CN112574300B - anti-SAR-COV-2 fully human monoclonal antibody and preparation method and application thereof - Google Patents
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Abstract
The invention relates to an anti-SAR-COV-2 antibody or an antigen binding fragment thereof and application thereof, and particularly discloses that an antibody 9O21 kappa has the sequence shown in SEQ ID No: 1-3, and SEQ ID No: 4-6; antibody 9O21 λ has the amino acid sequence of SEQ ID No: 11-13, and SEQ ID No: 14-16, and light chain complementarity determining regions. The antibody of the present invention is a humanized antibody, and has low side effects and high affinity and specificity.
Description
Technical Field
The invention belongs to the field of immunology, and particularly relates to an anti-SAR-COV-2 (COVID-19) fully human monoclonal antibody, and a preparation method and application thereof.
Background
In 2018, 8 of ten worldwide popular drugs are all humanized or humanized monoclonal antibody drugs. The first one is Humira, a fully human monoclonal antibody, which is a monoclonal antibody and is sold in 100 hundred million over 6 years. Since the first monoclonal antibody drug was marketed in 1986, the monoclonal antibody drugs underwent the stages of murine monoclonal antibody drugs (e.g., ontology OKT3), chimeric monoclonal antibody drugs (Rituximab), humanized monoclonal antibody drugs (Herceptin), and fully human monoclonal antibody drugs (Humira). Because human bodies have anti-mouse antibody reaction (HAMA), murine monoclonal antibody drugs and chimeric monoclonal antibody drugs are gradually eliminated, and the monoclonal antibody drugs occupying the market at present are all humanized monoclonal antibody drugs. Compared with the internationally advanced human antibody production technology, Shenzhen and even China have great gap, mainly manifested in the weak innovation ability of the human antibody drug field, few varieties of independent research and development, no report of the market of original humanized monoclonal antibody drug exists at present, and the huge antibody drug market is occupied by foreign drug enterprises. China changes the lagging situation and strives for antibody drug markets with huge consumption potential at home and abroad, and needs to overcome the fully humanized monoclonal antibody technology urgently.
The human monoclonal antibody has high specificity and obvious curative effect on inflammation, cancer, influenza, especially coronavirus, etc. COVID-19 is an acute respiratory infectious disease caused by a SAR-COV-2 coronavirus, and effective medicines and vaccines are still lacked so far. When the new coronavirus invades cells, the specific molecules expressed by the virus are required to be combined with receptors on human cells so as to infect the cells and further expand the cells. The human antibody for neutralizing the virus is a certain specific antibody generated by human B lymphocyte, and can be combined with the antigen on the surface of the virus, so that the virus is prevented from adhering to a target cell receptor, the virus is prevented from invading the cell, and the SAR-COV-2 influenza can be effectively prevented and treated.
Disclosure of Invention
To solve the above problems, the present invention provides an anti-SAR-COV-2 antibody or an antigen-binding fragment thereof, which specifically binds to the S protein of SAR-COV-2.
In one aspect, the invention provides an isolated anti-SAR-COV-2 antibody or antigen-binding fragment thereof; it Has Complementarity Determining Regions (HCDRs) of three heavy chain variable regions and complementarity determining regions (LCDRs) of three light chain variable regions of any one of the following groups:
9O21κ-HCDR1：GFTFSSYS SEQ ID No：1；
9O21κ-HCDR2：ISSSGTFI SEQ ID No：2；
9O21κ-HCDR3：ARERFVGVLDI SEQ ID No：3；
9O21κ-LCDR1：QSISRY SEQ ID No：4；
9O21 kappa-LCDR: 2: AAS SEQ ID No: 5; and
9O21κ-LCDR3：QQSYTNTWT SEQ ID No：6；
or
9O21λ-HCDR1：GFTFSSYS SEQ ID No：1；
9O21λ-HCDR2：ISSSGTFI SEQ ID No：2；
9O21λ-HCDR3：ARERFVGVLDI SEQ ID No：3；
9O21λ-LCDR1：SSDVGGYNY SEQ ID No：11；
9O21 λ -LCDR: 2: DVG SEQ ID No: 12; and
9O21λ-LCDR3：SSYTGSSTLEGVI SEQ ID No：13。
in another aspect of the present invention, there is provided an isolated anti-SAR-COV-2 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region and the light chain variable region of antibody 9O21 are:
9O21 kappa heavy chain variable region:
EVQLLESGGGLVKPGGSLRLSCAASGFTFSSYSMKWVRQAPGKGLEWVSTISSSGTFIKYADSLQGRFTITRDNAKTAVYLQMNSLRVEDTAVYYCARERFVGVLDIWGQGTMVTVSS SEQ ID No：7
9O21 κ light chain variable region:
STIMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKVLIYAASSLQSGVPSRFSGSGSGTDFTLIISNLQPEDFATYYCQQSYTNTWTFGRGTKVEIKR SEQ ID No：8；
alternatively, the heavy chain variable region and the light chain variable region of antibody 9O21 λ are:
9O21 λ heavy chain variable region:
EVQLLESGGGLVKPGGSLRLSCAASGFTFSSYSMKWVRQAPGKGLEWVSTISSSGTFIKYADSLQGRFTITRDNAKTAVYLQMNSLRVEDTAVYYCARERFVGVLDIWGQGTMVTVSS SEQ ID No：7
9O21 λ light chain variable region:
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVGKRPSGVSNRFSGSKSGNTASLIISGLQAEDEADYYCSSYTGSSTLEGVIFGGGTKLTVTV SEQ ID No:15
in the technical scheme of the invention, the antibody or the antigen-binding fragment thereof is a humanized antibody, and more preferably a fully humanized antibody.
In the technical scheme of the invention, the antibody is an IgG1 antibody.
In the technical scheme of the invention, the antibody or the antigen binding fragment thereof specifically binds to SAR-COV-2 surface S protein.
In the technical scheme of the invention, the antibody is a monoclonal antibody or a polyclonal antibody, and preferably a monoclonal antibody.
In the technical scheme of the invention, the antibody or the antigen binding fragment thereof specifically binds to SAR-COV-2 surface S protein.
In a further aspect of the invention there is provided a nucleotide sequence encoding an antibody or antigen-binding fragment thereof as hereinbefore described.
In a further aspect of the invention there is provided a vector comprising the nucleotide sequence as hereinbefore described.
In a further aspect the invention provides a host cell comprising the aforementioned vector or vectors, preferably the host cell is prokaryotic or eukaryotic, more preferably selected from the group consisting of yeast cells, mammalian cells or other cells suitable for the production of antibodies or antigen binding fragments thereof.
In a further aspect of the invention, there is provided a kit comprising an antibody or antigen-binding fragment thereof as described above.
In yet another aspect, the invention provides a detection reagent comprising an antibody or antigen-binding fragment thereof as described above.
In a further aspect, the present invention provides the use of an antibody or antigen-binding fragment thereof as described above as a detection reagent for: enzyme-linked immunosorbent assay (ELISA), immunoblotting (Western Blot), flow cytometry (FACS), Immunohistochemical (IHC) assay, or immuno-PCR.
In the above-mentioned immunological assay, the antibody or antigen-binding fragment thereof may be attached alone or in combination with a conjugate such as horseradish peroxidase (HRP), Alkaline Phosphatase (AP), Biotin (Biotin), Fluorescein Isothiocyanate (FITC), Cy3, Cy5, magnetic beads and agarose, by coupling via a chemical bond, electrostatic adsorption or hydrophilic-hydrophobic adsorption.
In the technical scheme of the invention, the detection reagent can be used for detection for non-diagnosis or treatment purposes.
In a further aspect of the invention, there is provided a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof as described above and a pharmaceutically acceptable excipient.
In an embodiment of the invention, wherein the antibody or antigen-binding fragment thereof blocks or reduces binding of S protein of SAR-COV-2 to a cell surface receptor of the subject, preferably a cellular angiotensin converting enzyme-related carboxypeptidase (ACE 2).
In a further aspect, the invention provides the use of an anti-SAR-COV-2 antibody or antigen-binding fragment thereof in the manufacture of a medicament for the prevention, treatment or amelioration of at least one symptom or indication of SAR-COV-2 infection.
In the technical scheme of the invention, the medicine is an oral or injection preparation.
In a further aspect, the invention provides a method of preventing, treating or ameliorating at least one symptom or indication of SAR-COV-2 infection, the method comprising administering to a subject an antibody or antigen-binding fragment thereof of any of the foregoing or a pharmaceutical composition of the foregoing.
In the technical solutions of the present invention, wherein the at least one symptom or indication is selected from the group consisting of: pulmonary inflammation, alveolar injury, fever, cough, dyspnea, hypoxemia, acute respiratory distress syndrome, septic shock, coagulation dysfunction, metabolic acidosis, nasal congestion, runny nose, sore throat, diarrhea, organ failure, septic shock, and death.
In the technical aspect of the present invention, wherein the pharmaceutical composition or the antibody or antigen-binding fragment thereof is administered in combination with a second therapeutic agent. Wherein the second therapeutic agent is selected from the group consisting of: anti-inflammatory drugs (such as corticosteroids and non-steroidal anti-inflammatory drugs), antiviral drugs, different antibodies against the S protein of SAR-COV-2, vaccines for SAR-COV-2, antibiotics, dietary supplements such as antioxidants and any other palliative treatment of SAR-COV-2 infection, drugs to alleviate the above symptoms or indications.
In the technical aspect of the present invention, wherein the pharmaceutical composition or the antibody or antigen-binding fragment thereof is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially.
Advantageous effects
(1) The anti-SAR-COV-2 antibody disclosed by the invention can be combined with the S protein of SAR-COV-2 virus in a targeted manner, has high specificity, and can effectively block the combination of the S protein on the surface of the SAR-COV-2 virus and a cell surface receptor of a subject.
(2) Compared with the mouse antibody, the gene of the fully human antibody is completely derived from the human gene, has no components of other species, does not generate toxic and side effects such as anti-mouse antibody and the like in a human body, has better biocompatibility, and is more suitable and has more potential to become a macromolecular medicament for treating influenza virus.
(3) Compared with the method for preparing the SAR-COV-2 virus resisting humanized monoclonal antibody by using the phage display technology provided by the prior art, the method for developing the SAR-COV-2 virus resisting antibody by using the single B cell has the advantages of simple and rapid operation, high affinity and specificity of the produced humanized antibody and the like.
Drawings
FIG. 1 is a graph showing the results of ELISA experiments in example 1.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below, but the present invention is not to be construed as being limited to the implementable range thereof.
For a more clear understanding of the technical features, objects and advantages of the present invention, reference is now made to the following detailed description of the embodiments of the present invention taken in conjunction with the accompanying drawings, which are included to illustrate and not to limit the scope of the present invention. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.
As used herein, the term "antibody" refers to a molecule comprising at least one antigen binding site that immunospecifically binds to a particular antigen target of interest. Thus, the term "antibody" includes, but is not limited to, full length antibodies and/or variants thereof, fragments thereof, peptide antibodies and variants thereof, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, human antibodies, humanized antibodies, and antibody mimetics that mimic the structure and/or function of an antibody or designated fragments or portions thereof, including single chain antibodies and fragments thereof. Binding of an antibody to a target can result in a variety of effects, such as, but not limited to, modulation, reduction, increase, antagonism, agonism, alleviation, slowing, blocking, inhibition, elimination and/or interference with at least one target activity or binding, or receptor activity or binding, of such binding in vitro, in situ and/or in vivo. Thus, antibodies of the present disclosure encompass antibody fragments capable of binding a biomolecule (e.g., an antigen or receptor) or a portion thereof, including but not limited to Fab, Fab ', and F (ab ')2, pFc ', Fd, single domain antibodies (sdAb), variable fragments (Fv), single chain variable fragments (scFv), or disulfide-linked fvs (sdfv); a bifunctional or bivalent bifunctional antibody; a linear antibody; a single chain antibody molecule; multispecific antibodies formed from antibody fragments. The antibody may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass.
As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor amounts of mutations that may naturally occur. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to its specificity, monoclonal antibodies also have the advantage that they can be synthesized uncontaminated with other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method.
As used herein, the term HCDR has the same meaning as the heavy chain complementarity determining region and LCDR has the same meaning as the light chain complementarity determining region.
As used herein, monoclonal antibodies include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular anti-class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and fragments of these antibodies, exhibit the desired biological activity.
As used herein, the term "SAR-COV-2," also known as "novel coronavirus," refers to a newly-occurring virus that causes novel coronavirus pneumonia (COVID-19).
As used herein, the S protein refers to the Spike protein (Spike protein) on coronavirus, and SARS-CoV-2 recognizes ACE2 on the cell surface in the human body through the Spike protein on the virus surface and infects host cells. The S protein on the surface of the coronavirus SARS-CoV-2 can be blocked to effectively inhibit the virus from adhering to a target cell receptor and prevent the virus from invading cells.
The term "humanized antibody" as used herein includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The humanized antibodies of the invention may comprise amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or in vitro site-specific mutagenesis or by in vivo somatic mutation).
The term anti-antigen-binding fragment, etc., as used herein includes any naturally occurring, enzymatically available, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The term "antigen-binding fragment" of an antibody as used herein refers to one or more fragments of the S protein of SAR-COV-2 that have the ability to bind.
In one aspect, the present invention provides a fully human monoclonal antibody against SAR-COV-2 or a biologically active fragment derived therefrom that specifically binds to SAR-COV-2, wherein the amino acid sequences of the heavy and light chain CDR1, CDR2 and CDR3 regions of the antibody 9O21 κ are as follows:
9O21κ-HCDR1：GFTFSSYS SEQ ID No：1；
9O21κ-HCDR2：ISSSGTFI SEQ ID No：2；
9O21κ-HCDR3：ARERFVGVLDI SEQ ID No：3；
9O21κ-LCDR1：QSISRY SEQ ID No：4；
9O21 kappa-LCDR: 2: AAS SEQ ID No: 5; and
9O21κ-LCDR3：QQSYTNTWT SEQ ID No：6。
or the amino acid sequences of the heavy and light chain CDR1, CDR2 and CDR3 regions of the antibody 9O21 lambda are respectively shown as follows:
9O21λ-HCDR1：GFTFSSYS SEQ ID No：1；
9O21λ-HCDR2：ISSSGTFI SEQ ID No：2；
9O21λ-HCDR3：ARERFVGVLDI SEQ ID No：3；
9O21λ-LCDR1：SSDVGGYNY SEQ ID No：11；
9O21 λ -LCDR: 2: DVG SEQ ID No: 12; and
9O21λ-LCDR3：SSYTGSSTLEGVI SEQ ID No：13。
in some embodiments, the heavy chain variable region amino acid sequence of the antibody 9O21 kappa is shown as SEQ ID NO. 7, or the amino acid sequence with equivalent functions formed by replacing, deleting or adding one or more amino acids in the sequence;
EVQLLESGGGLVKPGGSLRLSCAASGFTFSSYSMKWVRQAPGKGLEWVSTISSSGTFIKYADSLQGRFTITRDNAKTAVYLQMNSLRVEDTAVYYCARERFVGVLDIWGQGTMVTVSS SEQ ID No：7
and is
The variable region amino acid sequence of the light chain of the antibody is shown as SEQ ID NO. 8, or the amino acid sequence STIMTQSPSSLSASVGDRVTITCRAS with the same function formed by replacing, deleting or adding one or more amino acids in the sequenceQS ISRYLNWYQQKPGKAPKVLIYAASSLQSGVPSRFSGSGSGTDFTLIISNLQPEDFATYYCQQSYTNTWTFGRGTKVEIKR SEQ ID No：8。
In some embodiments, the heavy chain variable region amino acid sequence of the antibody 9O21 λ is shown in SEQ ID No. 7, or an amino acid sequence with equivalent functions formed by substituting, deleting or adding one or more amino acids in the sequence;
EVQLLESGGGLVKPGGSLRLSCAASGFTFSSYSMKWVRQAPGKGLEWVSTISSSGTFIKYADSLQGRFTITRDNAKTAVYLQMNSLRVEDTAVYYCARERFVGVLDIWGQGTMVTVSS SEQ ID No：7
and is
The light chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 15, or the amino acid sequence QSALTQPASVSGSPGQSITISCTGT with the same function formed by replacing, deleting or adding one or more amino acids in the sequenceS SDVGGYNYVSWYQQHPGKAPKLMIYDVGKRPSGVSNRFSGSKSGNTASLIISGLQAEDEADYYCSSYTGSSTLEGV IFGGGTKLTVTV SEQ ID No:15。
ELISA experiments prove that the SAR-COV-2 resistant fully human monoclonal antibody can be combined with S protein of SAR-COV-2 virus in a targeted mode. Compared with a mouse antibody, the gene of the fully human antibody is completely derived from the human gene, has no other species of components, does not generate toxic and side effects such as anti-mouse anti-antibody and the like in a human body, has better biocompatibility, and is more suitable and has more potential to become a macromolecular medicament for treating influenza virus.
In another aspect, the present invention provides a gene encoding an anti-SAR-COV-2 fully human monoclonal antibody of the present invention. In some embodiments, the gene comprises a nucleotide sequence encoding an amino acid having the amino acids set forth above.
In some embodiments, the nucleotide sequence is as follows (the following sequences are exemplary only, and one skilled in the art can design other nucleotide sequences that can be translated into the desired amino acid sequence depending on the particular amino acid sequence):
the nucleotide sequence encoding the heavy chain variable region of antibody 9O21 kappa or 9O21 lambda is:
Gaggtgcagctgttggagtcggggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtagttatagtatgaagtgggtccgccaggctccagggaaggggctggagtgggtctcaaccatcagtagtagtggtactttcataaagtatgcagactcactgcagggccgattcaccatcaccagagacaacgccaagaccgcagtgtatctgcaaatgaacagcctgagagtcgaggacacggctgtttattactgtgcgagaga acgattcgttggtgttttggatatctggggccaagggacaatggtcaccgtctcttca SEQ ID No：9
the nucleotide sequence encoding the variable region of the light chain of antibody 9O21 κ is:
TcgacgattatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcaggtatttaaattggtatcagcaaaaaccagggaaagcccctaaggtcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcatcatcagcaatctgcaacctgaagattttgcaacttactactgtcaacagagttacactaacacgtggacgttcggccgagggaccaaggtggaaatcaagCGA SEQ ID No：10
the nucleotide sequence encoding the variable region of the light chain of antibody 9O21 λ is:
cagtctgCCctgactcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatctcctgcactggaaccagcagtgacgttggtggttataactatgtctcctggtaccaacaacacccaggcaaagcccccaaactcatgatttatgatgtcggtaagcggccctcaggggtttctaatcgcttctctggctccaagtctggcaacacggcctccctgatcatctctgggctccaggctgaggacgaggctgattattactgcagctcatatacaggcagcagcactct cgagggggtaatattcggcggagggaccaagctgaccgtcacagTA SEQ ID No：14
in the heavy chain variable region and light chain variable region sequences of the antibody of the present invention, CDR regions are shown in the underlined regions.
In another aspect, the present invention provides a vector comprising the gene as described above.
In a further aspect, the invention provides a cell comprising a gene as described above or a vector as described above.
In still another aspect, the present invention provides a method for producing the SAR-COV-2-resistant fully human monoclonal antibody or a bioactive fragment derived from the monoclonal antibody and capable of specifically binding to SAR-COV-2, comprising culturing genetically engineered cells containing the above genes or the above vectors encoding the heavy and light chains of the SAR-COV-2-resistant fully human monoclonal antibody or directly culturing the above cells, collecting and purifying to obtain the SAR-COV-2-resistant fully human monoclonal antibody.
In the prior art, a method for preparing an anti-SAR-COV-2 virus humanized monoclonal antibody by adopting a phage display technology exists, although the method has the advantages of low production cost and no complicated work such as immunization, cell fusion and the like, the method has obvious defects, and the antibody obtained from a non-immune antibody library is often insufficient in affinity, limited by the conversion rate of foreign genes, insufficient in library capacity of the antibody library to cover the antibody diversity of animals and the like. The present invention separates B cell secreting functional antibody from patient's blood, extracts RNA and synthesizes cDNA, clones the gene secreting destination antibody, and recombines and expresses fully humanized monoclonal antibody. The technology is simple and quick to operate, the produced humanized antibody has high affinity and specificity, and in addition, the technology of the improved monoclonal antibody with the virus neutralizing or tumor killing function separated from the memory B cells can be further adopted, so that the complicated operation and cost are greatly reduced.
In another aspect, the invention provides a pharmaceutical composition comprising the anti-SAR-COV-2 fully human monoclonal antibody of the invention or a biologically active fragment derived from the monoclonal antibody and capable of specifically binding SAR-COV-2.
In another aspect, the invention provides an application of the anti-SAR-COV-2 fully human monoclonal antibody or a bioactive fragment which is derived from the monoclonal antibody and can specifically bind to SAR-COV-2, or the pharmaceutical composition in preparation of a medicine for treating diseases caused by SAR-COV-2 virus.
On the other hand, the invention provides a kit for detecting the level of SAR-COV-2 virus, which contains the anti-SAR-COV-2 fully human monoclonal antibody or a bioactive fragment which is derived from the monoclonal antibody and can specifically bind to SAR-COV-2; in some embodiments, the kit further comprises a second antibody and an enzyme for detection or a fluorescent or radioactive label, and a buffer; the second antibody is, for example, an anti-antibody against the monoclonal antibody of the present invention.
For a more clear understanding of the technical features, objects and advantages of the present invention, reference is now made to the following detailed description of the embodiments of the present invention taken in conjunction with the accompanying drawings, which are included to illustrate and not to limit the scope of the present invention. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.
Example 1
(1) Construction of NTH-3T3 cell line stably expressing CD40L (3T3-CD40L)
Lentivirus was used to establish 3T3-CD40L feeder cells. Constructing a lentivirus expression vector pLVX-CD40L, transfecting 293T cells, and collecting virus supernatant on the fourth day of transfection. NIH-3T3 cells were activated, cultured for 3 passages, infected with lentivirus, cultured further and passaged 3 times. Sorting with flow cytometryCells with FITC fluorescence intensity near MFI were re-added to the flask at 37 deg.C with 5% CO23T3 cells expressing CD40L and 3T3 cells transfected with the empty vector pLVX (with ZxGreen) were stained with anti-CD 40L with APC, respectively, and then analyzed by flow cytometry in an incubator. As a result, all 3T3-CD40L feeder cells were found to express CD 40L. When the cells grow to 80% -90%, the cells are collected by digestion at a concentration of 1X 10 per ml7A cell. Placing in an irradiator for 5000rads irradiation, and resuspending the cells in the frozen stock solution at a concentration of 3.5 × 10/ml7The cells are packed in 1mL of freezing tubules and frozen in liquid nitrogen (can be stored for 2 years).
(2) Sorting and activation of memory B cells
Separating and freezing PBMC of recovered patients infected with SAR-COV-2 virus by using lymph separation liquid, wherein each tube is 10-50 multiplied by 106Cells, frozen in liquid nitrogen tank. PBMC flow staining solutions were prepared, and the components thereof are shown in Table 1 below
TABLE 1 PBMC flow staining solution
| Antibodies | Volume (μ L) |
| CD19-PE-Cy7 | 0.5 |
| IgM-PE | 1.0 |
| IgA-APC | 2.5 |
| IgD-FITC | 2.5 |
| PBS-1％(wt/vol)BSA | 43.5 |
Thawing PBMC, adding the above PBMC flow staining solution, sorting on flow cytometer, and sorting out CD19+IgM–IgA–IgD–The purity of the memory B cells is required to be more than 90%, and if the purity of the memory B cells is less than 90%, the sorting process is repeated. A mixed medium for activating B cells was prepared as shown in table 2 below:
TABLE 2
| Components | Volume of |
| Complete IMDM Medium | 336mL |
| IL-2(10,000U mL-1) | 3.5mL |
| IL-21(100μg mL-1) | 175μL |
| 3T3-CD40L obtained in step (1) | 10mL |
Adding memory B cells into mixed culture medium, mixing, diluting in 384-well plate with 1 cell per well and 50 μ l volume, standing at 37 deg.C and 5% CO2And (5) standing and culturing in an incubator. After 13 days, the supernatant was takenPerforming ELISA to obtain the human monoclonal antibody.
(3) Experiment of binding of human monoclonal antibody to surface antigen S protein of SAR-COV-2 virus
The surface antigen S protein is purchased from Cassia procumbens, has immunogenicity, and the anti-S protein antibody can be SAR-COV-2 influenza virus. Performing ELISA experiment on the obtained supernatant human monoclonal antibody, specifically:
(1) coating 100ng/100 mu L of HA protein of SAR-COV-2 virus in a 96-well enzyme label plate, wherein each well is 100 mu L;
(2) standing in a refrigerator at 4 deg.C overnight;
(3) washing with PBST solution for three times, adding 5% skimmed milk powder solution 200 μ L per well, 37%℃Incubating for 1 hour;
(4) three washes with PBST solution, 100 μ l of normal human serum without virus infection (negative control) or supernatant, triplicate each;
(5) incubation for 1 hour at 37 ℃ followed by three washes with PBST solution;
(6) adding 100 mu L of HRP-containing anti-human IgG antibody (abcam) into an enzyme label plate at a dilution ratio of 1: 5000;
(7) incubation for 1 hour at 37 ℃ followed by three washes with PBST solution;
(8) add 100. mu.L of TMB substrate solution (Thermo Scientific) to each well for 5min at 37 ℃;
(9) the stop solution 2M sulfuric acid 100. mu.L is added to each well, and the absorbance is immediately detected in a microplate reader at a wavelength of 450 nm. The result is shown in figure 1, and an ELISA experiment shows that the human monoclonal antibody obtained by the invention can be combined with S protein of SAR-COV-2 virus in a targeted mode.
EXAMPLE 2 cloning of humanized monoclonal antibody Gene
The B cell capable of secreting antibody binding to SAR-COV-2 virus obtained in example 1 was lysed, and the lysate was taken for reverse transcription of RNA to obtain PCR template cDNA of human antibody gene. Primers for cloning antibody genes were designed and synthesized, genes for heavy and light chains of the antibody were cloned using cDNA as a template, and sequenced by Jinzhi corporation. Specifically, the method comprises the following steps:
(1) the lysed B cell fluid was transferred to a 96-well plate (Eppendorf, 030133366).
(2) Reverse transcription system: 150ng random primer (Invitrogen,48190-
III reverse transcriptase (Invitrogen,18080-044), DEPC water was supplied to 14. mu.L/well.
(3) Reverse transcription reaction procedure: 42 ℃ for 10 min; at 25 ℃ for 10 min; 50 ℃ for 60 min; 94 ℃ for 5 min.
(4) The cDNA was stored at-20 ℃.
(5) Design and synthesis of primers:
(6) heavy and light chains of the antibody gene were PCR amplified using the KOD-Plus-Neo (TOYOBO, KOD401) kit, respectively, in a 40. mu.L system: 3.5. mu.L of cDNA, 20nM mixed primer, 4. mu.L of buffer (buffer), 4. mu.L of 2mM dNTPs, 2.4. mu.L of MgSO 24，1μL KOD。
(7) Reaction procedure: 94 ℃ for 2 min; 45 cycles: 10s at 98 ℃; at 58 ℃ for 30 s; 68 ℃ for 28 s.
(8) The amplification products were subjected to agarose gel.
(9) The sequencing result of the PCR product of the heavy chain variable region of the antibody gene is as follows:
the nucleotide sequence of the heavy chain variable region of the antibody 9O21 kappa is shown as a sequence in SEQ ID No. 9:
SEQ ID No:9
Gaggtgcagctgttggagtcggggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtagttatagtatgaagtgggtccgccaggctccagggaaggggctggagtgggtctcaaccatcagtagtagtggtactttcataaagtatgcagactcactgcagggccgattcaccatcaccagagacaacgccaagaccgcagtgtatctgcaaatgaacagcctgagagtcgaggacacggctgtttattactgtgcgagaga acgattcgttggtgttttggatatctggggccaagggacaatggtcaccgtctcttca
the amino acid sequence of the heavy chain variable region of the antibody gene is shown as SEQ ID No. 7
SEQ ID No:7
EVQLLESGGGLVKPGGSLRLSCAASGFTFSSYSMKWVRQAPGKGLEWVSTISSSGTFIKYADSLQGRFTITRDNAKTAVYLQMNSLRVEDTAVYYCARERFVGVLDIWGQGTMVTVSS
The nucleotide sequence of the light chain variable region of the antibody 9O21 kappa is shown as SEQ ID No. 10,
SEQ ID No:10
TcgacgattatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcaggtatttaaattggtatcagcaaaaaccagggaaagcccctaaggtcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcatcatcagcaatctgcaacctgaagattttgcaacttactactgtcaacagagttacactaacacgtggacgttcggccgagggaccaaggtggaaatcaagCGA
the amino acid sequence of the antibody gene light chain variable region is shown as the sequence of SEQ ID No. 8;
SEQ ID No:8
STIMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKVLIYAASSLQSGVPSRFSGSGSGTDFTLIISNLQPEDFATYYCQQSYTNTWTFGRGTKVEIKR
correspondingly, the CDR region sequences are shown as follows:
9O21κ-HCDR1：GFTFSSYS SEQ ID No：1；
9O21κ-HCDR2：ISSSGTFI SEQ ID No：2；
9O21κ-HCDR3：ARERFVGVLDI SEQ ID No：3；
9O21κ-LCDR1：QSISRY SEQ ID No：4；
9O21κ-LCDR:2：AAS SEQ ID No：5；
9O21κ-LCDR3：QQSYTNTWT SEQ ID No：6。
the nucleotide sequence of the heavy chain variable region of the antibody 9O21 lambda is shown as SEQ ID No. 9:
SEQ ID No:9
Gaggtgcagctgttggagtcggggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtagttatagtatgaagtgggtccgccaggctccagggaaggggctggagtgggtctcaaccatcagtagtagtggtactttcataaagtatgcagactcactgcagggccgattcaccatcaccagagacaacgccaagaccgcagtgtatctgcaaatgaacagcctgagagtcgaggacacggctgtttattactgtgcgagaga acgattcgttggtgttttggatatctggggccaagggacaatggtcaccgtctcttca
the amino acid sequence of the heavy chain variable region of the antibody gene is shown as SEQ ID No. 7
SEQ ID No:7
EVQLLESGGGLVKPGGSLRLSCAASGFTFSSYSMKWVRQAPGKGLEWVSTISSSGTFIKYADSLQGRFTITRDNAKTAVYLQMNSLRVEDTAVYYCARERFVGVLDIWGQGTMVTVSS
The nucleotide sequence of the variable region of the light chain of the antibody 9O21 lambda is shown as SEQ ID No. 14,
SEQ ID No:14
cagtctgCCctgactcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatctcctgcactggaaccagcagtgacgttggtggttataactatgtctcctggtaccaacaacacccaggcaaagcccccaaactcatgatttatgatgtcggtaagcggccctcaggggtttctaatcgcttctctggctccaagtctggcaacacggcctccctgatcatctctgggctccaggctgaggacgaggctgattattactgcagctcatatacaggcagcagcactct cgagggggtaatattcggcggagggaccaagctgaccgtcacagTA
the amino acid sequence of the antibody gene light chain variable region is shown as the sequence of SEQ ID No. 15;
SEQ ID No:15
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVGKRPSGVSNRFSGSKSGNTASLIISGLQAEDEADYYCSSYTGSSTLEGVIFGGGTKLTVTV
correspondingly, the CDR region sequences are shown as follows:
9O21λ-HCDR1：GFTFSSYS SEQ ID No：1；
9O21λ-HCDR2：ISSSGTFI SEQ ID No：2；
9O21λ-HCDR3：ARERFVGVLDI SEQ ID No：3；
9O21λ-LCDR1：SSDVGGYNY SEQ ID No：11；
9O21λ-LCDR:2：DVG SEQ ID No：12；
9O21λ-LCDR3：SSYTGSSTLEGVI SEQ ID No：13
the above results show that the supernatant contains antibodies capable of binding to SAR-COV-2 virus.
Finally, the description is as follows: although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention and it is intended to cover any modifications or equivalents as may fall within the scope of the invention.
SEQUENCE LISTING
<110> Shenzhen advanced technology research institute
<120> SAR-COV-2 resistant fully human monoclonal antibody and preparation method and application thereof
<130> CP120010818C
<160> 15
<170> PatentIn version 3.3
<210> 1
<211> 8
<212> PRT
<213> 9O21κ-HCDR1
<400> 1
Gly Phe Thr Phe Ser Ser Tyr Ser
1 5
<210> 2
<211> 8
<212> PRT
<213> 9O21κ-HCDR2
<400> 2
Ile Ser Ser Ser Gly Thr Phe Ile
1 5
<210> 3
<211> 11
<212> PRT
<213> 9O21κ-HCDR3
<400> 3
Ala Arg Glu Arg Phe Val Gly Val Leu Asp Ile
1 5 10
<210> 4
<211> 6
<212> PRT
<213> 9O21κ-LCDR1
<400> 4
Gln Ser Ile Ser Arg Tyr
1 5
<210> 5
<211> 3
<212> DNA
<213> 9O21κ-LCDR:2
<400> 5
aas 3
<210> 6
<211> 9
<212> PRT
<213> 9O21κ-LCDR3
<400> 6
Gln Gln Ser Tyr Thr Asn Thr Trp Thr
1 5
<210> 7
<211> 118
<212> PRT
<213> 9O21 kappa heavy chain variable region
<400> 7
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ser Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Thr Ile Ser Ser Ser Gly Thr Phe Ile Lys Tyr Ala Asp Ser Leu
50 55 60
Gln Gly Arg Phe Thr Ile Thr Arg Asp Asn Ala Lys Thr Ala Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Arg Phe Val Gly Val Leu Asp Ile Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210> 8
<211> 108
<212> PRT
<213> 9O21 kappa light chain variable region
<400> 8
Ser Thr Ile Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Ile Ile Ser Asn Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Asn Thr Trp
85 90 95
Thr Phe Gly Arg Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 9
<211> 354
<212> PRT
<213> nucleotide sequence encoding the variable region of heavy chain of antibody 9O 21K or 9O21
<400> 9
Gly Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Thr Thr Gly Gly
1 5 10 15
Ala Gly Thr Cys Gly Gly Gly Gly Gly Gly Ala Gly Gly Cys Cys Thr
20 25 30
Gly Gly Thr Cys Ala Ala Gly Cys Cys Thr Gly Gly Gly Gly Gly Gly
35 40 45
Thr Cys Cys Cys Thr Gly Ala Gly Ala Cys Thr Cys Thr Cys Cys Thr
50 55 60
Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly Gly Ala Thr Thr
65 70 75 80
Cys Ala Cys Cys Thr Thr Cys Ala Gly Thr Ala Gly Thr Thr Ala Thr
85 90 95
Ala Gly Thr Ala Thr Gly Ala Ala Gly Thr Gly Gly Gly Thr Cys Cys
100 105 110
Gly Cys Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Gly Gly Ala Ala
115 120 125
Gly Gly Gly Gly Cys Thr Gly Gly Ala Gly Thr Gly Gly Gly Thr Cys
130 135 140
Thr Cys Ala Ala Cys Cys Ala Thr Cys Ala Gly Thr Ala Gly Thr Ala
145 150 155 160
Gly Thr Gly Gly Thr Ala Cys Thr Thr Thr Cys Ala Thr Ala Ala Ala
165 170 175
Gly Thr Ala Thr Gly Cys Ala Gly Ala Cys Thr Cys Ala Cys Thr Gly
180 185 190
Cys Ala Gly Gly Gly Cys Cys Gly Ala Thr Thr Cys Ala Cys Cys Ala
195 200 205
Thr Cys Ala Cys Cys Ala Gly Ala Gly Ala Cys Ala Ala Cys Gly Cys
210 215 220
Cys Ala Ala Gly Ala Cys Cys Gly Cys Ala Gly Thr Gly Thr Ala Thr
225 230 235 240
Cys Thr Gly Cys Ala Ala Ala Thr Gly Ala Ala Cys Ala Gly Cys Cys
245 250 255
Thr Gly Ala Gly Ala Gly Thr Cys Gly Ala Gly Gly Ala Cys Ala Cys
260 265 270
Gly Gly Cys Thr Gly Thr Thr Thr Ala Thr Thr Ala Cys Thr Gly Thr
275 280 285
Gly Cys Gly Ala Gly Ala Gly Ala Ala Cys Gly Ala Thr Thr Cys Gly
290 295 300
Thr Thr Gly Gly Thr Gly Thr Thr Thr Thr Gly Gly Ala Thr Ala Thr
305 310 315 320
Cys Thr Gly Gly Gly Gly Cys Cys Ala Ala Gly Gly Gly Ala Cys Ala
325 330 335
Ala Thr Gly Gly Thr Cys Ala Cys Cys Gly Thr Cys Thr Cys Thr Thr
340 345 350
Cys Ala
<210> 10
<211> 324
<212> DNA
<213> nucleotide sequence encoding variable region of antibody 9O21 kappa light chain
<400> 10
tcgacgatta tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaagtca gagcattagc aggtatttaa attggtatca gcaaaaacca 120
gggaaagccc ctaaggtcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180
aggttcagtg gcagtggatc tgggacagat ttcactctca tcatcagcaa tctgcaacct 240
gaagattttg caacttacta ctgtcaacag agttacacta acacgtggac gttcggccga 300
gggaccaagg tggaaatcaa gcga 324
<210> 11
<211> 9
<212> PRT
<213> 9O21λ-LCDR1
<400> 11
Ser Ser Asp Val Gly Gly Tyr Asn Tyr
1 5
<210> 12
<211> 3
<212> PRT
<213> 9O21λ-LCDR:2
<400> 12
Asp Val Gly
1
<210> 13
<211> 13
<212> PRT
<213> 9O21λ-LCDR3
<400> 13
Ser Ser Tyr Thr Gly Ser Ser Thr Leu Glu Gly Val Ile
1 5 10
<210> 14
<211> 342
<212> DNA
<213> nucleotide sequence encoding variable region of light chain of antibody 9O21 lambda
<400> 14
cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60
tcctgcactg gaaccagcag tgacgttggt ggttataact atgtctcctg gtaccaacaa 120
cacccaggca aagcccccaa actcatgatt tatgatgtcg gtaagcggcc ctcaggggtt 180
tctaatcgct tctctggctc caagtctggc aacacggcct ccctgatcat ctctgggctc 240
caggctgagg acgaggctga ttattactgc agctcatata caggcagcag cactctcgag 300
ggggtaatat tcggcggagg gaccaagctg accgtcacag ta 342
<210> 15
<211> 114
<212> PRT
<213> 9O21 lambda light chain variable region amino acid sequence
<400> 15
Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45
Met Ile Tyr Asp Val Gly Lys Arg Pro Ser Gly Val Ser Asn Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Ile Ile Ser Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Gly Ser
85 90 95
Ser Thr Leu Glu Gly Val Ile Phe Gly Gly Gly Thr Lys Leu Thr Val
100 105 110
Thr Val
Claims (12)
1. An isolated anti-SAR-COV-2 antibody or antigen-binding fragment thereof, having three heavy chain complementarity determining regions and three light chain complementarity determining regions of any one of the following groups:
antibody 9O21 κ:
9O21κ-HCDR1：GFTFSSYS SEQ ID No：1；
9O21κ-HCDR2：ISSSGTFI SEQ ID No：2；
9O21κ-HCDR3：ARERFVGVLDI SEQ ID No：3；
9O21κ-LCDR1：QSISRY SEQ ID No：4；
9O21 kappa-LCDR: 2: AAS SEQ ID No: 5; and
9O21κ-LCDR3：QQSYTNTWT SEQ ID No：6；
or antibody 9O21 lambda
9O21λ-HCDR1：GFTFSSYS SEQ ID No：1；
9O21λ-HCDR2：ISSSGTFI SEQ ID No：2；
9O21λ-HCDR3：ARERFVGVLDI SEQ ID No：3；
9O21λ-LCDR1：SSDVGGYNY SEQ ID No：11；
9O21 λ -LCDR: 2: DVG SEQ ID No: 12; and
9O21λ-LCDR3：SSYTGSSTLEGVI SEQ ID No：13。
2. the isolated anti-SAR-COV-2 antibody or antigen-binding fragment thereof of claim 1, wherein the sequences of the heavy chain variable region and the light chain variable region of antibody 9O21 κ, respectively;
9O21 kappa heavy chain variable region:
EVQLLESGGGLVKPGGSLRLSCAASGFTFSSYSMKWVRQAPGKGLEWVSTISSSGTFIKYADSLQGRFTITRDNAKTAVYLQMNSLRVEDTAVYYCARERFVGVLDIWGQGTMVTVSSSEQ ID No：7
9O21 κ light chain variable region:
STIMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKVLIYAASSLQSGVPSRFSGSGSGTDFTLIISNLQPEDFATYYCQQSYTNTWTFGRGTKVEIKR
SEQ ID No：8；
alternatively, the heavy chain variable region and the light chain variable region of antibody 9O21 λ have the sequences respectively;
9O21 λ heavy chain variable region:
EVQLLESGGGLVKPGGSLRLSCAASGFTFSSYSMKWVRQAPGKGLEWVSTISSSGTFIKYADSLQGRFTITRDNAKTAVYLQMNSLRVEDTAVYYCARERFVGVLDIWGQGTMVTVSSSEQ ID No：7
9O21 λ light chain variable region:
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVGKRPSGVSNRFSGSKSGNTASLIISGLQAEDEADYYCSSYTGSSTLEGVIFGGGTKLTVTV SEQ ID No:15。
3. the antibody or antigen-binding fragment thereof according to any one of claim 1 or claim 2, which is a humanized antibody or antigen-binding fragment thereof.
4. A nucleotide sequence characterized in that: encoding the antibody or antigen binding fragment thereof of any one of claims 1-3.
5. A carrier, characterized by: comprising the nucleotide sequence of claim 4.
6. A host cell, characterized in that: comprising the vector of claim 5.
7. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3.
8. A detection reagent comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3.
9. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 in the preparation of a test agent for: enzyme linked immunosorbent assay, immunoblotting, flow cytometry, immunohistochemical assay or immuno-PCR.
10. A pharmaceutical composition of the isolated antibody or antigen-binding fragment thereof of any one of claims 1-3 and a pharmaceutically acceptable excipient.
11. Use of an anti-SAR-COV-2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or a pharmaceutical composition according to claim 10, for the manufacture of a medicament for preventing, treating or ameliorating at least one symptom or indication of SAR-COV-2 infection.
12. The use of claim 11, wherein the at least one symptom or indication is selected from the group consisting of: novel coronavirus pneumonia, pulmonary inflammation, alveolar injury, fever, cough, dyspnea, hypoxemia, acute respiratory distress syndrome, septic shock, coagulation dysfunction, metabolic acidosis, nasal obstruction, watery nasal discharge, angina, diarrhea, organ failure, septic shock and death.
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