CN114805557B - Humanized anti-SARS-CoV-2 monoclonal antibody and its application - Google Patents

Humanized anti-SARS-CoV-2 monoclonal antibody and its application Download PDF

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CN114805557B
CN114805557B CN202111658564.0A CN202111658564A CN114805557B CN 114805557 B CN114805557 B CN 114805557B CN 202111658564 A CN202111658564 A CN 202111658564A CN 114805557 B CN114805557 B CN 114805557B
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CN114805557A (en
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严景华
黄庆瑞
史瑞
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Institute of Microbiology of CAS
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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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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A—HUMAN NECESSITIES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6839—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting material from viruses
    • A61K47/6841—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting material from viruses the antibody targeting a RNA virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
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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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
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    • C07K2317/00—Immunoglobulins specific features
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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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    • C07K2317/56—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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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
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    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Abstract

The invention relates to a humanized anti-SARS-CoV-2 monoclonal antibody HUR33 and application thereof. HUR33 humanized antibody comprises HCDR1, HCDR2 and HCDR3 comprising the heavy chain variable region shown as SEQ ID NO. 1, and LCDR1, LCDR2 and LCR3 comprising the light chain variable region shown as SEQ ID NO. 2, preferably further comprises heavy chain FR1 shown as SEQ ID NO. 17, heavy chain FR2 shown as SEQ ID NO. 18, heavy chain FR3 shown as SEQ ID NO. 19, heavy chain FR4 shown as SEQ ID NO. 20, light chain FR1 shown as SEQ ID NO. 21, light chain FR2 shown as SEQ ID NO. 22, light chain FR3 shown as SEQ ID NO. 23 and light chain FR4 shown as SEQ ID NO. 24, has high neutralizing activity against SARS-CoV-2 live virus.

Description

Humanized anti-SARS-CoV-2 monoclonal antibody and its application
Technical Field
The invention belongs to the fields of biological medicine and virology, and relates to a SARS-CoV-2 virus monoclonal antibody and application thereof.
Background
At present, new coronaries pneumonia (covd-19) caused by SARS-CoV-2 infection has been a global pandemic for a year and caused infections of 1 million people worldwide, 300 tens of thousands of people to die. SARS-CoV-2 virus is very contagious and is prone to severe and death in elderly people with underlying disease. SARS-CoV-2 is a serious hazard to human life and health and has profound effects on global economic activity and human life.
Neutralizing antibodies refer to antibodies that, upon binding to a virus, eliminate the ability of the virus to infect. In addition to being used for infection diagnosis or developing antigen detection kits, the humanized neutralizing antibody can also be used for clinical treatment or prevention of SARS-CoV-2 infection patients. The HUR33 antibodies of the invention have high neutralizing activity (IC) 50 The value is 0.038 mug/ml), and has great clinical development potential.
Disclosure of Invention
The invention provides an anti-SARS-Cov-2 antibody or antigen binding fragment thereof, which is characterized in that the antibody or antigen fragment thereof has high affinity to RBD and does not block the binding of human ACE2 to RBD.
Thus, the anti-SARS-Cov-2 antibody or antigen binding fragment thereof of the invention can be paired with an antibody that blocks the binding of human ACE2 to RBD, and used in the clinical treatment or prevention of SARS-Cov-2 infection in patients.
In particular, the invention relates to the following aspects:
1. an anti-SARS-Cov-2 antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2 and HCDR3 comprised by the heavy chain variable region as shown in SEQ ID No. 1, and LCDR1, LCDR2 and LCR3 comprised by the light chain variable region as shown in SEQ ID No. 2;
preferably, the antibody comprises, according to IMGT numbering system:
HCDR1 comprising or consisting of an amino acid sequence having at least 80%, preferably 81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity to the sequence shown in SEQ ID No. 3 or having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence,
HCDR2 comprising or consisting of an amino acid sequence having at least 80%, preferably 81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity to the sequence shown in SEQ ID No. 4 or having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, and
HCDR3 comprising or consisting of an amino acid sequence of at least 80%, preferably 81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity to the sequence shown in SEQ ID No. 5 or having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence and the antibody further comprises:
LCDR1 comprising or consisting of an amino acid sequence having at least 80%, preferably 81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity to the sequence depicted in SEQ ID NO. 6 or having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence,
LCDR2 comprising or consisting of an amino acid sequence of at least 80%, preferably 81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% sequence identity with the amino acid sequence shown in SEQ ID No. 7 or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, and
LCDR3 comprising or consisting of a sequence as set out in SEQ ID NO 8 or a sequence having at least 80%, preferably 81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or 99% or more sequence identity thereto or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence.
2. The anti-SARS-CoV-2 antibody or antigen-binding fragment thereof of item 1, further comprising heavy chain FR1 shown by SEQ ID NO. 17, heavy chain FR2 shown by SEQ ID NO. 18, heavy chain FR3 shown by SEQ ID NO. 19, SEQ ID NO. 31 or SEQ ID NO. 32, heavy chain FR4 shown by SEQ ID NO. 20, light chain FR1 shown by SEQ ID NO. 21, light chain FR2 shown by SEQ ID NO. 22, light chain FR3 shown by SEQ ID NO. 23 and light chain FR4 shown by SEQ ID NO. 24.
3. The anti-SARS-CoV-2 antibody or antigen-binding fragment thereof of item 1, wherein said antibody comprises:
a heavy chain variable region comprising or consisting of:
the amino acid sequence shown in SEQ ID NO 9, SEQ ID NO 33 or SEQ ID NO 34, or
A sequence having at least 85%, preferably 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or more than 99% sequence identity to the sequence shown in SEQ ID NO 9, SEQ ID NO 33 or SEQ ID NO 34, or
An amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO 9, SEQ ID NO 33 or SEQ ID NO 34, and
A light chain variable region comprising or consisting of:
an amino acid sequence shown in SEQ ID NO. 10 or SEQ ID NO. 39, or
A sequence having at least 85%, preferably 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98% or more than 99% sequence identity to the sequence shown in SEQ ID NO. 10 or SEQ ID NO. 39, or
An amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence shown in SEQ ID NO. 10 or SEQ ID NO. 39.
4. The anti-SARS-CoV-2 antibody or antigen-binding fragment thereof of any of clauses 1-3, wherein said anti-SARS-CoV-2 antibody further comprises a heavy chain constant region (preferably having the sequence shown as SEQ ID NO: 41) and a light chain constant region (preferably having the sequence shown as SEQ ID NO: 42), preferably said heavy chain amino acid sequence is shown as SEQ ID NO:11, SEQ ID NO:35 or SEQ ID NO:36, said light chain amino acid sequence is shown as SEQ ID NO:12 or SEQ ID NO:39, more preferably said heavy chain nucleotide sequence is shown as SEQ ID NO:13, SEQ ID NO:37 or SEQ ID NO:38, and said light chain nucleotide sequence is shown as SEQ ID NO:14 or SEQ ID NO: 40.
5. The anti-SARS-CoV-2 antibody or antigen-binding fragment thereof of any of clauses 1-4, said antigen-binding fragment being selected from the group consisting of Fab, fab ', F (ab') 2 Fd, fv, dAb, fab/c, complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), diabodies, or domain antibodies.
6. A polynucleotide molecule comprising a nucleotide sequence encoding a heavy chain variable region and/or a light chain variable region of the antibody or antigen binding fragment thereof of any one of items 1-5.
7. A vector comprising the polynucleotide molecule of item 6.
8. A host cell comprising the polynucleotide molecule of item 6, or the vector of item 6.
9. A method of preparing the anti-SARS-CoV-2 antibody or antigen-binding fragment thereof of any of items 1-5, comprising the steps of culturing the host cell of item 8 under suitable conditions, and recovering the antibody or antigen-binding fragment thereof from the cell culture.
10. An antibody conjugate comprising an anti-SARS-CoV-2 antibody or antigen-binding fragment thereof of any one of clauses 1-5, and a conjugate moiety conjugated to the anti-SARS-CoV-2 antibody or antigen-binding fragment thereof, the conjugate moiety being a purification tag (e.g., his tag), a cytotoxic agent, or a detectable label. Preferably, the coupling moiety is a radioisotope, luminescent substance, colored substance, enzyme or polyethylene glycol.
11. A multispecific antibody, preferably a bispecific antibody, comprising an anti-SARS-CoV-2 antibody or antigen-binding fragment thereof of any one of clauses 1-5, and an antibody or antigen-binding fragment directed against another antigen and/or other epitope.
12. A fusion protein comprising the anti-SARS-CoV-2 antibody or antigen-binding fragment thereof of any one of items 1-5.
13. A kit comprising the anti-SARS-CoV-2 antibody or antigen-binding fragment thereof of any one of items 1-5, or the antibody conjugate of item 10, the multispecific antibody of item 11, or the fusion protein of item 11.
14. The kit of item 13, further comprising a second antibody that specifically recognizes the anti-SARS-CoV-2 antibody or antigen-binding fragment thereof; optionally, the second antibody further comprises a detectable label, such as a radioisotope, luminescent material, colored material, enzyme, or polyethylene glycol.
15. Use of the anti-SARS-CoV-2 antibody or antigen-binding fragment thereof of any one of items 1-5, the antibody conjugate of item 10, the multispecific antibody of item 11, or the fusion protein of item 11 to detect the presence or level of SARS-CoV-2 in a sample, or to prepare a kit to detect the presence or level of human SARS-CoV-2 in a sample.
16. A pharmaceutical composition comprising the anti-SARS-CoV-2 antibody or antigen-binding fragment thereof of any one of clauses 1-5, the antibody conjugate of clause 10, the multispecific antibody of clause 11, or the fusion protein of clause 12; optionally, it further comprises a pharmaceutically acceptable carrier and/or excipient.
17. The use of the anti-SARS-CoV-2 antibody or antigen-binding fragment thereof of any one of clauses 1-5, the antibody conjugate of clause 10, the multispecific antibody of clause 11, or the fusion protein of clause 12, for treating a disease caused by SARS-CoV-2 infection, or for the manufacture of a medicament for treating a disease caused by SARS-CoV-2.
18. The use of item 17, wherein the medicament is in a form suitable for injection, preferably a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection or intralesional injection.
19. A method of treating a disease caused by a SARS-CoV-2 infection comprising the step of administering to a subject in need thereof an effective amount of a cell comprising the anti-SARS-CoV-2 antibody or antigen binding fragment thereof of any one of items 1-5, the antibody conjugate of item 10, the multispecific antibody of item 11, or the fusion protein of item 12.
Since the presence of free cysteine C in an antibody may cause post-translational modification or intermolecular disulfide bond formation, thereby affecting the function of the antibody, mutation modification of the existing cysteine C in the antibody of the present invention helps to maintain the stability of the antibody, etc. Specifically, in the present invention, the amino acid of heavy chain FR3 region (sequence shown as SEQ ID NO: 19) is mutated, specifically, the 18 th C of SEQ ID NO:19 is replaced with S or N, to obtain HUR33 humanized antibody mutant FR3 (sequence shown as SEQ ID NO:31 or SEQ ID NO: 32) and HUR33 humanized antibody mutant heavy chain variable region (sequence shown as SEQ ID NO:33 or SEQ ID NO: 34).
According to the prior art and literature information (US 8775090B2; wines BD et al, J immunol.2000May 15;164 (10): 5313-8), the introduction of a specific position mutation, such as the M252Y/S254T/T256E (EU numbering, abbreviated as "YTE" mutation), into therapeutic IgG provides a number of advantages, such as the increased binding affinity of IgG1/Fc to FcRn, thereby improving the serum half-life of the corresponding host and thus reducing the frequency and/or dosage of administration. Specifically, in the present invention, the Fc region of the HUR33 humanized antibody heavy chain (SEQ ID NO: 11) was mutated, specifically, M at position 252 (positioned according to the EU numbering system numbering) or 265 (positioned according to the Kabat numbering system numbering) was substituted for Y, S at position 254 (positioned according to the EU numbering system numbering) or 267 (positioned according to the Kabat numbering system) was substituted for T, and T at position 256 (positioned according to the EU numbering system numbering) or 269 (positioned according to the Kabat numbering system numbering) was substituted for E, to obtain the heavy chain (sequence shown as SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO: 38) of the HUR33 humanized antibody mutant.
Referring to the new crown antibodies already marketed (e.g., BRII-196 mAb), when the light chain is a kappa chain, conventional humanized modifications typically employed include deletion of the last 1 amino acid S at the C-terminus of the light chain. Specifically, in the invention, the last S amino acid of the HUR33 humanized antibody light chain is deleted to obtain a HUR33 humanized antibody light chain mutant (the sequence is shown as SEQ ID NO:39 or SEQ ID NO: 40).
Thus, a HUR33 humanized antibody mutation was obtained: HUR33-1 and HUR33-2, wherein the heavy chain sequence of HUR33-1 is shown as SEQ ID NO. 35 or SEQ ID NO. 37, and the light chain sequence of HUR33-1 is shown as SEQ ID NO. 39 or SEQ ID NO. 40; the heavy chain sequence of HUR33-2 is shown as SEQ ID NO. 36 or SEQ ID NO. 38, and the light chain sequence of HUR33-2 is shown as SEQ ID NO. 39 or SEQ ID NO. 40.
Definition:
it should be noted that an indefinite number of entity definitions shall refer to one or more of the entities; for example, "bispecific antibody" is understood to mean one or more bispecific antibodies. Likewise, the terms "one or more" and "at least one" are used interchangeably herein, as not defined in an explicit number.
"homology" or "identity" or "similarity" refers to the degree of sequence similarity between two peptide chain molecules or between two nucleic acid molecules. Homology can be determined by comparing the positions in each sequence, and can be compared by alignment. When there are identical bases or amino acids at positions in the sequences being compared, the molecules at that position are homologous. Homology between sequences is a function of the number of paired or homologous sites shared by the sequences. The "unrelated" or "non-homologous" sequences have less than 40% homology, but preferably less than 25% homology, to one of the sequences of the present application.
A polynucleotide or polynucleotide region (or polypeptide region) has a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of "sequence identity" with another sequence, meaning that when aligned, the percentage of bases (or amino acids) is the same when the two sequences are compared. Such alignments and percent homology or sequence identity may be determined using software programs known in the art, such as those described by Ausubel et al (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment. BLAST is an alignment program that uses default parameters. Specifically, programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect=10; matrix = BLOSUM62; descriptive = 50sequences; sort by = HIGH SCORE; databases = non-redundant, genbank+embl+ddbj+pdb+ GenBank CDS translations +swissprotein+pir. Details of these programs are available at the following internet addresses: http:// www.ncbi.nlm.nih.gov/blast. Cgi, last visit at month 5, 2008, 21. Biologically equivalent polynucleotides are polynucleotides that have the specified percentage of homology noted above and encode polypeptides having the same or similar biological activity.
The term "encoding" when applied to a polynucleotide refers to a polynucleotide that is considered to "encode" a polypeptide, which may be transcribed and/or translated to produce an mRNA and/or fragment thereof for the polypeptide, either in its native state or when manipulated by methods well known to those skilled in the art. The antisense strand is the complement of such a nucleic acid from which the coding sequence can be deduced.
The term "antibody fragment" or "antigen-binding fragment" as used herein is a portion of an antibody, such as F (ab') 2 、F(ab) 2 Fab', fab, fv, fd, fv, dAb, fab/c, complementarity Determining Region (CDR) fragments, disulfide-linked Fvs (sdFv), single chain antibodies (e.g., scFv), diabodies, or domain antibodies, and the like. The term "antibody fragment" also includes any synthetic or genetically engineered protein that, like antibodies, can bind to a particular antigen to form a complex. The term "diabody" is a small antigen-binding fragment having two antigen-binding sites.
"Single chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin. In certain aspects, these regions are linked with short linker peptides of 10 to about 25 amino acids. The linker may be glycine-rich to be flexible, also serine or threonine-rich to be soluble, and be able to link the N-terminus of the VH to the C-terminus of the VL, and vice versa. The protein retains the original immunoglobulin properties except that the constant region is removed and a linker is introduced. ScFv molecules are known in the art and are described in U.S. Pat. No. 5,892,019.
Antibodies, antigen binding polypeptides, variants or derivatives thereof of the present application include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primatized antibodies, or chimeric antibodies, single chain antibodies, antigen epitope binding fragments, e.g., fab 'and F (ab') 2, fd, fvs, single chain Fvs (scFv), single chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising a VL domain or VH domain, fragments produced by a Fab expression library, and anti-idiotype (anti-Id) antibodies. The immunoglobulin molecules or antibody molecules of the present application may be of any type (e.g., igG, igE, igM, igD, igA and IgY), any class (e.g., igGl, igG2, igG3, igG4, igAl, and IgA 2) or subclass of immunoglobulin molecules.
The amino acids of the CDRs and framework regions can be readily identified by those of skill in the art for any given heavy or light chain variable region as they are already well-defined (see, "Sequences of Proteins of Immunological Interest," Kabat, e., et al, U.S. department of health and public service (u.s.device of Health and Human Services,), (1983); chothia and Lesk, j.moi.biol.,196:901-917 (1987), which is incorporated herein by reference in its entirety).
Where a term is used and/or is acceptable in the art, where there are two or more definitions of that term, the definition of term used herein is intended to include all meanings unless explicitly stated to the contrary. One specific example is the use of the term "complementarity determining regions" ("CDRs") to describe non-contiguous antigen binding sites present in both the variable regions of heavy and light chain polypeptides. Such specific areas are described by Kabat et al in the U.S. department of health and public service, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al and J.MoI.biol.196:901-917 (1987), which are incorporated herein by reference in their entirety. According to the definition of Kabat and Chothia, CDRs comprise overlapping amino acid residues, or amino acid substructures, when compared to each other. However, each application of definition of a CDR with respect to an antibody or variant thereof will be within the scope of the terms defined and used herein. Suitable amino acid residues comprising CDRs as defined by each of the references cited above are listed in the following table for comparison. The exact number of residues comprising a particular CDR will vary with the sequence and size of the CDR. Given the variable region amino acid sequence of the antibody, one skilled in the art can generally determine which residues comprise a particular CDR.
TABLE 1 definition of antibody variable regions
Kabat Chothia
CDR-H1 31-35 26-32
CDR-H2 50-65 52-58
CDR-H3 95-102 95-102
CDR-L1 24-34 26-32
CDR-L2 50-56 50-52
CDR-L3 89-97 91-96
Kabat et al also define a numbering system for variable domain sequences that is applicable to either antibody. The skilled person can undoubtedly apply this "Kabat numbering" system to any variable domain sequence, without relying on any experimental data outside the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al, the contents of which are described in the U.S. department of health and public service, "Sequence of Proteins of Immunological Interest" (1983).
In addition to the above table, the CDR regions described by the Kabat numbering system are as follows: CDR-H1 begins at about amino acid 31 (i.e., about 9 residues after the first cysteine residue), includes about 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at residue 15 after the end of CDR-H1, including about 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at about amino acid residue 33 after the end of CDR-H2; comprising 3-25 amino acids; and terminates at the sequence W-G-X-G, wherein X is any amino acid. CDR-L1 begins at about residue 24 (i.e., after a cysteine residue); comprising about 10-17 residues; and terminates at the next tryptophan residue. CDR-L2 begins about 16 residues after the end of CDR-L1 and includes about 7 residues. CDR-L3 begins at about residue 33 after the end of CDR-L2 (i.e., after the cysteine residue); comprising about 7-11 residues and terminating at the sequence F or W-G-X-G, wherein X is any amino acid.
The term "heavy chain constant region" as used herein includes amino acid sequences from immunoglobulin heavy chains. The polypeptide comprising a heavy chain constant region comprises at least one of a: CH1 domain, a hinge (e.g., upper hinge region, middle hinge region, and/or lower hinge region) domain, CH2 domain, CH3 domain, or variant or fragment thereof. For example, an antigen binding polypeptide as used herein may 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 structure, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptides of the present application include polypeptide chains having a CH3 domain. In addition, antibodies 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 one of ordinary skill in the art will appreciate that the heavy chain constant regions may be modified such that they differ in amino acid sequence from naturally occurring immunoglobulin molecules.
"light chain-heavy chain pair" refers to a collection of light and heavy chains that can form dimers through disulfide bonds between the CL and CH1 domains of the light chain.
The term "chimeric antibody" as used herein will be used to refer to any of the following antibodies: wherein the immunoreactive region or locus thereof is obtained or derived from a first species and the constant region thereof (which may be intact, partial or modified according to the present application) is obtained from a second species. In certain embodiments the target binding region or site is from a non-human source (e.g., mouse or primate) and the constant region is from a human.
As used herein, "percent humanization" is calculated by: the number of framework amino acid differences (i.e., non-CDR differences) between the humanized domain and the germline domain is determined, subtracted from the total number of amino acids, divided by the total number of amino acids, and multiplied by 100.
The term "treatment" or "treatment" as used herein refers to therapeutic treatment and prophylactic or preventative measures, wherein the subject is prevented or slowed down (lessened) by the development of an undesirable physiological change or disease, such as cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (e.g., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or not. "treatment" may also refer to an increase in survival compared to the expected survival when not receiving treatment. Those conditions in need of treatment include those already with the disorder or condition and those prone to have the disorder or condition or those in which the disorder or condition is to be prevented.
Any of the above antibodies or polypeptides may also include additional polypeptides, forming conjugates or fusion proteins, e.g., a polypeptide encoded as described herein, a signal peptide at the N-terminus of an antibody, which signal peptide is used to direct secretion, or other heterologous polypeptides as described herein.
It will also be appreciated by those of ordinary skill in the art that the antibodies described herein may be modified such that their amino acid sequences differ from naturally occurring binding polypeptides from which they are derived. For example, a polypeptide or amino acid sequence from a given protein may be similar to the starting sequence, e.g., have a certain percentage of identity to the starting sequence, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the starting sequence.
In addition, nucleotide or amino acid substitutions, deletions, or insertions may be made to make conservative substitutions or changes in the "nonessential" amino acid region. For example, the polypeptide or amino acid sequence from a given protein may be identical to the starting sequence except for one or more independent amino acid substitutions, insertions, or deletions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more independent amino acid substitutions, insertions, or deletions. In certain embodiments, the polypeptide or amino acid sequence from a given protein has 1 to 5, 1 to 10, 1 to 15, or 1 to 20 independent amino acid substitutions, insertions, or deletions relative to the starting sequence.
In other embodiments, the antigen binding polypeptides of the present application may comprise conservative amino acid substitutions.
A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, non-essential amino acid residues of an immunoglobulin polypeptide are preferably replaced with other amino acid residues from the same side chain family. In another embodiment, a series of amino acids may be replaced by a structurally similar series of amino acids, the latter differing in order and/or composition of the side chain family.
Non-limiting examples of conservative amino acid substitutions are provided in the following table, wherein a similarity score of 0 or higher indicates that there is a conservative substitution between the two amino acids.
TABLE 2 non-limiting list of conservative amino acid substitutions
Examples of techniques that may be used to generate single chain Fvs (scFvs) and antibodies include those described in U.S. Pat. nos. 4,946,778 and 5,258,498; methods in Enzymology203:46-88 (1991) of Huston et al; proc.Natl.Sci.USA 90:1995-1999 (1993) by Shu et al; and Skerra et al Science 240:1038-1040 (1988). For certain applications, including the use of antibodies in humans and in vitro assay, it may be preferable to use chimeric, humanized or human antibodies. Chimeric antibodies are molecules in which different parts of the antibody are derived from different animal species, e.g., antibodies that contain variable regions derived from murine monoclonal antibodies and human immunoglobulin constant regions. Methods of producing chimeric antibodies are known in the art. See, e.g., morrison, science 229:1202 (1985); bioTechniques 4:214 of Oi et al (1986); gillies et al J.Immunol. Methods125:191-202 (1989); U.S. patent No. 5,807,715;4,816,567 and 4,816397, the entire contents of which are incorporated herein by reference.
Humanized antibodies are antibody molecules derived from a non-human species and which bind to the desired antigen, the antibody molecules having one or more Complementarity Determining Regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Typically, framework residues in the human framework region will be altered by replacement of corresponding residues from the CDR donor antibody, preferably to increase antigen binding capacity. These framework substitutions are identified by methods known in the art, for example, by modeling the interactions of CDRs with framework residues to identify framework residues that are important for antigen binding and sequence, to find aberrant framework residues at particular positions. (see, e.g., U.S. Pat. No. 5,585,089 to Queen et al; nature 332:323 to Riechmann et al (1988), the entire contents of which are incorporated herein by reference). The antibodies can be humanized using a variety of techniques known in the art, including, for example, CDR-grafting (EP 239,400; PCT publication No. WO 91/09967; U.S. Pat. Nos. 5,225,539;5,530,101 and 5,585,089), veneering (veneering) or surface substitution (resurfacing) (EP 592,106;EP 519,596;Padlan,Molecular Immunology 28 (4/5): 489-498 (1991); studnicka et al, protein Engineering (6): 805-814 (1994); proc. Natl. Sci. USA91:969-973 (1994)), and chain shuffling (resuffling) (U.S. Pat. No. 5,565,332), the entire contents of which are incorporated herein by reference).
One or more CDRs of the antigen binding polypeptides of the present application can be inserted into a framework region, e.g., into a human framework region, using conventional recombinant DNA techniques to humanize a non-human antibody. The framework regions may be naturally occurring or consensus framework regions, and are preferably human framework regions (see, e.g., J.mol. Biol.278:457-479 (1998), chothia et al, list of human framework regions). Preferably, the polynucleotide produced by the combination of the framework regions and CDRs encodes a polypeptide that specifically binds to at least one epitope of the desired polypeptide, e.g., LIGHT. Preferably, one or more amino acid substitutions may be made within the framework region, and preferably, the amino acid substitutions increase the antigen binding capacity of the antibody. In addition, such methods can be used to obtain amino acid substitutions or deletions of one or more variable region cysteine residues that are involved in intrachain disulfide bond formation, thus producing antibody molecules lacking one or more intrachain disulfide bonds. Other modifications to the polynucleotide are within the scope of the present application and within the skill of the art.
Furthermore, techniques for producing "chimeric antibodies" by gene splicing from mouse antibody molecules (Morrison et al, proc. Natl. Acad. Sci. USA:851-855 (1984); neuberger et al, nature 372:604-608 (1984); takeda et al, nature314:452-454 (1985)) may be used, together with a human antibody molecule gene having suitable biological activity, with appropriate antigen specificity. As used herein, a chimeric antibody is one in which different parts are derived from different animal species, e.g., an antibody containing a variable region from a murine monoclonal antibody and a human immunoglobulin constant region.
Yet another efficient method for producing recombinant antibodies is disclosed in Newman, biotechnology 10:1455-1460 (1992). In particular, this technique results in the production of primatized antibodies containing a monkey variable domain and a human constant sequence. This document is incorporated by reference in its entirety. In addition, such techniques are also described in commonly assigned U.S. Pat. nos. 5,658,570, 5,693,780 and 5,756,096, each of which is incorporated herein by reference.
Alternatively, antibody-producing cell lines may be selected and cultured using techniques well known to those skilled in the art. Such techniques are described in various laboratory manuals and major publications. In this regard, techniques suitable for use herein are described, for example, in Current Protocols in Immunology, code Coligan et al, green Publishing Associates and Wiley-Interscience, john Wiley and Sons, new York (1991), which are incorporated herein by reference in their entirety, including supplementary references.
Furthermore, standard techniques well known to those skilled in the art may be used to introduce mutations in the nucleotide sequences encoding the antibodies of the present application, including, but not limited to, site-directed mutagenesis and PCR-mediated mutations, which result in amino acid substitutions. Preferably, the variant (including derivative) encodes less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to a reference variable heavy chain region, CDR-H1, CDR-H2, CDR-H3, light chain variable region, CDR-L1, CDR-L2, or CDR-L3. Alternatively, mutations may be randomly introduced along all or part of the coding sequence, for example by saturation mutagenesis, and the resulting mutants may be screened for biological activity to determine mutations that retain activity.
Drawings
FIG. 1.PHRNT vector map.
FIG. 2 shows a map of the mRNA transcription template plasmid pHRNT-RBD cloned with RBD.
FIG. 3 RBDmRNA vaccine immunization induced high levels of SARS-CoV-2 virus specific antibodies.
FIG. 4. Real virus neutralizing antibody titer determination.
FIG. 5 RBD group mice memory B cell sorting.
FIG. 6 affinity of R33 antibody for SARS-CoV-2 RBD.
FIG. 7 shows the blocking activity of HUR33 antibody against human ACE2 binding SARS-CoV-2 RBD.
FIG. 8 affinity of HUR33 antibody for SARS-CoV-2 RBD.
FIG. 9 neutralization activity of negative control antibodies (unrelated isotype IgG antibodies).
FIG. 10 neutralization activity of HUR33 antibodies.
FIG. 11 neutralizing activity of positive control antibody CB 6.
Detailed Description
The invention will be further illustrated with reference to specific examples. It should be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, which do not address specific conditions in the following examples, are generally carried out according to conventional conditions such as molecular cloning guidelines (third edition published by scientific press) written by j. Reagents used in the experiments, unless otherwise specified, are commercially available.
Example 1: mRNA vaccine preparation
(1) Obtaining a target gene: the amino acid sequence of RBD of SARS-CoV-2 receptor binding domain is derived from Genebank MN908947, its amino acid sequence is shown as SEQ ID NO. 29, after codon optimization, its nucleotide sequence is shown as SEQ ID NO. 30, and after synthesis by Beijing qing mill, new Biotechnology Co., ltd, it is cloned into mRNA transcription template pHRNT vector (for example, from Beijing qing mill, new Biotechnology Co., ltd.) respectively (FIG. 1), and the mRNA transcription template plasmid pHRNT-RBD is obtained (FIG. 2).
(2) mRNA transcription
The mRNA transcription template pHRNT-RBD was subjected to enzyme-tangential digestion with the restriction enzyme BamHI, and subjected to agarose gel electrophoresis to confirm whether linearization was complete, and then the linearized transcription template was recovered with a gel recovery kit.
An mRNA in vitro transcription reaction system (enzymes and reagents used were purchased from NEB company, usa) was prepared as follows:
the total volume of RNase-free water and DNA template totaled 7.5ul.
After 4 hours of reaction at 37℃1. Mu.l of RNase-free DNase I was added and the reaction was carried out for 15 minutes at 37 ℃.
Then, RNA was isolated and purified. There are various methods for isolating and purifying RNA, such as ammonium acetate precipitation, liCl precipitation, organic solvent extraction-ammonium acetate precipitation, and RNA-binding column purification. Taking LiCl precipitation as an example:
a) Adding 7.5M LiCl into the RNA solution to make the final concentration of the LiCl be 2.5M;
b) -20 ℃ overnight;
c) Centrifuging at 12000rpm/min for 15 min, and discarding the solution;
d) Adding 75% ethanol pre-cooled at-20deg.C into the precipitate, cleaning the precipitate, centrifuging at 12000rpm/min for 1 min, discarding ethanol solution, and repeatedly cleaning for three times;
e) The RNA pellet was dried at room temperature and then the RNA was dissolved in RNase-free water. After RNA concentration was determined using Nanodrop, the samples were stored at-80 ℃.
(2) mRNA capping
mRNA can be capped by the following method 7 Gppp(m 2′-O ) N1, the specific method is as follows:
a) In vitro transcribed mRNA (50-60. Mu.g) was diluted to 67. Mu.l with RNase-free water;
b) Incubating at 65 ℃ for 5-10 minutes, and then cooling on ice;
c) The reaction system mixture (enzymes and reagents used were purchased from NEB company, usa) was prepared as follows;
d) The mRNA cooled in b) was added to the mixture of c) before the reaction was started, and 4. Mu.l of capping enzyme was added thereto and reacted at 37℃for half an hour.
And then separating and purifying the capped mRNA. Specific methods As described above, mRNA-RBD was finally obtained.
(3) mRNA nanoparticle packaging
Nanoparticle packaging is carried out on mRNA by a microfluidic technology. The aqueous phase is mRNA solution (50 mM sodium acetate buffer solution, pH 4.0), the ethanol phase is lipid mixed solution, and the lipid mixed solution is prepared from the ditolyl-methyl-4-dimethylaminobutyrate, distearoyl phosphatidylcholine, cholesterol and PEG2K-DMPE according to the molar ratio of 50:10:38.5:1.5. The total flow rate of the water phase and the ethanol phase during packaging is 12ml/min, and the volume ratio of the water phase to the ethanol phase is 3:1. after the packed mRNA vaccine was replaced with PBS using a dialysis bag, mRNA vaccine RBD was obtained. The encapsulated mRNA concentration was then determined using RiboGreen reagent and stored at 4℃until use.
Example 2: vaccine-induced antibody level assessment
10 female 6-8 week old BALB/c mice were divided into 2 groups of 5 mice each, each of which were given intramuscular placebo (the packaging method was the same as the vaccine group but coated with polycytidylic acid lipid nanoparticles, wherein polycytidylic acid (purchased from Sigma) and RBD (15. Mu.g) mRNA vaccine. The immunization was boosted at week 4 after immunization. Blood was taken at weeks 4 and 8 after the initial immunization, serum was isolated at 4℃and inactivated at 56℃for 30 minutes before storage at-80℃for use.
(1) Antigen-specific antibody titer determination
SARS-CoV-2RBD protein (available from Beijing Yiqiao Biotechnology Co., ltd.) was diluted to 2. Mu.g/ml with ELISA coating solution, 100. Mu.l per well of ELISA plate was added and left overnight at 4 ℃. The following day after blocking the ELISA plates for 1 hour, the mouse serum was diluted in a 2-fold gradient, added to ELISA plates for 1 hour at 37℃and then washed three times with PBS (PBST) containing 0.05% Tween 20, then added with goat anti-mouse HRP secondary antibody (purchased from Kyoto Meter Cunninghamia Biotechnology Co.) and incubated for 1 hour at 37℃and then washed five times with PBST, added with TMB chromogenic solution and stopped with 2M hydrochloric acid, and read with OD450 on a microplate reader. ELISA results showed that RBDmRNA vaccine induced high levels of SARS-CoV-2 virus specific antibodies after one-needle immunization (FIG. 3), and antibody levels were further raised 170-fold after booster immunization (FIG. 3).
(2) Real virus neutralizing antibody titer determination
The serum of the mice is diluted in a gradient manner according to the ratio of 2 times and is mixed with 100TCID 50 Wild type SARS-CoV-2 real virus (HB 01 strain from China academy of sciences microbiological institute P3 laboratory) was mixed in equal volume, incubated at 37℃for 1 hour, and 100. Mu.l of the mixed solution was added to a 100. Mu.l of the mixed solution having a density of 1.5X10 5 /mL of Vero E6 cells. After incubation at 37℃for 72 hours, the cells were observed under a microscope for lesions. Finally, calculating the serum dilution factor for protecting 50% of cells from virus infection by Karber method, namely the real virus neutralizing antibodyTiter NT 50 Values. The results indicate that the primary vaccination with RBD mRNA vaccine resulted in neutralizing antibody titres NT 50 263, neutralizing antibody titer NT after booster immunization 50 Is improved by 222 times (fig. 4).
Example 3: screening of memory B cells in mice after immunization
(1) Obtaining of lymphocytes
8 weeks after the initial immunization, mice were anesthetized and sacrificed, dissected to remove lymphocytes. 8 lymph nodes (superficial parotid, axillary, subiliac and popliteal) were taken from each rat. Lymph nodes were removed and placed in 1640 medium containing 1% fetal bovine serum, ground and filtered using a 0.45 μm filter.
(2) Lymphocyte staining
The lymphocytes after filtration were centrifuged using a refrigerated centrifuge, centrifuged at 400g for 10 minutes at 4℃and the supernatant removed, resuspended in 1ml of PBS containing 0.04% BSA (i.e.staining buffer) and transferred into a 1.5ml EP tube. The cells were resuspended in staining buffer remaining at the bottom of the tube by centrifugation again for 10min, and the supernatant removed. Mu.l of cells were removed, 400. Mu.l of staining buffer was added, and after mixing, the mixture was aliquoted into 8 EP tubes, 50. Mu.l per tube. Of these, 7 tube was used as Shan Yangguan (FITC anti-murine GL7 antigen, PE anti-murine CD138, PE/Cyanine7 anti-murine CD38, APC anti-murine CD93, brilliant Violet 421 anti-murine CD45R/B220, brilliant Violet 510 anti-murine IgD or strep-BV711 antibodies, all available from Biolegend Co.) and 1 tube was used as negative control tube (neither antibody was stained). The remaining cells were resuspended by adding 250. Mu.l staining buffer and mixed well for use as a sample tube. Biotin-labeled SARS-CoV-2RBD protein (final concentration 400nM, protein from Biotechnology Co., beijing Yiqiao Shenzhou Co., ltd.) was added to the sample tube and strep-BV711 single-stained tube and incubated at 4℃for 30 minutes in the absence of light. The cells were washed 2 times with staining buffer, the corresponding antibodies (using concentration reference instructions) were added to the cells and incubated at 4℃for 30 minutes in the absence of light. The cells were washed 2 times with staining buffer, resuspended in 2ml staining buffer and filtered through a 0.45 μm filter, transferred into a flow tube, and ready for loading.
(3) Sorting of antigen-specific memory B cells
Cells which simultaneously meet FITC-GL7 positive, PE-CD138 negative, PE/Cy7-CD38 negative or weak positive, APC-CD93 negative, BV421-B220 strong positive, BV510-IgD negative and strep-BV711 positive are selected by a flow cytometer (BD Biosciences), and are antigen-specific memory B cells.
The results showed that RBD mRNA vaccine fractions were sorted to approximately 20000 target cells (fig. 5), and finally the number of cells reaching the viability was detected on-machine to be approximately 9000, respectively.
Example 4: high throughput sequencing and acquisition of antibody sequences
The monoclonal memory B cell BCR sequencing sample treatment was performed according to the 10 x Genomics company chromomum single cell 5' library construction manual of the united states. After memory B cells were sorted by flow cytometry, the cells were centrifuged, resuspended in PBS buffer containing 3% fetal bovine serum (purchased from Sigma-Aldrich), and then cell number and quality control was performed using a cell counter. The treated B cells require greater than 70% cell viability. After determining the cell density and mass, cells were loaded into three channels to ensure approximately 1000-3000 cells per channel. Gel beads per channel of the chip were combined with single cell formed oil droplets (GEMs) in a 10 x chromasum machine and collected for GEM reverse transcription. After GEMs have been demulsified, the GEM-RT product is amplified by circular PCR and purified using SPRIselect beads (available from Beckman Coulter).
Single cell BCR V (D) J libraries were prepared according to the 10 x Genomics user guide. The next operation was then performed using Bioanalyzer High Sensitivity DNA kit (available from Agilent Technologies). The quantitative procedure was then performed using Kapa Library Quantification Kit (available from Kapa Biosystems). The final prepared library was sequenced on Illumina NovaSeq using paired-end sequencing. BCL data is converted to FASTQ files using Illumina BCL2FASTQ 2.20.
We retained the first 26 bases for reading a single cell containing a 16nt cell barcode and a 10nt Unique Molecular Identifier (UMI). The FASTQ file was then analyzed. Bar code treatment and single cell V (D) J sequence analysis were performed using Cell Ranger Single-Cell Software Suite (version 3.1.0). Then FASTQ file processing was performed using Cell Ranger V (D) J pipeline. First, reads were filtered for potent cell barcodes and UMIs, and the filtered reads were spliced into contigs by alignment with the GRCm 38V (D) J reference genome, then V, D and J fragments were defined as 1 contig, and CDR3 sequences were identified and whether contigs were readable or not was determined from these data, meaning that it might correspond to a functional B cell receptor. Finally, the barcode is determined to be the target cell if the following three requirements are met. 1) It must be possible to read through, confident contigs, and if there is only one such contig, there must be more than one UMI supporting its J region. 2) There must be at least three filtered UMIs, with at least two read pairs per UMI. 3) The N50 value of the number of read heads per UMI in all barcodes is calculated. If the maximum read numbers of filtered UMIs for a given bar code is less than 3% of N50, the bar code is not referred to as a single cell. By exact nucleotide matching, the collection of cells that are identical to the read-through CDR3 sequences is defined as clonotypes. V (D) J sequences and clonotypes in single cell 5' data generated by 10 x Chromium were analyzed, searched and visualized using the LoupTem V (D) J browser, and the sequences were further annotated and analyzed using IgBLAST V1.6.1 to identify variable region gene fragments and somatic mutations.
The results showed that 4060 cells were detected in the RBD vaccine group after cell priming, of which 3615 heavy chain sequences, 4163 light chain sequences, 3048 light and heavy chain sequences were paired, and cells with identical light and heavy chain CDR3 regions were defined as one clonotype, for a total of 1611 clones.
Example 5: construction, expression and Activity determination of antibodies
(1) Construction of antibody heavy and light chain plasmids
The first 100 clones with highest frequency (corresponding antibodies are named as R1, R2, … and R100) are selected from RBD vaccine antibody libraries, the amino acid sequence of a monoclonal antibody variable region is subjected to codon optimization by the Nanjing Jinsrui company, and then a signal peptide sequence and a mouse IgG2a antibody constant region are respectively added at the 5 'end and the 3' end of a gene, and then complete gene synthesis is carried out. The amino acid sequence and the nucleotide sequence of the signal peptide are shown as SEQ ID NO. 15 and SEQ ID NO. 16 respectively, the amino acid sequence and the nucleotide sequence of a constant region of a mouse IgG2a light chain are shown as SEQ ID NO. 25 and SEQ ID NO. 26 respectively, the amino acid sequence and the nucleotide sequence of a constant region of a mouse IgG2a heavy chain are shown as SEQ ID NO. 27 and SEQ ID NO. 28 respectively, the whole gene synthesis of the light chain and the heavy chain of the antibody is respectively constructed into pCAGGS vectors (for example, the plasmid can be purchased from Feng Hui organisms), and finally the complete light chain and heavy chain expression plasmid of the monoclonal antibody is obtained.
(2) Determination of antibody concentration in cell expression supernatants
Heavy and light chain plasmids paired with the same antibody were used as 2:3, and after 4-6 hours of transfection, the cells were washed 2 times with PBS and changed to serum-free DMEM medium for culture. Cell supernatants were harvested 3 days after transfection, centrifuged to remove cell debris and antibody supernatants were obtained.
The antibody concentration in the supernatant was determined using the Mouse IgG2a Elisa Kit (purchased from Multi Sciences). First, a mouse anti-IgG 2a standard in the kit or a cell supernatant to be assayed is added to a commercial Elisa plate pre-coated with an anti-mouse IgG2a monoclonal antibody in the kit and incubated at room temperature for 2 hours. PBST was washed 6 times, HRP conjugated detection antibody was added and incubated for 1 hour at room temperature. After 6 washes of PBST, TMB was added for color development and stopped with 2M sulfuric acid, and the OD450 read on a microplate reader. And calculating a standard curve according to the concentration of the standard substance and the reading value, and calculating the concentration of the antibody in the cell supernatant according to the standard curve and the absorbance value of the sample.
(3) Neutralization Activity evaluation of antibody expression supernatants
Diluting the antibody to different concentration ranges according to the quantitative result of the concentration of the antibody in the cell supernatant>6. Mu.g/ml, 1-6. Mu.g/ml, 0.1-0.6. Mu.g/ml and <0.1. Mu.g/ml), 4 replicates per concentration gradient, 50. Mu.l each replicate, and 50. Mu.l 100TCID for each sample 50 Is incubated for 1 hour at 37℃with an equal volume of wild-type SARS-CoV-2 real virus (HB 01 strain). 100 μl of the mixture was then added to the mixture to give a density of 1.5X10 5 /mL of Vero E6 cells. The cells were incubated at 37℃for 72 hours and examined under a microscope for lesions. IC for defining antibodies 50-100％ The value is the minimum antibody concentration range required to inhibit 50% and more of the onset of lesions in cells (i.e., 2 replicates), if the antibody is>At a concentration of 6. Mu.g/ml, the cytopathic rate was still above 50%, and the antibody was defined as having no neutralizing activity. Finally, R33 antibody with strong neutralizing activity is screened, and IC thereof 50-100％ Less than<0.1. Mu.g/ml. The amino acid and nucleotide sequences of the light and heavy chain variable region of the neutralizing antibody and the amino acid sequences of the CDR regions are shown in SEQ ID NO. 1 to SEQ ID NO. 10.
Example 7: expression, isolation and purification of R33 antibodies
The cells with larger cell density are separated into plates (for example, a plate of 10cm culture dishes which are 100% full of 293T cells is passaged at 1:3) 14-16 hours before transfection, and the transfection can be carried out after the cell density reaches more than 70% after 14-16 hours.
The heavy chain and light chain expression plasmids of the R33 antibody of example 5 (1) were expressed as 2:3, and after 4-6 hours of transfection, the cells were washed 2 times with PBS and changed to serum-free DMEM medium for culture. Cell supernatants were harvested on days 3 and 7, respectively, after transfection, centrifuged to remove cell debris, and the antibody supernatants obtained in two times were mixed for subsequent purification of the target protein.
Protein G (5 ml) HP affinity column (GE company) was attached to AKTA Purifier/Explorer/FPLC/START (GE company) and the machine was operated as follows: the column is equilibrated by washing 20% ethanol in the column with water, buffer solution of 20mM Na3PO4,pH 7.0, after the conductivity on the instrument is 4.5% and is stable, the antibody supernatant is injected into the column to be combined with Protein G by a 10ml loop sample loading mode, the flow rate is 2ml/min, after UV is stable, 1M Tris pH9.0 buffer solution is added into a subsequent collecting tube for about 0.8ml (the collecting volume is about 3.2 ml), then the antibody hung on the column is eluted by changing the procedure to 100% 0.1M Gly pH3.0, the eluted sample is collected, then the antibody buffer solution is replaced by PBS by adopting a concentrated liquid replacement method, and the obtained product is directly used or packaged and stored in a refrigerator at-80 ℃.
Example 9: affinity assay for R33 antibodies
Measuring R33 antibody affinity by SPR (surface plasmon resonance) technique, and selecting Biacore8k to complete sample detection(available from GE company, USA). The chip surface was equilibrated with HBS-EP buffer at a flow rate of 10. Mu.l/min for 5 min, followed by activation of the chip by injection of a 1:1 mixture of "NHS+EDC" at a flow rate of 10. Mu.l/min for 100 seconds, coupling was performed by injection of anti-mouse IgG Fc (available from GE Co. Ltd. In the United states) diluted in 10mM sodium acetate at a flow rate of 10. Mu.l/min for about 180 seconds, and finally surface blocking was performed by injection of ethanolamine at a flow rate of 10. Mu.l/min for 200 seconds. Three pre-cycles were performed with HBS-EP buffer as sample to equilibrate the chip to stabilize the baseline. The capture antibody was injected at a flow rate of 30. Mu.l/min for 120 seconds, followed by 100nM SARS-CoV-2RBD-his protein (available from Biotechnology Co., ltd. In Beijing) at a flow rate of 30. Mu.l/min for 240 seconds for binding, followed by 30. Mu.l/min for 300 seconds for dissociation, and 30. Mu.l/min for 10mM Gly-HCl at a flow rate of 10mM, pH1.7 for three times, 30 seconds for regeneration, with one cycle ending. The antibody concentration was varied and the next cycling assay was performed at gradient concentrations up to all gradient concentrations (6.125 nM, 12.5nM, 25nM, 50nM, 100 nM). After double-deduction (control channel and zero concentration) of experimental data, fitting of a "1:1 binding" model was performed in Biacore8K evaluation software (GE corporation), and binding kinetic parameters were analyzed and affinity constants (kD) were calculated. The results show that the affinity kD value of the R33 antibody for SARS-CoV-2RBD is 1.04×10 -9 M (FIG. 6).
Example 10: humanization of R33 murine antibodies
According to the sequence homology of the R33 antibody, the humanized antibody HUR33 is obtained by replacing a humanized antibody skeleton on the basis of keeping a CDR region of the antibody. The HUR33 antibody light and heavy chain gene was synthesized by the whole gene of new biotechnology limited company of the family beijing, cloned and constructed on a mammalian expression vector pCAGGS (for example, purchased from Feng Hui organism), and the humanized antibody HUR33 was expressed by transiently transfecting 293T cells.
SEQ ID No. 1R 33 murine antibody heavy chain variable region amino acid sequence
SEQ ID No. 2R 33 murine antibody light chain variable region amino acid sequence
SEQ ID No. 3R 33 murine antibody heavy chain variable region CDR1 amino acid sequence
SEQ ID No.4, amino acid sequence of CDR2 of heavy chain variable region of R33 murine antibody
SEQ ID No. 5R 33 murine antibody heavy chain variable region CDR3 amino acid sequence
SEQ ID No. 6R 33 murine antibody light chain variable region CDR1 amino acid sequence
SEQ ID No.7, amino acid sequence of CDR2 of light chain variable region of R33 murine antibody
SEQ ID No.8, amino acid sequence of CDR3 of light chain variable region of R33 murine antibody
SEQ ID No.9 HUR33 humanized antibody heavy chain variable region amino acid sequence
SEQ ID No.10 amino acid sequence of light chain variable region of HUR33 humanized antibody
SEQ ID No.11 HUR33 humanized antibody heavy chain amino acid sequence
SEQ ID No.12 HUR33 humanized antibody light chain amino acid sequence
SEQ ID No.13 HUR33 humanized antibody heavy chain nucleotide sequence
SEQ ID No.14 HUR33 humanized antibody light chain nucleotide sequence
SEQ ID No.15 HUR33 humanized antibody light-heavy chain signal peptide amino acid sequence
SEQ ID No.16 HUR33 humanized antibody light-heavy chain signal peptide nucleotide sequence
SEQ ID NO.17 HUR33 humanized antibody heavy chain FR1 amino acid sequence
SEQ ID NO.18 HUR33 humanized antibody heavy chain FR2 amino acid sequence
SEQ ID NO.19 HUR33 humanized antibody heavy chain FR3 amino acid sequence
SEQ ID NO.20 HUR33 humanized antibody heavy chain FR4 amino acid sequence
SEQ ID NO.21 HUR33 humanized antibody light chain FR1 amino acid sequence
SEQ ID NO.22 HUR33 humanized antibody light chain FR2 amino acid sequence
SEQ ID NO.23 HUR33 humanized antibody light chain FR3 amino acid sequence
SEQ ID NO.24 HUR33 humanized antibody light chain FR4 amino acid sequence
Example 11: in vitro recombinant expression of humanized antibody HUR33
The heavy and light chain plasmids of the HUR33 antibodies were combined at 2:3, and after 4-6 hours of transfection, the cells were washed 2 times with PBS and changed to serum-free DMEM medium for culture. Cell supernatants were harvested on days 3 and 7, respectively, after transfection, centrifuged to remove cell debris, and the antibody supernatants obtained in two times were mixed for subsequent purification of the target protein.
Antibodies were purified by Protein A (5 ml) HP affinity column (GE company) and either used directly or stored in sub-packaging at-80 ℃.
Example 12: HUR33 antibody blocking function identification
The full-length coding sequence of human ACE2 (Genbank Accession number BAJ 21180) is synthesized by the total gene of the entrusted Kirschner Biotechnology Co., ltd.) and cloned into an expression vector pEGFP-N1 (from the Kirschner Biotechnology Co., ltd.) through two enzyme cutting sites of HindIII at the 5 'end and BamHI at the 3' end, so as to construct a transmembrane eukaryotic expression plasmid pEGFP-hACE2 of the human ACE2 protein. The recombinant expression plasmid pEGFP-hACE2 was used to transfect HEK 293T cells, and 24h later the expression of human ACE2 membrane protein (co-expressed with EGFP) was observed using a fluorescence microscope. The culture broth was discarded, HEK 293T cells were trypsinized down, centrifuged and resuspended in PBS. A5 ul concentration of 50. Mu.g/ml SARS-CoV-2RBD-his protein (from Beijing Yiqiao China Biotechnology Co., ltd.) was mixed with 10 times the amount of antibody, incubated at 37℃for 30 minutes, wherein the positive control antibody was CB6 (the CB6 light and heavy chain amino acid sequence was derived from GenBank database, the success codes were MT470196 and MT470197, respectively; the neutralizing activity of CB6 antibody against SARS-CoV-2 virus was found in the articles Shi, R., shan, C., duan, X.et al. A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2.Nature 584,120-124, 2020), and the negative control antibody was an irrelevant isotype IgG antibody (the national academy of microorganisms). The 293T cells expressing the human ACE2 protein are separated into 96-well plates, the supernatant is discarded after centrifugation, and the SARS-CoV-2RBD-his protein and antibody mixture is added for incubation at 4℃for 30 minutes. 600g was centrifuged for 5 min, the supernatant was spun off, 200. Mu.L PBS was added to wash the cells, and 600g was centrifuged for 5 min, and the above steps were repeated 2 times. mu.L of 1:200 dilution of anti-his-APC mouse mAb (available from Meitian and Gentle Co.) was added to each well and incubated at 4℃for 30 minutes in the absence of light. 600g was centrifuged for 5 min, the supernatant was spun off, 200. Mu.L PBS was added to wash the cells, 600g was centrifuged for 5 min, the above steps were repeated 2 times, finally 200. Mu.L PBS buffer was added to resuspend the cells and transferred to the flow tube, and finally the sample detection was completed on a BD FACSCalibur flow meter. The final data analysis was done by FlowJo software. The results show that the unrelated isotype IgG antibody does not block the binding of human ACE2 to SARS-CoV-2RBD, the positive control CB6 antibody can completely block the binding of human ACE2 to RBD, while the HUR33 antibody does not block the binding of human ACE2 to RBD, indicating that the HUR33 antibody does not overlap the binding region of human ACE2 to RBD (fig. 7).
Example 13: HUR33 antibody affinity assay
The affinity of HUR33 antibody was determined by SPR (surface plasmon resonance) technique, and sample detection (available from GE company, USA) was accomplished by selecting Biacore 8 k. The chip surface was equilibrated with HBS-EP buffer at a flow rate of 10. Mu.l/min for 5 min, followed by activation of the chip by injection of a 1:1 mixture of "NHS+EDC" at a flow rate of 10. Mu.l/min for 100 seconds, coupling was performed by injection of anti-mouse IgG Fc (available from GE Co. Ltd. In the United states) diluted in 10mM sodium acetate at a flow rate of 10. Mu.l/min for about 180 seconds, and finally surface blocking was performed by injection of ethanolamine at a flow rate of 10. Mu.l/min for 200 seconds. Three pre-cycles were performed with HBS-EP buffer as sample to equilibrate the chip to stabilize the baseline. The capture antibody was injected at a flow rate of 30. Mu.l/min for 120 seconds, followed by 100nM SARS-CoV-2RBD-his protein (available from Biotechnology Co., ltd. In Beijing) at a flow rate of 30. Mu.l/min for 240 seconds for binding, followed by 30. Mu.l/min for 300 seconds for dissociation, and 30. Mu.l/min for 10mM Gly-HCl at a flow rate of 10mM, pH1.7 for three times, 30 seconds for regeneration, with one cycle ending. The antibody concentration was varied and the next cycling assay was performed at gradient concentrations up to all gradient concentrations (6.125 nM, 12.5nM, 25nM, 50nM, 100 nM). After the experimental data were double-subtracted (control channel and zero concentration), a "1:1 binding" model fit was performed in Biacore 8K evaluation software. The affinity of the antibodies for SARS-CoV-2RBD-his was determined using Biacore 8K. The results show that HUR33 antibody affinity kD value is 1.74×10 -9 M, affinity with humanized pre-murine antibody R33 (kD value 1.04X 10 -9 M) are equivalent (fig. 8).
Example 14: neutralizing Activity of HUR33 antibodies
Antibody-fold ratio serial dilutions were performed in 8 replicates per concentration gradient, 50 μl each replicate, and 50 μl 100TCID for each sample 50 Is obtained by mixing wild type SARS-CoV-2 real virus (HB 01 strain derived from P3 laboratory of national academy of sciences of microorganisms) in equal volume and incubating at 37℃for 1 hour. Wherein the positive control antibody is CB6, and the negative control antibody is an irrelevant isotype IgG antibody (China academy of sciences of microorganisms). 100 μl of the mixture was then added to the mixture to give a density of 1.5X10 5 /mL of Vero E6 cells. The cells were incubated at 37℃for 72 hours and examined under a microscope for lesions. Counting the number of parallel sample hole lesions of each concentration, calculating the neutralization inhibition rate=100% -the number of lesion holes/total number of holes is 100%, fitting an antibody concentration-inhibition rate curve by using a biological software Graphpad according to the result of the neutralization inhibition rate, and calculating the IC 50 Values. The results showed that the negative control antibody had no neutralizing activity (FIG. 9), HUR33 antibody IC 50 The value was 0.038. Mu.g/ml (FIG. 10), and the neutralizing activity was stronger than that of the positive control antibody CB6 (IC 50 The value was 0.2. Mu.g/ml) (FIG. 11).
Example 15: HUR33 humanized antibody mutation
Since the presence of free cysteine C in an antibody may result in post-translational modification or intermolecular disulfide bond formation, thereby affecting the function of the antibody, mutating the existing cysteine C in an antibody helps to maintain the stability of the antibody, etc. Specifically, in the present invention, the amino acid of heavy chain FR3 region (sequence shown as SEQ ID NO: 19) is mutated, specifically, the 18 th C of SEQ ID NO:19 is replaced with S or N, to obtain HUR33 humanized antibody mutant FR3 (sequence shown as SEQ ID NO:31 or SEQ ID NO: 32) and HUR33 humanized antibody mutant heavy chain variable region (sequence shown as SEQ ID NO:33 or SEQ ID NO: 34).
According to the prior art and literature information (US 8775090B2; wines BD et al, J immunol.2000May 15;164 (10): 5313-8), the introduction of a specific position mutation, such as the M252Y/S254T/T256E (EU numbering, abbreviated as "YTE" mutation), into therapeutic IgG provides a number of advantages, such as the increased binding affinity of the IgG1/Fc fragment to FcRn, thereby improving the serum half-life of the corresponding host and thus reducing the frequency and/or dosage of administration. Specifically, in the present invention, the Fc region of the HUR33 humanized antibody heavy chain (SEQ ID NO: 11) was mutated, M at position 252 (positioned according to the EU numbering system numbering) or 265 (positioned according to the Kabat numbering system numbering) was substituted for Y, S at position 254 (positioned according to the EU numbering system numbering) or 267 (positioned according to the Kabat numbering system) was substituted for T, and T at position 256 (positioned according to the EU numbering system numbering) or 269 (positioned according to the Kabat numbering system) was substituted for E, to obtain the heavy chain of the HUR33 humanized antibody mutant (sequence shown as SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO: 38). Referring to the new crown antibodies already marketed (e.g., BRII-196 mAb), when the light chain is a kappa chain, conventional humanized modifications typically employed include deletion of the last 1 amino acid S at the C-terminus of the light chain. Specifically, in the invention, the last S amino acid of the HUR33 humanized antibody light chain is deleted to obtain a HUR33 humanized antibody light chain mutant (the sequence is shown as SEQ ID NO:39 or SEQ ID NO: 40).
Thus, a HUR33 humanized antibody mutation was obtained: HUR33-1 and HUR33-2, wherein the heavy chain sequence of HUR33-1 is shown as SEQ ID NO. 35 or SEQ ID NO. 37, and the light chain sequence of HUR33-1 is shown as SEQ ID NO. 39 or SEQ ID NO. 40; the heavy chain sequence of HUR33-2 is shown as SEQ ID NO. 36 or SEQ ID NO. 38, and the light chain sequence of HUR33-2 is shown as SEQ ID NO. 39 or SEQ ID NO. 40.
Sequence listing
R33 murine antibody heavy chain variable region amino acid sequence SEQ ID NO.1
R33 murine antibody light chain variable region amino acid sequence SEQ ID NO. 2
R33 murine antibody heavy chain variable region CDR1 amino acid sequence SEQ ID NO. 3
R33 murine antibody heavy chain variable region CDR2 amino acid sequence SEQ ID NO. 4
R33 murine antibody heavy chain variable region CDR3 amino acid sequence SEQ ID NO. 5
R33 murine antibody light chain variable region CDR1 amino acid sequence SEQ ID NO. 6
R33 murine antibody light chain variable region CDR2 amino acid sequence SEQ ID NO. 7
R33 murine antibody light chain variable region CDR3 amino acid sequence SEQ ID NO. 8
HUR33 humanized antibody heavy chain variable region amino acid sequence SEQ ID No.9:
HUR33 humanized antibody light chain variable region amino acid sequence SEQ ID No.10:
HUR33 humanized antibody heavy chain amino acid sequence SEQ ID No.11:
HUR33 humanized antibody light chain amino acid sequence SEQ ID No.12:
HUR33 humanized antibody heavy chain nucleotide sequence SEQ ID No.13:
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HUR33 humanized antibody light chain nucleotide sequence SEQ ID No.14:
HUR33 humanized antibody light and heavy chain signal peptide amino acid sequence SEQ ID NO. 15
HUR33 humanized antibody light and heavy chain signal peptide nucleotide sequence SEQ ID NO. 16
HUR33 humanized antibody heavy chain variable region FR1 amino acid sequence SEQ ID No.17:
HUR33 humanized antibody heavy chain variable region FR2 amino acid sequence SEQ ID No.18:
HUR33 humanized antibody heavy chain variable region FR3 amino acid sequence SEQ ID No.19:
HUR33 humanized antibody heavy chain variable region FR4 amino acid sequence SEQ ID No.20:
HUR33 humanized antibody light chain variable region FR1 amino acid sequence SEQ ID No.21:
HUR33 humanized antibody light chain variable region FR2 amino acid sequence SEQ ID No.22:
HUR33 humanized antibody light chain variable region FR3 amino acid sequence SEQ ID No.23:
HUR33 humanized antibody light chain variable region FR4 amino acid sequence SEQ ID No.24:
amino acid sequence of mouse IgG2a light chain constant region SEQ ID NO. 25
Mouse IgG2a light chain constant region nucleotide sequence SEQ ID NO. 26
Amino acid sequence of heavy chain constant region of mouse IgG2a SEQ ID NO. 27
The nucleotide sequence of the heavy chain constant region of the mouse IgG2a is SEQ ID NO. 28
Amino acid sequence of SARS-CoV-2 receptor binding domain RBD SEQ ID NO. 29
Nucleotide sequence of SARS-CoV-2 receptor binding domain RBD SEQ ID NO. 30
HUR33 humanized antibody heavy chain variable region FR3 mutant amino acid sequence SEQ ID No.31
HUR33 humanized antibody heavy chain variable region FR3 mutant amino acid sequence SEQ ID No.32
HUR33 humanized antibody heavy chain variable region mutant amino acid sequence SEQ ID No.33
HUR33 humanized antibody heavy chain variable region mutant amino acid sequence SEQ ID No.34
HUR33 humanized antibody heavy chain mutant amino acid sequence SEQ ID NO. 35
Or HUR33 humanized antibody heavy chain mutant amino acid sequence SEQ ID NO. 36
HUR33 humanized antibody mutant heavy chain nucleotide sequence SEQ ID NO. 37
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Or (b)
HUR33 humanized antibody mutant heavy chain nucleotide sequence SEQ ID NO. 38
HUR33 humanized antibody mutant light chain amino acid sequence SEQ ID NO. 39
HUR33 humanized antibody mutant light chain nucleotide sequence SEQ ID NO. 40
Human IgG1 heavy chain constant region amino acid sequence SEQ ID NO 41
The amino acid sequence of the light chain constant region of human IgG1 SEQ ID NO. 42
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SEQUENCE LISTING
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<120> humanized anti-SARS-CoV-2 monoclonal antibody and use thereof
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Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
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Gly Trp Ile Asn Thr Tyr Ser Gly Glu Pro Thr Tyr Ala Asp Asp Phe
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Arg Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
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Leu Gln Ile Cys Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
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Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
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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
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405 410 415
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Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
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<223> HUR33 humanized antibody light chain amino acid sequence
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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
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Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Asn Phe
20 25 30
Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Ala Ser Gln Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Thr Trp Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu 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 Ser
210 215
<210> 13
<211> 1350
<212> DNA
<213> Artificial Sequence
<220>
<223> HUR33 humanized antibody heavy chain nucleotide sequence
<400> 13
caagtgcagc tggtccagag cggcagcgag ctgaagaagc ccggcgcttc tgtgaaggtg 60
tcttgtaaag cctccggcta caccttcacc gactacggca tgaactgggt gcggcaggcc 120
cctggacagg gcctggaatg gatgggctgg atcaacacct acagcggcga gcctacatac 180
gccgacgact ttagaggcag attcgtgttc agcctggaca ccagcgtgtc tacagcctac 240
ctgcagatct gcagcctgaa ggctgaagat accgccgtgt actactgcgc cagaggcgga 300
aattgggatt ggtatttcga cgtgtggggc cagggaaccc tggttacagt gtccagcgcc 360
agcaccaaag gcccgagcgt gtttccgctg gcgccgagca gcaaaagcac cagcggcggc 420
accgcggcgc tgggctgcct ggtgaaagat tattttccgg aaccggtgac cgtgagctgg 480
aacagcggcg cgctgaccag cggcgtgcat acctttccgg cggtgctgca gagcagcggc 540
ctgtatagcc tgagcagcgt ggtgaccgtg ccgagcagca gcctgggcac ccagacctat 600
atttgcaacg tgaaccataa accgagcaac accaaagtgg ataaacgcgt ggagcccaaa 660
tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 720
tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 780
gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 840
gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 900
acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 960
tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac tatctccaaa 1020
gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggatgagctg 1080
accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1140
gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200
gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag 1260
caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1320
aagagcctct ccctgtctcc gggtaaatga 1350
<210> 14
<211> 648
<212> DNA
<213> Artificial Sequence
<220>
<223> HUR33 humanized antibody light chain nucleotide sequence
<400> 14
gacatccaga tgacccagag ccccagcagc ctgagcgcct ctgtcggcga tagagtgacc 60
atcacctgtc gggccagcca aagcgtgtcc aacttcctgc actggtatca gcagaaaccc 120
ggcaaggccc ctaagctgct gatctactac gccagccagt ccatcagcgg cgtgccatct 180
agattcagcg gatctggcag cggaaccgac ttcaccctga caatttccag cctccagcct 240
gaggactttg ctacatacta ctgccagcaa tctaacacct ggcctctgac cttcggccag 300
ggcacaaagc tggaaatcaa gcgaactgtg gctgcaccat ctgtcttcat cttccctccc 360
agcgacgagc agctgaagag cggcaccgcc agcgtggtct gtctcctgaa caacttctat 420
cccagggagg ccaaggtcca gtggaaagtg gacaacgccc tgcaaagcgg caatagccag 480
gagtccgtca cagagcagga cagcaaggac agcacctaca gcctgtccag caccctgacc 540
ctcagcaagg ccgactacga gaagcacaag gtgtacgctt gcgaggtgac ccatcagggc 600
ctgtccagcc ccgtgaccaa gtccttcaac aggggcgaat gcagctaa 648
<210> 15
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> HUR33 humanized antibody light and heavy chain Signal peptide amino acid sequence
<400> 15
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp
20
<210> 16
<211> 63
<212> DNA
<213> Artificial Sequence
<220>
<223> HUR33 humanized antibody light-heavy chain Signal peptide nucleotide sequence
<400> 16
atggagacgg atacgctgct cctgtgggtt ttgctgctct gggttccagg ttccactggt 60
gac 63
<210> 17
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> HUR33 humanized antibody heavy chain variable region FR1 amino acid sequence
<400> 17
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
20 25 30
<210> 18
<211> 14
<212> PRT
<213> Artificial Sequence
<220>
<223> HUR33 humanized antibody heavy chain variable region FR2 amino acid sequence
<400> 18
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
1 5 10
<210> 19
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> HUR33 humanized antibody heavy chain variable region FR3 amino acid sequence
<400> 19
Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr Leu Gln
1 5 10 15
Ile Cys Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
20 25 30
<210> 20
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> HUR33 humanized antibody heavy chain variable region FR4 amino acid sequence
<400> 20
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
1 5 10
<210> 21
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<223> HUR33 humanized antibody light chain variable region FR1 amino acid sequence
<400> 21
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
20
<210> 22
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> HUR33 humanized antibody light chain variable region FR2 amino acid sequence
<400> 22
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
1 5 10 15
<210> 23
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> HUR33 humanized antibody light chain variable region FR3 amino acid sequence
<400> 23
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
1 5 10 15
Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
20 25 30
<210> 24
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> HUR33 humanized antibody light chain variable region FR4 amino acid sequence
<400> 24
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
1 5 10
<210> 25
<211> 107
<212> PRT
<213> Mus musculus
<400> 25
Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu
1 5 10 15
Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
20 25 30
Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg
35 40 45
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
65 70 75 80
Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser
85 90 95
Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
100 105
<210> 26
<211> 324
<212> DNA
<213> Mus musculus
<400> 26
cgagctgatg ctgcaccaac tgtatccatc ttcccaccat ccagtgagca gttaacatct 60
ggaggtgcct cagtcgtgtg cttcttgaac aacttctacc ccaaagacat caatgtcaag 120
tggaagattg atggcagtga acgacaaaat ggcgtcctga acagttggac tgatcaggac 180
agcaaagaca gcacctacag catgagcagc accctcacgt tgaccaagga cgagtatgaa 240
cgacataaca gctatacctg tgaggccact cacaagacat caacttcacc cattgtcaag 300
agcttcaaca ggaatgagtg ttag 324
<210> 27
<211> 330
<212> PRT
<213> Mus musculus
<400> 27
Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly
1 5 10 15
Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr
20 25 30
Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu
50 55 60
Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile
65 70 75 80
Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys
85 90 95
Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys
100 105 110
Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro
115 120 125
Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys
130 135 140
Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp
145 150 155 160
Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg
165 170 175
Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln
180 185 190
His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn
195 200 205
Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly
210 215 220
Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu
225 230 235 240
Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met
245 250 255
Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu
260 265 270
Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe
275 280 285
Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn
290 295 300
Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr
305 310 315 320
Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys
325 330
<210> 28
<211> 993
<212> DNA
<213> Mus musculus
<400> 28
gccaaaacaa cagccccatc ggtctatcca ctggcccctg tgtgtggaga tacaactggc 60
tcctcggtga ctctaggatg cctggtcaag ggttatttcc ctgagccagt gaccttgacc 120
tggaactctg gatccctgtc cagtggtgtg cacaccttcc cagctgtcct gcagtctgac 180
ctctacaccc tcagcagctc agtgactgta acctccagca cctggcccag ccagtccatc 240
acctgcaatg tggcccaccc ggcaagcagc accaaggtgg acaagaaaat tgagcccaga 300
gggcccacaa tcaagccctg tcctccatgc aaatgcccag cacctaacct cttgggtgga 360
ccatccgtct tcatcttccc tccaaagatc aaggatgtac tcatgatctc cctgagcccc 420
atagtcacat gtgtggtggt ggatgtgagc gaggatgacc cagatgtcca gatcagctgg 480
tttgtgaaca acgtggaagt acacacagct cagacacaaa cccatagaga ggattacaac 540
agtactctcc gggtggtcag tgccctcccc atccagcacc aggactggat gagtggcaag 600
gagttcaaat gcaaggtcaa caacaaagac ctcccagcgc ccatcgagag aaccatctca 660
aaacccaaag ggtcagtaag ggctccacag gtatatgtct tgcctccacc agaagaagag 720
atgactaaga aacaggtcac tctgacctgc atggtcacag acttcatgcc tgaagacatt 780
tacgtggagt ggaccaacaa cgggaaaaca gagctaaact acaagaacac tgaaccagtc 840
ctggactctg atggttctta cttcatgtac agcaagctga gagtggaaaa gaagaactgg 900
gtggaaagaa atagctactc ctgttcagtg gtccacgagg gtctgcacaa tcaccacacg 960
actaagagct tctcccggac tccgggtaaa tga 993
<210> 29
<211> 242
<212> PRT
<213> Artificial Sequence
<220>
<223> amino acid sequence of SARS-CoV-2 receptor binding Domain RBD
<400> 29
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Asn Leu Thr Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn
20 25 30
Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe
35 40 45
Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala
50 55 60
Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys
65 70 75 80
Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val
85 90 95
Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala
100 105 110
Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp
115 120 125
Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser
130 135 140
Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser
145 150 155 160
Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala
165 170 175
Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro
180 185 190
Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro
195 200 205
Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr
210 215 220
Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val
225 230 235 240
Asn Phe
<210> 30
<211> 729
<212> DNA
<213> Artificial Sequence
<220>
<223> nucleotide sequence of SARS-CoV-2 receptor binding Domain RBD
<400> 30
atgttcgtgt tcctcgtgct cctgcctctg gtgtctagcc agtgcgtgaa cctgacccgg 60
gtgcagccta cagagtctat tgtgcggttc ccaaacatca caaacctgtg ccctttcggc 120
gaggtgttca acgccacccg gttcgcctct gtgtacgcct ggaaccggaa gcggatctct 180
aactgcgtgg ccgactactc cgtgctgtac aactccgcct ctttctctac attcaagtgc 240
tacggcgtgt cccctacaaa gctgaacgac ctgtgcttca ccaacgtgta cgccgactct 300
ttcgtgatta gaggcgacga ggtgaggcag attgcccccg gccagacagg caagatcgcc 360
gactacaact acaagctgcc cgacgacttc acaggctgcg tgatcgcctg gaactctaac 420
aacctggact ctaaggtggg cggcaactac aactacctgt acagactgtt ccggaagtct 480
aacctgaagc cattcgagag ggacattagc accgagattt accaggccgg ctctacccca 540
tgcaacggcg tggagggctt caactgctac ttcccactgc agtcctacgg cttccagcct 600
acaaacggcg tgggctacca gccttaccgg gtggtggtgc tgtctttcga gctgctccac 660
gcccccgcca cagtgtgcgg cccaaagaag agcacaaacc tcgtgaagaa caagtgcgtg 720
aacttctaa 729
<210> 31
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> HUR33 humanized antibody heavy chain variable region FR3 mutant amino acid sequence
<400> 31
Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr Leu Gln
1 5 10 15
Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
20 25 30
<210> 32
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> HUR33 humanized antibody heavy chain variable region FR3 mutant amino acid sequence
<400> 32
Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr Leu Gln
1 5 10 15
Ile Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
20 25 30
<210> 33
<211> 119
<212> PRT
<213> Artificial Sequence
<220>
<223> HUR33 humanized antibody heavy chain variable region mutant amino acid sequence
<400> 33
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Ser Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60
Arg Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Asn Trp Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 34
<211> 119
<212> PRT
<213> Artificial Sequence
<220>
<223> HUR33 humanized antibody heavy chain variable region mutant amino acid sequence
<400> 34
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Ser Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60
Arg Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Asn Trp Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 35
<211> 449
<212> PRT
<213> Artificial Sequence
<220>
<223> HUR33 humanized antibody heavy chain mutant amino acid sequence
<400> 35
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Ser Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60
Arg Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Asn Trp Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly
100 105 110
Thr Leu 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 Arg 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 Glu Glu Met 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 Met 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> 36
<211> 449
<212> PRT
<213> Artificial Sequence
<220>
<223> HUR33 humanized antibody heavy chain mutant amino acid sequence
<400> 36
Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Ser Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60
Arg Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Asn Trp Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly
100 105 110
Thr Leu 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 Arg 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 Glu Glu Met 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 Met 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> 37
<211> 1347
<212> DNA
<213> Artificial Sequence
<220>
<223> HUR33 humanized antibody mutant heavy chain nucleotide sequence
<400> 37
caggtgcagc tggtgcagag cggcagcgaa ctgaaaaaac cgggcgcgag cgtgaaagtg 60
agctgcaaag cgagcggcta tacctttacc gattatggca tgaactgggt gcgccaggcg 120
ccgggccagg gcctggaatg gatgggctgg attaacacct atagcggcga accgacctat 180
gcggatgatt ttcgcggccg ctttgtgttt agcctggata ccagcgtgag caccgcgtat 240
ctgcagatta gcagcctgaa agcggaagat accgcggtgt attattgcgc gcgcggcggc 300
aactgggatt ggtattttga tgtgtggggc cagggcaccc tggtgaccgt gagcagcgcg 360
agcaccaaag gcccgagcgt gtttccgctg gcgccgagca gcaaaagcac cagcggcggc 420
accgcggcgc tgggctgcct ggtgaaagat tattttccgg aaccggtgac cgtgagctgg 480
aacagcggcg cgctgaccag cggcgtgcat acctttccgg cggtgctgca gagcagcggc 540
ctgtatagcc tgagcagcgt ggtgaccgtg ccgagcagca gcctgggcac ccagacctat 600
atttgcaacg tgaaccataa accgagcaac accaaagtgg ataaacgcgt ggaaccgaaa 660
agctgcgata aaacccatac ctgcccgccg tgcccggcgc cggaactgct gggcggcccg 720
agcgtgtttc tgtttccgcc gaaaccgaaa gataccctgt atattacccg cgaaccggaa 780
gtgacctgcg tggtggtgga tgtgagccat gaagatccgg aagtgaaatt taactggtat 840
gtggatggcg tggaagtgca taacgcgaaa accaaaccgc gcgaagaaca gtataacagc 900
acctatcgcg tggtgagcgt gctgaccgtg ctgcatcagg attggctgaa cggcaaagaa 960
tataaatgca aagtgagcaa caaagcgctg ccggcgccga ttgaaaaaac cattagcaaa 1020
gcgaaaggcc agccgcgcga accgcaggtg tataccctgc cgccgagccg cgaagaaatg 1080
accaaaaacc aggtgagcct gacctgcctg gtgaaaggct tttatccgag cgatattgcg 1140
gtggaatggg aaagcaacgg ccagccggaa aacaactata aaaccacccc gccggtgctg 1200
gatagcgatg gcagcttttt tctgtatagc aaactgaccg tggataaaag ccgctggcag 1260
cagggcaacg tgtttagctg cagcgtgatg catgaagcgc tgcataacca ttatacccag 1320
aaaagcctga gcctgagccc gggcaaa 1347
<210> 38
<211> 1347
<212> DNA
<213> Artificial Sequence
<220>
<223> HUR33 humanized antibody mutant heavy chain nucleotide sequence
<400> 38
caggtgcagc tggtgcagag cggcagcgaa ctgaaaaaac cgggcgcgag cgtgaaagtg 60
agctgcaaag cgagcggcta tacctttacc gattatggca tgaactgggt gcgccaggcg 120
ccgggccagg gcctggaatg gatgggctgg attaacacct atagcggcga accgacctat 180
gcggatgatt ttcgcggccg ctttgtgttt agcctggata ccagcgtgag caccgcgtat 240
ctgcagatta acagcctgaa agcggaagat accgcggtgt attattgcgc gcgcggcggc 300
aactgggatt ggtattttga tgtgtggggc cagggcaccc tggtgaccgt gagcagcgcg 360
agcaccaaag gcccgagcgt gtttccgctg gcgccgagca gcaaaagcac cagcggcggc 420
accgcggcgc tgggctgcct ggtgaaagat tattttccgg aaccggtgac cgtgagctgg 480
aacagcggcg cgctgaccag cggcgtgcat acctttccgg cggtgctgca gagcagcggc 540
ctgtatagcc tgagcagcgt ggtgaccgtg ccgagcagca gcctgggcac ccagacctat 600
atttgcaacg tgaaccataa accgagcaac accaaagtgg ataaacgcgt ggaaccgaaa 660
agctgcgata aaacccatac ctgcccgccg tgcccggcgc cggaactgct gggcggcccg 720
agcgtgtttc tgtttccgcc gaaaccgaaa gataccctgt atattacccg cgaaccggaa 780
gtgacctgcg tggtggtgga tgtgagccat gaagatccgg aagtgaaatt taactggtat 840
gtggatggcg tggaagtgca taacgcgaaa accaaaccgc gcgaagaaca gtataacagc 900
acctatcgcg tggtgagcgt gctgaccgtg ctgcatcagg attggctgaa cggcaaagaa 960
tataaatgca aagtgagcaa caaagcgctg ccggcgccga ttgaaaaaac cattagcaaa 1020
gcgaaaggcc agccgcgcga accgcaggtg tataccctgc cgccgagccg cgaagaaatg 1080
accaaaaacc aggtgagcct gacctgcctg gtgaaaggct tttatccgag cgatattgcg 1140
gtggaatggg aaagcaacgg ccagccggaa aacaactata aaaccacccc gccggtgctg 1200
gatagcgatg gcagcttttt tctgtatagc aaactgaccg tggataaaag ccgctggcag 1260
cagggcaacg tgtttagctg cagcgtgatg catgaagcgc tgcataacca ttatacccag 1320
aaaagcctga gcctgagccc gggcaaa 1347
<210> 39
<211> 214
<212> PRT
<213> Artificial Sequence
<220>
<223> HUR33 humanized antibody mutant light chain amino acid sequence
<400> 39
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 Ser Gln Ser Val Ser Asn Phe
20 25 30
Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Ala Ser Gln Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Thr Trp Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu 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> 40
<211> 642
<212> DNA
<213> Artificial Sequence
<220>
<223> HUR33 humanized antibody mutant light chain nucleotide sequence
<400> 40
gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc 60
attacctgcc gcgcgagcca gagcgtgagc aactttctgc attggtatca gcagaaaccg 120
ggcaaagcgc cgaaactgct gatttattat gcgagccaga gcattagcgg cgtgccgagc 180
cgctttagcg gcagcggcag cggcaccgat tttaccctga ccattagcag cctgcagccg 240
gaagattttg cgacctatta ttgccagcag agcaacacct ggccgctgac ctttggccag 300
ggcaccaaac tggaaattaa acgcaccgtg gcggcgccga gcgtgtttat ttttccgccg 360
agcgatgaac agctgaaaag cggcaccgcg agcgtggtgt gcctgctgaa caacttttat 420
ccgcgcgaag cgaaagtgca gtggaaagtg gataacgcgc tgcagagcgg caacagccag 480
gaaagcgtga ccgaacagga tagcaaagat agcacctata gcctgagcag caccctgacc 540
ctgagcaaag cggattatga aaaacataaa gtgtatgcgt gcgaagtgac ccatcagggc 600
ctgagcagcc cggtgaccaa aagctttaac cgcggcgaat gc 642
<210> 41
<211> 330
<212> PRT
<213> Homo sapiens
<400> 41
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly 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 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
Arg 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 Met Ile Ser Arg Thr 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 Met 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> 42
<211> 108
<212> PRT
<213> Homo sapiens
<400> 42
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Ser
100 105

Claims (34)

1. An anti-SARS-Cov-2 antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2 and HCDR3 comprised by the heavy chain variable region as shown in SEQ ID No. 1, and LCDR1, LCDR2 and LCR3 comprised by the light chain variable region as shown in SEQ ID No. 2;
according to IMGT numbering system, the antibody comprises:
HCDR1, which consists of the sequence shown in SEQ ID NO. 3,
HCDR2, which consists of the sequence shown in SEQ ID NO. 4, and
HCDR3, which consists of the sequence shown in SEQ ID NO. 5,
and the antibody further comprises:
LCDR1, which consists of the amino acid sequence shown in SEQ ID NO. 6,
LCDR2, which consists of the amino acid sequence shown in SEQ ID NO:7, and
LCDR3, which consists of the amino acid sequence shown in SEQ ID NO. 8,
the antigen binding fragment is selected from the group consisting of Fab, fab ', F (ab') 2 Fd, fv, dAb, fab/c, complementarity determining region fragments, single chain antibodies, diabodies or domain antibodies.
2. The anti-SARS-CoV-2 antibody or antigen binding fragment thereof according to claim 1, wherein said anti-SARS-CoV-2 antibody further comprises heavy chain FR1 shown in SEQ ID No. 17, heavy chain FR2 shown in SEQ ID No. 18, heavy chain FR3 shown in SEQ ID No. 19, SEQ ID No. 31 or SEQ ID No. 32, heavy chain FR4 shown in SEQ ID No. 20, light chain FR1 shown in SEQ ID No. 21, light chain FR2 shown in SEQ ID No. 22, light chain FR3 shown in SEQ ID No. 23 and light chain FR4 shown in SEQ ID No. 24.
3. The anti-SARS-CoV-2 antibody or antigen-binding fragment thereof of claim 1 or 2, wherein said anti-SARS-CoV-2 antibody comprises:
a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO. 9, and
a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO. 10.
4. The anti-SARS-Cov-2 antibody or antigen binding fragment thereof of claim 1 or 2, wherein said anti-SARS-Cov-2 antibody further comprises a heavy chain constant region and a light chain constant region of human IgG 1.
5. The anti-SARS-Cov-2 antibody or antigen binding fragment thereof as specified in claim 4 wherein the heavy chain constant region sequence of human IgG1 is as depicted by SEQ ID NO. 41.
6. The anti-SARS-Cov-2 antibody or antigen binding fragment thereof as specified in claim 4 wherein the human IgG1 light chain constant region sequence is set forth in SEQ ID NO. 42.
7. The anti-SARS-Cov-2 antibody or antigen-binding fragment thereof according to claim 4, wherein the heavy chain amino acid sequence of said anti-SARS-Cov-2 antibody is shown in SEQ ID NO. 11 and the light chain amino acid sequence is shown in SEQ ID NO. 12.
8. The anti-SARS-Cov-2 antibody or antigen-binding fragment thereof of claim 4 wherein the heavy chain nucleotide sequence of said anti-SARS-Cov-2 antibody is shown in SEQ ID NO. 13 and the light chain nucleotide sequence is shown in SEQ ID NO. 14.
9. The anti-SARS-Cov-2 antibody or antigen-binding fragment thereof of claim 1, wherein the single chain antibody is an scFv.
10. A polynucleotide molecule comprising nucleotide sequences encoding the heavy chain variable region and the light chain variable region of the anti-SARS-Cov-2 antibody or antigen binding fragment thereof according to any one of claims 1 to 9.
11. A vector comprising the polynucleotide molecule of claim 10.
12. A host cell comprising the polynucleotide molecule of claim 10, or the vector of claim 11.
13. A method of preparing an anti-SARS-Cov-2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 comprising the steps of culturing the host cell of claim 12 under suitable conditions and recovering said anti-SARS-Cov-2 antibody or antigen-binding fragment thereof from the cell culture.
14. An antibody conjugate comprising the anti-SARS-Cov-2 antibody or antigen-binding fragment thereof of any one of claims 1-9, and a conjugate moiety conjugated to the anti-SARS-Cov-2 antibody or antigen-binding fragment thereof, the conjugate moiety being a purification tag, a cytotoxic agent, or a detectable label.
15. The antibody conjugate of claim 14, wherein the purification tag is a His tag.
16. The antibody conjugate of claim 14, wherein the conjugate moiety is a luminescent substance or polyethylene glycol.
17. The antibody conjugate of claim 14, wherein the conjugate moiety is a radioisotope.
18. The antibody conjugate of claim 14, wherein the conjugate moiety is an enzyme.
19. The antibody conjugate of claim 14, wherein the conjugate moiety is a colored substance.
20. A multispecific antibody comprising an anti-SARS-Cov-2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, and an antibody or antigen-binding fragment directed against another antigen and/or another epitope.
21. The multispecific antibody of claim 20 which is a bispecific antibody.
22. A fusion protein comprising the anti-SARS-Cov-2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.
23. A kit comprising the anti-SARS-Cov-2 antibody or antigen binding fragment thereof according to any one of claims 1 to 9 or the antibody conjugate according to any one of claims 14 to 19, the multispecific antibody according to claim 20 or 21 or the fusion protein according to claim 22.
24. The kit of claim 23, further comprising a second antibody that specifically recognizes the anti-SARS-Cov-2 antibody or antigen binding fragment thereof.
25. The kit of claim 24, wherein the second antibody further comprises a detectable label.
26. The kit of claim 25, wherein the detectable label is a radioisotope.
27. The kit of claim 25, wherein the detectable label is a luminescent substance or polyethylene glycol.
28. The kit of claim 25, wherein the detectable label is a colored substance.
29. The kit of claim 25, wherein the detectable label is an enzyme.
30. Use of the anti-SARS-Cov-2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the antibody conjugate according to any one of claims 14 to 19, the multispecific antibody according to claim 20 or 21 or the fusion protein according to claim 22 in the preparation of a kit for detecting the presence or level of human SARS-Cov-2 in a sample.
31. A pharmaceutical composition comprising the anti-SARS-Cov-2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the antibody conjugate according to any one of claims 14 to 19, the multispecific antibody according to claim 20 or 21, or the fusion protein according to claim 22; optionally, it further comprises a pharmaceutically acceptable carrier and/or excipient.
32. Use of an anti-SARS-Cov-2 antibody or antigen binding fragment thereof according to any one of claims 1 to 9, an antibody conjugate according to any one of claims 14 to 19, a multispecific antibody according to claim 20 or 21 or a fusion protein according to claim 22 in the manufacture of a medicament for the treatment of a disease caused by SARS-Cov-2.
33. The use of claim 32, wherein the medicament is in a form suitable for injection.
34. The use of claim 33, wherein the medicament is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, or intralesional injection.
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CN111732655B (en) * 2020-07-01 2021-10-22 中国人民解放军军事科学院军事医学研究院 RBD-targeted high-neutralization-activity anti-SARS-CoV-2 fully-humanized monoclonal antibody and application thereof
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