibodies of the invention against SARS-CoV and/or SARS-CoV-2RBD proteins and compositions thereof to a patient having one or more disease symptoms for which the therapeutic agent is known to have a therapeutic effect. Typically, the patient is administered an amount of the therapeutic agent (therapeutically effective amount) effective to alleviate one or more symptoms of the disease.
As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that there may be, but need not be, 1, 2, or 3 antibody heavy chain variable regions of a particular sequence.
"sequence identity" as used herein refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate substitutions, insertions, or deletions of mutations. The sequence identity between the sequences described in the present invention and sequences with which it has identity may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.
Coronavirus (Coronavir, coV)
Coronaviruses belong to the genus Coronaviridae (Coronavirus) of the family Coronaviridae (Nidovirales) of the order Coronavirales in the phylogenetic classification. Coronaviruses are enveloped RNA viruses whose genome is linear single-stranded plus strand, and are a broad class of viruses that are widely found in nature. Coronaviruses have diameters of about 80 to 120nm, a methylated cap structure at the 5 'end of the genome, a poly (A) tail at the 3' end, and a genome of about 27 to 32kb in total length, and are currently known as the largest genome viruses among RNA viruses. It infects only vertebrates, such as humans, mice, pigs, cats, dogs, wolves, chickens, cattle, birds.
The polyclonal fully human monoclonal antibodies of the invention target the relatively conserved core region of the receptor binding region of coronaviruses, thus allowing broad-spectrum binding and neutralization of SARS-CoV-2 and SARS-CoV.
Antibodies to
As used herein, the term "antibody" or "immunoglobulin" is an iso-tetralin protein of about 150000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H), having identical structural features. Each light chain is linked to the heavy chain by a 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 a variable region (VH) at one end followed by a plurality of constant regions. One end of each light chain is provided with a variable region (VL) and the other end is provided with a constant region; 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. Specific amino acid residues form an 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 fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming a connecting loop, which in some cases may form a partially folded structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH publication No.91-3242, vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.
The "light chain" of a vertebrate antibody (immunoglobulin) can be classified into one of two distinct classes (called kappa and lambda) depending on the amino acid sequence of its constant region. Immunoglobulins can be assigned to different classes based on the amino acid sequence of their heavy chain constant region. 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 IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring any particular method for producing the antibody.
The invention also includes monoclonal antibodies having the corresponding amino acid sequences of the SARS-CoV and/or SARS-CoV-2RBD protein monoclonal antibodies, monoclonal antibodies having the variable region chains of the SARS-CoV and/or SARS-CoV-2RBD protein monoclonal antibodies, and other proteins or protein conjugates and fusion expression products having these chains. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a light chain and a heavy chain comprising a hypervariable region (complementarity determining region, CDR), provided that the hypervariable region is identical or at least 90% homologous, preferably at least 95% homologous, to the hypervariable regions of the light chain and heavy chain of the invention.
Immunoconjugates and fusion expression products include, as known to those of skill in the art: conjugates of drugs, toxins, cytokines (cytokines), radionuclides, enzymes and other diagnostic or therapeutic molecules in combination with the SARS-CoV and/or SARS-CoV-2RBD protein anti-monoclonal antibodies or fragments thereof. The invention also includes cell surface markers or antigens that bind to the anti-SARS-CoV and/or SARS-CoV-2RBD protein monoclonal antibodies or fragments thereof.
The term "antigen-binding sheet of an antibodyA fragment "(or simply" antibody fragment ") refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. Fragments of full length antibodies have been shown to be useful for performing the antigen binding function of antibodies. Examples of binding fragments included in the term "antigen-binding fragment of an antibody" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F (ab') 2 A fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bridge on the longer chain region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VH and VL domains of a single arm of an antibody. Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and have a minimal antibody fragment of the entire antigen binding site. Generally, fv antibodies also comprise a polypeptide linker between the VH and VL domains, and are capable of forming the structures required for antigen binding.
The invention includes not only intact monoclonal antibodies but also immunologically active antibody fragments such as Fab or (Fab') 2 Fragments; antibody heavy chain; an antibody light chain.
The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes typically comprise at least 3,4,5,6,7,8,9,10,11,12,13,14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation.
The terms "specific binding," "selective binding," "selectively binding," and "specifically binding" refer to binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody is present at about less than 10 -7 M, e.g. less than about 10 -8 M、10 -9 M or l0 -10 Affinity (KD) binding of M or less. As used herein, the term "epitope" refers to a discrete, three-dimensional spatial site on an antigen that is recognized by an antibody or antigen-binding fragment of the invention.
The invention includes not only whole antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.
In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared by 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 prepared using DNA recombination techniques well known in the art. The term "murine antibody" is used herein to refer to monoclonal antibodies against SARS-CoV and/or SARS-CoV-2RBD protein, made according to the knowledge and skill of the art. The term "chimeric antibody (chimeric antibody)" refers to an antibody in which a variable region of a murine antibody is fused to a constant region of a human antibody, and which can reduce an immune response induced by the murine antibody. The term "humanized antibody (humanized antibody)", also known as CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting murine CDR sequences into the framework of human antibody variable regions, i.e., the framework sequences of different types of human germline antibodies. Humanized antibodies can overcome the heterologous response induced by chimeric antibodies that carry large amounts of murine protein components. Such framework sequences may be obtained from public DNA databases including germline antibody gene sequences or published references. To avoid a decrease in immunogenicity while at the same time causing a decrease in activity, the human antibody variable region framework sequences may be subjected to minimal reverse or back-mutations to maintain activity.
In the present invention, antibodies may be monospecific, bispecific, trispecific, or more multispecific.
As used herein, the term "heavy chain variable region" is used interchangeably with "VH".
As used herein, the term "variable region" is used interchangeably with "complementarity determining region (complementarity determining region, CDR)".
The term "CDR" refers to one of the 6 hypervariable regions within the variable domain of an antibody that contribute primarily to antigen binding. One of the most common definitions of the 6 CDRs is provided by Kabat E.A et al, (1991) Sequences of proteins of immunological interface.
In a preferred embodiment of the present invention, the heavy chain of the antibody comprises the heavy chain variable region described above and a heavy chain constant region, which may be of murine or human origin.
As used herein, the term "light chain variable region" is used interchangeably with "VL".
In a preferred embodiment of the invention, the heavy chain variable region and the light chain variable region of the antibody comprise three complementarity determining regions CDRs (amino acid sequence and nucleotide sequence) respectively as shown in table a below:
table A
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In another preferred embodiment, the amino acid sequences of the heavy chain variable regions are shown in SEQ ID NOS.10-12, respectively.
In another preferred embodiment, the amino acid sequences of the light chain variable regions are shown in SEQ ID NOS.22-24, respectively.
In a preferred embodiment of the present invention, the light chain of the antibody comprises the light chain variable region described above and a light chain constant region, which may be murine or human in origin.
The function of the antibody is determined by the gene specific gene sequences of the light chain and heavy chain variable region of the antibody, can be specifically combined with RBD proteins of SARS-CoV and/or SARS-CoV-2, and can prevent SARS-CoV and/or SARS-CoV-2 from infecting susceptible cells. Using the antibody variable region gene or Complementarity Determining Region (CDR) gene, different forms of engineered antibodies can be engineered and produced in any expression system utilizing prokaryotic and eukaryotic cells.
In the present invention, the terms "antibody of the invention", "protein of the invention", or "polypeptide of the invention" are used interchangeably to refer to antibodies that specifically bind SARS-CoV and/or SARS-CoV-2RBD protein, such as a protein or polypeptide having a heavy chain variable region (e.g., the amino acid sequence set forth in SEQ ID NO. 10-12) and/or a light chain variable region (e.g., the amino acid sequence set forth in SEQ ID NO. 22-24). They may or may not contain an initiating methionine.
In another preferred embodiment, the antibody is a murine or human murine chimeric monoclonal antibody against SARS-CoV and/or SARS-CoV-2RBD protein, the heavy chain constant region and/or light chain constant region of which may be a humanized heavy chain constant region or light chain constant region. More preferably, the humanized heavy chain constant region or light chain constant region is a heavy chain constant region or light chain constant region of human IgG1, igG2, or the like.
In general, the antigen binding properties of antibodies can be described by 3 specific regions located 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, the β -sheets formed by the FR therebetween 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 the same type of antibody.
The variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest because they are involved, at least in part, in binding to an antigen. Thus, the invention includes those molecules having monoclonal antibody light and heavy chain variable regions with CDRs, so long as the CDRs are 90% or more (preferably 95% or more, most preferably 98% or more) homologous to the CDRs identified herein. The invention includes not only intact monoclonal antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.
As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that retain substantially the same biological function or activity of an antibody of the invention. The polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (i i) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a sequence used to purify the polypeptide or a pro-protein sequence, or a fusion protein with a 6His tag. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.
The antibody of the present invention refers to a polypeptide having SARS-CoV and/or SARS-CoV-2RBD protein binding activity, comprising the above CDR regions. The term also includes variants of polypeptides comprising the above-described CDR regions that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal end. For example, in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. As another example, the addition of one or more amino acids at the C-terminus and/or N-terminus typically does not alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.
The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA which hybridizes under high or low stringency conditions with the encoding DNA of an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.
The invention also provides other polypeptides, such as fusion proteins comprising a human antibody or fragment thereof. In addition to nearly full length polypeptides, the invention also includes fragments of the antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids, preferably at least about 60 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the antibody of the invention.
In the present invention, a "conservative variant of an antibody of the present invention" refers to a polypeptide in which 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 of similar or similar nature, as compared to the amino acid sequence of the antibody of the present invention. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table B.
Table B
Initial residues Representative substitution Preferred substitution
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
The invention also provides polynucleotide molecules encoding the antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to the coding region sequence set forth in SEQ ID No. 25-48 or a degenerate variant. As used herein, "degenerate variant" refers in the present invention to a nucleic acid sequence encoding a polypeptide having the same amino acid sequence as the polypeptide of the present invention, but which differs from the coding region sequences set forth in SEQ ID NOS.25-48.
Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; a coding sequence for a mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) of the mature polypeptide, and non-coding sequences.
The term "polynucleotide encoding a polypeptide" may include polynucleotides encoding the polypeptide, or may include additional coding and/or non-coding sequences.
The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturing agents such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42℃and the like during hybridization; or (3) hybridization only occurs when the identity between the two sequences is at least 90% or more, more preferably 95% or more. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID No. 1-24.
The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the heavy chain coding sequence and the expression tag (e.g., 6 His) may be fused together to form a fusion protein.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. 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. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules that exist in an isolated form.
At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.
The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of 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. Representative examples are: coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; animal cells of CHO, COS7, 293 cells, and the like.
Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which can take up DNA, can be obtained after the exponential growth phase and then treated with CaCl 2 The process is carried out using procedures well known in the art. Another approach is to use MgCl 2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.
The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.
The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.
The antibodies of the invention may be used alone or in combination or coupling with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or enzymes capable of producing a detectable product.
Couplable therapeutic agents include, but are not limited to: insulin, IL-2, interferon, calcitonin, GHRH peptide, intestinal peptide analog, albumin, antibody fragments, cytokines, and hormones.
Therapeutic agents that may also be bound or conjugated to the antibodies of the invention include, but are not limited to: 1. a radionuclide; 2. biological toxicity; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; 5. a viral particle; 6. a liposome; 7. nano magnetic particles; 8. the prodrug activates the enzyme; 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticle, and the like.
The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof or fusion protein thereof as described above, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may 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: oral, respiratory, intratumoral, intraperitoneal, intravenous, or topical administration.
The pharmaceutical composition of the present invention can be directly used to bind to novel coronavirus RBD protein molecules, and thus can be used to extend the half-life of a drug, and in addition, other therapeutic agents can be used simultaneously.
The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the monoclonal antibodies (or conjugates thereof) of the 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 formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The 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 10 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.
When a pharmaceutical composition is used, 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 per kilogram of body weight, and in most cases no more than about 8 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
Carrier body
Nucleic acid sequences encoding a desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from vectors known to include the gene, or by direct isolation from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest may be produced synthetically.
The invention also provides vectors into which the expression cassettes of the invention are inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, as they allow long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.
In brief summary, the expression cassette or nucleic acid sequence of the invention is typically operably linked to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration of eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.
The expression constructs of the invention may also be used in nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Pat. nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the invention provides a gene therapy vector.
The nucleic acid may be cloned into many types of vectors. For example, the nucleic acid may be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe-generating vectors, and sequencing vectors.
Further, the expression vector may be provided to the cell in the form of a viral vector. Viral vector techniques are well known in the art and are described, for example, in Sambrook et al (2001,Molecular Cloning:ALaboratory Manual,Cold Spring Harbor Laboratory,New York) and other virology and molecular biology manuals. Viruses that may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, suitable vectors include an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism (e.g., WO01/96584; WO01/29058; and U.S. Pat. No. 6,326,193).
Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus may then be isolated and delivered to a subject cell in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenovirus vector is used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.
Additional promoter elements, such as enhancers, may regulate the frequency of transcription initiation. Typically, these are located in the 30-110bp region upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so as to maintain promoter function when the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50bp before the activity begins to decrease. Depending on the promoter, it appears that individual elements may act cooperatively or independently to initiate transcription.
One example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is extended growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV 40) early promoter, the mouse mammary carcinoma virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the ebustan-balr (Epstein-Barr) virus immediate early promoter, the ruses sarcoma virus promoter, and human gene promoters such as but not limited to the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that is capable of switching on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or switching off expression when expression is undesired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.
To assess expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cell may also comprise either or both a selectable marker gene or a reporter gene to facilitate identification and selection of the expressing cell from a population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on a single piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.
The reporter gene is used to identify potentially transfected cells and to evaluate the functionality of the regulatory sequences. Typically, the reporter gene is the following gene: which is not present in or expressed by the recipient organism or tissue and which encodes a polypeptide whose expression is clearly indicated by some readily detectable property, such as enzymatic activity. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is assayed at the appropriate time. Suitable reporter genes may include genes encoding luciferases, beta-galactosidases, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., ui-Tei et al 2000FEBS Letters479:79-82). Suitable expression systems are well known and can be prepared using known techniques or commercially available. Typically, constructs with a minimum of 5 flanking regions that show the highest level of reporter gene expression are identified as promoters. Such promoter regions can be linked to reporter genes and used to evaluate agents for their ability to regulate promoter-driven transcription.
Methods for introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, the vector may be readily introduced into a host cell, e.g., a mammalian, bacterial, yeast or insect cell, by any method known in the art. For example, the expression vector may be transferred into the host cell by physical, chemical or biological means.
Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, for example, sambrook et al (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York). A preferred method of introducing the polynucleotide into a host cell is calcium phosphate transfection.
Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method of inserting genes into mammalian, e.g., human, cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. patent nos. 5,350,674 and 5,585,362.
Chemical means for introducing the polynucleotide into a host cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery tool (del ivery vehicle) is a liposome (e.g., an artificial membrane vesicle).
In the case of non-viral delivery systems, an exemplary delivery means is a liposome. Lipid formulations are contemplated for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid can be associated with a lipid. The nucleic acid associated with the lipid may be encapsulated into the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution comprising the lipid, mixed with the lipid, associated with the lipid, contained in the lipid as a suspension, contained in or complexed with the micelle, or otherwise associated with the lipid. The lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution. For example, they may exist in a bilayer structure, as micelles or with a "collapsed" structure. They may also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example, lipids include fat droplets, which naturally occur in the cytoplasm as well as in such compounds comprising long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
In a preferred embodiment of the invention, the vector is a plasmid, such as a conventional mammalian expression plasmid.
Formulations
The invention provides a pharmaceutical composition comprising 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, a recombinant protein according to the sixth aspect of the invention, a CAR construct according to the seventh aspect of the invention, an immune cell according to the eighth aspect of the invention, or an antibody drug conjugate according to the ninth aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection.
In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, or the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The formulations of the present invention are preferably formulated for intravenous administration.
Detection application and kit
The antibodies of the invention may be used in detection applications, for example for detecting samples, thereby providing diagnostic information.
In the present invention, the samples (specimens) used include cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to a person skilled in the art. Thus biopsies used in the present invention may include tissue samples prepared, for example, by endoscopic methods or by puncture or needle biopsy of an organ.
Samples for use in the present invention include fixed or preserved cell or tissue samples.
The invention also provides a kit comprising an antibody (or fragment thereof) of the invention, which in a preferred embodiment of the invention further comprises a container, instructions for use, buffers, etc. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.
The main advantages of the invention include:
(1) The invention develops a kind of antibodies with high specificity and high affinity to novel coronavirus SARS-CoV and/or SARS-CoV-2RBD proteins.
(2) The present invention surprisingly results in monoclonal antibodies against novel coronaviruses with extremely excellent affinity and specificity. The results of the invention show that 3 monoclonal antibodies (named PW5-4, PW5-5 and PW 5-535) prepared by the invention have strong neutralization effect on various tested SARS-CoV, SARS-CoV-2 and mutant pseudoviruses thereof, and the broad-spectrum neutralization capability is proved.
(3) The present invention isolates individual memory B cells from peripheral blood lymphocytes of volunteers receiving five-needle new crown vaccine. The volunteer immunization protocol included 2 intramuscular injections of the psilosis crown mRNA vaccine BNT162b29 and 3 intramuscular injections (beijing) of the kexing midwifery crown inactivated vaccine. On day 14 after the immunization scheme is completed, 100ml of volunteer venous blood is collected, single memory B cells in peripheral blood of a vaccine inoculator are separated, sequence information is obtained through a single cell sequencing method, and a broad-spectrum anti-coronavirus monoclonal antibody is obtained through further screening.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
Unless otherwise specified, materials and reagents used in the examples of the present invention are commercially available products.
General method
Flow cell sorting and BCR sequencing: 10ml RPM 1640 medium containing 50% FBS was pre-warmed to 37C and used to thaw frozen PBMC samples, followed by centrifugation at 400Xg for 5 minutes. After discarding the supernatant, the cells were resuspended in 5ml FACS buffer (PBS, 2%FBS,2mM EDTA). Cell surface marker specific fluorescent labeled antibodies were labeled with 1:100 dilutions were prepared as a premix in FACS buffer for CD3 (APC-Cy 7), CD19 (BV-421), CD27 (APC) staining of the PBMC samples. Meanwhile, SARS-CoV-2Omicron S protein with His-tag and His-tag MERS-CoV S protein were incubated with PBMC for 1h. Cells were then incubated with antibodies to PE-anti His for 30 minutes in the dark, washed with FACS buffer, the stained cells resuspended in 500ml FACS buffer per 10-2000 tens of thousands of cells, filtered through a 70mm cap into FACS tubes, and the S protein-specific memory B cells were sorted using a BD FACSMelody sorter. Flow cytometry cell sorting was initiated. The sorting strategy focused on live memory B lymphocytes of CD3-, cd19+ and cd27+. The last step focused on cells that bound SARS-CoV-2 omnikom and the middle east respiratory syndrome coronavirus spike trimer. Single cells were first circled from PBMCs and then CD3 was selected - Excluding the effect of T cells. NextCD19 is circled + Is used to find out B cells. Further finding CD27 + And (3) high-expression cells, thereby finding out memory B cells. Finally, memory B cells bound to MERS-CoV or Omicon spike trimer were circled, and a total of 2412 memory B cells were sorted out. The sorted cells were added to a 10XChromium chip of a 5' single cell immunoassay assay (10X genomics). Library preparation and quality control were performed according to the manufacturer's protocol and sequencing was performed on a NextSeq 500 sequencer (Illumina).
Expression and purification of antibodies: monoclonal antibodies tested in this study were constructed and produced at the university of double denier. For each antibody, the variable gene was codon optimized for human cell expression and synthesized by HuaGeneTM (Shanghai, china) as plasmids (gWiz or pcdna 3.4) encoding the constant regions of human IgG1 heavy or light chains. Expression of heavy and light chain plasmids by cotransfection with polyethylenimine antibodies were expressed in an Expi293F (Sieimer femll, A14527) and at 125rpm and 8% CO at 37 ℃ 2 The cells were cultured with shaking. On day 5, the antibodies were purified using affinity chromatography using MabSelectTM PrismA (cytova, 17549801).
ELISA of antibodies antigen protein (2. Mu.g/mL) was added to ELISA microwell plates and incubated overnight at 4 ℃. After washing with PBS containing 0.2% Tween-20 (PBST), blocking was performed in PBST with 2% BSA at 37℃for 1 hour. After washing with PBST, monoclonal antibodies were added to the plates and incubated for 1 hour at 37 ℃. Monoclonal antibodies were assayed at an initial concentration of 1 μg/mL and 7 additional quadruple serial dilutions. For monoclonal antibodies, plates were washed and then HRP-conjugated anti-human IgG secondary antibody was used at 1:10 Dilutions of 000 were incubated. After washing, TMB substrate was added to the plate and incubated for 6min at room temperature, using 2M H 2 SO 4 The reaction was stopped. Absorbance was read at 450nm/630nm using a microplate reader.
Preparation of pseudoviruses: plasmids encoding SARS-CoV, MERS-CoV, WT (D614G) SARS-CoV-2 spike and the apomi Rong Ya lineage spike protein, etc. are synthesized. Expi293F cells were grown to 3X10 using polyethylenimine (Polyscience) using the indicated spike genes prior to transfection 6 /mL. Cell inAt 37℃with 8% CO 2 Cells in DMEM were incubated overnight and infected with VSV-G pseudo DG-luciferase (G x DG-luciferase, kerafast) at 5-fold multiple infection for 4 hours, and then washed three times with 1 xDPBS. The next day, the transfection supernatant was collected and clarified by centrifugation at 300g for 10 minutes. Each virus stock was then incubated with 20% I1 hybridoma (anti-VSV-G; ATCC, CRL-2700) supernatant at 37C for 1 hour to neutralize the contaminating VSV-G pseudotype DG-luciferase virus, and then titers were measured and aliquots made for storage at 80 ℃.
Neutralization experiments of antibodies neutralization assays were performed by incubating pseudoviruses with serial dilutions of monoclonal antibodies or serum and scored by reduction of luciferase gene expression. Briefly, vero E6 cells were seeded in 96-well plates at a concentration of 2x10 per well 4 Individual cells. Pseudoviruses were incubated the next day, test samples were serially diluted and incubated in triplicate for 30 minutes at 37 ℃. The mixture was added to the cultured cells and incubated for an additional 24 hours. Luminescence was measured by the luciferase assay system (Beyotime). IC (integrated circuit) 50 Defined as the 50% reduction in relative light units compared to the virus control wells (virus + cells) after subtraction of the background in the control group with cells only. IC5 0 Values were calculated using nonlinear regression in GraphPad Prism.
Example 1 antibody sequences
We obtained 2414 individual memory B cells of Omicon cross-binding MERS-CoV and SARS-CoV-2 by flow cytometry sorting. After BCR sequencing and bioinformatic prediction, the heavy and light chains of the antibody sequences were synthesized on mammalian expression vectors, respectively, and 100 pairs of antibodies were expressed in total using mammalian 293F cells. The following 3 pairs of antibodies PW5-4, PW5-5, and PW5-535 were finally identified by a series of in vitro experiments. They have good neutralizing ability to different degrees against 2 pseudostrains of human highly pathogenic novel coronaviruses SARS-CoV and SARS-CoV-2. The amino acid sequence information of the heavy chain and the light chain of the antibody sequences is shown in Table 1.
TABLE 1
Example 2 binding Capacity of antibodies to different coronavirus spike proteins
We tested 3 for the binding capacity of antibodies to spike protein trimers of 2 novel coronaviruses SARS-CoV and SARS-CoV-2. ELISA was used to test the binding capacity of spike protein trimers coated with 2 novel coronaviruses in ELISA plates to the following 3 pairs of antibodies diluted in double, OD values at 450nm were read using ELISA apparatus and the concentration of half-effective (EC 50 ) The corresponding results are shown in FIG. 1. The experimental results are shown in table 2. The results show that 3 pairs of antibodies can be effectively combined with different coronavirus spike proteins, and have broad binding spectrum.
TABLE 2
The following 3 pairs of antibodies, each having an initial concentration of 100. Mu.g/ml and a five-fold dilution, were incubated overnight with 2 coronavirus spike protein trimers, respectively, using an ELISA plate, washed and incubated with HRP-labeled anti-human IgG antibodies, developed with a substrate, OD values at 450nm were read using an ELISA plate to determine the binding capacity of the antibodies to the different coronavirus spike proteins, and half the effective concentration (EC 50 ) And (3) representing. FIG. 1 (A) shows the binding capacity of antibodies to spike protein trimer of coronavirus SARS-CoV, wherein PW5-5 has the best binding capacity to SARS-CoV, EC 50 0.002. Mu.g/ml, the remaining antibodies EC 50 From 0.006 to 0.008 μg/ml; FIG. 1 (B) shows the binding capacity of antibodies to spike protein trimers of coronavirus SARS-CoV-2, wherein PW5-4, PW5-5 and PW5-535 have an EC on SARS-CoV-2 50 0.003,0.002 and 0.002. Mu.g/ml, respectively.
EXAMPLE 3 neutralizing Capacity of antibodies against 2 highly pathogenic coronavirus pseudoviruses
Next, we tested 3 the neutralizing capacity of antibodies against pseudoviruses of the spike proteins of the three coronaviruses SARS-CoV and SARS-CoV-2. We used pseudoviruses expressing the spike proteins of SARS-CoV and SARS-CoV-2 established based on the Vesicular Stomatitis Virus (VSV) system to further evaluate the cross-protective capacity of antibodies against 2 highly pathogenic coronavirus pseudoviruses by detecting the protective effect of antibodies against Vero-E6 in African green monkey kidney cells and using half-inhibitory concentrations (IC 50 ) The corresponding results are shown in Table 3 and FIG. 2. We define IC 50 Antibodies with the numerical value not more than 100 mug/ml have the neutralizing capacity for inhibiting the pseudovirus, and experimental results show that the antibodies have a certain capacity for inhibiting 2 coronaviruses in a broad spectrum. Wherein PW5-4, PW5-5 and PW5-535 have good neutralizing ability against pseudoviruses of spike proteins of SARS-CoV and SARS-CoV-2.
TABLE 3 Table 3
Experiments employed pseudoviruses expressing the spike proteins of SARS-CoV and SARS-CoV-2 established based on the Vesicular Stomatitis Virus (VSV) system. Firstly, the constructed pseudovirus is incubated with the antibody diluted in a gradient manner, then the Vero-E6 cells of the African green monkey kidney cells are infected, the infection level of the pseudovirus is detected by utilizing luciferase after 16-24 hours, and the cross protection capability of the antibody to various coronaviruses is evaluated by the neutralization percentage, so that the broad spectrum of the pseudovirus is judged. FIG. 2 (A) shows the neutralizing ability of antibodies against SARS-CoV pseudovirus, wherein PW5-5 has the best neutralizing ability against SARS-CoV, IC 50 0.011 μg/ml, the remaining antibody ICs 50 From 0.211 to 0.624 μg/ml; panel (B) shows the neutralizing ability of the antibody against the pseudovirus of coronavirus SARS-CoV-2, and three pairs of antibodies each having the neutralizing ability of the pseudovirus of SARS-CoV-2, antibody IC 50 At 1-2. Mu.g/ml.
EXAMPLE 4 neutralizing Capacity of antibodies against SARS-CoV-2 five VOCs pseudoviruses
Next, we examined the neutralizing potential of antibodies against the SARS-CoV-2 five interesting Variant Strains (VOCs) pseudoviruses Alpha, beta, gamma, deltaForce. We further evaluated the cross-protective ability of antibodies against coronavirus SARS-CoV-2 mutant using pseudoviruses established based on the Vesicular Stomatitis Virus (VSV) system that express five variant strains of interest (VOCs) pseudoviral spike proteins, and used half-inhibitory concentrations (IC 50 ) The corresponding results are shown in Table 4 and FIG. 3. We define IC 50 The antibody with the value not more than 100 mug/ml has the neutralizing capacity for inhibiting the pseudovirus, and the experimental result shows that the 3 pairs of antibodies have a certain capacity for resisting SARS-CoV-2 mutant strain in a broad spectrum.
TABLE 4 Table 4
Experiments utilized pseudoviruses established based on the Vesicular Stomatitis Virus (VSV) system that expressed the spike proteins of five mutant strains SARS-CoV-2. Firstly, the constructed pseudovirus is incubated with a gradient diluted antibody to infect Vero-E6 cells of African green monkey kidney cells, and the infection level of the pseudovirus is detected by utilizing luciferase after 16-24 hours, and the cross-protective capacity of the antibody to five mutant strains Alpha (A), beta (B), gamma (C), delta (D) and Omicron BA.1 (E) of SARS-CoV-2 is evaluated by the neutralization percentage. These 3 antibodies all have a certain capacity of broad-spectrum anti SARS-CoV-2 mutant strain.
EXAMPLE 5 neutralizing Capacity of antibodies against SARS-CoV-2Omicron subline mutant pseudovirus
Next, we examined the neutralizing ability of the antibodies against the SARS-CoV-2Omicron subline mutant pseudovirus. We further assessed the cross-protective capacity of antibodies against coronavirus SARS-CoV-2Omicron subline mutant strains using pseudoviruses established based on the Vesicular Stomatitis Virus (VSV) system expressing spike proteins of the four SARS-CoV-2Omicron subline mutant strains, and using half-inhibitory concentrations (IC 50 ) The corresponding results are shown in Table 5 and FIG. 4. We define IC 50 Antibodies having a value of not more than 100. Mu.g/ml have neutralizing ability against the pseudovirusExperimental results show that antibodies generally generate immune escape to the Omicron sub-line mutant pseudovirus, but still maintain the neutralizing capacity to part of SARS-CoV-2Omicron sub-line mutant pseudovirus.
TABLE 5
Experiments utilized pseudoviruses established based on the Vesicular Stomatitis Virus (VSV) system that expressed spike proteins of the four Omicron sub-line mutants of SARS-CoV-2. First, the constructed pseudoviruses were incubated with the gradient diluted antibodies and then infected with Vero-E6 cells of african green monkey kidney cells, and the infection level of the pseudoviruses was detected by using luciferase after 16-24 hours, and the cross-protective ability of the antibodies against four Omicron subline mutants of SARS-CoV-2, ba.2 (a), ba.5 (B), xbb.1 (C), bq.1.1 (D) was evaluated by the neutralization percentage. Each of these three pairs of antibodies has neutralizing capacity against the SARS-CoV-2Omicron subline mutant pseudovirus.
Example 6 binding epitope of part of antibody is located on RBD
We used the enzyme-linked immunosorbent assay (ELISA) method to detect and verify the binding capacity of potential part of antibodies to the cell receptor binding domain (Receptor binding domain, RBD) and N-terminal domain (NTD) proteins of the S protein domain of coronavirus SARS-CoV-2, and the corresponding results are shown in FIG. 5. Experimental results show that the OD450 values of antibodies PW5-4, PW5-5 and PW5-535 for RBD protein binding are higher along with the increase of the concentration of the antibodies, which indicates that the antibodies are more firmly bound with the RBD proteins, and the antibodies are the RBD type binding antibodies; while the partial antibodies do not show binding capacity to NTD proteins.
The cell Receptor Binding Domain (RBD) and N-terminal domain (NTD) proteins of the S protein domain of SARS-CoV-2 were coated overnight with an ELISA plate, and antibodies PW5-4, PW5-5, and PW5-535 diluted at an initial concentration of 1 μg/ml and five-fold ratio were incubated, respectively, after washing, HRP-labeled anti-human IgG antibodies were incubated, and after development of the substrate, OD values at 450nm were read using an ELISA plate to determine binding of the antibodies to the cell Receptor Binding Domain (RBD) (A) and N-terminal domain (NTD) (B) of the S protein domain of SARS-CoV-2. Panel (A) shows the binding capacity of the antibody to the cell Receptor Binding Domain (RBD) of the S protein domain of SARS-CoV-2. Antibodies PW5-4, PW5-5 and PW5-535 have good binding effect on RBD; panel (B) shows that antibodies W5-4, W5-5, and W5-535, which show good binding ability to the N-terminal domain (NTD) protein of SARS-CoV-2 and have good binding effect to RBD protein, have no binding ability to NTD within 1. Mu.g.
Example 7 distribution of part of antibodies in RBD internal epitopes
By using an enzyme-linked immunosorbent assay (ELISA) method, a competitive ELISA binding experiment is performed on the potential part of antibodies in RBD internal epitope and the antibodies related to the known antigen epitope, so that the potential binding epitope of the potential antibodies can be obtained initially, and the result is shown in FIG. 6. According to the corresponding experimental detection results, the antibodies PW5-4 and PW5-5 have the competition relationship of epitope, and the antibodies PW5-4 or PW5-5 and PW5-535 have no obvious competition relationship of epitope. In competition binding with antibodies with known epitopes, experimental data show that antibody PW5-5 has competition relationship with CR3022, suggesting that the antibody PW5-5 may be four classes of antibodies (class IV) of RBD; antibodies PW5-535 are in a competing relationship with CR3022, and antibodies PW5-535 may be four classes of antibodies (class IV) for RBD.
The antibodies shown in the vertical columns are respectively coated overnight by using an ELISA plate, the antibodies shown in the horizontal lines are added into the ELISA plate after incubation with SARS-CoV-2 spike protein trimer, the HRP-marked anti-human IgG antibodies are incubated after washing, and the substrate is read by using an ELISA instrument after color development. The competition results are expressed in percentages, red for complete competition, white for no competition, and gradient red for squares. Panel (A) shows the results of a competitive ELISA assay between the RBD class binding antibodies PW5-4, PW5-5PW 5-535. The results show that the antibodies and the autoantibodies have a competition relationship, and the PW5-4 and the PW5-5 have a competition relationship, and the other antibodies have no obvious competition relationship (the obvious competition relationship refers to the competition percentage of 50%). Panel B shows a competition ELISA binding experiment of RBD binding antibodies and antibodies related to known epitopes. CB6, a class of SARS-CoV-2RBD (class I) antibodies; LY-CoV555, SARS-CoV-2RBD class I antibody; s309, three classes (class I II) antibodies to SARS-CoV-2 RBD; CR3022, four classes (class IV) antibodies to SARS-CoV-2 RBD. The results showed that antibodies PW5-5 were in a competitive relationship with CR3022 and PW5-535 were in a competitive relationship with CR3022, except that the antibodies were in a competitive relationship with the autoantibodies.
Example 8 part of the antibodies have a competitive relationship with the receptor ACE2
We used a competitive enzyme-linked immunosorbent assay (ELISA) method to test the ability of antibodies to compete with ACE2 protein for binding to SARS-CoV-2 spike protein trimer, the corresponding results are shown in FIG. 7. Experimental results show that antibodies PW5-4, PW5-5 and ACE2 do not compete for SARS-CoV-2 spike protein trimer; while PW5-535 competes with ACE2 for SARS-CoV-2 spike protein trimer, and competes with ACE2 more strongly with increasing concentration.
The ACE2 protein is coated by an ELISA plate overnight, the antibody and SARS-CoV-2 spike protein trimer are added into the ELISA plate after incubation, the HRP-marked anti-human IgG antibody is incubated after washing, and the substrate is read by an ELISA instrument after color development. The competition results are expressed as percentages (A and B), FIG. 7A illustrates that antibodies PW5-4 and PW5-5 do not affect the binding of ACE2 to the SARS-CoV-2 spike protein trimer, whereas PW5-535 affects the binding of ACE2 to the SARS-CoV-2 spike protein trimer. The ordinate above represents that there is a competing relationship, and the ordinate below represents that there is no competing relationship. Antibody PW5-535 was able to present a concentration-dependent competition with ACE2 for SARS-CoV-2 spike protein trimer (fig. 7B).
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims (15)

1. An antibody heavy chain variable region, wherein said heavy chain variable region comprises the following three complementarity determining region CDRs:
CDR1 as shown in SEQ ID NO 1 or 2 or 3,
CDR2 as shown in SEQ ID NO 4 or 5 or 6, and
CDR3 as shown in SEQ ID NO 7 or 8 or 9.
2. An antibody heavy chain having the heavy chain variable region of claim 1.
3. An antibody light chain variable region comprising the following three complementarity determining region CDRs:
CDR1' shown in SEQ ID NO 13 or 14 or 15,
CDR2' as shown in SEQ ID NO 16 or 17 or 18, and
CDR3' shown in SEQ ID NO 19 or 20 or 21.
4. An antibody light chain having the light chain variable region of claim 3.
5. An antibody, comprising:
(1) The heavy chain variable region of claim 1; and/or
(2) The light chain variable region of claim 3.
6. A recombinant protein, said recombinant protein comprising:
(i) A heavy chain variable region of claim 1, a heavy chain of claim 2, a light chain variable region of claim 3, a light chain of claim 4, or an antibody of claim 5; and
(ii) Optionally a tag sequence to assist expression and/or purification.
7. A CAR construct, wherein the antigen binding region of the CAR construct is an scFv that specifically binds to SARS-CoV and/or SARS-CoV-2RBD proteins, and wherein the scFv has the heavy chain variable region of claim 1 and the light chain variable region of claim 3.
8. A recombinant immune cell expressing the CAR construct of claim 7 exogenously; or the immune cells express or are exposed outside the cell membrane with the antibody of claim 5.
9. An antibody drug 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, or the antibody of claim 5, 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, drug, toxin, cytokine, radionuclide, enzyme, gold nanoparticle/nanorod, nanomagnetic particle, viral coat protein or VLP, or a combination thereof.
10. Use of an active ingredient, characterized in that the active ingredient is 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, or the antibody of claim 5, the recombinant protein of claim 6, or a combination thereof, for use in (a) preparing a diagnostic reagent or kit for novel coronavirus infection; and/or (b) preparing a medicament for preventing and/or treating a novel coronavirus infection.
11. A pharmaceutical composition comprising:
(i) An active ingredient 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, or the antibody of claim 5, the recombinant protein of claim 6, the immune cell of claim 8, the antibody drug conjugate of claim 9, or a combination thereof; and
(ii) A pharmaceutically acceptable carrier.
12. A polynucleotide encoding a polypeptide selected from the group consisting of:
(1) A heavy chain variable region of claim 1, a heavy chain of claim 2, a light chain variable region of claim 3, a light chain of claim 4, or an antibody of claim 5; or (b)
(2) The recombinant protein of claim 6;
(3) The CAR construct of claim 7.
13. A vector comprising the polynucleotide of claim 12.
14. A genetically engineered host cell comprising the vector of claim 13 or the polynucleotide of claim 12 integrated into the genome.
15. A pharmaceutical combination comprising:
(i) A first active ingredient 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, or the antibody of claim 5, the recombinant protein of claim 6, the immune cell of claim 8, the antibody drug conjugate of claim 9, or a combination thereof;
(ii) A second active ingredient comprising other agents for treating novel coronavirus infections.
CN202310607960.3A 2023-05-26 2023-05-26 Preparation and application of broad-spectrum anti-coronavirus monoclonal antibody Pending CN117088970A (en)

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