CN114790239A

CN114790239A - Antibody for resisting coronavirus N protein and application thereof

Info

Publication number: CN114790239A
Application number: CN202210317800.0A
Authority: CN
Inventors: 魏化伟; 张伦
Original assignee: Suzhou Dongkang Biotechnology Co ltd
Current assignee: Suzhou Dongkang Biotechnology Co ltd
Priority date: 2022-03-29
Filing date: 2022-03-29
Publication date: 2022-07-26
Anticipated expiration: 2042-03-29
Also published as: CN114790239B

Abstract

The invention discloses an antibody for resisting coronavirus N protein and application thereof, wherein the antibody comprises SEQ ID NO: 2, HCDR1 of SEQ ID No. 3, HCDR2 of SEQ ID No. 4, and LCDR3 of SEQ ID No. 10, LCDR1 of SEQ ID No. 11, and LCDR3 of SEQ ID No. 12; the antibodies have a higher affinity.

Description

Antibody for resisting coronavirus N protein and application thereof

Technical Field

The invention belongs to the fields of cell biotechnology and immunology, and relates to an antibody for resisting coronavirus N protein and application thereof.

Background

The laboratory detection of pathogens comprises real-time fluorescence RT-PCR, virus gene sequencing and serum immunology detection, although RT-PCR and virus gene sequencing are diagnostic gold standards, the detection time is long, the laboratory biological safety requirement standard is high, and the like, and the serum immunology detection has certain clinical significance as an index for early disease screening due to short detection time and simple operation.

Disclosure of Invention

In order to remedy the deficiencies of the prior art, it is an object of the present invention to provide an antibody against the N protein of coronavirus which has a high activity of binding to the N protein.

In a first aspect, the present invention provides a monoclonal antibody against the N protein, the monoclonal antibody comprising:

the amino acid sequence of SEQ ID NO: 2 HCDR1, SEQ id no:3 and HCDR2 of SEQ id no:4 and HCDR3 of SEQ id no:10, LCDR1, SEQ id no:11 and LCDR2 of SEQ id no: LCDR3 of 12.

Further, the monoclonal antibody also comprises a monoclonal antibody having an amino acid sequence substantially identical to SEQ id no: 5. 6, 7, 8, HFR2, HFR3, HFR4, and HFR1, which have at least 90% sequence identity to the amino acid sequence set forth in seq id No. 2; and a peptide having a sequence identical to SEQ id no: 13. 14, 15, 16, LFR1, LFR2, LFR3, and LFR4, which have at least 90% sequence identity to the amino acid sequence set forth in seq id no.

Further, the heavy chain variable region of the monoclonal antibody has a sequence identical to that of SEQ ID NO: 9, and preferably 95%, and the light chain variable region has a VH with at least 90% sequence identity, preferably 95% sequence identity, to the amino acid sequence set forth in SEQ id no:17, and preferably at least 90% sequence identity, preferably 95% sequence identity, to the amino acid sequence set forth in VL.

Further, the VH of the monoclonal antibody has the amino acid sequence of SEQ id no: 9; the VL of the monoclonal antibody has SEQ id no:17, or a pharmaceutically acceptable salt thereof.

Further, the monoclonal antibody further comprises a heavy chain constant region and a light chain constant region.

A second aspect of the invention provides a substance according to any one of the following:

1) a nucleic acid molecule encoding the monoclonal antibody according to the first aspect of the invention or a functional fragment thereof;

2) a recombinant expression vector comprising the nucleic acid molecule of 1);

3) a host cell comprising the recombinant expression vector of 2);

4) a drug conjugate comprising a monoclonal antibody according to the first aspect of the invention or a functional fragment thereof;

5) a product for detecting N protein of coronavirus, said product comprising a monoclonal antibody according to the first aspect of the invention or a functional fragment thereof; or a nucleic acid molecule, recombinant expression vector, host cell, drug conjugate encoding the monoclonal antibody or functional fragment thereof according to the first aspect of the invention;

6) a composition comprising a monoclonal antibody according to the first aspect of the invention or a functional fragment thereof, or a nucleic acid molecule encoding a monoclonal antibody according to the first aspect of the invention or a functional fragment thereof, a recombinant expression vector, a host cell, a drug conjugate.

Further, the recombinant expression vector has a signal peptide operably linked to an antibody.

Further, the recombinant expression vector further comprises a transcriptional regulatory element.

Further, the product may further comprise a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction.

Further, the reagent for performing the antigen-antibody reaction includes a buffer, a salt, and the like.

Further, the drug conjugate further comprises a coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, or enzyme.

A third aspect of the invention provides a pharmaceutical composition comprising a monoclonal antibody according to the first aspect of the invention or a substance according to the second aspect of the invention; and/or a pharmaceutically acceptable carrier.

A fourth aspect of the invention provides a method as defined in any one of the following:

1) a method of preparing a monoclonal antibody according to the first aspect of the invention, the method comprising: culturing the host cell of the second aspect of the invention, optionally isolating the antibody from the host cell and/or the medium in which the host cell is grown;

2) a method of detecting N protein in a sample, the method comprising: contacting a test sample with the monoclonal antibody of the first aspect of the invention and determining the presence or level of N protein in the test sample.

Further, the method of 1) further comprises purifying the monoclonal antibody.

Further, the host cell is selected from mammalian cells.

Further, the cell is selected from HEK293 cells.

A fourth aspect of the invention provides a use as claimed in any one of:

1) use of a monoclonal antibody according to the first aspect of the invention, a substance according to the second aspect of the invention for detecting N protein;

2) use of a monoclonal antibody according to the first aspect of the invention, or a substance according to the second aspect of the invention, in the manufacture of a product for detecting coronavirus infection;

3) use of a monoclonal antibody according to the first aspect of the invention, or a substance according to the second aspect of the invention, in the preparation of a product for detecting a disease associated with a coronavirus infection;

4) use of a monoclonal antibody according to the first aspect of the invention, a substance according to the second aspect of the invention or a pharmaceutical composition according to the third aspect of the invention for the manufacture of a medicament for the prevention and/or treatment of a disease associated with a coronavirus infection.

Further, the product comprises a kit.

Further, the kit comprises: colloidal gold immunoassay kit, chemiluminescence kit, radioimmunoassay kit, enzyme linked immunosorbent assay (ELISA), fluorescence immunoassay kit and microfluid chip.

Further, the coronavirus is selected from SARS-CoV-2, MERS-CoV, and SARS-CoV.

Further, the coronavirus is SARS-CoV-2.

Further, the disease related to coronavirus infection is COVID.

Further, the disease related to coronavirus infection is COVID-19.

Drawings

FIG. 1 is an electrophoretogram that detects monoclonal antibody 8D 7;

FIG. 2 is an HPLC chart for detection of monoclonal antibody 8D 7;

FIG. 3 is a graph of the binding activity of monoclonal antibody 8D7 detected by ELISA.

Detailed Description

Before the present methods are described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.

The term "coronavirus" or "CoV" refers to any virus of the coronavirus family, including but not limited to SARS-CoV-2, MERS-CoV, and SARS-CoV. SARS-CoV-2 refers to a coronavirus that was identified as emerging. SARS-CoV-2 is also known as 2019-nCoV. It binds to the human host cell receptor angiotensin converting enzyme 2(ACE2) via the viral spike protein.

As used herein, the term "coronavirus infection" or "CoV infection" refers to an infectious coronavirus such as SARS-CoV-2, MERS-CoV, or SARS-CoV. The term encompasses coronavirus respiratory infections, typically in the lower respiratory tract. Symptoms may include high fever, dry cough, shortness of breath, pneumonia, gastrointestinal symptoms (such as diarrhea), organ failure (renal failure and dysfunction), septic shock and death in severe cases.

As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, or bispecific antibody that binds to a particular antigen. Natural intact antibodies comprise two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as α, δ, ε, γ and μ, each heavy chain consisting of a variable region (VH) and first, second, third and optionally fourth constant regions (CH 1, CH2, CH3, CH4, respectively); mammalian light chains are classified as λ or κ, whereas each light chain consists of a variable region (VL) and a constant region. The antibody is "Y" shaped, wherein the stem of Y consists of the second and third constant regions of two heavy chains that are joined together by disulfide bonds. Each arm of Y comprises the variable and first constant regions of a single heavy chain in combination with the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains typically contain three highly variable loops, called Complementarity Determining Regions (CDRs) (light chain CDRs include LCDR1, LCDR2 and LCDR3, and heavy chain CDRs include HCDR1, HCDR2, HCDR 3). The CDR boundaries of the antibodies and antigen binding fragments disclosed in the present invention may be defined or identified by the convention of Kabat, IMGT, Chothia or Al-Lazikani as three CDRs are inserted between flanking segments (stretch) called Framework Regions (FR) (light chain FR includes LFR1, LFR2, LFR3 and LFR4, heavy chain FR includes HFR1, HFR2, HFR3 and HFR4) which are more conserved than CDRs and form a scaffold supporting highly variable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of the antibody heavy chain constant region. The five major classes or isotypes of antibodies are the large immunoglobulins a (iga), IgD, IgE, IgG and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma and mu heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1(γ 1 heavy chain), IgG2(γ 2 heavy chain), IgG3(γ 3 heavy chain), IgG4 (γ 4 heavy chain), IgA1(α 1 heavy chain), or IgA2(α 2 heavy chain).

In certain embodiments, the antibodies provided herein encompass any antigen binding fragment thereof. As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed from a portion of an antibody that comprises one or more CDRs or any other antibody fragment that binds an antigen but does not comprise the entire native antibody structure. Examples of antigen binding fragments include, but are not limited to, diabodies, fabs, Fab ', F (ab ')2, Fv fragments, disulfide stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv '), disulfide stabilized diabodies (ds diabodies), single chain antibody molecules (scFv), scFv dimers (diabodies), bispecific antibodies, multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. The antigen binding fragment is capable of binding to the same antigen as the parent antibody.

The term "operably linked" refers to the joining of two or more biological sequences of interest, with or without a spacer or linker, such that they are in a relationship that allows them to function in the intended manner. When used with respect to a polypeptide, it means that the polypeptide sequences are linked in a manner that allows the linked product to have the intended biological function. The term may also be used with respect to polynucleotides. For example, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.), it means that the polynucleotide sequences are linked in a manner that allows for the regulation of expression of the polypeptide by the polynucleotide. In one embodiment, the operably linked nucleotide sequences are contiguous (e.g., in the case of a signal sequence). Alternatively, operably linked nucleotide sequences may not be contiguous (e.g., in the case of an enhancer).

Antibodies against coronavirus N protein

The invention provides a monoclonal antibody directed against the N protein, said antibody comprising SEQ id no: 2 HCDR1, SEQ id no:3 and HCDR2 of SEQ id no:4 and HCDR3 of SEQ id no:10, LCDR1, SEQ id no:11 LCDR2 and SEQ id no: LCDR3 of 12.

In one embodiment, the monoclonal antibody further comprises a heavy chain variable region having a sequence identical to SEQ id no: 5. 6, 7, 8, HFR2, HFR3, HFR4, and HFR1, which have at least 90% sequence identity to the amino acid sequence set forth in seq id No. 2; and has a sequence identical to SEQ id no: 13. 14, 15, 16, and LFR1, LFR2, LFR3, and LFR4, which are light chain variable region framework regions having at least 90% sequence identity to the amino acid sequences depicted in figures 14, 15, and 16.

In yet another embodiment, the heavy chain variable region of the monoclonal antibody has a sequence identical to SEQ id no: 9, preferably a VH with at least 90% sequence identity, preferably 95% sequence identity, to the amino acid sequence set forth in SEQ id no:17, and preferably at least 90% sequence identity, preferably 95% sequence identity, to the amino acid sequence set forth in VL.

As a preferred embodiment, the VH of the monoclonal antibody has the amino acid sequence of SEQ id no: 9; the VL of the monoclonal antibody has an amino acid sequence shown in SEQ ID NO. 17.

In some embodiments, the antibodies and antigen binding fragments thereof provided herein comprise all or a portion of a heavy chain variable domain and/or all or a portion of a light chain variable domain. In one embodiment, the antibodies and antigen binding fragments thereof provided herein are single domain antibodies consisting of all or a portion of a heavy chain variable domain provided herein.

In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein further comprise an immunoglobulin (Ig) constant region, which optionally further comprises a heavy chain and/or light chain constant region. In certain embodiments, the heavy chain constant region comprises a CH1, a hinge, and/or a CH2-CH3 region (or optionally a CH2-CH3-CH4 region). In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgM. In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise the heavy chain constant region of human IgG 1. In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise the heavy chain constant region of human IgG 4. In certain embodiments, the light chain constant region comprises Ckappa (C κ) or Clambda (C λ). The constant regions of the antibodies and antigen-binding fragments thereof provided herein can be identical in sequence to, or differ from, the wild-type constant region by one or more mutations.

The antibodies and antigen-binding fragments thereof provided herein also encompass various variants of the antibody sequences provided herein.

In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise one or more amino acid residue substitutions in one or more of the CDR sequences and/or in one or more of the FR sequences. In certain embodiments, the affinity variant comprises no more than 20, 15, 10, 9, 8, 7, 6,5, 4, 3, 2, or 1 substitutions in total in the CDR sequence and/or FR sequence.

In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise 1, 2, or 3 CDR sequences that have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to that (or those) CDR sequences listed herein, but still retain specific binding to the coronavirus N protein at a level similar to or even higher than that of its parent antibody.

In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise one or more variable region sequences that have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to that (or those) of the variable region sequences listed herein, but still retain a level of specific binding affinity for the coronavirus N protein that is similar to, or even higher than, that of its parent antibody. In some embodiments, the mutation occurs in a region outside the CDR (e.g., in the FR).

Recombinant expression vector

As used herein, the term "vector" refers to a vehicle into which a polynucleotide encoding a protein can be operably inserted to cause expression of the protein. The vector may be used to transform, transduce or transfect a host cell so that it expresses the carried genetic element in the host cell. Examples of vectors include plasmids; phagemid; cosmids and artificial chromosomes, such as Yeast Artificial Chromosome (YAC), Bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); bacteriophages, such as lambda bacteriophage or M13 bacteriophage; and animal viruses. Classes of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, and papovaviruses (e.g., SV 40). The vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain an origin of replication. The term "origin of replication" refers to a sequence which, when present in a vector, initiates replication. The origin of replication may be recognized by the replication initiating factor or alternatively by a DNA helicase. The vector may also include materials that facilitate its entry into a cell, including, but not limited to, viral particles, liposomes, or protein envelopes.

The vector may be a recombinant expression vector or a cloning vector. The invention provides vectors (e.g., expression vectors) comprising a nucleic acid sequence provided herein encoding an anti-N protein antibody, at least one promoter operably linked to the nucleic acid sequence, and/or at least one selectable marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV40), lambda phage, and M13 phage, plasmids such as pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, EMEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO 865, pSELECT, pUNO, Psg5L, pBABE, pWPXL, pBI, p 15-L, pPro18, pTDD, pGS 10, pLexA, pACTPT 2.2, pCTMV-SCRIPT 6. 8, pDNAx, pAVR 1.25, pAVR, pDFF 1.25, pDFF, pDFRE, pDFE, pFU, pDFO, pDFE, pFU, pDFT, pDFE, pDFP, pDFE, pDFP, pDFE, pDFP 1.2.2.2.2, pDFT, pDFP, pDFE, pDFP, pDFE, pDFP, pDFE, pDFP, pDFE, pDFP, pDFE, pDFP, pDFE, pDFP, pDFE, pDFP, pDFE, pDFP, pDFE, p.

A "recombinant expression vector" is a nucleic acid molecule encoding a gene that is expressed in a host cell and that, in addition, contains the necessary elements to control the expression of the gene. Typically, an expression vector comprises a transcription promoter, a gene of interest, and a transcription terminator.

In certain embodiments, the recombinant expression vector is a viral-based vector. In certain embodiments, the recombinant expression vector is a lentiviral vector. In certain embodiments, the recombinant expression vector is an adeno-associated virus (AAV) vector.

In certain embodiments, the nucleic acid sequence encoding the anti-N protein antibody or antigen-binding fragment thereof provided herein is codon optimized. As used herein, the term "codon-optimized" refers to the alteration of a nucleic acid sequence to enhance expression in a vertebrate (e.g., a human) of interest by replacing at least one, more than one, or a substantial number of codons of the native sequence with codons that are more frequently or most frequently used in the gene of the vertebrate, but which do not alter the amino acid sequence of the original translated protein. Various species exhibit specific preferences for certain codons for particular amino acids.

In certain embodiments, the nucleic acid sequence encoding the anti-N protein antibody is codon optimized. In certain embodiments, the nucleic acid sequence encoding the heavy chain variable region of the anti-N protein antibody is codon optimized. In certain embodiments, the nucleic acid sequence encoding the light chain variable region of the anti-N protein antibody is codon optimized. In certain embodiments, the nucleic acid sequence encoding the heavy chain constant region of the anti-N protein antibody is codon optimized. In certain embodiments, the nucleic acid sequence encoding the light chain constant region of the anti-N protein antibody is codon optimized.

Host cell

Vectors comprising polynucleotide sequences encoding the antibodies may be introduced into host cells for cloning or gene expression. Suitable host cells for cloning or expressing the DNA in the vectors of the invention are prokaryotes, yeast or higher eukaryote cells. Suitable prokaryotes for this purpose include eubacteria, such as gram-negative or gram-positive organisms, for example of the family Enterobacteriaceae (Enterobacteriaceae), such as the genus Escherichia (Escherichia), for example Escherichia coli; enterobacter (Enterobacter); erwinia (Erwinia); klebsiella (Klebsiella); proteus (Proteus); salmonella (Salmonella), such as Salmonella typhimurium (Salmonella typhimurium); serratia species (Serratia), such as Serratia marcescens (Serratia marcescens); and Shigella (Shigella), and bacillus (bacillus), such as bacillus subtilis and bacillus licheniformis (b.licheniformis); pseudomonas (Pseudomonas), such as Pseudomonas aeruginosa (p. aeruginosa); and Streptomyces (Streptomyces).

In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts for the expression of anti-coronavirus N proteins. Saccharomyces cerevisiae (Saccharomyces cerevisiae) or common baker's yeast is the most commonly used among lower eukaryotic host microorganisms. However, a variety of other genera, species and strains are generally available and suitable for use in the present invention, such as Schizosaccharomyces pombe (Schizosaccharomyces pombe); kluyveromyces hosts such as Kluyveromyces lactis (k.lactis), Kluyveromyces fragilis (k.fragilis) (ATCC 12,424), Kluyveromyces bulgaricus (k.bulgaricus) (ATCC 16,045), Kluyveromyces vachelli (k.wickraimi) (ATCC 24,178), Kluyveromyces lactis (k.waltii) (ATCC 56,500), Kluyveromyces drosophilus (k.drosophilarium) (ATCC 36,906), Kluyveromyces thermotolerans (k.the rmotoleras), and Kluyveromyces marxianus (k.marxianus); yarrowia (EP 402,226); pichia pastoris (EP 183,070); candida (Candida); trichoderma reesei (Trichoderma reesei) (EP 244,234); neurospora crassa (Neurospora crassa); schwanniomyces (Schwanniomyces), such as Schwanniomyces occidentalis (Schwanniomyces occidentalis); and filamentous fungi, such as Neurospora (Neurospora), Penicillium (Penicillium), torticollis (Tolypocladium), and Aspergillus (Aspergillus) hosts, such as Aspergillus nidulans (a. nidulans) and Aspergillus niger (a. niger).

Suitable host cells for expressing the antibodies or antigen fragments provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains and variants have been identified as well as corresponding permissive insect host cells from: spodoptera frugiperda (Spodoptera frugiperda) (caterpillars), Aedes aegypti (mosquitoes), Aedes albopictus (mosquitoes), Drosophila melanogaster (Drosophila melanogaster), and Bombyx mori (Bombyx mori). A variety of viral strains for transfection are publicly available, for example the L-1 variant of Autographa californica (Autographa californica) NPV and the Bm-5 viral strain of Bombyx mori NPV, and the viruses can be used according to the present invention as the viruses in the present invention, in particular for transfecting Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco may also be used as hosts.

However, vertebrate cells have also attracted considerable attention, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of suitable mammalian host cell lines are monkey kidney CV1 strain transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney lines (293 or 293 cells subcloned for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR; mouse support cells (TM 4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat (buffalo rat) liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CCL 51); TRI cells; MRC 5 cells; FS4 cells; and human liver tumor lines (Hep G2).

Host cells are transformed with the expression or cloning vectors described above to produce anti-N protein antibodies and cultured in modified conventional nutrient media, as appropriate, to induce promoters, select transformants, or amplify genes encoding the desired sequences. In another embodiment, the antibody can be made by homologous recombination as known in the art.

Pharmaceutical compositions and methods of administration

The invention further provides pharmaceutical compositions comprising a recombinant expression vector expressing an antibody against coronavirus N protein, or an antigen-binding fragment thereof, provided by the invention, and one or more pharmaceutically acceptable carriers.

The term "pharmaceutically acceptable" indicates that the specified carrier is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with its recipient.

Pharmaceutically acceptable carriers for use in the compositions of the present invention may include, but are not limited to, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous vehicles (e.g., sodium chloride injection, ringer's injection, isotonic glucose injection, sterile water injection, or ringer's glucose and lactate injection), non-aqueous vehicles (e.g., non-volatile oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil), antimicrobial agents, isotonic agents (e.g., sodium chloride or dextrose), buffers (e.g., phosphate or citrate buffers), antioxidants (e.g., sodium bisulfate), anesthetics (e.g., procaine hydrochloride), suspending/dispersing agents (e.g., sodium carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone), chelating agents (e.g., EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid)), an emulsifier such as polysorbate 80(Tween-80), a diluent, adjuvant, excipient, or nontoxic auxiliary substance, other components known in the art, or various combinations thereof. Suitable components may include, for example, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickening agents, coloring agents, or emulsifying agents.

The present invention provides an agent for treating or preventing a disease associated with an antigen (e.g., N protein) -positive cell of interest, which comprises the anti-N protein antibody or an antigen-binding fragment thereof of the present invention as an active ingredient, and a therapeutically effective amount or a prophylactically effective amount of the agent can be administered to a subject in need thereof to treat or prevent a coronavirus positive disease. The anti-N protein antibody or antigen binding fragment thereof can inhibit coronavirus-induced disease-associated activity or eliminate or reduce the number of coronaviruses.

In addition, the present invention relates to a method for immunodetection or measurement of a target antigen (e.g., N protein), a reagent for immunodetection or measurement of a target antigen (e.g., N protein), a method for immunodetection or measurement of a coronavirus expressing a target antigen (e.g., N protein), and a diagnostic agent for diagnosis of a disease associated with infection with a target antigen (e.g., N protein) coronavirus, which comprises the antibody or antibody fragment specifically recognizing binding to a target antigen (e.g., human N protein) of the present invention as an active ingredient.

In the present invention, the method for detecting or determining the amount of the target antigen (e.g., N protein) may be any known method. For example, it includes immunodetection or assay methods.

The immunoassay or measurement method is a method of detecting or measuring the amount of an antibody or the amount of an antigen using a labeled antigen or antibody. Examples of the immunological detection or measurement method include a radioactive substance-labeled immune antibody method (RIA), an enzyme immunoassay (EIA or ELISA), a Fluorescence Immunoassay (FIA), a luminescence immunoassay, a western immunoblotting method, a physicochemical method, and the like.

The above-mentioned diseases associated with coronavirus infection can be diagnosed by detecting or measuring the expression N protein with the antibody or antibody fragment of the present invention.

For detecting cells expressing the polypeptide, known immunodetection methods can be used, and immunoprecipitation, fluorescent cell staining, immunohistological staining, and the like are preferably used. In addition, a fluorescent antibody staining method using FMAT8100HTS (Applied biosystems) and the like can be used.

In the present invention, the sample for detecting or measuring the target antigen (e.g., N protein) is not particularly limited as long as it has a possibility of containing the expression of the target antigen (e.g., N protein), such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, interstitial fluid, or culture fluid.

The diagnostic agent containing the monoclonal antibody or antibody fragment thereof of the present invention may further contain a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction, depending on the desired diagnostic method. Reagents for performing antigen-antibody reactions include buffers, salts, and the like. The reagent for detection includes reagents generally used in immunodetection or assay methods, such as a labeled second antibody recognizing the monoclonal antibody, an antibody fragment thereof or a binding substance thereof and a substrate corresponding to the label, and the like.

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.

EXAMPLE 1 screening of monoclonal antibodies

1. Synthesis of recombinant novel crown N protein

Synthesizing an N protein sequence of SARS-CoV-2 virus, and constructing into a pEM5.1 vector; extracting plasmids for transfection; transfecting to HEK293 cells, and culturing the cells for 7 days; and (3) harvesting the supernatant, purifying by using a Ni column, and concentrating and replacing a buffer solution to obtain the recombinant new crown N protein, wherein the sequence of the recombinant new crown N protein is derived from Uniprot P0DTC 9. The sequence is shown as SEQ ID NO:1 is shown.

2. Immunization

The first time, 100 mug of Freund's complete adjuvant is used, the injection is carried out in the abdominal cavity, the total dose is 0.5 ml/piece, and the second immunization is carried out at the interval of 3 weeks; the second time of using Freund's incomplete adjuvant with the dose of 50 mug/0.5 ml/piece, and carrying out the third immunization at the interval of 2 weeks; cell fusion was prepared 10 days after the third injection;

taking feeder cells, and pressing 10 ⁵ Perforamen, plating 10 the day before fusion ⁵ Each well is 100 mu l; mice immunized spleen cells were fused with prepared myeloma cells using the fusion agent PEG, and plated on 96-cell culture plates to which feeder cells had been added at 100. mu.l/well.

3. Screening and cloning of hybridoma cells

Screening positive holes by an ELISA detection method, and laying recombinant expression N protein overnight; washing the plate, adding skimmed milk powder, sealing, and standing at 37 deg.C for 1 hr; washing the plate, adding 100 mul of culture solution supernatant of 96 holes, and incubating for 1h at 37 ℃; washing the plate, adding a goat anti-mouse secondary antibody marked by HRP, and incubating for 30min at 37 ℃; washing the plate, adding a color development solution, developing for 10min, adding a stop solution, and reading the numerical value of OD 450; and screening high expression cell strains for subclone culture.

4. Sequencing

Collecting cells, extracting total RNA by Trizol, and carrying out reverse transcription by using oligo (dT)20 as a primer to generate cDNA. Then, specific primers are used for PCR amplification of the heavy chain variable region gene and the light chain variable region gene respectively. And (3) after the PCR product is subjected to electrophoretic purification, inserting the PCR product into a vector through TA cloning, converting, and selecting positive clones for sequencing.

5. As a result, the

The monoclonal antibody 8D7 against SARS-CoV-2 was selected, and its sequence is shown in Table 1.

TABLE 1 sequence of monoclonal antibody 8D7

Example 2 functional study of monoclonal antibody 8D7

1. Expression and purification of monoclonal antibodies

1) The selected sequence is chemically synthesized and cloned into a eukaryotic expression vector.

2) And (5) amplifying and extracting the plasmid.

3) The plasmid encoding the antibody was transiently transfected into mammalian cells HEK 293.

4) Collecting the supernatant, and purifying by affinity chromatography to obtain the monoclonal antibody.

5) As a result, the expression amount of the purified antibody was 225 mg/L.

2. Detection of physicochemical Properties of monoclonal antibody

2.1 gel electrophoresis detection of purity of monoclonal antibodies

1) Instrumentation and equipment

Name (R)	Manufacturer of the product	Type number
			Chemiluminescence imager	Tanon	Tanon-5200
Electrophoresis apparatus	BIO-RAD	poweerpac basic
			Electrophoresis tank	BIO-RAD	DYC-Mini4

2) Primary reagent

3) Sample preparation

Mixing 20 μ l sample with 5 μ l5 × reducing buffer, heating at 95 deg.C for 5min, and cooling;

mu.l of the sample was mixed well with 5. mu.l of 5 Xnon-reducing buffer.

4) Electrophoresis

Preparing gel, adding a proper amount of electrophoresis buffer solution, adding sample, and performing electrophoresis.

5) Dyeing and bleaching

After electrophoresis, putting the gel into a proper amount of Coomassie brilliant blue staining solution, and staining for 1 hour or more at room temperature; pouring out the staining solution, adding appropriate amount of Coomassie brilliant blue staining decolorization solution, and decolorizing at room temperature for 4-24 hr. After completion of decolorization, ddH was used ₂ And soaking in O, comparing with an unstained gel according to the Marker protein, cutting off the gel of the required protein component, and collecting. The protein to be purified is then separated from the gel.

6) As a result, the

The results are shown in FIG. 1, and the bands from left to right are marker, reduction band and non-reduction band; the detection purity of the monoclonal antibody is more than 95 percent.

2.2HPLC detection of purity of monoclonal antibodies

1) Instrumentation and equipment

2) Principal agent

Name (R)	Manufacturer of the product	Specification of	Goods number
				Dipotassium hydrogen phosphate trihydrate	SINOPHARM CHEMICAL REAGENT Co.,Ltd.	500 g/bottle	10017592
Potassium dihydrogen phosphate	SINOPHARM CHEMICAL REAGENT Co.,Ltd.	500 g/bottle	10017692
				Potassium chloride	SINOPHARM CHEMICAL REAGENT Co.,Ltd.	500 g/bottle	10016392

3) Fluid phase system

Dipotassium phosphate trihydrate, potassium dihydrogen phosphate and potassium chloride are added into about 900ml of purified water, stirred and dissolved, the volume is adjusted to 1L, and the pH value is determined to be between 6.2 +/-0.1 by measuring with a pH meter. Filtering with 0.22 μm filter membrane, and storing at room temperature.

4) Sample preparation

System applicability sample: MIL62 standard substance diluted with mobile phase to 2mg/ml

And (3) testing the sample: the sample to be tested was diluted to 2mg/ml with mobile phase.

5) Chromatographic conditions

6) As a result, the

As shown in FIG. 2, the purity of monoclonal antibody was greater than 95%.

3. Detection of binding Activity of monoclonal antibodies

1) Coating: diluting antigen N protein to 2 μ g/ml with coating solution, mixing well, adding 96-well coated plate, 100 μ l/well, sealing with membrane, and standing at 4 deg.C overnight.

2) The plate washer washes 3 times, no liquid can remain on the plate in the last time, and the liquid on the surface of the plate is patted dry by using absorbent paper.

3) And (3) sealing: add 5% milk powder (0.5g milk powder in 10ml DPBS), 300. mu.l/well, incubate 1h at 37 ℃ and wash plate 3 times as per step 2).

4) The antibody was diluted in a gradient, 100. mu.l/well, reacted at 37 ℃ for 1h, and the plate was washed 3 times according to step 2).

5) Adding a secondary antibody: diluting with DPBS according to a ratio of 1:2000, adding into a 96-well plate, reacting at a temperature of 100 mu l/well for 1h, and washing the plate for 3 times according to the step 2).

6) Color development: TMB was added at 100. mu.l/well and developed in the dark at room temperature for 10 min.

7) And (4) terminating: 2N H was added ₂ SO ₄ 100 μ l/well.

8) Detecting OD450 with enzyme-labeling instrument within 10min

9) Results

As a result, as shown in FIG. 3, the monoclonal antibody 8D7 specifically binds to the antigen N protein and exhibits a concentration dependence, EC50 being 0.01505. mu.g/ml.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that it would be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit of the invention, and these modifications and variations also fall within the scope of the claims of the present invention.

Sequence listing

<110> eastern Biotechnology Ltd

<120> antibody against coronavirus N protein and application thereof

<141> 2022-03-29

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Claims

1. An anti-N protein monoclonal antibody, comprising:

the amino acid sequence of SEQ ID NO: 2, HCDR1 of SEQ ID NO:3 and HCDR2 of SEQ ID NO:4, HCDR3 of SEQ ID NO:10, LCDR1 of SEQ ID NO:11 and LCDR2 of SEQ ID NO: LCDR3 of 12.

2. The monoclonal antibody of claim 1, further comprising a heavy chain variable region having an amino acid sequence substantially identical to SEQ ID NO: 5. 6, 7, 8, HFR2, HFR3, HFR4, which are heavy chain variable region framework regions HFR1, HFR2, HFR3, which have at least 90% sequence identity; and has a sequence identical to SEQ ID NO: 13. 14, 15, 16, LFR1, LFR2, LFR3, and LFR4, which have at least 90% sequence identity to the amino acid sequence set forth in seq id no.

3. The monoclonal antibody of claim 1 or 2, wherein the heavy chain variable region of the monoclonal antibody has an amino acid sequence identical to SEQ ID NO: 9, preferably a VH with at least 90% sequence identity, preferably 95% sequence identity, to the amino acid sequence set forth in SEQ ID NO:17, and preferably at least 90% sequence identity, and preferably at least 95% sequence identity, to the VL.

4. The monoclonal antibody of claim 1 or 2, wherein the monoclonal antibody further comprises a heavy chain constant region and a light chain constant region.

5. A substance according to any one of the following:

1) a nucleic acid molecule encoding the monoclonal antibody or functional fragment thereof according to any one of claims 1-4;

2) a recombinant expression vector comprising the nucleic acid molecule of 1);

3) a host cell comprising the recombinant expression vector of 2).

4) A drug conjugate comprising the monoclonal antibody or functional fragment thereof according to any one of claims 1-4;

5) a product for detecting N protein of coronavirus, said product comprising the monoclonal antibody or functional fragment thereof according to any one of claims 1 to 4, the nucleic acid molecule according to 1) of claim 5, the recombinant expression vector according to 2), the host cell according to 3) or the drug conjugate according to 4);

6) a product for detecting coronavirus infection, said product comprising the monoclonal antibody or functional fragment thereof of any one of claims 1-4, the nucleic acid molecule of 1) of claim 5, the recombinant expression vector of 2), the host cell of 3) or the drug conjugate of 4);

7) a composition comprising the monoclonal antibody or functional fragment thereof according to any one of claims 1-4, the nucleic acid molecule described in 1), the recombinant expression vector described in 2), and the host cell described in 3).

6. The agent according to claim 5, wherein the recombinant expression vector has a signal peptide operably linked to an antibody;

preferably, the recombinant expression vector further comprises a transcriptional regulatory element.

7. The agent according to claim 5, wherein the drug conjugate further comprises a coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, or enzyme.

8. A pharmaceutical composition comprising the monoclonal antibody of any one of claims 1-4 or the substance of any one of claims 5-7; and/or a pharmaceutically acceptable carrier.

9. The method of any one of:

1) a method of making the monoclonal antibody of any one of claims 1-4, the method comprising: culturing the host cell of claim 5, optionally isolating the antibody from the host cell and/or the medium in which the host cell is grown;

preferably, the method further comprises purifying the monoclonal antibody;

preferably, the host cell is selected from mammalian cells;

preferably, the cell is selected from HEK293 cells;

2) a method of detecting N protein in a sample, the method comprising: contacting the monoclonal antibody of claim 1 with a test sample and determining the presence or level of N protein in the test sample.

10. Use according to any one of the following:

1) use of the monoclonal antibody of any one of claims 1 to 4, the substance of any one of claims 5 to 7 for detecting N protein;

2) use of a monoclonal antibody according to any one of claims 1 to 4, an agent according to any one of claims 5 to 7 for the manufacture of a product for the detection of a coronavirus infection;

3) use of the monoclonal antibody of any one of claims 1 to 4, the substance of any one of claims 5 to 7 for the preparation of a product for the diagnosis of a disease associated with a coronavirus infection;

4) use of a monoclonal antibody according to any one of claims 1 to 4, a substance according to any one of claims 5 to 7 or a pharmaceutical composition according to claim 8 for the manufacture of a medicament for the prophylaxis and/or treatment of a disease associated with a coronavirus infection.