CN115197317A - Monoclonal antibody for resisting SARS-CoV-2 virus and its application - Google Patents

Monoclonal antibody for resisting SARS-CoV-2 virus and its application Download PDF

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CN115197317A
CN115197317A CN202210313645.5A CN202210313645A CN115197317A CN 115197317 A CN115197317 A CN 115197317A CN 202210313645 A CN202210313645 A CN 202210313645A CN 115197317 A CN115197317 A CN 115197317A
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antibody
ser
amino acid
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sequence
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李强
孙见宇
武翠
周利
董炤
刁家升
张晓峰
马心鲁
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Anyuan Pharmaceutical Technology Shanghai Co ltd
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    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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    • 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
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • 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
    • C07K2317/567—Framework region [FR]
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • 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

Abstract

The invention relates to the field of therapeutic antibodies and molecular immunology, and particularly provides an antibody for SARS-CoV-2 coronavirus S protein and application thereof in preparing a medicament for treating novel coronavirus pneumonia COVID-19. The antibody can specifically recognize and bind with high affinity to SARS-CoV-2 coronavirus S protein, which ensures that the antibody can block the infection of SARS-CoV-2 to human cells.

Description

Monoclonal antibody for resisting SARS-CoV-2 virus and its application
Technical Field
The present invention relates to the field of therapeutic antibody and molecular immunology, in the concrete, it relates to a recombinant monoclonal antibody of SARS-CoV-2 coronavirus S protein and application of said antibody, in particular, it relates to the application in the treatment, prevention and diagnosis of COVID-19 disease caused by SARS-CoV-2.
Background
The novel coronavirus SARS-CoV-2, a newly emerging human pathogen, causes severe respiratory disease and COVID-19 pneumonia with fever, asthenia, and dry cough as the main manifestations. The novel pathogen was shown to be a novel member of the genus beta coronavirus, based on genomic nucleic acid sequence. SARS-CoV-2 has high similarity with bat coronavirus RaTG13, and the homology of genome sequence reaches 96.2% (Zhou P et al,2020, nature, 579. SARS-CoV-2 is closely related to two bat SARS-like coronaviruses bat-SL-CoVZC45 (homology 88%) and bat-SL-CoVZXC21 (homology 87%), but is relatively distantly related to SARS-CoV (homology 79%) and MERS-CoV (homology 50%) (Lu R et al,2020, lancet, 395.
Like other coronaviruses, the novel coronaviruse SARS-CoV-2 is a positive-sense RNA virus encoding several major proteins, S, M, N and E, an RNA-dependent RNA polymerase RDRP, and dozens of non-structural proteins. Where the S, M, N and E proteins are used for packaging viral structures, RDRP and a dozen non-structural proteins are used for viral genomic RNA replication and synthesis of individual protein mRNAs. SARS-CoV-2 is similar to SARS-CoV virus in amino acid sequence homology, and its S, M, N, E and RDRP protein amino acid number and SARS-CoV homology are 1273 (76%), 222 (91%), 419 (91%), 75 (95%), 932 (96%), respectively. Similar to SARS-CoV virus, SARS-CoV-2 virus is spherical, enveloped and arranged with coronary spikes. The spike S protein of SARS-CoV-2 forms a trimer (Wrapp D et al,2020, science,6483, 1260-1263) shaped like a mushroom, which is embedded in the outer surface membrane of the virus. The S protein is the main antigen component of the virus and is responsible for the binding of the virus to the receptor ACE2 of invaded host cells and the fusion of the virus and cells. Similar to the SARS-CoV virus S protein (Yuan Y et al,2017, nat Commun,8, 15092), the SARS-CoV-2 coronavirus S protein is largely divided into two domains, S1 (1-685) and S2 (686-1122), as well as a short transmembrane region and a cytoplasmic tail. In the S protein trimer of types such as mushrooms, the three S1 domains form the "mushroom cap" and the three S2 domains form the "mushroom stem". Among them, the RBD domain (amino acids 331-527) in S1 is responsible for binding to the invaded host cell Receptor ACE2, and S2 is responsible for fusion with host cells. The S2 domain is usually present in a folded or coiled-compressed conformation in the overall S protein, and when the virus fuses with host cells after S1 shedding, S2 displays an extended conformation for insertion into the host cell membrane (Walls AC et al,2017, proc Natl Acad Sci USA,114, 11157-11162 Harrison SC,2008, nat Struct Mol biol, 15. The binding affinity of the S protein of SARS-CoV-2 virus to human cell receptors is reported to be much higher than that of the S protein of SARS-CoV virus (Wrapp D et al,2020, science,6483, 1260-1263, walls AC et al,2020, cell, 181. Another difference from SARS-CoV virus S protein is that SARS-CoV-2S protein has a Furin cleavage site RRAR (amino acids 682-685), which divides S protein into two parts, S1 and S2, and S1 and S2 are linked together in non-covalent bond form after cleavage. Because of the Furin cleavage site between S1/S2 and the extensive expression of Furin enzyme in eukaryotic tissues and cells, and the fact that Furin sites containing polybase amino acids are degraded by other lysine or arginine targeted enzymes, such as cell surface enzyme TMPRSS2, endosomal cathepsin L enzyme or possibly Trypsin (trpsin) etc. (Hoffmann M et al,2020, cell,181 (2): 271-280.e8 shang J et al,2020, proc Natl Acad Sci usa, 117. Whereas the SARS-CoV virus S1/S2 is linked only by one basic amino acid arginine, the S protein here is cleaved enzymatically by the cell surface enzyme TMPRSS2 and cathepsin L in endosomes to infect host cells (Belouzard S et al,2012, viruses,4, belouzard S et al,2009, proc Natl Acad Sci USA, 106. Therefore, the two differences, i.e., the presence of Furin cleavage site and high affinity to human receptor ACE2, may be responsible for the high infectivity of SARS-CoV-2 coronavirus. Since the S protein is responsible for binding to human host cell receptors and fusion with host cells, the S protein is the primary target for therapeutic neutralizing antibodies to SARS-CoV and SARS-CoV-2 coronavirus.
The neutralizing antibody can prevent virus transmission by blocking virus invasion to host cells, thereby achieving the purpose of treating diseases. Under the situation of severe new crown epidemic situation, the SARS-CoV-2 coronavirus S protein neutralizing antibody with high specificity, good clinical efficacy and lower treatment cost is developed, which provides more drug options for patients.
Disclosure of Invention
The invention provides an antibody capable of specifically recognizing and binding SARS-CoV-2 coronavirus S protein with high affinity, wherein the antibody can block SARS-CoV-2 infection of host cells. The S protein antibodies disclosed herein can be used (alone or in combination with other agents or therapeutic methods) to treat, prevent and/or diagnose diseases caused by SARS-CoV-2, such as COVID-19.
In a first aspect of the invention, there is provided an antibody or antigen-binding fragment thereof capable of specifically binding to the S protein of SARS-CoV-2 coronavirus, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising at least one, two, or three Complementarity Determining Regions (CDRs) selected from the group consisting of:
(i) HCDR1 having the amino acid sequence as set forth in SEQ ID NO: 1.7, 12 or 18, or a sequence having one or several amino acid substitutions, deletions or additions (e.g. 1,2 or 3 substitutions, deletions or additions) compared to any of the above sequences;
(ii) HCDR2 having the sequence as set forth in SEQ ID NO: 2. 8, 13, 19, 33 or 35, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 substitutions, deletions or additions) as compared to any of the above sequences; and
(iii) HCDR3 having the amino acid sequence as set forth in SEQ ID NO: 3. 9, 14, 20, 34 or 36, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 substitutions, deletions or additions) compared to any of the above sequences;
and/or, it comprises a light chain variable region (VL) comprising at least one, two or three Complementarity Determining Regions (CDRs) selected from the group consisting of:
(iv) LCDR1 having the amino acid sequence as set forth in SEQ ID NO: 4. 10, 15 or 21, or a sequence having one or several amino acid substitutions, deletions or additions (e.g. 1,2 or 3 substitutions, deletions or additions) compared to any of the above sequences;
(v) LCDR2 having the amino acid sequence as set forth in SEQ ID NO: 5. 11, 16 or 22, or a sequence having one or several amino acid substitutions, deletions or additions (e.g. 1,2 or 3 substitutions, deletions or additions) compared to any of the above sequences; and
(vi) LCDR3 having the amino acid sequence as set forth in SEQ ID NO:6 or 17, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 substitutions, deletions or additions) compared to the above sequence.
In certain preferred embodiments, the substitution recited in any one of (i) - (vi) is a conservative substitution.
In certain preferred embodiments, the HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region, and/or the LCDR1, LCDR2 and LCDR3 contained in the light chain variable region are defined by the Kabat or IMGT numbering system. Table 1 in example 5 exemplarily shows the CDR amino acid sequences of the murine antibody as defined by the Kabat or IMGT numbering system.
In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises 3 VH variable region CDRs and 3 VL variable region CDRs selected from the group of 6:
(i) The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NO: 1. 2, 3, 4, 5 or 6, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 substitutions, deletions or additions) as compared to any of the above sequences;
(ii) The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NO: 7. 8, 9, 10, 11 or 6, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 substitutions, deletions or additions) compared to any of the above sequences;
(iii) The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequences shown in SEQ ID NO: 12. 13, 14, 15, 16 or 17, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 substitutions, deletions or additions) as compared to any of the above sequences;
(iv) The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NO: 18. 19, 20, 21, 22 or 17, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 substitutions, deletions or additions) compared to any of the above sequences;
(v) The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequences shown in SEQ ID NO: 12. 33, 34, 15, 16 or 17, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 substitutions, deletions or additions) as compared to any of the above sequences;
(vi) The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NO: 18. 35, 36, 21, 22 or 17, or a sequence having one or several amino acid substitutions, deletions or additions (e.g. 1,2 or 3 substitutions, deletions or additions) compared to any of the above sequences.
In certain embodiments, the antibody or antigen-binding fragment thereof is murine or chimeric, and the heavy chain variable region thereof comprises a heavy chain FR region of murine IgG1, igG2, igG3, or a variant thereof; and the light chain variable region comprises the light chain FR region of a murine kappa, lambda chain or variant thereof. The amino acid sequence numbering of the variable regions of preferred murine antibodies is given in table 2 in example 5.
In certain preferred embodiments, the murine or chimeric antibody or antigen binding fragment thereof comprises VH and VL sequences selected from group 2:
(i) The VH domain comprises the amino acid sequence set forth in SEQ ID NO:23, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or has one or more amino acid substitutions (e.g., conservative substitutions)) to the sequences described above; and the VL domain comprises the amino acid sequence as set forth in SEQ ID NO:24, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or has one or more amino acid substitutions (e.g., conservative substitutions)) to the sequences described above;
(ii) The VH domain comprises the amino acid sequence set forth in SEQ ID NO:25, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or has one or more amino acid substitutions (e.g., conservative substitutions)) to the sequences described above; and the VL domain comprises the amino acid sequence set forth in SEQ ID NO:26, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or has one or more amino acid substitutions (e.g., conservative substitutions)) to the sequences described above.
In certain embodiments, the antibody or antigen-binding fragment thereof is humanized. The basic scheme for the humanization strategy is given in example 5, the CDR amino acid sequences of the preferred humanized antibodies as defined by the Kabat or IMGT numbering system are exemplarily given in table 1, and the variable region amino acid sequence numbering of the preferred humanized antibodies is given in table 2.
In certain preferred embodiments, the humanized antibody or antigen-binding fragment thereof comprises VH and VL sequences selected from the group consisting of:
(i) The VH domain comprises the amino acid sequence set forth in SEQ ID NO:37, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or has one or more amino acid substitutions (e.g., conservative substitutions)) to the sequences described above; and the VL domain comprises the amino acid sequence as set forth in SEQ ID NO:38, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or has one or more amino acid substitutions (e.g., conservative substitutions)) to the sequences described above;
(ii) The VH domain comprises the amino acid sequence set forth in SEQ ID NO:39, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or has one or more amino acid substitutions (e.g., conservative substitutions)) to the sequences described above; and the VL domain comprises the amino acid sequence set forth in SEQ ID NO:40, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or has one or more amino acid substitutions (e.g., conservative substitutions)) to the sequences described above.
In certain embodiments, the antibody comprises a heavy chain constant region and a light chain constant region derived from a human immunoglobulin.
More preferably, the antibody comprises a human kappa appa chain constant region amino acid sequence (amino acid sequence shown in SEQ ID NO: 27).
More preferably, the antibody comprises a heavy chain constant region selected from the group consisting of human IgG1, igG2, igG3, igG4, igM, igA1, igA2, igD, and IgE; more preferably, a heavy chain constant region selected from the group consisting of human IgG1, igG2 and IgG 4; and, the heavy chain constant region has a native sequence or a sequence having substitution, deletion or addition of one or more amino acids compared to the native sequence from which it is derived. For example, in one embodiment, the humanized antibody molecule comprises the heavy chain constant region of wild-type human IgG1 (amino acid sequence shown in SEQ ID NO: 28). In another embodiment, the humanized antibody molecule comprises the heavy chain constant region of human IgG1 (amino acid sequence shown in SEQ ID NO: 29) mutated at M252Y, S254T, T256E, and M428L according to EU numbering. In another embodiment, the humanized antibody molecule comprises the heavy chain constant region of wild-type human IgG2 (amino acid sequence shown in SEQ ID NO: 30). In one embodiment, the humanized antibody molecule comprises human IgG2 modified at the hinge region according to EU numbering (e.g., deletion of ERKCC, amino acid sequence shown in SEQ ID NO: 31), see Chinese patent No. CN104177496B. In another embodiment, the humanized antibody molecule comprises human IgG4 (amino acid sequence shown in SEQ ID NO: 32) mutated at position 228 (e.g., S to P) according to EU numbering.
In certain preferred embodiments, the heavy chain of the antibody has the amino acid sequence as set forth in SEQ ID NO: 41; or a sequence having one or several substitutions, deletions or additions (e.g., 1,2, 3, 4 or 5 substitutions, deletions or additions) as compared to any of the above sequences; or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identity compared to any of the above sequences; and/or the light chain of the antibody has the amino acid sequence shown as SEQ ID NO: 42; or a sequence having one or several substitutions, deletions or additions (e.g., 1,2, 3, 4 or 5 substitutions, deletions or additions) as compared to any of the above sequences; or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identity compared to any of the above sequences.
In certain preferred embodiments, the heavy chain of the antibody has the amino acid sequence as set forth in SEQ ID NO: 43; or a sequence having one or several substitutions, deletions or additions (e.g., 1,2, 3, 4 or 5 substitutions, deletions or additions) compared to any of the above sequences; or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identity compared to any of the above sequences; and/or the light chain of the antibody has the amino acid sequence shown as SEQ ID NO: 44; or a sequence having one or several substitutions, deletions or additions (e.g., 1,2, 3, 4 or 5 substitutions, deletions or additions) compared to any of the above sequences; or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identity compared to any of the above sequences.
In certain preferred embodiments, the substitutions described above are conservative substitutions.
In a second aspect of the invention, there is provided a DNA molecule encoding the above antibody or antigen binding fragment thereof.
In a preferred embodiment of the invention, the DNA molecule encoding the heavy chain of said antibody has the amino acid sequence as shown in SEQ ID NO:45 or 47, and a DNA molecule encoding the light chain of said antibody has the nucleotide sequence set forth in SEQ ID NO:46 or 48.
In a third aspect of the invention, there is provided a vector comprising the DNA molecule described above.
In a fourth aspect of the present invention, there is provided a host cell comprising the above-described vector; the host cell comprises a prokaryotic cell, a yeast or a mammalian cell, such as a CHO cell, NS0 cell or other mammalian cell, preferably a CHO cell.
In a fifth aspect of the invention, there is provided a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described above and a pharmaceutically acceptable excipient, carrier or diluent.
In a sixth aspect of the invention, there is also provided a method of making an antibody or antigen-binding fragment thereof of the invention, comprising: (a) Obtaining the gene of the antibody or the antigen binding fragment thereof, and constructing an expression vector of the antibody or the antigen binding fragment thereof; (b) Transfecting the expression vector into a host cell by a genetic engineering method; (c) Culturing the above host cell under conditions that allow production of the antibody or antigen-binding fragment thereof; (d) Isolating and purifying the antibody or antigen-binding fragment thereof produced.
Wherein, the expression vector in the step (a) is selected from one or more of plasmids, bacteria and viruses, and preferably, the expression vector is pcDNA3.1;
wherein, the constructed vector is transfected into a host cell by a genetic engineering method in the step (b), and the host cell comprises prokaryotic cells, yeast or mammalian cells, such as CHO cells, NS0 cells or other mammalian cells, preferably CHO cells.
Wherein step (d) separates, purifies the antibody or antigen-binding fragment thereof by conventional immunoglobulin purification methods, including protein a affinity chromatography and ion exchange, hydrophobic chromatography, or molecular sieve methods.
In a seventh aspect of the invention, there is provided the use of the antibody or antigen-binding fragment thereof in the manufacture of a medicament for the treatment and prevention of a disease caused by SARS-CoV-2 coronavirus.
The eighth aspect of the present invention provides an immunoassay method for detecting or measuring the presence of SARS-CoV-2 virus or an antigen thereof in a biological sample or quantifying the amount thereof using the above-mentioned antibody; the method comprises incubating a biological sample to be tested with the anti-SARS-CoV-2 virus S protein monoclonal antibody or antigen binding fragment thereof of the invention to form an antigen-antibody complex, and performing qualitative detection and quantitative determination on the formed binding complex, wherein the existence or amount of the complex indicates the existence or content of SARS-CoV-2 virus; specifically, the method comprises the following steps:
(1) Incubating a biological sample to be tested with at least one monoclonal antibody or antigen-binding fragment thereof of the invention under suitable conditions;
(2) Detecting the presence of the bound complex in the step.
The monoclonal antibody or antigen-binding fragment thereof according to the present invention can be used in the above immunoassay method independently of the label used (e.g., enzyme, fluorescence, etc.) and independently of the detection mode (e.g., fluorescence immunoassay, enzyme-linked immunosorbent assay, chemiluminescence assay, etc.) or assay principle (e.g., sandwich method, competition method, etc.); examples of such antigen-binding fragments include, but are not limited to, F (ab') 2 Fab', fab and Fv.
The above immunoassay methods, including enzyme immunoassay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, western blot, immunochromatography, latex agglutination assay, etc.; furthermore, the above-mentioned immunoassay methods can be used for measuring a target antigen in a biological sample by a competitive method or a sandwich method using an antigen or an antibody labeled with a labeling substance.
The competitive method is based on the quantitative competitive binding reaction of SARS-CoV-2 virus in the detected specimen and labeled SARS-CoV-2 virus S protein and the monoclonal antibody or antigen binding fragment thereof of the invention; specifically, the competition method includes: embedding a predetermined amount of the monoclonal antibody of the present invention against SARS-CoV-2 virus S protein or an antigen-binding fragment thereof on a solid phase carrier, then adding a biological sample containing SARS-CoV-2 virus to be detected and a predetermined amount of SARS-CoV-2 virus S protein labeled with a labeling substance, and incubating under appropriate conditions for a sufficient period of time; washing said solid phase extensively after the reaction and detecting the signal value of the label retained on the support or not retained on the support; the measured signal value is then compared to a predetermined amount of a control sample measured in parallel to determine the presence and relative amount of SARS-CoV-2 virus in the sample; preferably, the labeled antigen and the biological sample to be detected are added at approximately the same time.
The sandwich method is based on the fact that the monoclonal antibody of the present invention or an antigen-binding fragment thereof as a capture antibody (or a solid-phase antibody) and a labeled antibody capable of being used in combination both specifically bind to SARS-CoV-2 virus in a biological sample, and the amount of SARS-CoV-2 virus in the sample is measured by quantifying the labeled antibody; specifically, the above sandwich method comprises: binding the specific monoclonal antibody or antigen binding fragment thereof aiming at SARS-CoV-2 virus S protein to a solid phase carrier to form a solid phase antibody (also called capture antibody or first antibody), then respectively adding a biological sample to be detected and a control sample to the coated solid phase carrier and incubating for a sufficient time under proper conditions; after the reaction, fully washing the solid phase, adding a second antibody which is marked by a proper amount of a marker and can be combined with S protein of SARS-CoV-2 virus, and incubating again; after the reaction, washing the solid phase sufficiently and detecting a signal value of the label bound to the second antibody by an appropriate method; the measured signal value is compared to a signal value of a control sample of a predetermined amount measured in parallel to determine the presence and relative amount of SARS-CoV-2 virus in the sample.
The second antibody may also be other polyclonal antibodies; preferably, the second antibody is a monoclonal antibody.
More preferably, the second antibody is selected from any of the monoclonal antibodies or antigen-binding fragments thereof of the present invention that can be used in conjunction with the first antibody.
Wherein the label may be a radioisotope (e.g., a radioisotope) 125 I) Enzymes, enzyme substrates, phosphorescent substances, fluorescent substances, biotin and coloring substances.
Preferably, the markers used in the present invention include, for example, alkaline phosphatase, horseradish peroxidase, β -galactosidase, urease and glucose oxidase; the label may also be a fluorescent substance such as fluorescein derivatives and rhodamine derivatives; alternatively, the label may be a rare earth element or rare earth element complex, such as europium or europium complexes, which allow time-resolved fluorescence measurements; in addition, the label may be a phosphorescent substance, such as acridinium ester and isoluminol; or a radioactive isotope such as 125 I、 3 H、 14 C and 32 p; in addition, the labeling substance may be a coloring substance such as latex particles and colloidal gold. That is, the present invention includes qualitative or quantitative determination of SARS-CoV-2 virus in a biological component by measuring color, fluorescence, time-resolved fluorescence, chemiluminescence, electrochemical fluorescence or radioactivityPresence or amount of (b).
When SARS-CoV-2 immunoassay is carried out by the above-mentioned competition method and sandwich method, it is necessary to wash the solid phase sufficiently to measure the activity of binding to the marker. When the label is a radioisotope, the measurement is performed using a well counter or a liquid scintillation counter. When the label is an enzyme, a substrate is added and the enzyme activity is measured colorimetrically or by fluorescence after color development. When the labeling substance is a fluorescent substance, a phosphorescent substance, or a colored substance, the measurement can be performed by a method known in the art, respectively.
The above-mentioned biological sample is selected from the group consisting of plasma, whole blood, mouthwash, throat swab, urine, stool, and bronchial perfusate.
The solid phase carriers mentioned above include, but are not limited to, nitrocellulose membranes, latex particles, magnetic particles, colloidal gold, beads or other materials such as glass, fiberglass or polymers (e.g., polystyrene or polyvinyl chloride) or fiber optic sensors.
The ninth aspect of the invention provides the use of the above antibody or antigen binding fragment thereof in the preparation of a SARS-CoV-2 virus detection kit.
In a tenth aspect of the present invention, there is provided a SARS-CoV-2 virus detection kit comprising at least one monoclonal antibody or an antigen-binding fragment thereof of the present invention; the monoclonal antibody used for preparing the detection reagent is not particularly limited, and may be any of the monoclonal antibodies of the present invention described above or an antigen-binding fragment thereof (e.g., F (ab') 2 Fab', and scFv) are used alone as one of a solid phase antibody or a labeled antibody; two monoclonal antibodies or antigen-binding fragments thereof against different epitopes of the present invention as described above may be used in combination as a solid phase antibody or a labeled antibody, respectively.
In a preferred embodiment of the present invention, the detection kit comprises:
(1) Selected from any one of:
a. a solid support and a first antibody;
b. a solid support coated with a first antibody;
the first antibody is selected from any one of the monoclonal antibodies or antigen binding fragments thereof;
(2) A second antibody;
the second antibody is optionally labeled appropriately and is selected from the group consisting of a monoclonal antibody or an antigen-binding fragment thereof of the present invention that can be used in combination with the first antibody of (1).
The monoclonal antibody or antigen-binding fragment thereof of the present invention contained in the detection reagent can be pre-immobilized on a solid phase carrier, such as but not limited to nitrocellulose membrane, latex particles, magnetic particles, colloidal gold, beads, or an optical sensor such as glass, fiberglass, or a polymer (e.g., polystyrene or polyvinyl chloride) or fiber; in a preferred embodiment of the present invention, the solid support is a microtiter plate.
The monoclonal antibody or antigen-binding fragment thereof of the present invention contained in the above-mentioned immunoassay reagent may be labeled with a label in advance to form a labeled antibody, the label including, but not limited to, a radioisotope (e.g., radioisotope) 125 I) Enzymes, enzyme substrates, phosphorescent substances, fluorescent substances, biotin and coloring substances; preferably, the enzymes include, for example, alkaline phosphatase, horseradish peroxidase, beta-galactosidase, urease, and glucose oxidase; the fluorescent substance includes, for example, fluorescein derivatives and rhodamine derivatives, and rare earth elements or rare earth element complexes, such as europium or europium complexes; the phosphorescent substances include such as acridine ester and isoluminol; the radioactive isotopes include 125 I、 3 H、 14 C and 32 p; the coloring matter includes, for example, latex particles and colloidal gold; in a preferred embodiment of the present invention, the marker is biotin.
In the eleventh aspect of the present invention, there is provided use of the above-mentioned immunoassay reagent for diagnosing a disease caused by SARS-CoV-2 virus infection. Preferably, the disease is novel coronavirus pneumonia.
The technical scheme disclosed by the invention achieves beneficial technical effects, and is summarized as follows:
1. by mouse hybridoma platform, using S protein asImmunizing mice with immune antigen to obtain a series of murine antibodies of SARS-CoV-2 coronavirus S protein, which can specifically recognize and bind with high affinity to S protein, K D Values reached in nM.
2. The results of in vitro competitive experiments of ACE2 show that the murine antibodies compete with human ACE2 receptor for binding site of S protein, IC 50 Values were as low as nM.
3. The humanized modification of murine antibody reduces its immunogenicity. The humanized antibody retains the affinity of the murine antibody and pseudovirus inhibitory activity, which is equivalent to nM. The characteristics lay a foundation for the clinical application of the antibody.
4. The antibody provided by the invention can also be used for detecting the existence of SARS-CoV-2 virus or corresponding antigen in a sample, and the detection sensitivity of the antibody is lower than 100pg/mL.
Detailed Description
Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols, and reagents described herein. 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 limit the scope of the present invention which 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.
Abbreviations and Definitions
CDR complementary-Determining Region, the Complementarity Determining Region in an immunoglobulin variable Region, is defined using the Kabat, IMGT, chothia or AbM numbering system (see the terms "hypervariable Region" or "CDR Region" or "Complementarity Determining Region").
ELISA enzyme-linked immunosorbent assay
FR antibody Framework region (Framework), immunoglobulin variable region excluding CDR region
HRP horse radish peroxidase
IC 50 Concentration giving 50% inhibition
IgG immunoglobulin G
The alignment and numbering system of immunoglobulin amino acid sequences advocated by Elvin A Kabat by Kabat.
mAb monoclonal antibodies
PCR polymerase chain reaction
V regions are IgG chain segments with variable sequences between different antibodies. It extends to Kabat residue 109 of the light chain and residue 113 of the heavy chain.
K D Equilibrium dissociation constant
k a Constant of binding rate
k d Off rate constant
The term "EC 50 "refers to the concentration of an antibody or antigen-binding fragment thereof that induces a 50% response in an in vitro or in vivo assay using the antibody or antigen-binding fragment thereof, i.e., the concentration at half-way between the maximal response and baseline.
The term "EU Numbering System" (EU Numbering System or Scheme): eu refers to the first human IgG1 immunoglobulin isolated and purified in the last 60's of the last century (1968-1969), and is designated Eu, whose amino acid sequence was determined and assigned by its number (Edelman GM et al,1969, proc Natl Acad USA, 63. The heavy chain constant regions of other immunoglobulins are aligned with the amino acid sequence of Eu, and the corresponding amino acid position is the Eu number. The Eu numbering system is primarily directed to immunoglobulin heavy chain constant regions, including CH1, CH2, CH3 and the hinge region.
The term "Kabat Numbering System" (Kabat Numbering System or Scheme): in 1979, kabat et al first proposed a standardized numbering scheme for human Immunoglobulin variable regions (Kabat EA, wu TT, bilofsky H, sequences of Immunoglobulin Chains: tasks and Analysis of Amino Acid Sequences of syndromes, V-regions, C-regions, J-Chains and β 2 Microglobulins.1979.Department of Health, edition, and Welfare, public Health Service, national Institutes of Health). In the book "immunologically relevant protein sequences" (Kabat EA, wu TT, perry HM, gottesman KS, foeller C.1991.Sequences of Proteins of ImmunologicalInterest,5th edition, bethesda, MD, U.S. patent of Health and Human services, national Institutes for Health), kabat et al aligned and numbered the amino acid sequences of the antibody light and heavy chains. They found that these analyzed sequences exhibited variable lengths, and that the default and inserted amino acids or amino acid fragments were only present at specific positions. Interestingly, the insertion point is mostly located within the CDRs, but may also occur at certain positions in the framework regions. In the Kabat numbering scheme, the light chain variable region is numbered to position 109, the heavy chain variable region is numbered to position 113, and the inserted amino acids of the light and heavy chains are identified and annotated by letters (e.g., 27a, 27b.). All Lambda light chains do not contain the residue at position 10, whereas Lambda and Kappa light chains are encoded by two different genes, located on different chromosomes. Lambda and Kappa light chains may be distinguished by differences in their constant region amino acid sequences. Unlike the EU numbering system which is directed only to the heavy chain constant region, the numbering range of the Kabat numbering system covers the full-length immunoglobulin sequences, including the variable and constant regions of the immunoglobulin light and heavy chains.
The term "epitope" or "antigenic determinant" refers to a particular chemical group or peptide sequence on a molecule that is antigenic (i.e., capable of eliciting a specific immune response), being the site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., the S protein of SARS-CoV-2). Epitopic determinants are typically composed of chemically active surface groups of the molecule (e.g., amino acids or glycosyl side chains) and typically have specific three-dimensional structural properties as well as specific charge properties. There are two epitopes or antigenic determinants (epitopes) of an antigen, a B cell epitope and a T cell epitope, recognized by B cells and T cells, respectively. By epitope we generally refer to B cell epitopes. B cell epitopes are located on the surface of antigenic molecules, and are antigenic sites that bind to B Cell Receptors (BCRs), an antibody located on the B cell membrane, and B cell epitopes are recognized directly by B cells without processing. The B cells then phagocytose antigenic molecules, process them into small peptides (about 15 amino acids in size, antigenic T cell epitopes) and present them on Th cells (helper T cells). Also, antigenic molecules may be produced by another route, e.g. phagocytosis by macrophages, plusProcessed into small peptides, presented to Th cells. Th is co-stimulated by B cells and macrophages, the three cells interact together, and the Th cells send feedback signals to the B cells to indicate the B cells to proliferate, differentiate into plasma cells and memory cells. The plasma cells have the function of secreting antibodies and mediate humoral adaptive immunity. Antibodies bind to antigen molecules through their variable region Fv portion and bind to receptor fcrs on various immune cells through their constant region Fc portion, thereby directing the various immune cells to kill the antigen molecules and perform ADCC (by NK cells), CDC (by complement) and ADCP (by macrophages) functions. Each B cell is specific and secretes only one antibody. B cell epitopes can be classified into continuous epitopes and conformational epitopes (or discontinuous epitopes) according to their continuity in the amino acid sequence of the protein. The size of the B cell epitope is variable and has 5-20 amino acids. T cell epitopes are recognized by T cells and, unlike B cell epitopes, T cell epitopes can be located anywhere in an antigenic molecule (e.g., a viral protein) and thus are within the sequence of the entire protein. T cell epitopes are continuous determinants, typically 10-20 amino acids in size. T-cell epitopes are bound to and presented on the cell surface by class I (MHC I) or class II (MHC II) MHC molecules, respectively, CD8, two different subsets of T-cells + T cells (killer T cells) and CD4 + T cell (helper Th cell) recognition. Thus, the T cell epitope is CD8 + And CD4 + T cell epitopes are two. MHC I molecules are expressed by almost all cells and can provide some conditions in the cells, for example, when the cells are infected by virus, small peptide molecules of virus fragments are suggested on the cell surface through MHC I and can be used for killing CD8 + T cells and the like for killing. MHC II molecules are mostly located on antigen presenting cells, such as macrophages and the like. Such MHC II molecules provide extra-cellular (e.g. humoral) conditions, such as bacterial invasion of tissues, and subsequent phagocytosis by macrophages, bacterial debris is presented to helper Th cells using MHC II, initiating an immune response. B cells and T cells can only identify and combine the antigen epitope of foreign antigen molecules and can only identify and combine the antigen fragments derived from organisms, such as protein molecules and fragments thereofAnd has no binding ability because B cells and T cells, which have high affinity for self protein molecules or fragments, are inhibited from developmental maturation or undergoing apoptosis during differentiation, development and maturation of the B cells and T cells.
The term "antibody" generally refers to a protein-binding molecule having a function such as an immunoglobulin. Typical examples of antibodies are immunoglobulins, as well as derivatives or functional fragments thereof, as long as they exhibit the desired binding specificity. Techniques for making antibodies are well known in the art. "antibodies" include natural immunoglobulins of different classes (e.g., igA, igG, igM, igD, and IgE) and subclasses (e.g., igG1, lgG2, igA1, igA2, etc.). "antibody" also includes non-natural immunoglobulins, including, for example, single chain antibodies, chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies), as well as antigen-binding fragments thereof (e.g., fab ', F (ab') 2 Fab, fv and rIgG). See also, e.g., pierce Catalog and Handbook,1994-1995 (Pierce Chemical Co, rockford, ill); kuby J, immunology,3rd Ed, WH Freeman&Co, new York,1997. Antibodies can bind to an antigen, termed "monospecific"; or bind to two different antigens, referred to as "bispecific"; or bind to more than one different antigen, referred to as "multispecific". Antibodies can be monovalent, bivalent, or multivalent, i.e., an antibody can bind to one, two, or more antigen molecules at a time. An antibody binds "monovalent" to a particular protein, i.e., a molecule of antibody binds only to one molecule of protein, but the antibody may also bind to a different protein. When an antibody binds only to each molecule of two different proteins, the antibody is "monovalent" binding to each protein, and the antibody is "bispecific" and "monovalent" binding to each of the two different proteins. An antibody may be "monomeric," i.e., it comprises a single polypeptide chain. An antibody can comprise multiple polypeptide chains ("multimeric") or can comprise two ("dimeric"), three ("trimeric") or four ("tetrameric") polypeptide chains. If the antibody is multimeric, the antibody may be homomultimeric (homomulitmer), i.e.Antibodies comprise more than one molecule of only one polypeptide chain, including homodimers, homotrimers, or homotetramers. Alternatively, the multimeric antibody may be a heteromultimer, i.e., the antibody comprises more than one different polypeptide chain, including a heterodimer, a heterotrimer, or a heterotetramer.
The term "monoclonal antibody (mAb)" refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprised by the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the phrase "monoclonal" means that the antibody is characterized as not being a mixture of discrete antibodies. Monoclonal antibodies are produced by methods known to those skilled in the art, such as by fusing myeloma cells and immune spleen cells to produce hybrid antibody producing cells. Synthesized by hybridoma culture, and is not contaminated by other immunoglobulins. Monoclonal antibodies can also be obtained using, for example, recombinant techniques, phage display techniques, synthetic techniques, or other techniques known in the art.
The term "whole antibody" refers to an antibody consisting of two antibody heavy chains and two antibody light chains. "intact antibody heavy chain" is composed of an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1 (CH 1), an antibody Hinge Region (HR), an antibody heavy chain constant domain 2 (CH 2), and an antibody heavy chain constant domain 3 (CH 3) in the N-terminal to C-terminal direction, abbreviated VH-CH1-HR-CH2-CH3; and, in the case of antibodies of the IgE subclass, optionally also antibody heavy chain constant domain 4 (CH 4). Preferably an "intact antibody heavy chain" is a polypeptide consisting of VH, CH1, HR, CH2 and CH3 in the N-terminal to C-terminal direction. An "intact antibody light chain" is a polypeptide consisting of an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), abbreviated VL-CL, in the N-terminal to C-terminal direction. The antibody light chain constant domain (CL) may be kappa (kappa) or lambda (lambda). Intact antibody chains are linked together by interpoly disulfide bonds between the CL and CH1 domains (i.e., between the light and heavy chains) and interpoly disulfide bonds between the hinge region of the intact antibody heavy chain. Examples of typical whole antibodies are natural antibodies such as IgG (e.g., igG1 and IgG 2), igM, igA, igD, and IgE.
The term "Antibody fragment" or "antigen-binding fragment" refers to antigen-binding fragments and Antibody analogs of antibodies that retain the ability to specifically bind to an antigen (e.g., the S protein of SARS-CoV-2 coronavirus), which typically include at least a portion of the antigen-binding or variable region of a parent Antibody (partial Antibody). Antibody fragments retain at least some of the binding specificity of the parent antibody. Usually, when molar units (K) are used D ) When active, the antibody fragment retains at least 10% of the parent binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% of the binding affinity of the parent antibody to the target. Antibody fragments include, but are not limited to: fab fragment, fab 'fragment, F (ab') 2 Fragments, fv fragments, fd fragments, complementarity Determining Region (CDR) fragments, disulfide bond stability proteins (dsFv), and the like; linear antibodies (Linear antibodies), single chain antibodies (e.g., scFv Single antibodies), monoclonal antibodies (Unibody, technology from Genmab), bivalent Single chain antibodies, single chain phage antibodies, single Domain antibodies (e.g., VH Domain antibodies), domain antibodies (domanis, technology from domanis), nanobodies (technology from Ablynx); multispecific antibodies formed from antibody fragments (e.g., three-chain antibodies, four-chain antibodies, etc.); and engineered antibodies such as Chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (Heteroconjugate antibodies), and the like. These antibody fragments are obtained by conventional techniques known to those skilled in the art and are screened for utility in the same manner as are intact antibodies.
The term "single chain Fv antibody" (or "scFv antibody") refers to an antibody fragment comprising the VH and VL domains of an antibody, a recombinant protein of the heavy chain variable region (VH) and the light chain variable region (VL) linked by a linker (linker) that allows the two domains to be cross-linked to form an antigen binding site, the linker sequence typically consisting of a flexible peptide, such as, but not limited to, G2 (GGGGS) 3 . The size of scFv is typically 1/6 of that of a whole antibody. Single chain antibodies are preferably a sequence of amino acids encoded by a single nucleotide chain. For a review of scFv see Pluckthun A,1994.Antibodies from Escherichia coli, in The PharmacThe biology of Monoclonal Antibodies, vol 113, rosenberg M and Moore GP (EDs.), springer-Verlag, new York, pp 269-315. See also International patent application publication No. WO 88/01649 and U.S. Pat. Nos. 4946778 and 5260203.
The term "VL domain" refers to the amino-terminal variable region domain of an immunoglobulin light chain.
The term "VH domain" refers to the amino-terminal variable region domain of an immunoglobulin heavy chain.
The term "functional domain" refers to a three-dimensional structure capable of specifically recognizing and/or binding to an epitope, such as an antibody or antibody fragment, including a natural intact antibody, a single chain antibody (scFv), an Fd fragment, an Fab fragment, an F (ab') 2 Fragments, single domain antibody fragments, isolated CDR fragments and derivatives thereof. By "single-stranded" is meant herein that the first and second domains are covalently linked and may be represented by a co-linear amino acid sequence encoded by a single nucleic acid molecule.
The term "Fab fragment" consists of the variable region of one heavy chain with the CH1 region and one light chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. The "Fab antibody" is 1/3 the size of the intact antibody, which contains only one antigen binding site.
The term "Fab' fragment" contains a light chain, the VH and CH1 domains of a heavy chain, and the constant region portion between the CH1 and CH2 domains.
The term "F (ab') 2 A fragment "contains the VH and CH1 domains of two light and two heavy chains and a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus, F (ab') 2 The fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.
The term "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant region, and is the smallest fragment that comprises the entire antigen recognition and binding site.
The term "heavy chain constant region" includes amino acid sequences from immunoglobulin heavy chains. A polypeptide comprising a heavy chain constant region comprises at least one of: a CH1 domain, a hinge (e.g., an upper hinge region, a middle hinge region, and/or a lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen binding polypeptide used herein can comprise a polypeptide chain having a CH1 domain; a polypeptide having a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain having a CH1 domain and a CH3 domain; a polypeptide chain having a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain having a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of the present application comprises a polypeptide chain having a CH3 domain. In addition, an antibody used in the present application may lack at least a portion of a CH2 domain (e.g., all or a portion of a CH2 domain). As described above, but as will be appreciated by those of ordinary skill in the art, the heavy chain constant regions may be modified such that they differ in amino acid sequence from the naturally occurring immunoglobulin molecule.
The term "light chain constant region" includes amino acid sequences from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain and a constant lambda domain.
The term "Fc region" or "Fc fragment" refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the hinge region, a CH2 domain, and a CH3 domain, which mediates binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component of the classical complement system (C1 q). The Fc region includes a native sequence Fc region and a variant Fc region.
Typically, the human IgG heavy chain Fc region is the region from the amino acid residue at position Cys 226 or Pro 230 to the carboxy terminus thereof, although the boundaries may vary. The C-terminal lysine of the Fc region (residue 447, according to the EU numbering system) may or may not be present. Fc may also refer to this region, either independently, or in the case of a protein polypeptide comprising Fc, such as "binding protein comprising an Fc region," also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesin). The native sequence Fc region in the antibodies of the invention is derived from IgG1, igG2 (IgG 2A, igG 2B), igG3, and IgG4, including mammalian (e.g., human). In certain embodiments, the two Fc polypeptide chains have a single amino acid substitution, insertion, and/or deletion of about 10 amino acids per 100 amino acids in the amino acid sequence relative to the mammalian Fc polypeptide amino acid sequence. In some embodiments, the above-described Fc region amino acid differences may be Fc alterations that extend half-life, alterations that increase FcRn binding, alterations that enhance fcgamma receptor (fcyr) binding, and/or alterations that enhance ADCC, ADCP and/or CDC.
In IgG, igA, and IgD antibody isotypes, the Fc region comprises the CH2 and CH3 constant domains of each of the two heavy chains of the antibody; the IgM and IgE Fc regions comprise three heavy chain constant domains (CH 2-4 domains) in each polypeptide chain.
The term "chimeric antibody" refers to a portion of the heavy and/or light chain that is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4816567; morrison SL et al,1984, proc Natl Acad Sci usa, 81. For example, the term "chimeric antibody" can include an antibody (e.g., a human murine chimeric antibody) in which the heavy and light chain variable regions of the antibody are from a first antibody (e.g., a murine antibody) and the heavy and light chain constant regions of the antibody are from a second antibody (e.g., a human antibody).
The term "human" antibody refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains constant regions, the constant regions are also derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
The term "humanized antibody" refers to a non-human antibody that has been genetically engineered to have an amino acid sequence modified to increase homology to the sequence of a human antibody. Most or all of the amino acids outside the CDR domain of a non-human antibody, e.g., a mouse antibody, are replaced with corresponding amino acids from a human immunoglobulin, while most or all of the amino acids within one or more CDR regions are unchanged. Amino acid additions, deletions, insertions, substitutions or modifications are permissible provided they do not abrogate the ability of the antibody to bind to a particular antigen. "humanized" antibodies retain antigen specificity similar to the original antibody. The source of the CDR is not particularly limited and may be derived from any animal. For example, CDR regions derived from a mouse antibody, a rat antibody, a rabbit antibody, or a non-human primate (e.g., cynomolgus monkey) antibody can be utilized. Framework regions human antibody germline sequences can be obtained by searching for IMGT antibody germline database (http:// www.imgt. Org/3D structure-DB/cgi/DomainGapAlign. Cgi), and generally human germline antibody sequences with high homology to the engineered non-human antibody are selected as framework regions for the humanized antibody.
The term "hypervariable region" or "CDR region" or "complementarity determining region" refers to the amino acid residues of an antibody which are responsible for antigen binding and are non-contiguous amino acid sequences. CDR region sequences can be defined by the methods of Kabat, chothia, IMGT (Lefranc et al,2003, dev company at Immunol, 27-77) and AbM (Martin ACR et al,1989, proc Natl Acad Sci USA,86 9268-9272) or any CDR region sequence determination method well known in the art. For example, the hypervariable region comprises the following amino acid residues: amino acid residues from "complementarity determining regions" or "CDRs" defined by sequence alignment (Kabat numbering system), e.g., residues 24-34 (LCDR 1), 50-56 (LCDR 2), and 89-97 (LCDR 3) of the light chain variable domain and residues 31-35 (HCDR 1), 50-65 (HCDR 2), and 95-102 (HCDR 3) of the heavy chain variable domain, see Kabat et al,1991, sequences of Proteins of Immunological interest,5th edition, public Health service, national Institutes of Health, bethesda, md.; and/or residues from the "hypervariable loops" (HVLs) defined by structure (Chothia numbering system), e.g., residues 26-32 (LCDR 1), 50-52 (LCDR 2) and 91-96 (LCDR 3) of the light chain variable domain and residues 26-32 (HCDR 1), 53-55 (HCDR 2) and 96-101 (HCDR 3) of the heavy chain variable domain, see Chothia C and Lesk AM,1987, J Mol biol,196 901-917; chothia C et al,1989, nature, 342. "framework" residues or "FR" residues are variable domain residues other than the hypervariable region residues defined herein. In certain embodiments, the CDRs contained by the antibodies or antigen binding fragments thereof of the present invention are preferably determined by the Kabat, IMGT, or Chothia numbering system. The person skilled in the art can unambiguously assign each numbering system to any variable domain sequence without relying on any experimental data beyond the sequence itself. For example, kabat residue numbering for a given antibody can be determined by aligning the antibody sequences to the regions of homology for each "standard" numbered sequence. The determination of the numbering of any variable region sequence in a sequence listing is well within the routine skill of those in the art based on the sequence numbering scheme provided herein.
The term "isolated antibody molecule" refers to an antibody molecule that has been recognized and separated and/or recovered from a component of its natural environment. Contaminant components of their natural environment are substances that would interfere with diagnostic or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
The term "isolated" as used herein with respect to a nucleic acid (e.g., DNA or RNA) refers to a molecule that is isolated from other DNA or RNA, respectively, that is present as a macromolecule from natural sources. The term "isolated" as used herein also refers to a nucleic acid or polypeptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when produced by chemical synthesis. Furthermore, "isolated nucleic acid" is meant to include nucleic acid fragments that are not naturally occurring fragments and are not found in the natural state. The term "isolated" is also used herein to refer to cells or polypeptides that are isolated from other cellular proteins or tissues. Isolated polypeptides are meant to include both purified and recombinant polypeptides.
The term "cross-reactive" refers to the ability of an antibody described herein to bind to an antigen from a different species. For example, an antibody described herein that binds to the S protein of SARS-CoV-2 coronavirus can also bind to an S protein from other species (e.g., the S protein of SARS-CoV). Cross-reactivity can be measured by detecting specific reactivity with purified antigen in a binding assay (e.g., SPR, ELISA), or binding to or otherwise interacting with the function of a cell that physiologically expresses the antigen. Examples of assays known in the art to determine binding affinity include surface plasmon resonance (e.g., biacore) or similar techniques (e.g., kinexa or Octet).
The terms "immunological binding" and "immunological binding properties" refer to a non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of an immunological binding interaction may be determined by the equilibrium dissociation constant (K) of the interaction D ) Is represented by, wherein K D Smaller values indicate higher affinities. The immunological binding properties of the selected polypeptide may be determined using methods well known in the art. One assay involves measuring the rate of antigen/antibody complex formation and dissociation. "binding Rate constant" (K) a Or K on ) And "dissociation rate constant" (K) d Or K off ) Both can be calculated from the concentration and the actual rate of association and dissociation (see Malmqvist M,1993, nature,361, 186-187). k is a radical of d /k a Is equal to the equilibrium dissociation constant K D (see Davies DR et al,1990, annual Rev biochem, 59. K can be measured by any effective method D 、k a And k d The value is obtained.
The term "host cell" refers to a cell, which may be prokaryotic or eukaryotic, in which a vector can be propagated and its DNA expressed. The term also includes any progeny of the subject host cell. It is understood that not all progeny may be identical to the parent cell, since mutations may occur during replication and such progeny are included. The host cell comprises a prokaryotic cell, a yeast or a mammalian cell, such as a CHO cell, NS0 cell or other mammalian cell.
The term "identity" is used to refer to the match in sequence between two polypeptides or between two nucleic acids. When a position in both of the sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of the total 6 positions match). Typically, the comparison is made when the two sequences are aligned to yield maximum identity. Such an alignment can be conveniently carried out by computer programs such as the Align program (DNAstar, inc.), by using the method of Needleman and Wunsch (Needleman SB and Wunsch CD,1970, J Mol biol,48, 443-453).
The terms "mutated", "mutant" and "mutation" refer to the substitution, deletion or insertion of one or more nucleotides or amino acids, respectively, as compared to the native nucleic acid or polypeptide (i.e., a reference sequence that may be used to define the wild-type).
Antibodies with conservative modifications
The term "conservative modification" is intended to mean that the amino acid modification does not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions refer to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been described in detail in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in a CDR region of an antibody of the invention can be replaced with other amino acid residues from the same side chain family.
Therapeutic uses of antibodies against SARS-CoV-2 coronavirus S protein
The term "prevention" refers to a method performed to prevent or delay the onset of a disease or disorder or condition (e.g., a tumor or infection) in a subject or if its effects are minimized.
The term "treatment" refers to a method performed in order to obtain a beneficial or desired clinical result. Beneficial or desired clinical results include, but are not limited to, decreasing the rate of disease progression, ameliorating or palliating the disease state, and regression or improved prognosis, whether detectable or undetectable. The amount of therapeutic agent effective to alleviate any particular disease symptom may vary depending on factors such as the disease state, age and weight of the patient, and the ability of the drug to elicit a desired response in the subject. Whether a symptom of a disease is alleviated can be assessed by any clinical measure that is typically used by a physician or other skilled healthcare provider to assess the severity or progression of the symptom.
Antibodies of the invention (which include bispecific, polyclonal, monoclonal, humanized antibodies) may be used as therapeutic agents. These agents may be used generally to treat or prevent novel coronary pneumonia COVID-19, increase vaccine efficacy or enhance innate immune response in a subject. Antibody preparations, preferably with high specificity and high affinity for their target antigen S protein, are administered to a subject and are generally effective for their binding to the target. Administration of the antibody can eliminate or inhibit or interfere with the activity of the SARS-CoV-2 coronavirus S protein. In the case of antibody fragments, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based on the variable region sequences of the antibody, which retain the ability to bind to the target protein sequence. Such peptides can be chemically synthesized and/or prepared by recombinant DNA techniques (see, e.g., marasco WA et al,1993, proc Natl Acad Sci USA, 90.
The antibody or fragment thereof of the present invention that specifically binds to SARS-CoV-2 coronavirus S protein can be administered in the form of a pharmaceutical composition. The formulations may contain more than one active compound, preferably those with complementary activities that do not adversely affect each other, as required for the particular indication being treated. Alternatively or additionally, the composition may comprise an agent that enhances its function.
Pharmaceutical composition
The antibodies of the invention, or nucleic acids or polynucleotides encoding the antibodies of the application, may be used to prepare pharmaceutical or sterile compositions, for example, by mixing the antibodies with a pharmaceutically acceptable carrier, excipient or stabilizer. The pharmaceutical composition may comprise one or a combination (e.g. two or more different) of the antibodies of the invention. For example, a pharmaceutical composition of the invention may comprise a combination of antibodies or antibody fragments (or immunoconjugates) with complementary activity that bind to different epitopes on a target antigen. Formulations of the therapeutic and diagnostic agents may be prepared by mixing with pharmaceutically acceptable carriers, excipients or stabilizers, for example, in the form of lyophilized powders, slurries, aqueous solutions or suspensions. The term "pharmaceutically acceptable" means that the molecular entity, molecular fragment, or composition does not produce an adverse, allergic, or other untoward reaction when properly administered to an animal or human. Specific examples of some substances that may serve as pharmaceutically acceptable carriers or components thereof include sugars (e.g., lactose), starch, cellulose and its derivatives, vegetable oils, gelatin, polyols (e.g., propylene glycol), alginic acid, and the like. The antibodies of the invention or nucleic acids or polynucleotides encoding the antibodies of the application may be used alone or may be used in conjunction with one or more other therapeutic agents, such as vaccines.
The term "pharmaceutically acceptable carrier and/or excipient and/or stabilizer" refers to a carrier and/or excipient and/or stabilizer that is pharmacologically and/or physiologically compatible with the subject and active ingredient and that is non-toxic to the cells or mammal to which it is exposed at the dosages and concentrations employed. Including but not limited to: pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, naCl, and the like. Agents that delay absorption include, but are not limited to, monostearate salts and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, for example, thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning commonly understood by those skilled in the art that are capable of stabilizing the desired activity of the active ingredient in a pharmaceutical, and include, but are not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or degradation products thereof (such as milk albumin hydrolysate), and the like.
Diagnostic use of antibodies against SARS-CoV-2 coronavirus S protein
The monoclonal antibody or antigen-binding fragment thereof of the present invention can be used for the detection or quantification of SARS-CoV-2 virus by immunoassay. The immunoassay method itself is well known, and any known immunoassay method can be used. That is, there are a sandwich method, a competition method, an aggregation method, a western blot method and the like if classification is performed in a measurement format, and there are a fluorescence method, an enzymatic method, a radiation method, a biotin method and the like if classification is performed with a label used, and these methods can be used. Diagnosis can also be made by immunohistological staining. When a labeled antibody is used in the immunoassay method, the method of labeling the antibody is known per se, and any known method can be used.
These immunoassays are known per se, and needless to say in this specification, for example, the sandwich method is a method in which an antibody or an antigen-binding fragment of the present invention is immobilized as a first antibody on a solid phase, reacted with a biological sample to be tested, rinsed, reacted with a second antibody, and rinsed, followed by measurement of the second antibody bound to the solid phase. The second antibody can be labeled with an enzyme, a fluorescent substance, a radioactive substance, biotin, or the like, and the second antibody bound to the solid phase can be measured. By measuring a plurality of standards of known concentrations by the above method, preparing a standard curve based on the relationship between the amount of the measured marker and the content of the standard, and comparing the measurement result of the test sample of unknown concentration with the standard curve, the SARS-CoV-2 virus antigen in the test sample can be quantified. The first antibody and the second antibody may also be substituted in the above description. In the agglutination method, the antibody or antigen-binding fragment thereof of the present invention is immobilized on particles such as latex, and reacted with a sample to measure the absorbance. By measuring a plurality of standards of known concentrations by the above method, preparing a standard curve based on the relationship between the amount of the measured marker and the content of the standard, and comparing the measurement result of the test sample of unknown concentration with the standard curve, the SARS-CoV-2 virus antigen in the test sample can be quantified.
The biological sample to be supplied to the immunoassay method is not particularly limited as long as it contains the S protein of SARS-CoV-2 virus, and examples thereof include human-and animal-derived serum, plasma, and whole blood, as well as body fluid extracts such as nasal swab (nasal swab), nasal aspirate, and pharyngeal swab (pharyngeal swab), saliva, respiratory secretions, urine, feces, cells, and tissue homogenate.
By using the monoclonal antibody of the present invention, a reagent for immunoassay of SARS-CoV-2 virus can be produced by using the antibody as at least one of the solid-phase antibody and the labeled antibody. The solid phase to which the monoclonal antibody is bound may be any of various solid phases conventionally used in immunoassays, and examples thereof include: ELISA plate, latex, gelatin particles, magnetic particles, polystyrene, glass and other various solid phase, beads, liquid-transmissible matrix and other insoluble carrier. The labeled antibody can be prepared by labeling an antibody with an enzyme, colloidal metal particles, colored latex particles, a light-emitting substance, a fluorescent substance, a radioactive substance, or the like. By combining these reagents such as the solid-phase antibody and/or the labeled antibody, a reagent used in enzyme-linked immunoassay, radioimmunoassay, fluorescence immunoassay, or the like can be prepared. These assay reagents are reagents for assaying a target antigen in a sample by a sandwich method or a competitive binding assay.
The reagent for immunoassay by the sandwich method may be the following reagents: for example, two kinds of monoclonal antibodies of the present invention are prepared, one of which is the labeled antibody and the other of which is a solid-phase antibody bound to the solid phase. First, a sample containing an antigen to be measured is reacted with the solid-phase antibody, and then a labeled antibody (second antibody) is reacted with the antigen captured to the solid-phase antibody, whereby the presence or activity of the label bound to the insoluble carrier is detected, whereby immunoassay can be performed. Similarly, an immunoassay can be performed by reacting a sample containing an antigen to be measured with a solid-phase antibody, subsequently reacting a labeled antibody (second antibody) with the antigen captured on the solid-phase antibody, and determining the presence or activity of the label bound to the insoluble carrier, that is, quantifying the amount of the antigen to be measured by the amount of the labeled antibody. In the immunoassay reagent of the sandwich method, one monoclonal antibody may be used as the solid phase antibody and the labeled antibody (for example, when the antigen is a polymer), but it is generally preferable to use 2 or more antibodies that can recognize two different epitopes of the antigen to be measured, respectively. Furthermore, any of the solid-phase antibody and the labeled antibody may be selected from 2 or more monoclonal antibodies and used in combination.
The immunoassay reagent used in the competitive binding assay method may be prepared, for example, as a predetermined amount of a virus antigen labeled with an enzyme, a colloidal metal particle, a colored latex particle, a luminescent substance, a fluorescent substance, a radioactive substance, or the like. The reagent can be used to conduct a competitive reaction with a sample containing a certain amount of the monoclonal antibody of the present invention, the labeled viral antigen and the antigen to be measured, and the amount of the antigen in the sample to be measured can be quantified from the amount of the labeled viral antigen bound or unbound to the antibody, thereby conducting immunoassay.
In the present invention, the antibody or antigen can be bound to a solid phase or a label by a physical adsorption method, a chemical binding method, or the like (see "protein nucleic acid enzyme", japanese patent application, no.31, 37-45 (1987)).
The labeled anti-SARS-CoV-2 virus monoclonal antibody can be prepared by binding the anti-SARS-CoV-2 virus monoclonal antibody to a labeling substance. The labeling substance may be an enzyme, a colloidal metal particle, a colored latex particle, a fluorescent latex particle, a luminescent substance, a fluorescent substance, or the like. The Enzyme may be various enzymes used in Enzyme-linked immunoassays (EIA), such as alkaline phosphatase, peroxidase, β -D-galactosidase, and the like; as the colloidal metal particles, for example, colloidal gold particles, colloidal selenium particles, and the like can be used.
The method for binding the marker to the monoclonal antibody against SARS-CoV-2 virus can be a known method for generating a covalent bond or a non-covalent bond. Examples of the method of bonding are: glutaraldehyde method, periodic acid method, maleimide method, dipyridyl disulfide method, method using various crosslinking agents, etc. (for example, "protein nucleic acid enzyme", no.31, 37-45 (1985)). In the binding method using a crosslinking agent, for example, N-succinimidyl-4-maleimidobutyric acid (GMBS), N-succinimidyl-6-maleimidocaproic acid, N-succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid, and the like can be used as the crosslinking agent. In the method of covalent bonding, depending on the use of a functional group present in the antibody, a labeled monoclonal antibody against SARS-CoV-2 virus can be produced by introducing a functional group such as thiol, amino, carboxyl, hydroxyl or the like into the antibody by a conventional method, and then binding the functional group to the label by the above-mentioned binding method. The non-covalent bonding method may be a physical adsorption method.
As the substrate, various chromogenic substrates, fluorescent substrates, luminescent substrates, etc., which correspond to the enzyme of the label and are represented as follows, can be used.
(a) Chromogenic substrate: 2,2' -azino-bis (3-ethylbenzothiazoline-6-sulfonic Acid) (ABTS), 3', 5' -Tetramethylbenzidine (TMB), diaminobenzidine (DAB) in combination with hydrogen peroxide for peroxidase; 5-bromo-4-chloro-3-indolyl phosphate (BCIP), p-nitrophenyl phosphate (p-NPP), sodium 5-bromo-4-chloro-3-indolyl phosphate (BCIP. Na) are used for alkaline phosphatase.
(b) Fluorescent substrate: 4-methylumbelliferyl phosphate (4-MUP) for alkaline phosphatase; 4-methylumbelliferyl-beta-D-galactoside (4 MUG) was used for beta-D-galactosidase.
(c) Luminescent substrate: 3- (2' -spiroadamantane) -4-methoxy-4- (3 "-phosphoryloxy) phenyl-1, 2-dioxetane.2sodium salt (AMPPD) for alkaline phosphatase; 3- (2' -spiroadamantane) -4-methoxy-4- (3 "-beta-D-galactopyranosyl) phenyl-1, 2-dioxetane (AMGPD) for beta-D-galactosidase; luminol, isoluminol obtained in combination with hydrogen peroxide is used in peroxidases.
By assaying various biological samples derived from human or animal using the monoclonal antibody against the S protein of SARS-CoV-2 virus of the present invention, the diagnosis of SARS-CoV-2 virus infection can be carried out.
Drawings
FIG. 1, measurement of binding Capacity of purified murine antibodies ST-10-4 and ST-35-4 to SARS-CoV-2ST
FIG. 2, murine antibodies ST-10-4 and ST-35-4 block binding of the spike S protein to 293T-ACE2 cells.
FIG. 3, the molecular docking model of murine antibody ST-10-4, the ACE2/RBD structure (PDB 6M 0J) and the antibody CR3022/RBD structure (PDB 6W 41).
FIG. 4 shows the overlay of three structures, namely a murine antibody ST-35-4 molecular docking model, an ACE2/RBD structure (PDB 6M 0J) and an antibody CR3022/RBD structure (PDB 6W 41).
FIG. 5, assay of the in vitro inhibitory activity of humanized antibodies hST-10-4 and hST-35-4 against pseudoviruses.
Detailed Description
EXAMPLE 1 preparation of anti-SARS-CoV-2 murine monoclonal antibody
Antigen preparation: the preparation process of SARS-CoV-2S trimer antigen: among them, 16-1213aa (S trimer, labeled ST) was selected based on the full-length amino acid sequence of the S protein of the novel coronavirus disclosed in Uniprot (Uniprot Entry P0DTC 2) and used as an antigen for screening an antibody in this example. In order to obtain a target protein with high expression efficiency, the coding gene of ST is artificially reconstructed and optimized, a eukaryotic expression vector pcDNA3.1-ST of the target gene is constructed according to a conventional molecular biology method, a recombinant expression plasmid with correct sequencing is transfected into a CHO cell, expression and purification are carried out according to a conventional method, and a purified antigen is obtained for immunization.
Animal immunization: the SARS-CoV-2ST protein antigen is fully emulsified by complete Freund's adjuvant, and then a multipoint immunization mode is adopted to immunize male Balb/C mice (Shanghai Si Laike laboratory animal, inc.), wherein each mouse is 50 mu g, and the immunization period is once in three weeks. On day 10 after the 3rd immunization, blood was drawn from the orbit, and the degree of immune response in mice was monitored by testing the serum anti-SARS-CoV-2 ST antibody titer by indirect ELISA and the serum SARS-CoV-2ST neutralizing antibody level by competitive ELISA as described in example 2.1. Mice that produced the highest anti-SARS-CoV-2 ST antibody titers and the highest levels of neutralizing antibodies were then boosted once 3 days prior to fusion. After 3 days, the mice were sacrificed and their spleens were removed and fused with a mouse myeloma Sp2/0 cell line.
Cell fusion and antibody screening: mixing 2X 10 8 Sp2/0 cells and 2X 10 cells 8 Splenocytes were fused in 50% polyethylene glycol (molecular weight 1450) and 5% Dimethylsulfoxide (DMSO) solution. Iscove's medium (containing 10% fetal calf serum, 100 units/mL penicillin, 100. Mu.g/mL streptomycin, 0.1mM hypoxanthine, 0.4. Mu.M aminopterin and 16. Mu.M thymidine) was used to adjust the number of spleen cells to 5X 10 5 0.3 mL/mL, added to wells of a 96-well plate and placed at 37 ℃ C. To 5% CO 2 In the incubator. After 10 days of culture, the antibody in the supernatant and the HRP-labeled hACE2 were detected by high-throughput ELISA-Fc (ACE 2 (18-740) -Fc) (ACRO Biosystems, same source below) competed for the ability to bind to SARS-CoV-2ST, thereby screening positive wells for competition with hACE2-Fc (see example 2.2 for methods). The above-mentioned fusion cells in the wells containing the monoclonal antibody capable of inhibiting the binding of hACE2-Fc to SARS-CoV-2ST were subcloned, and hybridoma cell lines expressing the high affinity murine monoclonal antibody were obtained by screening using the competitive ELISA method. Of these 2 cell lines were later demonstrated to be cell lines expressing the following antibodies: # ST-10-4 and # ST-35-4. Specific antibody-producing clones were cultured in RPMI 1640 medium supplemented with 10% FCS. When the cell density reaches about 5X 10 5 At individual cells/mL, the medium was replaced with serum-free medium. After 2 to 4 days, the cultured medium was centrifuged, and the culture supernatant was collected. The antibody was purified using a protein A column and the monoclonal antibody eluate was dialyzed against 150mM NaCl. The dialyzed solution was filter-sterilized through a 0.2 μm filter to obtain purified murine monoclonal antibodies ST-10-4 and ST-35-4 to be tested.
Example 2 functional characterization of anti-SARS-CoV-2 murine monoclonal antibody
2.1, indirect ELISA method for determining the binding Capacity of murine antibody to SARS-CoV-2ST antigen
The plate was coated with SARS-CoV-2ST (ACRO Biosystems) overnight at room temperature. The coating solution was discarded, blocked with skim milk powder dissolved in PBS buffer for 1h, and the plates were washed with PBST (PBS containing 0.05% Tween 20, pH 7.4). Then 100. Mu.L of purified anti-SARS-CoV-2 murine antibodies ST-10-4 and ST-35-4 were added to each well, incubated at room temperature for 1H, washed with PBST, then 100. Mu.L of HRP-labeled goat anti-mouse IgG polyclonal antibody (Jackson Laboratory) was added to each well as the detection antibody, after washing the plates with PBST, substrate TMB was added for 10min, followed by 0.2M H 2 SO 4 The reaction was terminated and the absorbance value (OD value) was read, and the result is shown in FIG. 1.
2.2 competitive ELISA method for determining the ability of murine antibody to block the binding of SARS-CoV-2ST to ACE2
SARS-CoV-2ST protein was diluted to 0.1. Mu.g/mL with PBS buffer and added to a 96-well plate at 100. Mu.L/well overnight at room temperature. Discard coating solution, add 200. Mu.L PBST/1% skim milk powder per well, chamberIncubate for 1h for blocking. The blocking solution was removed and after washing the plate 3 times with PBST buffer, 100. Mu.L of a mixture of hACE2-Fc labeled with horseradish peroxidase (HRP) and antibodies ST-10-4, ST-35-4, respectively, was added to each well. PBST was used as a blank control. After sufficient incubation, unbound HRP-labeled hACE2-Fc was washed off with PBS and incubated at room temperature for 1h. After washing the plate with PBST, 100. Mu.L of TMB was added to each well and incubated at room temperature for 5-10min. Finally, 50. Mu.L of 0.2M H was added to each well 2 SO 4 The reaction was stopped and the OD read with a microplate reader at a dual wavelength of 450/620 nm.
Example 3 anti-SARS-CoV-2 ST murine antibody blocks the binding of the spike S protein to 293T-ACE2 cells
On the cellular level, measuring the binding of an antibody to be tested competitive blocking spike S-mouse Fc fusion protein (S-mFc) and 293T-ACE2 cells by a FACS method, detecting the average fluorescence intensity of the S-mFc bound on the cells by using a fluorescence-labeled goat anti-mouse secondary antibody, and calculating the IC of the antibody to be tested blocking the binding of the S protein and the ACE2 on the cell surface 50 And evaluating the blocking effect of the antibody to be detected.
The preparation process of the S-mFc antigen comprises the following steps: based on the full-length amino acid sequence of the novel coronavirus S protein disclosed in Uniprot (Uniprot Entry P0DTC 2), a full-length segment of the S protein was selected, and a murine IgG2a Fc fragment (Uniprot Entry P01863 (107-330 aa)) was ligated to the C-terminus of the S protein, to construct an antigen S-mFc fusion protein used as an evaluation neutralizing antibody in this example. In order to obtain the target protein with high-efficiency expression, the coding gene of the S-mFc is artificially modified and optimized, the eukaryotic expression vector pcDNA3.1-S-mFc of the target gene is constructed according to a conventional molecular biology method, the recombinant expression plasmid with correct sequencing is transfected into CHO cells, and the expression and purification are carried out according to a conventional method.
The blocking experiment of the murine antibody in vitro comprises the following specific steps. 293T-ACE2 cells (Shanghai assist saint Biotech Co., ltd.) were trypsinized at a cell density of 1X 10 6 Adding 100 mu L/well of the cells/mL into a 96-well U-shaped bottom plate, and incubating for 30 minutes at 4 ℃; diluting S-mFc to a concentration by 1% PBSB; diluting a murine antibody sample ST-10-4 to be detected to 8 mu g/mL, diluting the murine antibody sample ST-10-4 by a 4-fold ratio, carrying out 9 gradients, and then mixing the diluted S-mFc and antibodies 1 with different concentration gradients: 1 mixing, pre-mixing at room temperatureIncubating for 30 minutes, adding into the 96-hole U-shaped bottom plate, and incubating for 1 hour at 4 ℃; centrifuging to remove supernatant, washing cells 3 times with 1% PBSB; AF 647-goat anti-mouse IgG Fc (Jackson Immuno) was purified by 1% PBSB at 1:400 dilution, 100 u L/hole, 4 degrees C were incubated for 1 hours; centrifuging to remove supernatant, washing cells 3 times with 1% PBSB; resuspend cells with 1% PBSB, 150. Mu.L/well; the flow cytometer detects the signal intensity. Then taking the average fluorescence intensity as the Y axis and the antibody concentration as the X axis, analyzing by software GraphPad Prism6, and calculating the IC of the anti-SARS-CoV-2 ST murine antibody for blocking the combination of the S-mFc protein and 293T-ACE2 cells 50 The value is obtained.
As shown in FIG. 2, in cellular level, anti-SARS-CoV-2 ST murine antibodies ST-10-4 and ST-35-4 can compete well for blocking the binding of S protein and ACE2, IC 50 16.55ng/mL and 11.79ng/mL, respectively.
Example 4 evaluation of the Effect of murine antibodies to ST protein on the binding Capacity between RBD/ACE2 Using computer molecular docking technology
The structural modeling of the murine antibodies ST-10-4 and ST-35-4 is carried out by adopting computer software Discovery Studio, the molecular docking space conformation of the murine antibodies and the RBD structural domain of the murine antibodies is simulated, the binding sites of the S protein murine antibodies ST-10-4 and ST-35-4 on the RBD structural domain are predicted, and the influence of the antibodies on the binding capacity between RBD/ACE2 is evaluated.
A Discovery Studio software is used for constructing three-dimensional structure models of the murine antibodies ST-10-4 and ST-35-4. The modeling takes 3 steps to perform: 1. and searching three-dimensional structure templates which respectively have high similarity of amino acid sequences with the variable regions of the light chain and the heavy chain of the murine antibody. Searching a three-dimensional structural template with high similarity of amino acid sequences with the variable region (combined light chain and heavy chain) of the whole murine antibody so as to determine the relative orientation of the light chain and the heavy chain in the variable region of the murine antibody; 2. constructing a framework region structure model of the murine antibody by using the 3 structure model templates obtained in the step 1 and the light and heavy chain amino acid sequences of the variable region of the murine antibody; 3. and (3) constructing a structural model of six CDR circular regions on the basis of the step 2. The RBD structure model in the molecular docking simulation calculation adopts protein database
Figure BDA0003569258090000211
High resolution RBD structure (PDB ID 6M 0J). By comparing the two RBD structures (PDB ID 6M0J and 6W 41), the side chain conformation of the F486 residue in 6M0J was adjusted to be identical to that in 6W41, i.e., the rotamer1 conformation. This conformation is the highest occupancy, and the side chain of F486 does not exist in this conformation due to RBD in 6M0J binding to ACE 2. The software for molecular docking adopts ZDCK software in the Discovery Studio software package. The parameters used in the molecular docking simulation experiment are all default values. The murine antibody is used as a molecular docking receptor, and the RBD is used as a molecular docking ligand. Acceptor blocked amino acids (Receptor blocked residues) are selected from the variable region amino acids which are located away from the CDR regions in the opposite spatial positions to those of the CDR regions. Receptor binding site amino acids (Receptor binding site residues) the top amino acid of the HCDR3 loop exposed on the protein surface was selected. The results of molecular docking of murine antibodies ST-10-4 and ST-35-4 with RBD are shown in FIGS. 3 and 4. The ACE2 (PDB 6M0J, lan J et al,2020, nature, 581. As can be seen from the figure, there are a large number of overlapping regions of murine anti-ST-10-4 and ST-35-4 with the binding site of ACE2 on the RBD domain, indicating that ST-10-4 and ST-35-4 compete directly with ACE2 for binding to RBD.
The molecular docking results show that the murine antibodies ST-10-4 and ST-35-4 compete with ACE2 to bind to RBD, can block the binding between RBD and ACE2, and provides a reasonable explanation on the molecular level for the antibodies to inhibit SARS-CoV-2 virus infection of host cells.
Example 5 humanization of a murine antibody to the ST protein of SARS-CoV-2 coronavirus
We used CDR grafting (CDR grafting) to humanize murine antibodies. The basic principle of CDR grafting is to transplant the CDR area of the mouse antibody to the template of the human antibody, and to introduce the stable CDR conformation and several or some key residues of the mouse anti-FR area important for antigen-antibody combination into the template of the human antibody (backbmutions), so as to achieve the aim of reducing the immunogenicity of the mouse antibody and maintaining the affinity of the mouse antibody. In addition to the above CDR-grafting procedures, we further calculated the isoelectric Point (PI), hydrophobic aggregation (aggregation), post-translational modification (PTM, such as glycosylation, fragmentation, isomerization site, etc.) and immunogenicity (immunogenicity) of the humanized antibody after CDR-grafting, and mutated the amino acids that cause the problems in this respect, so that the humanized antibody can sufficiently exert its pharmacological effects in clinical use.
The specific procedure for antibody humanization is as follows. The human antibody germline database of the IMGT website (IMGT human antibody germline database, http:// www.imgt.org/3D structure-DB/cgi/DomainGapAlign.cgi) was searched for human antibody templates with high similarity to murine antibodies. CDR region annotation was performed on murine and humanized antibody templates using Discovery Studio, defining CDR regions according to the Kabat or IMGT protocol. Six CDR regions of the humanized antibody template were replaced with six CDR regions of a murine antibody, respectively. Each individual CDR region of the grafted 6 CDR regions may be an amino acid region as defined by Kabat, or an amino acid region as defined by IMGT. After CDR-grafting, back-mutation from the murine antibody to the humanized template FR region was performed. The key murine anti-FR region amino acids that stabilize antibody CDR region conformation and are important for antigen-antibody binding include the 4 classes of amino acid residues: 1) CDR region
Figure BDA0003569258090000222
Amino acids buried beneath the surface of the antibody; 2) CDR region
Figure BDA0003569258090000223
Amino acids that are internally exposed at the surface of the antibody; 3) Interfacial amino acids between antibody light and heavy chain domains; and 4) Vernier zone resins that stabilize the conformation of the CDR regions of the antibody (Foote J and Winter G,1992, J Mol biol, 224. The above 4 types of key murine anti-FR region residues were determined by modeling the murine anti-FR three-dimensional structure. For these 4 types of amino acids of the human-derived template that do not correspond to the sequence of the mouse antibody, amino acids important for maintaining the CDR conformation and antigen-antibody binding are selected by three-dimensional structural analysis, and amino acid transplantation or substitution from the mouse antibody to the human-derived template is performed. Then, the humanized antibody generated after the 4-class amino acid transplantation is subjected to further calculation of isoelectric point and sparseWater aggregation, post-translational modification and immunogenicity, the problem amino acids are mutated to give the final humanized antibody sequence.
According to the above method, humanized antibodies were constructed based on the CDRs of the murine antibodies ST-10-4 and ST-35-4, and the variable regions of the murine and humanized antibodies contained CDR amino acid sequences as shown in Table 1 and the heavy and light chain variable region amino acid sequences as shown in Table 2.
TABLE 1 CDR regions in murine and humanized antibody variable regions
Figure BDA0003569258090000221
Figure BDA0003569258090000231
TABLE 2 humanized antibodies corresponding to murine antibodies and their variable region amino acid sequences
Antibody numbering VH amino acid sequence VL amino acid sequence
ST-10-4 SEQ ID NO:23 SEQ ID NO:24
ST-35-4 SEQ ID NO:25 SEQ ID NO:26
hST-10-4 SEQ ID NO:37 SEQ ID NO:38
hST-35-4 SEQ ID NO:39 SEQ ID NO:40
To obtain a full-length antibody sequence consisting of two heavy chains and two light chains, the VH and VL sequences shown in Table 2 are spliced or assembled using conventional techniques with antibody heavy chain constant region (preferably from human IgG1, igG2 or IgG 4) and light chain constant region (preferably from human kappa light chain, with the amino acid sequences shown in SEQ ID NO: 27) sequences. For example, in one embodiment, the humanized antibody molecule comprises the heavy chain constant region of wild-type human IgG1 (amino acid sequence shown in SEQ ID NO: 28). In another embodiment, the humanized antibody molecule comprises the heavy chain constant region of human IgG1 mutated at M252Y, S254T, T256E, and M428L according to EU numbering (amino acid sequence shown in SEQ ID NO: 29). In another embodiment, the humanized antibody molecule comprises the heavy chain constant region of wild-type human IgG2 (amino acid sequence shown in SEQ ID NO: 30). Or with a modified human IgG2 constant region sequence, in one embodiment, the humanized antibody molecule comprises human IgG2 modified at the hinge region according to EU numbering (e.g., deletion of ERKCC, amino acid sequence shown in SEQ ID NO: 31). Or a modified human IgG4 constant region sequence, in one embodiment, the humanized antibody molecule comprises human IgG4 (amino acid sequence shown in SEQ ID NO: 32) mutated at position 228 according to EU numbering (e.g., S to P).
Example 6 construction of humanized antibody expression vector and protein expression
The cDNA coding for the heavy chain and light chain of the humanized antibody obtained in the above method is inserted into PcDNA3.1 or its derived plasmid, or other eukaryotic expression vector to construct a humanized antibody expression vector. Preferably, the vector plasmid used should contain the cytomegalovirus early gene promoter-enhancer required for high level expression in mammalian cells. Meanwhile, the vector plasmid contains a selectable marker gene to confer ampicillin resistance in bacteria and G418 resistance in mammalian cells. In addition, the vector plasmid contains the DHFR gene, and in a suitable host cell, the humanized antibody gene and the DHFR gene can be co-amplified with Methotrexate (MTX, sigma) (see, for example, patent CN 103333917B).
The above-constructed recombinant expression vector plasmid is transfected into a mammalian host cell line to express a humanized antibody. For stable high level expression, a preferred host cell line is a dihydrofolate reductase (DHFR) -deficient Chinese Hamster Ovary (CHO) cell (see, e.g., U.S. patent No. 4818679 to Chasin, l. The preferred method of transfection is electroporation, although other methods may be used, including calcium phosphate co-precipitation, lipofection, protoplast fusion, and the like. In electroporation, 2X 107 cells suspended in 0.8mL of PBS were added to a cuvette using a Gene Pulser (Bio-Rad Laboratories) set at 250V electric field and 960. Mu. Fd capacitance, and contained 10. Mu.g of expression vector plasmid DNA linearized with PvuI (Takara). 2 days after transfection, the cells were transfected with 0.2mg/mL G418 and 200nM methotrexate (methotrexate or MTX). To achieve higher levels of expression, the transfected humanized antibody gene was co-amplified with the DHFR gene inhibited by the MTX drug. The secretion rate of each cell line was measured by limiting dilution of the subcloned transfectants and ELISA, and cell lines expressing the humanized antibody at a high level were selected. Conditioned media of humanized antibodies were collected for determination of their biological activity in vitro and in vivo.
For example, the nucleotide sequences encoding the heavy and light chains of humanized antibodies hST-10-4 and hST-35-4 shown in Table 3 were inserted into the expression vectors constructed above, and cell lines stably and highly expressing the target antibodies were selected by pressure, subcloned, cultured and purified to obtain the respective target antibodies.
TABLE 3 amino acid sequences of humanized antibody heavy and light chains and nucleotide sequences encoding same
Antibody numbering HC amino acid sequence LC amino acid sequence HC nucleotide sequence LC nucleotide sequence
hST-10-4 SEQ ID NO：41 SEQ ID NO：42 SEQ ID NO：45 SEQ ID NO：46
hST-35-4 SEQ ID NO：43 SEQ ID NO：44 SEQ ID NO：47 SEQ ID NO：48
Example 7 functional characterization of humanized antibodies
7.1 measurement of binding Capacity of humanized antibody to SARS-CoV-2S protein by ELISA method
SARS-CoV S protein (ACRO Biosystems) was diluted to 0.1. Mu.g/mL with PBS buffer, added to a 96-well plate in a volume of 100. Mu.L/well, and left at 4 ℃ for 16 to 20 hours. The supernatant was aspirated off, the plate washed 1 time with PBST buffer, and then blocked by adding 200. Mu.L 1% PBSB solution per well and incubating at room temperature for 1h. Remove blocking solution, wash plate 3 times with PBST buffer, add a series ofHumanized antibodies hST-10-4 and hST-35-4 were incubated at a concentration of 100. Mu.L/well for 1.5h at room temperature. The reaction was removed and after 3 washes of the plates with PBST, 50. Mu.L/well 1: a4000-dilution HRP-labeled goat anti-mouse IgG secondary antibody (The Jackson Laboratory) was incubated at room temperature for 1h. After washing the plate with PBST for 3 times, 100. Mu.L of TMB was added to each well, and the plate was incubated at room temperature for 5-10min for color development. Finally, 50. Mu.L of 0.2M H was added to each well 2 SO 4 The reaction was stopped and the OD read with a microplate reader at a dual wavelength of 450/620 nm. The OD450/620nm absorbance was taken as the Y-axis and the antibody concentration as the X-axis, and the half-binding concentration (EC) was calculated by analyzing with the software GraphPad Prism6 and fitting a four-parameter curve 50 ) The value is obtained.
The results show that, at molecular level, the humanized antibodies hST-10-4 and hST-35-4 can both well and specifically bind to SARS-CoV-2S protein and hST-10-4 binding EC 50 Binding EC of hST-35-4 at 2.3ng/mL 50 It was 1.8ng/mL.
7.2 competitive ELISA method for determining the ability of the humanized antibody to block the binding of SARS-CoV-2S protein to ACE2
SARS-CoV-2S protein (His tag) was diluted to 0.1. Mu.g/mL with PBS buffer, added to a 96-well plate at 100. Mu.L/well, and coated overnight at 4 ℃. The coating solution was discarded, 200. Mu.l of 1% PBSB was added to each well, and the wells were incubated at room temperature for 1h for blocking. The blocking solution was removed and the plate washed with PBST buffer. The hACE2-Fc labeled with horseradish peroxidase (HRP) was added at a rate of 1: diluted at a ratio of 700, mixed with a series of concentrations of humanized antibodies hST-10-4 and hST-35-4 at a ratio of 1; the mixture was then added to a 96-well plate at 100. Mu.L per well and incubated at room temperature for 1 hour. After washing the plate 3 times with PBST, 100. Mu.L of TMB was added to each well and developed for 5min at room temperature. 50 μ L of 0.2M H was added 2 SO 4 The reaction was stopped and the OD read with a microplate reader at a dual wavelength of 450/620 nm. The OD450/620nm absorbance was taken as the Y-axis and the antibody concentration as the X-axis, and the half maximal Inhibitory Concentration (IC) was calculated by analyzing with the software GraphPad Prism6, fitting a curve with four parameters, and 50 ) The value is obtained.
The results show that in the molecular level, humanized antibodies hST-10-4 and hST-35-4 can well block the IC of SARS-CoV-2S protein binding to ACE2 and hST-10-4 50 A value of 256.8ngIC of/mL, hST-35-4 50 The value was 194.3ng/mL.
7.3 anti-SARS-CoV-2 humanized antibody blocks the binding of spike S protein to 293T-ACE2 cell
See example 3 for experimental methods for blocking binding of spike S protein to 293T-ACE2 cells. The average fluorescence intensity was used as the Y-axis and the antibody concentration as the X-axis, and the IC of blocking the binding of S-mFc protein to 293T-ACE2 cells by humanized antibodies hST-10-4 and hST-35-4 was calculated by analysis using the software GraphPad Prism6 50 The value is obtained.
The results show that the humanized antibodies hST-10-4 and hST-35-4 can well block the IC of the SARS-CoV-2S protein for binding ACE2 and hST-10-4 on the cellular level 50 IC with value of 12.3ng/mL, hST-35-4 50 The value was 16.3ng/mL.
7.4 anti-SARS-CoV-2 humanized monoclonal antibody in vitro pseudovirus inhibition experiment
Infecting 293T-ACE2 cells after incubating the new crown S protein pseudovirus and the antibody to be detected, detecting the luminescence value RLU of Luciferase by a chemiluminescence method, calculating the pseudovirus inhibition rate of the antibody to be detected according to the reading value of the RLU, and evaluating the neutralization effect of the antibody to be detected. The new corona S protein pseudoviral genome encodes firefly luciferase, and when the viral genome enters the cell for integration, the expression and activity of the firefly luciferase are directly proportional to the number of transduced cells. Pseudoviruses infect cells only once compared to euviruses.
The humanized antibody is used for inhibiting the infection of wild pseudovirus or mutant pseudovirus in the following steps. 293T-ACE2 cells (Shanghai assist saint organisms) were cultured with DMEM +10% FBS medium to logarithmic growth phase, seeded into 384-well plates at 3000 cells/well, and charged at 37 ℃ and 5% CO 2 The culture was carried out overnight in an incubator. The antibody samples hST-10-4 and hST-35-4 to be tested were diluted with DMEM containing 10% FBS, starting at a concentration of 10. Mu.g/mL, 5-fold dilution, 9 gradient; wild-type pseudovirus or mutant pseudovirus (B.1.1.7, B.1.351, B.1.429, B.1.617 or B.1.617.2) (Jiman organism) is taken out at-80 deg.C, re-melted at 4 deg.C, and diluted 125 times to obtain working solution; adding the diluted antibody to be detected and the pseudovirus working solution into the sample solution at a rate of 50 mu L/hole respectivelyUniformly mixing the mixture in a U-shaped bottom plate with 96 holes, and pre-incubating the mixture for 30 minutes at 37 ℃; then 20 μ L/well was added to 384-well white plates plated with cells the day before, positive control: the pseudovirus working solution and 10-vol FBS-containing DMEM medium were each added to a 384-well whiteboard at 10. Mu.L/well. Negative control group: DMEM medium containing 10% FBS was added to a 384-well whiteboard at 20. Mu.L/well, and 4 wells were set. After 24 hours, 30. Mu.L/well of DMEM medium containing 10% FBS was added, and the mixture was further placed in a cell culture chamber and cultured for 24 hours; the supernatant was carefully aspirated off by a pipette, a fresh luciferase developing solution was added at 30. Mu.L/well, incubated at room temperature for 5 minutes, and the 384-well plate was placed in a microplate reader to read the chemiluminescence signal from each well. Inhibition (%) = [1- (sample RLU signal value-negative control RLU signal value)/(positive control RLU signal value-negative control RLU signal value)]X 100. Then, the inhibition ratio is used as an axis Y, the antibody concentration is used as an axis X, the quantitative effect curve of the antibody is obtained by analyzing through software GraphPad Prism6, and the half Inhibition Concentration (IC) is calculated by using a nonlinear regression curve 50 )。
As shown in FIG. 5 and Table 4, the humanized antibody hST-10-4 can well inhibit the infection of the wild type and the new coronavirus mutant strains B.1.1.7, B.1.351, B.1.429, B.1.617 or B.1.617.2, and the humanized antibody hST-35-4 has a good inhibitory effect on the wild type, the wild type B.1.1.7, the wild type B.1.351, the wild type B.1.429 and the wild type B.1.617.
TABLE 4 IC inhibition of wild type pseudoviruses or pseudovirus mutant strains by anti-SARS-CoV-2 humanized antibodies 50 Results
Figure BDA0003569258090000261
All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Anyuan pharmaceutical technology (Shanghai) Co., ltd
<120> monoclonal antibody against SARS-CoV-2 virus and application
<130> 2021
<150> 202110361333.7
<151> 2021-04-02
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Asp Ile Val Leu Thr Gln Ser Pro Thr Ser Leu Pro Val Ser Leu Gly
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<400> 26
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
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20 25 30
Leu Thr Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile
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Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
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Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
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Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
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Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
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Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
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Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
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Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
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Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
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His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
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Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
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<213> amino acid sequence of modified human IgG1 constant region ()
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Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
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Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
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35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 30
<211> 326
<212> PRT
<213> wild-type human IgG2 constant region amino acid sequence ()
<400> 30
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro
100 105 110
Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
115 120 125
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
130 135 140
Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
145 150 155 160
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
165 170 175
Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp
180 185 190
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
195 200 205
Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu
210 215 220
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
225 230 235 240
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
245 250 255
Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
260 265 270
Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
275 280 285
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
290 295 300
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
305 310 315 320
Ser Leu Ser Pro Gly Lys
325
<210> 31
<211> 321
<212> PRT
<213> modified human IgG2 constant region amino acid sequence ()
<400> 31
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Thr Val Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro
100 105 110
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
115 120 125
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
130 135 140
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
145 150 155 160
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val
165 170 175
Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu
180 185 190
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys
195 200 205
Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
210 215 220
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
225 230 235 240
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu
245 250 255
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu
260 265 270
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
275 280 285
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
290 295 300
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
305 310 315 320
Lys
<210> 32
<211> 327
<212> PRT
<213> modified human IgG4 constant region amino acid sequence ()
<400> 32
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
100 105 110
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320
Leu Ser Leu Ser Leu Gly Lys
325
<210> 33
<211> 17
<212> PRT
<213> HCDR2
<400> 33
Ala Val Asn Pro Gly Asn Ala Asp Thr Ser Tyr Ser Gln Lys Phe Lys
1 5 10 15
Gly
<210> 34
<211> 11
<212> PRT
<213> HCDR3
<400> 34
Asp Ala Tyr Pro Tyr Phe Tyr Ala Leu Asp Ser
1 5 10
<210> 35
<211> 8
<212> PRT
<213> HCDR2
<400> 35
Val Asn Pro Gly Asn Ala Asp Thr
1 5
<210> 36
<211> 13
<212> PRT
<213> HCDR3
<400> 36
Ala Arg Asp Ala Tyr Pro Tyr Phe Tyr Ala Leu Asp Ser
1 5 10
<210> 37
<211> 119
<212> PRT
<213> VH amino acid sequence ()
<400> 37
Glu Val Thr Leu Lys Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln
1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Asp Ser Ser
20 25 30
Asn Met Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
35 40 45
Trp Leu Ala His Ile Tyr Trp Gly Asp Asp Asn Arg Tyr Asn Pro Ser
50 55 60
Leu Lys Ser Arg Leu Thr Val Ser Lys Asp Thr Ser Lys Asn Gln Val
65 70 75 80
Val Leu Thr Met Thr Asn Met Glu Pro Val Asp Thr Ala Thr Tyr Tyr
85 90 95
Cys Ala Arg Lys Trp Asn Trp Asp Trp Phe Pro Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Val Thr Val Ser Ser
115
<210> 38
<211> 111
<212> PRT
<213> VL amino acid sequence ()
<400> 38
Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Lys Ser Val Ser Ala Ser
20 25 30
Gly Tyr Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Ser Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Arg Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln His Ser Arg
85 90 95
Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 39
<211> 120
<212> PRT
<213> VH amino acid sequence ()
<400> 39
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Thr Tyr
20 25 30
Thr Ile His Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ala Val Asn Pro Gly Asn Ala Asp Thr Ser Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Ala Tyr Pro Tyr Phe Tyr Ala Leu Asp Ser Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 40
<211> 107
<212> PRT
<213> VL amino acid sequence ()
<400> 40
Asp Ile Gln 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 Gly Gln Glu Ile Ser Ser Tyr
20 25 30
Leu Thr Trp Leu Gln Gln Lys Pro Glu Gly Thr Ile Lys Arg Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 41
<211> 449
<212> PRT
<213> HC amino acid sequence ()
<400> 41
Glu Val Thr Leu Lys Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln
1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Asp Ser Ser
20 25 30
Asn Met Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
35 40 45
Trp Leu Ala His Ile Tyr Trp Gly Asp Asp Asn Arg Tyr Asn Pro Ser
50 55 60
Leu Lys Ser Arg Leu Thr Val Ser Lys Asp Thr Ser Lys Asn Gln Val
65 70 75 80
Val Leu Thr Met Thr Asn Met Glu Pro Val Asp Thr Ala Thr Tyr Tyr
85 90 95
Cys Ala Arg Lys Trp Asn Trp Asp Trp Phe Pro Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr
245 250 255
Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 42
<211> 218
<212> PRT
<213> LC amino acid sequence ()
<400> 42
Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Lys Ser Val Ser Ala Ser
20 25 30
Gly Tyr Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Ser Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Arg Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln His Ser Arg
85 90 95
Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 43
<211> 450
<212> PRT
<213> HC amino acid sequence ()
<400> 43
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Thr Tyr
20 25 30
Thr Ile His Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Ala Val Asn Pro Gly Asn Ala Asp Thr Ser Tyr Ser Gln Lys Phe
50 55 60
Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Ala Tyr Pro Tyr Phe Tyr Ala Leu Asp Ser Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile
245 250 255
Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 44
<211> 214
<212> PRT
<213> LC amino acid sequence ()
<400> 44
Asp Ile Gln 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 Gly Gln Glu Ile Ser Ser Tyr
20 25 30
Leu Thr Trp Leu Gln Gln Lys Pro Glu Gly Thr Ile Lys Arg Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 45
<211> 1347
<212> DNA
<213> hST-10-4 heavy chain nucleotide sequence ()
<400> 45
gaagtgactc taaaagagtc aggacctgct ctggtcaagc ctacccagac cctgacactc 60
acctgcacct tctccggctt ctccctggat tcctccaata tgggcgtgtc ttggatcaga 120
cagcctcctg gcaagggcct ggaatggctg gcccacatct actggggcga cgacaaccgg 180
tacaacccct ccctgaagtc ccggctgacc gtctccaagg atacctctaa gaaccaggtg 240
gtgctgacca tgaccaacat ggaacctgtg gacaccgcaa cctactactg cgcccggaag 300
tggaactggg actggttccc ttactgggga cagggcacca ccgtgaccgt gtcttccgct 360
tctaccaagg gcccatccgt gtttcccctg gcccctagct ctaagtctac ctccggagga 420
acagccgctc tgggatgtct ggtgaaggat tacttccccg agcctgtgac cgtgtcttgg 480
aactccggcg ctctgacctc tggcgtgcac acatttcctg ccgtgctgca gtccagcggc 540
ctgtattccc tgtcttccgt ggtgaccgtg ccaagctctt ccctgggcac ccagacatac 600
atctgcaacg tgaatcacaa gccctctaat acaaaggtgg acaagaaggt ggagcctaag 660
tcctgtgata agacccatac atgcccccct tgtccagctc cagagctgct gggaggacca 720
tccgtgttcc tgtttccacc caagcccaag gacacactgt atatcacacg ggagcctgag 780
gtgacatgcg tggtggtgga cgtgtcccac gaggacccag aggtgaagtt caactggtac 840
gtggatggcg tggaggtgca taatgccaag accaagccta gagaggagca gtataactct 900
acataccgcg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 960
tacaagtgca aggtgagcaa taaggctctg cctgccccaa tcgagaagac aatctctaag 1020
gctaagggcc agccaaggga gccccaggtg tataccctgc ctccaagccg ggacgagctg 1080
acaaagaacc aggtgtctct gacctgtctg gtgaagggct tctacccatc tgatatcgcc 1140
gtggagtggg agtccaatgg ccagcccgag aacaattata agaccacacc ccctgtgctg 1200
gacagcgatg gatccttctt tctgtactcc aagctgaccg tggacaagag caggtggcag 1260
cagggcaacg tgtttagctg ctctgtgctg catgaggctc tgcacaatca ttacacacag 1320
aagtccctga gcctgtctcc tggcaag 1347
<210> 46
<211> 654
<212> DNA
<213> hST-10-4 light chain nucleotide sequence ()
<400> 46
gacatcgtgc tgacacagtc tcctgacagc ctggccgtgt ccctgggcga gagggctacc 60
atcaactgtc gggcctccaa gagcgtgtct gcctctggct actccttcat gcattggtac 120
cagcagaagc ccggacaacc acctaagctg ctgatctctc tggcttccaa cctggaatcc 180
ggcgtgcctg ctagattcag cggcagcgga agcggcaccg acttcaccct gaccatttcc 240
cgcctgcaag ctgaggacgt ggccgtgtac tactgccagc actccagaga gctgcctctg 300
acctttggag ctggcacaaa actggaaatc aagcgcaccg tggctgcccc ttccgtgttc 360
atctttcctc catccgacga gcagctgaag tctggcacag cctccgtggt gtgcctgctg 420
aacaatttct atccaagaga ggctaaggtg cagtggaagg tggataacgc cctgcagtct 480
ggcaattccc aggagagcgt gacagagcag gactctaagg attccaccta cagcctgtcc 540
agcaccctga cactgagcaa ggctgattat gagaagcaca aggtgtacgc ctgcgaggtg 600
acacatcagg gcctgtcttc ccccgtgacc aagagcttca accgcggcga gtgt 654
<210> 47
<211> 1350
<212> DNA
<213> hST-35-4 heavy chain nucleotide sequence ()
<400> 47
gaggttcagc tggtgcagtc cggcgccgag gtaaagaagc caggctcttc tgtgaaagtg 60
tcctgcaaga cctctggcta tacctttacc acatacacca tccactggat ccggcaggcc 120
cctggccagg gcctcgagtg gatcggcgct gtgaatcctg gcaacgccga tacttcctac 180
tcccagaagt tcaagggcag agccactttg accgccgaca agagctccaa caccgcttac 240
atggaactgt cctctctgag atccgaggac actgccgtgt actactgtgc cagagacgcc 300
tacccctact tctacgccct ggactcctgg ggccaaggca caaccgtcac cgtgtcttcc 360
gcttctacca agggcccatc cgtgtttccc ctggccccta gctctaagtc tacctccgga 420
ggaacagccg ctctgggatg tctggtgaag gattacttcc ccgagcctgt gaccgtgtct 480
tggaactccg gcgctctgac ctctggcgtg cacacatttc ctgccgtgct gcagtccagc 540
ggcctgtatt ccctgtcttc cgtggtgacc gtgccaagct cttccctggg cacccagaca 600
tacatctgca acgtgaatca caagccctct aatacaaagg tggacaagaa ggtggagcct 660
aagtcctgtg ataagaccca tacatgcccc ccttgtccag ctccagagct gctgggagga 720
ccatccgtgt tcctgtttcc acccaagccc aaggacacac tgtatatcac acgggagcct 780
gaggtgacat gcgtggtggt ggacgtgtcc cacgaggacc cagaggtgaa gttcaactgg 840
tacgtggatg gcgtggaggt gcataatgcc aagaccaagc ctagagagga gcagtataac 900
tctacatacc gcgtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaag 960
gagtacaagt gcaaggtgag caataaggct ctgcctgccc caatcgagaa gacaatctct 1020
aaggctaagg gccagccaag ggagccccag gtgtataccc tgcctccaag ccgggacgag 1080
ctgacaaaga accaggtgtc tctgacctgt ctggtgaagg gcttctaccc atctgatatc 1140
gccgtggagt gggagtccaa tggccagccc gagaacaatt ataagaccac accccctgtg 1200
ctggacagcg atggatcctt ctttctgtac tccaagctga ccgtggacaa gagcaggtgg 1260
cagcagggca acgtgtttag ctgctctgtg ctgcatgagg ctctgcacaa tcattacaca 1320
cagaagtccc tgagcctgtc tcctggcaag 1350
<210> 48
<211> 642
<212> DNA
<213> hST-35-4 light chain nucleotide sequence ()
<400> 48
gatatccaga tgacccagag cccttcttct ctgtctgctt ccgtgggcga cagagtgacc 60
atcacctgta gagctggcca agagatctcc tcttacctga cctggctgca gcagaaacct 120
gagggcacca tcaagagact gatctacgcc gcttctacac tggattctgg cgtgcccaag 180
cggttctctg gctccagatc tggctccgac tacaccctga caatctccag cctgcagccc 240
gaggattttg ccacatatta ctgcctgcag tacgcttctt acccttggac cttcggcggc 300
ggaaccaagg tggaaatcaa gcgcaccgtg gctgcccctt ccgtgttcat ctttcctcca 360
tccgacgagc agctgaagtc tggcacagcc tccgtggtgt gcctgctgaa caatttctat 420
ccaagagagg ctaaggtgca gtggaaggtg gataacgccc tgcagtctgg caattcccag 480
gagagcgtga cagagcagga ctctaaggat tccacctaca gcctgtccag caccctgaca 540
ctgagcaagg ctgattatga gaagcacaag gtgtacgcct gcgaggtgac acatcagggc 600
ctgtcttccc ccgtgaccaa gagcttcaac cgcggcgagt gt 642

Claims (20)

1. An antibody or antigen-binding fragment thereof capable of specifically binding to the SARS-CoV-2 coronavirus S protein, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising at least one, two, or three Complementarity Determining Regions (CDRs) selected from the group consisting of seq id nos:
(i) HCDR1 having the amino acid sequence as set forth in SEQ ID NO: 1.7, 12 or 18, or a sequence having one or several amino acid substitutions, deletions or additions (e.g. 1,2 or 3 substitutions, deletions or additions) compared to any of the above sequences;
(ii) HCDR2 having the sequence as set forth in SEQ ID NO: 2. 8, 13, 19, 33 or 35, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 substitutions, deletions or additions) compared to any of the above sequences; and
(iii) HCDR3 having the amino acid sequence as set forth in SEQ ID NO: 3. 9, 14, 20, 34 or 36, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 substitutions, deletions or additions) compared to any of the above sequences;
and/or, it comprises a light chain variable region (VL) comprising at least one, two or three Complementarity Determining Regions (CDRs) selected from the group consisting of:
(iv) LCDR1 having the amino acid sequence as set forth in SEQ ID NO: 4. 10, 15 or 21, or a sequence having one or several amino acid substitutions, deletions or additions (e.g. 1,2 or 3 substitutions, deletions or additions) compared to any of the above sequences;
(v) LCDR2 having the amino acid sequence as set forth in SEQ ID NO: 5. 11, 16 or 22, or a sequence having one or several amino acid substitutions, deletions or additions (e.g. 1,2 or 3 substitutions, deletions or additions) compared to any of the above sequences; and
(vi) LCDR3 having the amino acid sequence as set forth in SEQ ID NO:6 or 17, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1,2 or 3 substitutions, deletions or additions) as compared to the above sequence;
wherein the 3 CDRs comprised by the heavy chain variable region and/or the 3 CDRs comprised by the light chain variable region are defined by the Kabat or IMGT numbering system;
wherein the substitution described in any one of (i) to (vi) is a conservative substitution.
2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises 3 VH variable region CDRs and 3 VL variable region CDRs selected from the group consisting of:
(i) The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NO: 1. 2, 3, 4, 5 or 6, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 substitutions, deletions or additions) compared to any of the above sequences;
(ii) The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NO: 7. 8, 9, 10, 11 or 6, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 substitutions, deletions or additions) as compared to any of the above sequences;
(iii) The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NO: 12. 13, 14, 15, 16 or 17, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 substitutions, deletions or additions) as compared to any of the above sequences;
(iv) The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequences shown in SEQ ID NO: 18. 19, 20, 21, 22 or 17, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 substitutions, deletions or additions) as compared to any of the above sequences;
(v) The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequences shown in SEQ ID NO: 12. 33, 34, 15, 16 or 17, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1,2 or 3 substitutions, deletions or additions) as compared to any of the above sequences;
(vi) The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have the sequences shown in SEQ ID NO: 18. 35, 36, 21, 22 or 17, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1,2 or 3 substitutions, deletions or additions) compared to any of the above sequences.
3. The antibody or antigen-binding fragment thereof of claim 2, wherein the antibody or antigen-binding fragment thereof is murine or chimeric and the heavy chain variable region comprises the heavy chain FR region of a murine IgG1, igG2, igG3, or variant thereof; and the light chain variable region comprises the light chain FR region of a murine kappa, lambda chain or variant thereof.
4. The antibody or antigen-binding fragment thereof of claim 3, wherein the antibody or antigen-binding fragment thereof comprises VH and VL sequences selected from the group consisting of SEQ ID NOs:
(i) The VH domain comprises the amino acid sequence set forth in SEQ ID NO:23, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or has one or more amino acid substitutions (e.g., conservative substitutions)) to the sequences described above; and the VL domain comprises the amino acid sequence as set forth in SEQ ID NO:24, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or has one or more amino acid substitutions (e.g., conservative substitutions)) to the sequences described above;
(ii) The VH domain comprises the amino acid sequence as set forth in SEQ ID NO:25, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or has one or more amino acid substitutions (e.g., conservative substitutions)) to the sequences described above; and the VL domain comprises the amino acid sequence set forth in SEQ ID NO:26, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or has one or more amino acid substitutions (e.g., conservative substitutions)) to the sequences described above.
5. The antibody or antigen-binding fragment thereof of claim 2, wherein the antibody or antigen-binding fragment thereof is humanized.
6. The antibody or antigen-binding fragment thereof of claim 5, wherein the antibody or antigen-binding fragment thereof comprises VH and VL sequences selected from the group consisting of seq id nos:
(i) The VH domain comprises the amino acid sequence set forth in SEQ ID NO:37, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or has one or more amino acid substitutions (e.g., conservative substitutions)) to the sequences described above; and the VL domain comprises the amino acid sequence as set forth in SEQ ID NO:38, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or has one or more amino acid substitutions (e.g., conservative substitutions)) to the sequences described above;
(ii) The VH domain comprises the amino acid sequence as set forth in SEQ ID NO:39, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or has one or more amino acid substitutions (e.g., conservative substitutions)) to the sequences described above; and the VL domain comprises the amino acid sequence as set forth in SEQ ID NO:40, or a sequence that is substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical or has one or more amino acid substitutions (e.g., conservative substitutions)) to the sequences described above.
7. The antibody of claim 6, wherein said antibody comprises a heavy chain constant region and a light chain constant region from a human immunoglobulin; more preferably, the heavy chain constant region is selected from the group consisting of human IgG1, igG2, igG3, igG4, igM, igA1, igA2, igD and IgE heavy chain constant regions; more preferably, the heavy chain constant region is selected from the group consisting of the heavy chain constant regions of human IgG1, igG2, igG3 and IgG 4; and, the heavy chain constant region has a native sequence or a sequence having substitution, deletion or addition of one or more amino acids compared to the native sequence from which it is derived; and the light chain constant region is preferably as set forth in SEQ ID NO:27, constant region of human kappa appa chain.
8. The antibody of claim 7, wherein said antibody comprises a heavy chain constant region selected from the group consisting of:
(i) As shown in SEQ ID NO:28, a heavy chain constant region of a wild-type human IgG 1;
(ii) As shown in SEQ ID NO:29 heavy chain constant region of human IgG1 containing the M252Y, S254T, T256E and M428L mutations;
(iii) As shown in SEQ ID NO:30 of a heavy chain constant region of wild-type human IgG 2;
(iv) As shown in SEQ ID NO:31, a heavy chain constant region of a hinge region modified human IgG 2;
(v) As shown in SEQ ID NO:32, heavy chain constant region of human IgG4 containing the S228P mutation.
9. The antibody of claim 7, wherein said antibody comprises a full-length amino acid sequence selected from the group consisting of SEQ ID NO:
(i) The heavy chain has an amino acid sequence shown as SEQ ID NO. 41, and the light chain has an amino acid sequence shown as SEQ ID NO. 42; or a sequence having one or several substitutions, deletions or additions (e.g., 1,2, 3, 4 or 5 substitutions, deletions or additions) compared to any of the above sequences; or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identity compared to any of the above sequences;
(ii) The heavy chain has an amino acid sequence shown as SEQ ID NO. 43, and the light chain has an amino acid sequence shown as SEQ ID NO. 44; or a sequence having one or several substitutions, deletions or additions (e.g., 1,2, 3, 4 or 5 substitutions, deletions or additions) compared to any of the above sequences; or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identity compared to any of the above sequences.
10. A DNA molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-9.
11. The DNA molecule of claim 10, wherein the DNA molecule encoding the heavy chain of said antibody has the amino acid sequence set forth in SEQ ID NO:45 or 47, and a DNA molecule encoding the light chain of said antibody has the nucleotide sequence set forth in SEQ ID NO:46 or 48.
12. A vector comprising the DNA molecule of claim 10 or 11.
13. A host cell comprising the vector of claim 12; the host cell comprises a prokaryotic cell, a yeast or a mammalian cell, preferably a CHO cell.
14. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-9 and a pharmaceutically acceptable excipient, carrier or diluent.
15. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-9, comprising: (a) Obtaining the gene of the antibody or the antigen binding fragment thereof, and constructing an expression vector of the antibody or the antigen binding fragment thereof; (b) Transfecting the expression vector into a host cell by a genetic engineering method; (c) Culturing the above host cell under conditions that allow production of the antibody or antigen-binding fragment thereof; (d) Isolating, purifying the antibody or antigen binding fragment thereof produced;
wherein, the expression vector in the step (a) is selected from one or more of plasmids, bacteria and viruses;
wherein, the constructed vector is transfected into a host cell by a genetic engineering method in the step (b), and the host cell comprises prokaryotic cells, yeast or mammalian cells, such as CHO cells, NS0 cells or other mammalian cells;
wherein step (d) separates, purifies the antibody or antigen-binding fragment thereof by conventional immunoglobulin purification methods, including protein a affinity chromatography and ion exchange, hydrophobic chromatography, or molecular sieve methods.
16. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 in the manufacture of a medicament for the treatment and prevention of a disease caused by SARS-CoV-2 coronavirus.
17. A method for detecting the presence of SARS-CoV-2 virus or a corresponding antigen in a sample, comprising the steps of:
(1) Incubating a biological sample to be tested with at least one monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 under suitable conditions;
(2) Detecting the presence of the bound complex in the step;
wherein the biological sample is selected from the group consisting of plasma, whole blood, mouthwash, throat swab, urine, stool, and bronchial perfusate;
wherein said antigen binding fragment is selected from the group consisting of F (ab') 2 Fab', fab and Fv.
18. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 in the preparation of a SARS-CoV-2 virus detection kit.
19. A test kit comprising at least one monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-9.
20. The test kit of claim 20, comprising:
(1) Selected from any one of:
a. a solid support and a first antibody;
b. a solid support coated with a first antibody;
the first antibody is selected from any one of the monoclonal antibodies or antigen binding fragments thereof of claims 1-9;
(2) A second antibody;
said second antibody is optionally suitably labeled and is selected from the group consisting of a monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-9 that is capable of being used in conjunction with the first antibody of (1).
CN202210313645.5A 2021-04-02 2022-03-28 Monoclonal antibody for resisting SARS-CoV-2 virus and its application Pending CN115197317A (en)

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CN202110361333 2021-04-02

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