CN112225806B - Neutralizing active monoclonal antibody of human source for resisting novel coronavirus (SARS-CoV-2) - Google Patents

Neutralizing active monoclonal antibody of human source for resisting novel coronavirus (SARS-CoV-2) Download PDF

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CN112225806B
CN112225806B CN202011265546.1A CN202011265546A CN112225806B CN 112225806 B CN112225806 B CN 112225806B CN 202011265546 A CN202011265546 A CN 202011265546A CN 112225806 B CN112225806 B CN 112225806B
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李亚峰
段琦
刘星玮
辛晓红
冯嘉炳
平鑫博
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Abstract

The invention provides a novel human coronavirus neutralizing active antibody and application thereof. The invention separates and screens the monoclonal antibody with neutralizing activity from the body of a rehabilitation patient infected by the new coronavirus, and the antibody has excellent affinity and specificity to the new coronavirus (SARS-CoV-2) and can effectively block the combination of the new coronavirus and a receptor protein.

Description

Neutralizing active monoclonal antibody of human source for resisting novel coronavirus (SARS-CoV-2)
Technical Field
The invention relates to the technical field of antibodies, in particular to a novel humanized coronavirus (SARS-CoV-2) neutralizing antibody.
Background
The novel coronavirus is a novel coronavirus of beta genus, and has envelope, round or elliptical particle, usually polymorphism, and diameter of 60-140 nm. The whole genome comparison shows that the homology of the novel coronavirus and severe acute respiratory syndrome virus (SARS-CoV) reaches 70%, and the sequence difference is mainly reflected in a key spike gene (coding S-protein) which acts with a host cell.
Pneumonia symptoms such as fever, hypodynamia, dry cough and the like are usually shown after the novel coronavirus infection, and a few patients are accompanied with upper respiratory tract and digestive tract symptoms such as nasal obstruction, watery nasal discharge, diarrhea and the like. Severe cases often develop dyspnea after 1 week, and severe cases rapidly progress to acute respiratory distress syndrome, septic shock, uncorrectable metabolic acidosis, and hemorrhagic coagulation dysfunction. Although some therapeutic drugs are reported at present, more clinical practices are needed to prove the effect, and the development of related vaccines is also under development, but the time is needed for clinical application.
The neutralizing antibody is a specific immunoglobulin for targeting pathogens and blocking pathogen invading cells, and the neutralizing antibody capable of screening novel coronavirus is the basis for developing new coronavirus therapeutic drugs
The invention is provided in view of the above.
Disclosure of Invention
The primary object of the present invention is to find a neutralizing monoclonal antibody which is effective in blocking the binding of the novel coronavirus (SARS-CoV-2) to the receptor protein.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides an isolated antibody or fragment thereof, wherein the antibody or fragment thereof specifically binds to a human novel coronavirus (SARS-CoV-2) RBD protein, the antibody or fragment thereof comprising a VH CDR1-3 as set forth in SEQ ID nos. 1-3, and a VL CDR1-3 as set forth in SEQ ID nos. 4-6; or the antibody or fragment thereof comprises a VH CDR1-3 as shown in SEQ ID Nos. 11-13 and a VL CDR1-3 as shown in SEQ ID Nos. 14-16. .
In some embodiments, the antibody or fragment thereof further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.
In some embodiments, the antibody or fragment thereof comprises a heavy chain variable region consisting of SEQ ID NO: 7-8 or 17-18; or with SEQ ID NO: 7-8 or 17-18, such as 70%, 80% or 90% homologous amino acid sequences.
In some preferred embodiments, the 70% homology is to the Fr region of the antibody or fragment thereof.
In some embodiments, the antibody or fragment thereof is of one of the isotypes of IgG, IgM, IgA, IgE or IgD, and in some particular embodiments, the antibody or fragment thereof is of the IgG4 subtype.
In some embodiments, the antibody or fragment thereof is a chimeric, humanized, fully human antibody, or the like.
In some embodiments, the antibody or fragment thereof is a Fab, Fab ', and F (ab') 2, Fvs, or single chain antibody.
In some embodiments, the antibody or fragment thereof comprises amino acid sequence modifications including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modifications.
The present invention also provides a polynucleotide encoding the above antibody or a fragment thereof.
In some embodiments, the polynucleotide sequence is set forth in SEQ ID nos. 9-10 or 19-20.
The present invention also provides a composition comprising the above antibody or fragment thereof.
In some embodiments, the composition is a pharmaceutical composition; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
The invention also provides a complex to which the above antibody or a fragment thereof is attached;
in some embodiments, the complex is a dual-antibody complex, which can be linked to another antibody sequence; preferably, the complex further comprises a pharmaceutically acceptable carrier.
The invention also provides an isolated cell comprising one or more polynucleotides encoding the antibody or fragment thereof described above.
The invention also provides a screening method comprising contacting the antibody or fragment thereof of claim with another solution, and performing screening based on the contacting.
The invention also provides a method of detection comprising contacting the antibody or fragment thereof of claim with another solution, and detecting based on the contacting.
In some preferred embodiments, the detection includes, but is not limited to, ELISA, WB, IP, IHC, CHIP, IP, or CBA detection.
The invention also provides application of the antibody or the fragment thereof in preparing medicines for diagnosing or treating diseases caused by the new coronavirus.
Compared with the prior art, the method for extracting, separating and purifying the trametes acid has the following beneficial effects:
1) the human monoclonal antibody prepared by the invention can be specifically combined with a novel coronavirus, and has high affinity.
2) The human anti-novel coronavirus antibody prepared by the invention can effectively block the combination of novel coronavirus and receptor protein, and is an effective neutralizing active monoclonal antibody.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 electrophorogram prepared from Spike protein;
FIG. 2 is a graph of the flow cytometric sorting results of antibody screening;
FIG. 3 partial results of heavy chain gene amplification gel run identification;
FIG. 4 partial results of light chain gene amplification gel run identification;
figure 5 antibody EC50 test values;
figure 6 antibody IC50 test values.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The following terms or definitions are provided only to aid in understanding the present invention. These definitions should not be construed to have a scope less than understood by those skilled in the art.
Unless defined otherwise below, all technical and scientific terms used in the detailed description of the present invention are intended to have the same meaning as commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.
As used herein, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If in the following a certain group is defined to comprise at least a certain number of embodiments, this should also be understood as disclosing a group which preferably only consists of these embodiments.
Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun.
The terms "about" and "substantially" in the present invention denote an interval of accuracy that can be understood by a person skilled in the art, which still guarantees the technical effect of the feature in question. The term generally denotes a deviation of ± 10%, preferably ± 5%, from the indicated value.
Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
The term "isolated" as used herein with respect to cells, nucleic acids, e.g., "isolated antibody or fragment thereof" refers to a molecule that is separated from other antibodies or fragments thereof, respectively, that are present in a natural source; it may also refer to nucleic acids or peptides that are substantially free of cellular material, viral material, or cell culture media when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Furthermore, "isolated" is intended to include not occurring in the natural state, and not occurring in the natural state. The term "isolated" is also used herein to refer to cells, polypeptides, or nucleic acids, etc., that are isolated from other cellular proteins or tissues. Isolated antibodies are intended to include both purified and recombinant polypeptides.
A polynucleotide consists of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and thymine (U) when the polynucleotide is RNA. Thus, the term "polynucleotide sequence" is a letter representation of a polynucleotide molecule. The alphabetical representation can be entered into a database in a computer having a central processing unit and used for bioinformatics applications, such as for functional genomics and homology searches. The term "polymorphism" refers to the coexistence of more than one form of a gene or a part thereof, and a part of a gene having at least two different forms (i.e., two different nucleotide sequences) is referred to as "polymorphic region of a gene". Polymorphic regions may be single nucleotides, which have different identities in different alleles.
The term "encoding" as applied to a polynucleotide refers to a polynucleotide that is said to "encode" a polypeptide, which if in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and/or translated to produce mRNA for the polypeptide and/or fragments thereof. The antisense strand is the complement of such a nucleic acid, from which the coding sequence can be deduced.
In the present invention, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. The antibody may be a whole antibody and any antigen binding fragment thereof or a single chain thereof. The term "antibody" thus includes protein-containing or peptide-containing molecules that contain at least a portion of an immunoglobulin molecule having biological activity that binds an antigen. This embodiment, including but not limited to, includes Complementarity Determining Regions (CDRs) of a heavy or light chain or ligand binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a Framework (FR) region or any portion thereof, or at least a portion of a binding protein.
In the present invention, the term "fragment" or "antigen-binding fragment" is a part of an antibody, such as F (ab ') 2, F (ab)2, Fab', Fab, Fv, scFv, and the like. Regardless of their structure, fragments bind to the same antigen that is recognized by an intact antibody. The term "fragment" includes aptamers, spiegelmers, and diabodies. The term "fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by forming a complex with a particular antigen.
In the present invention, the term "single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin. In some aspects, these regions are linked to a short linker peptide of 10 to about 25 amino acids. The linker may be glycine rich to increase flexibility and serine or threonine rich to increase solubility and may link the N-terminus of VH and the C-terminus of VL, or vice versa. Although the protein has the constant region removed and the linker introduced, it retains the specificity of the original immunoglobulin. ScFv molecules are known in the art and are described in, for example, U.S. Pat. No. 5,892,019.
The term "antibody" includes a wide variety of polypeptides that can be biochemically distinguished. Those skilled in the art will appreciate that the class of heavy chains includes gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses (e.g., γ 1- γ 4). The nature of this chain determines the "class" of the antibody as IgG, IgM, IgA, IgG or IgE, respectively. The immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, and the like, have been well characterized and the functional specificities conferred are also known. Each of these classes and isotypes will readily occur to those of ordinary skill in the art and are therefore within the scope of the present disclosure, and all immunoglobulin classes are clearly within the scope of the present disclosure, with the following discussion generally directed to IgG classes of immunoglobulin molecules. With respect to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides having a molecular weight of about 23,000 daltons and two identical heavy chain polypeptides having a molecular weight of about 53,000 and 70,000. These four chains are typically linked in a "Y" configuration by disulfide bonds, with the light chain beginning at the "Y" mouth and continuing through the variable region surrounding the heavy chain.
The antibodies, antigen binding polypeptides, fragments disclosed herein include, but are not limited to, monoclonal, fully human, humanized or chimeric antibodies, single chain antibodies, epitope binding fragments such as Fab, Fab 'and F (ab') 2, Fd, Fvs, single chain Fvs (scFv), single chain antibodies, disulfide linked Fvs (sdFv), fragments comprising a Fab or VH domain, fragments produced from a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including anti-Id antibodies such as the LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules disclosed herein may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin.
Light chains can be classified as kappa or lambda (. kappa.,. lamda.). Each heavy chain may be associated with a kappa or lambda light chain. In general, when immunoglobulins are produced by hybridomas, B cells or genetically engineered host cells, the light and heavy chains are joined by covalent bonds and the "tail" portions of the two heavy chains are joined by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the forked end of the Y configuration to the C-terminus of the bottom of each chain.
Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used in accordance with function. It is understood that the variable regions of the light (V κ) and heavy (VH) chain portions determine antigen recognition and specificity. In contrast, the constant regions of the light (CK) and heavy (CH1, CH2, or CH3) chains confer important biological properties, such as secretion, transplacental movement, Fc receptor binding, complement fixation, and the like. The N-terminal part is a variable region and the C-terminal part is a constant region; the CH3 and CK domains actually comprise the carboxy-termini of the heavy and light chains, respectively.
As described above, the variable regions enable the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the VK domain and VH domain of the antibody, or a subset of the Complementarity Determining Regions (CDRs), combine to form variable regions that define a three-dimensional antigen-binding site. The antibody quaternary structure forms an antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three CDRs in each of the VH and VK chains (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3). In certain cases, such as certain camelid derived or engineered immunoglobulin molecules based on camelid immunoglobulins, the intact immunoglobulin molecule may consist of only heavy chains, without light chains. See, e.g., camera-Casterman et al, Nature 363: 446-448(1993).
CDRs defined according to Kabat and Chothia include overlaps or subsets of amino acid residues when compared to each other. Nevertheless, it is within the scope of the terms defined and used herein to apply either definition to refer to the CDRs of an antibody or variant thereof. The exact residue number comprising a particular CDR will vary depending on the sequence and size of the CDR. One skilled in the art can routinely determine which residues comprise a particular CDR based on the amino acid sequence of the variable region of an antibody.
In the present invention, the term "heavy chain constant region" includes amino acid sequences derived 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, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment. For example, the antigen binding polypeptides disclosed herein can include a polypeptide chain comprising a CH1 domain, a polypeptide chain comprising a CH1 domain and at least a portion of a hinge region and a CH2 domain, a polypeptide chain comprising a CH1 domain and a CH3 domain, a polypeptide chain comprising a CH1 domain, at least a portion of a hinge region, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge region, and a CH2 domain and a CH3 domain. In another embodiment, the disclosed polypeptides include a polypeptide chain comprising a CH3 domain. Furthermore, an antibody used in the invention may lack at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). As described above, it will be appreciated by those of ordinary skill in the art that the heavy chain constant regions may be modified such that the amino acid sequence of their naturally occurring immunoglobulin molecules is altered.
In the present invention, the term "light chain constant region" includes amino acid sequences from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain.
In the present invention, the term "specifically binds" or "specific for … …" generally refers to an antibody that binds to an epitope through its antigen binding domain, and that binding requires complementarity between the antigen binding domain and the epitope. According to this definition, an antibody, when bound to the epitope via its antigen binding domain, binds to the epitope more readily than it binds to a random, unrelated epitope, which is said to "specifically bind" to the epitope. The term "specificity" is used herein to define the relative affinity of an antibody for binding to an epitope. For example, antibody "a" can be considered to have a higher specificity for a particular epitope than antibody "B", or antibody "a" can be considered to bind epitope "C" with a higher specificity than to bind the relevant epitope "D".
In the present invention, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the object of which is to prevent or slow down (lessen) the progression of an undesired physiological change or disorder, such as an autoimmune disease. Beneficial or desired clinical results include, but are not limited to, results, whether detectable or undetectable, including alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total). "treatment" also means an extended life span compared to the life span expected when not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those susceptible to the condition or disorder, or those in need of prevention of the condition or disorder.
Anti-neocoronavirus (SARS-CoV-2) antibody
The invention provides an anti-human new coronavirus (SARS-CoV-2) antibody with high affinity, which is derived from recovered volunteers. The antibodies tested exhibit potent binding and inhibitory activity and are useful for therapeutic and diagnostic purposes. The combination blocks the combination of the new coronavirus RBD protein and a receptor protein ACE 2.
According to one embodiment of the present disclosure, there is provided an antibody comprising heavy and light chain variable domains having CDR regions as defined by sequences SEQ ID nos. 1-6, as shown in the following table.
Figure GDA0002828173300000051
As shown in the experiments in the examples, antibodies containing these CDR regions have potent binding and inhibitory activity against the novel coronavirus RBD protein. In some embodiments, the anti-neocoronavirus RBD protein antibodies disclosed herein comprise a VH and a VL listed in the following table.
Figure GDA0002828173300000052
According to one embodiment of the present disclosure, there is provided an antibody comprising heavy and light chain variable domains having CDR regions as defined by sequences SEQ ID nos. 11-16, as shown in the following table.
Figure GDA0002828173300000053
Figure GDA0002828173300000061
As shown in the experiments in the examples, antibodies containing these CDR regions have potent binding and inhibitory activity against the novel coronavirus RBD protein. In some embodiments, the anti-neocoronavirus RBD protein antibodies disclosed herein comprise a VH and a VL listed in the following table.
Figure GDA0002828173300000062
In some embodiments, the light and heavy chains may include amino acid modifications, which may be additions, deletions, or substitutions of amino acids. The modification may be a substitution in each CDR or FR region. In some embodiments, the modification is a substitution at one, two, or three such residues. In one embodiment, the modification is a substitution at one residue. In some embodiments, such substitutions are conservative substitutions.
"conservative amino acid substitution" refers to a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acids 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, it is preferred to replace a non-essential amino acid residue in an immunoglobulin polypeptide with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a string of amino acids that are structurally similar in order and/or composition to be different in the same side chain family member.
It will also be appreciated by those of ordinary skill in the art that the antibodies disclosed herein may be modified to have an amino acid sequence that differs from the amino acid sequence of the naturally occurring binding polypeptide from which the antibody is derived. For example, a polypeptide or amino acid sequence derived from the same given protein may be similar to the starting sequence, e.g., have a certain percent identity, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% percent identical to the starting sequence.
In some embodiments, the antibodies and fragments thereof of the present disclosure can be monospecific or bispecific antibodies or fragments. For bispecific antibodies, the other specificity may be for a different epitope of interest or a different target protein that may be used for a particular use (e.g., therapeutic use).
In some embodiments, the antibody comprises an amino acid sequence or one or more groups that are not normally associated with an antibody. For example, the antibody may comprise a flexible linker sequence, or may be modified to add a functional moiety (e.g., PEG, drug, toxin, or tag).
In some embodiments, the antibody may include glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modifications, and the like.
Polynucleotides encoding the antibodies and methods of making the antibodies.
Isolated polynucleotides or nucleic acid molecules encoding the antibodies, variants, and derivatives thereof of the invention are also disclosed. The polynucleotides disclosed herein may encode the entire heavy and light chain variable regions of the antigen-binding polypeptide, variant or derivative on the same polynucleotide molecule or on different polynucleotide molecules. In addition, the polynucleotides disclosed herein may encode portions of the heavy and light chain variable regions of the antigen-binding polypeptide, variant or derivative on the same polynucleotide molecule or on different polynucleotide molecules.
In some embodiments, the anti-neocoronavirus RBD protein-encoding antibodies disclosed herein comprise VH and VL polynucleotide sequences listed in the following table.
Figure GDA0002828173300000071
In some embodiments, the anti-neocoronavirus RBD protein-encoding antibodies disclosed herein comprise VH and VL polynucleotide sequences listed in the following table.
Figure GDA0002828173300000072
Methods for making antibodies are well known in the art. In certain embodiments, the variable and constant regions of the antibodies disclosed herein are all human and can be prepared using recombinant DNA techniques that are conventional in the art.
Diagnostic treatment methods and uses
In the present invention, the antibodies, variants or derivatives of the invention are useful in certain diagnostic and therapeutic methods. It is expected that the neutralizing antibody of the present invention can be used for preventing or treating diseases caused by a novel coronavirus, and specifically can inhibit the binding of the novel coronavirus to an in vivo receptor by neutralizing activity, thereby preventing viral infection and exerting disease preventing and ameliorating effects. In addition, the neutralizing antibody of the present invention can be applied to the detection or diagnosis of a novel coronavirus based on an antigen-antibody assembly reaction, for example, by ELISA, WB, IP, IHC, CHIP, IP, CBA, or the like.
The following are specific example experiments.
Example 1 antigen preparation
Cloning, expression and purification of Spike antigens of novel coronaviruses
Obtaining a sequence of the new coronavirus Spike protein from NCBI, constructing into a pcDNA3.1 vector through codon optimization, obtaining transfectable plasmids from the vector by using an EndoFree Plasmid Kit endotoxin-free Plasmid extraction Kit of Qiagen, transiently transfecting 293T cells by using lipo3000, transiently expressing 7d at 37 ℃, harvesting cell expression supernatant, and carrying out affinity purification by using a Ni column to obtain the new coronavirus Spike protein with the purity of more than 80%, wherein the purification result is shown in figure 1.
Example 2 antibody isolation screening
The prepared Spike antigen is marked with FITC fluorescent dye and then purified to obtain S-FITC; separating peripheral blood lymphocytes from peripheral blood of a recovered volunteer, and enriching B cells by a magnetic bead scheme; staining B cells by utilizing anti-IgG PE, anti-IgM PerCP-Cy5.5 and S-FITC, and directly sorting IgG + IgM-S + group new coronavirus S antigen specific memory B cell subsets into a 96-hole PCR plate added with cell lysate by flow sorting, wherein each hole is provided with one cell.
Magnetic bead enrichment sorting of single cells into 96-well plates: magnetic bead manufacturer stemcell
1. Cell recovery: frozen recovered PBMC cells and healthy human PBMC cells were cultured in 1 cell each (cell count 1 x 10)7cells) was rapidly thawed at 37 degrees, frozen cells were added to 8ml buffer1 (PBS + 2% FBS) pre-warmed at 37 degrees, and centrifuged at 500g for 5 min;
2. resuspend with 10ml buffer1, filter with 40 μm cell sieve, centrifuge at 500g for 5min, sample and count;
3. the magnetic bead-passed healthy and recovered human specimens were resuspended in 5X 10 by adding buffer2(PBS + 2% FBS +1nM EDTA)7The concentration of/ml, the volume is between 0.25 and 2ml, and the mixture is transferred into a flow tube;
4. adding Cocktail Enhancer according to the amount of 50 mu L/mL of sample;
5. adding the Isolation Cocktail in an amount of 50. mu.L/mL of sample;
6. mixing, and incubating at room temperature for 5 min;
7. mixing Vortex RapidSpheresTMVortex for 30s and add RapidSpheres in an amount of 50. mu.L/mL of sampleTMMixing uniformly;
8. immediately adding buffer to 2.5ml volume, reversing the upper part and the lower part, uniformly mixing for 2-3 times, putting the tube into a magnetic frame, and incubating for 3min at room temperature;
9. pouring the supernatant into a new flow tube, placing the new flow tube into a magnetic frame, and incubating at room temperature for 1 min;
serial number Flow antibody
1 blank (without any antibody) Healthy human specimens, however, magnetic beads, 1X 106/ml，100μl
2 IgG(PE) Healthy human specimens, however, magnetic beads, 1X 106/ml，100μl
3 S protein(FITC) Healthy human specimens, however, magnetic beads, 1X 106/ml，100μl
4 IgM(PerCP-Cy5.5) Healthy human specimens, however, magnetic beads, 1X 106/ml，100μl
5 IgG+IgM Healthy human sample, Permagnetic bead, 1X 106/ml，100μl
6 IgG+IgM+S protein Healthy human sample, Permagnetic bead, 1X 106/ml，100μl
7 S protein(AF488) Samples of rehabilitees, not magnetic beads
8 IgG+IgM The sample of the rehabilitee is processed by magnetic beads,1×106/ml，100μl
9 IgG+IgM+S protein rehabilitative sample, Perbeads, 1X 106/ml，100μl
10. Pouring the supernatant into a new flow tube, sampling and counting;
11.500 g, centrifuging for 5min, resuspending the recovered cells with buffer1, adding IgG, IgM, and S proteins (IgG, IgM: 3. mu.l/106 cells, S protein: 1. mu.g/106 cells, respectively, at this time, the volume is increased, but all are increased in equal proportion) according to the cell number;
reacting at 12.4 ℃ for 40min, adding 1ml of buffer and 500g of the mixture respectively, and centrifuging for 5 min;
13. washing is repeated for one time;
14. resuspending the cells to 5X 10 according to the cell numbers in steps 12 and 136Density per ml, on-machine sorting, BD FACSAria III, pure format, sorting into 96-well plates (lysate components from vazyme N711 kit have been added), for a total of 3 plates.
The flow sorting results are shown in FIG. 2, which circles out the B cell population and IgG + IgM-S + cell population, respectively, and screens the target antibody to obtain 23D145 and 23D142 cells.
Example 3 antibody sequence determination
The variable region genes of the light and heavy chains of the antibody of a single B cell in each well were taken by single cell reverse transcription.
The single cell reverse transcription obtains double-chain cDNA according to the steps of a vazyme N711 kit, and the double-chain cDNA is used as a template to regulate light and heavy chain genes of a single B cell antibody, wherein the system comprises the following steps: 25 ul vazyme P515, 2 ul of each of the upstream and downstream primers, 1 ul of template cDNA, and 20 ul of DDW, and the PCR conditions are 95 ℃, 3min, 95 ℃, 15s, 60 ℃, 15s, 72 ℃, 30s, 72 ℃ and 7 min. Through running glue identification, the positive rate of antibody gene PCR is more than 85%.
FIG. 3 shows the result of the heavy chain gene amplification part, and FIG. 4 shows the result of the light chain gene amplification part. Finally obtaining the variable region sequence of the antibody 23D145 gene from the recovered volunteers, as shown in SEQ ID NO.7-8, the CDR sequence as shown in SEQ ID NO.1-6, and the coding polynucleotide sequence as shown in SEQ ID NO. 9-10; the antibody 23D142 gene variable region sequence is shown as SEQ ID NO.17-18, the CDR sequence is shown as SEQ ID NO.11-16, and the coding polynucleotide sequence is shown as SEQ ID NO. 19-20.
Example 4 antibody protein preparation
The method comprises the steps of respectively obtaining CMV and polyA fragments by taking a PEE12.4 vector as a template, obtaining an IgG4-CH-polyA fragment by taking a constructed PEE-IgG4-CH vector as a template, respectively obtaining transfectable fragments containing CMV, antibody genes and the polyA fragment by an overlap PCR method, transiently transferring CHO cells in a deep-well plate, expressing for 8 days at 32 ℃, obtaining antibody expression supernatant by centrifugation, and purifying proteinA to obtain antibody protein with the purity of more than 90%.
Example 5 antibody affinity assay
The variable region sequence of each single B cell from recovered volunteers was engineered as IgG4 subtype as in example 4, and the binding ability of the antibodies 23D145 and 23D142 to the new coronavirus RBD antigen was confirmed by ELISA binding assay.
ELISA binding experiments:
1. reagent preparation
2. Preparing a carbonate buffer solution:
Figure GDA0002828173300000091
preparation of 1 × PBST wash:
Figure GDA0002828173300000092
preparation of PBS buffer:
Figure GDA0002828173300000093
blocking solution/antibody dilutions (PBST buffer with 1% BSA)
BSA 1g
PBST 100ml
3. Coating specific antigens
1) Diluting antigen protein (expressed RBD protein) to 0.1-10 ug/ml (coating concentration is 1ug/ml generally, and test selection is required under optimal coating condition), adding diluted antigen into 96-well enzyme label plate (100 ul/well), washing plate with 1 XPBST 2 times at 4 deg.C overnight, 2 min/time, and drying.
2) Preparing a sealing liquid: 1% BSA (w/v) was prepared in PBS buffer.
3) And (3) sealing: adding 200ul of blocking solution into each well, incubating at 37 deg.C for 2h-3h, washing the plate with 1 XPBST for 2 times, 2 min/time, and spin-drying for use.
3. Diluting the antibody to be detected
Diluting the antibody to be detected to 5000ng/ml with an antibody diluent (confining liquid), and fully and uniformly mixing;
diluting with 11 concentration gradients according to 2-time gradients, and adding diluent into the last hole;
adding sample into each 100 μ l well, incubating the ELISA plate at 37 deg.C after adding sample, washing the plate with PBST washing solution for 4 times (2 min/time) after 1h, and spin-drying.
4. Diluted enzyme-labeled secondary antibody
Diluting the enzyme-labeled secondary antibody by 10000 times by using an antibody diluent, adding 100 ul/hole of the diluted enzyme-labeled secondary antibody into the enzyme-labeled plate obtained in the step 2.2.4, incubating for 40min at 37 ℃, washing the plate for 4 times and 2 min/time by using PBST washing liquor, and drying by spinning.
5. Developing color, adding 100ul of TMB developing solution into each hole, and reacting at 37 ℃ for 5-20 min.
6. Stop, stop with 50ul 2M sulfuric acid per well.
7. And (4) measuring the light absorption value of each hole at 450nm by using a microplate reader.
23D145 23D142 Negative 1 Negative 2
EC50(ng/ml) 14.32 14.65 6380 3870
Negative 1 and negative 2 are other existing new crown antibodies respectively, and have certain binding activity with RBD, but do not inhibit the binding of RBD and ACE 2.
The results are shown in fig. 5, with the 23D145 and 23D142 antibodies having the best EC50 values compared to the others, indicating the best antigen binding activity.
Example 6 antibody Competition test
The variable region sequence of each single B cell from recovered volunteers was constructed as IgG4 subtype by genetic engineering method as in example 4, and the blocking ability of antibody 23D145 against the new coronavirus was confirmed by HTRF competition experiment.
HTRF competition experiment
1. RBD-tag1 antigen was diluted to a concentration of 100nM with Diluent buffer (50mM Hepes, 0.1% Tween20, 0.2% BSA, 0.5% o P300, pH 7.0).
2. The ACE2-tag2 antigen was diluted with Diluent buffer to a concentration of 80 nM.
3. Anti-tag1-Eu was diluted with Detection buffer (50mM Hepes, 250mM KF, 0.1% Tween20, 0.2% BSA, 0.5% o P300, pH7.0) to a concentration of 20 nM.
4. Anti-tag2-A2 was diluted with Detection buffer to a concentration of 50 nM.
5. Uniformly mixing the diluted anti-tag1-Eu and anti-tag2-A2 at a ratio of 1: 1.
6. Antibody was diluted with Diluent gradient (initial concentration 5.3 x 10)-7M), 2-fold gradient dilution.
7. Mu.l of the antibody was added to a 384 well plate, 4. mu.l of ACE2-tag2 was added, 4. mu.l of RBD-tag1 was added, and 8. mu.l of the receptor prepared by the 5 th step was added.
8. Incubate at room temperature for 2 h.
9. And (5) detecting by using a microplate reader.
23D145 23D142 Negative 1 Negative 2
IC50(ug/ml) 0.1033 0.1019 Without inhibition Without inhibition
The results are shown in fig. 6, and the activity of the antibodies 23D145 and 23D142 for blocking the binding of the novel coronavirus RBD protein and the receptor protein ACE2 is determined by using HTRF (homogeneous time-resolved fluorescence) methodology, and the results show that both the antibodies 23D145 and 23D142 have significant blocking activity.
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Sequence listing
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Ile Tyr Ser Gly Gly Ser Thr
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Ala Arg Glu Arg Gly Thr Thr Gly Tyr Asp Tyr
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Gln Asp Ile Ser Asn Tyr
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Gly Ala Ser
1
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Gln Gln Tyr Asp Asn Leu Pro Pro Thr
1 5
<210> 7
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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Thr Val Ser Ser Asn
20 25 30
Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Leu Ile Tyr Ser Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Glu Arg Gly Thr Thr Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu
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Val Thr Val Ser Ser
115
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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 Gln Ala Ser Gln Asp Ile Ser Asn Tyr
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Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
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Gly Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Gly Thr Gly Gly
1 5 10 15
Ala Gly Thr Cys Thr Gly Gly Ala Gly Gly Ala Gly Gly Cys Thr Thr
20 25 30
Gly Ala Thr Cys Cys Ala Gly Cys Cys Thr Gly Gly Gly Gly Gly Gly
35 40 45
Thr Cys Cys Cys Thr Gly Ala Gly Ala Cys Thr Cys Thr Cys Cys Thr
50 55 60
Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly Gly Gly Gly Thr
65 70 75 80
Cys Ala Cys Cys Gly Thr Cys Ala Gly Thr Ala Gly Cys Ala Ala Cys
85 90 95
Thr Ala Cys Ala Thr Gly Ala Ala Cys Thr Gly Gly Gly Thr Cys Cys
100 105 110
Gly Cys Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Gly Gly Ala Ala
115 120 125
Gly Gly Gly Gly Cys Thr Gly Gly Ala Ala Thr Gly Gly Gly Thr Cys
130 135 140
Thr Cys Ala Cys Thr Thr Ala Thr Thr Thr Ala Thr Ala Gly Thr Gly
145 150 155 160
Gly Thr Gly Gly Thr Ala Gly Cys Ala Cys Ala Thr Thr Cys Thr Ala
165 170 175
Cys Gly Cys Ala Gly Ala Cys Thr Cys Cys Gly Thr Gly Ala Ala Gly
180 185 190
Gly Gly Cys Cys Gly Ala Thr Thr Cys Ala Cys Cys Ala Thr Cys Thr
195 200 205
Cys Cys Ala Gly Ala Gly Ala Cys Ala Ala Thr Thr Cys Cys Ala Gly
210 215 220
Gly Ala Ala Cys Ala Cys Ala Cys Thr Gly Thr Ala Thr Cys Thr Thr
225 230 235 240
Cys Ala Ala Ala Thr Gly Ala Ala Cys Ala Gly Cys Cys Thr Gly Ala
245 250 255
Gly Ala Gly Cys Cys Gly Ala Ala Gly Ala Cys Ala Cys Gly Gly Cys
260 265 270
Cys Gly Thr Gly Thr Ala Thr Thr Ala Thr Thr Gly Thr Gly Cys Gly
275 280 285
Ala Gly Gly Gly Ala Ala Cys Gly Gly Gly Gly Thr Ala Cys Thr Ala
290 295 300
Cys Thr Gly Gly Thr Thr Ala Thr Gly Ala Cys Thr Ala Cys Thr Gly
305 310 315 320
Gly Gly Gly Cys Cys Ala Gly Gly Gly Ala Ala Cys Cys Cys Thr Gly
325 330 335
Gly Thr Cys Ala Cys Cys Gly Thr Cys Thr Cys Cys Thr Cys Ala
340 345 350
<210> 10
<211> 321
<212> PRT
<213> homo sapiens
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Gly Ala Cys Ala Thr Cys Cys Ala Gly Ala Thr Gly Ala Cys Cys Cys
1 5 10 15
Ala Gly Thr Cys Thr Cys Cys Ala Thr Cys Cys Thr Cys Cys Cys Thr
20 25 30
Gly Thr Cys Thr Gly Cys Ala Thr Cys Thr Gly Thr Gly Gly Gly Ala
35 40 45
Gly Ala Cys Ala Gly Ala Gly Thr Cys Ala Cys Cys Ala Thr Cys Ala
50 55 60
Cys Thr Thr Gly Cys Cys Ala Gly Gly Cys Gly Ala Gly Thr Cys Ala
65 70 75 80
Gly Gly Ala Cys Ala Thr Thr Ala Gly Cys Ala Ala Cys Thr Ala Thr
85 90 95
Thr Thr Ala Ala Ala Thr Thr Gly Gly Thr Ala Thr Cys Ala Gly Cys
100 105 110
Ala Gly Ala Ala Ala Cys Cys Ala Gly Gly Gly Ala Ala Ala Gly Cys
115 120 125
Cys Cys Cys Thr Ala Ala Gly Cys Thr Cys Cys Thr Gly Ala Thr Cys
130 135 140
Thr Ala Cys Gly Gly Thr Gly Cys Ala Thr Cys Cys Ala Ala Thr Thr
145 150 155 160
Thr Gly Gly Ala Ala Thr Cys Ala Gly Gly Gly Gly Thr Cys Cys Cys
165 170 175
Ala Thr Cys Ala Ala Gly Gly Thr Thr Cys Ala Gly Thr Gly Gly Ala
180 185 190
Ala Gly Thr Gly Gly Ala Thr Cys Thr Gly Gly Gly Ala Cys Ala Gly
195 200 205
Ala Thr Thr Thr Thr Ala Cys Thr Thr Thr Cys Ala Cys Cys Ala Thr
210 215 220
Cys Ala Gly Cys Ala Gly Cys Cys Thr Gly Cys Ala Gly Cys Cys Thr
225 230 235 240
Gly Ala Ala Gly Ala Thr Ala Thr Thr Gly Cys Ala Ala Cys Ala Thr
245 250 255
Ala Thr Thr Ala Cys Thr Gly Thr Cys Ala Ala Cys Ala Gly Thr Ala
260 265 270
Thr Gly Ala Thr Ala Ala Thr Cys Thr Cys Cys Cys Thr Cys Cys Gly
275 280 285
Ala Cys Thr Thr Thr Thr Gly Gly Cys Cys Ala Gly Gly Gly Gly Ala
290 295 300
Cys Cys Ala Ala Gly Cys Thr Gly Gly Ala Gly Ala Thr Cys Ala Ala
305 310 315 320
Ala
<210> 11
<211> 8
<212> PRT
<213> homo sapiens
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Gly Leu Thr Val Ser Ser Asn Tyr
1 5
<210> 12
<211> 7
<212> PRT
<213> homo sapiens
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Ile Tyr Ser Gly Gly Ser Thr
1 5
<210> 13
<211> 15
<212> PRT
<213> homo sapiens
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Ala Arg Gly Asp Ser Tyr Asp Asn Tyr Tyr Tyr Gly Leu Asp Val
1 5 10 15
<210> 14
<211> 9
<212> PRT
<213> homo sapiens
<400> 14
Ser Ser Asn Ile Gly Ala Gly Tyr Asp
1 5
<210> 15
<211> 3
<212> PRT
<213> homo sapiens
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Gly Asn Thr
1
<210> 16
<211> 11
<212> PRT
<213> homo sapiens
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Gln Ser Tyr Asp Ser Ser Leu Ser Gly Ser Ile
1 5 10
<210> 17
<211> 121
<212> PRT
<213> homo sapiens
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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Val Ser Ser Asn
20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gly Asp Ser Tyr Asp Asn Tyr Tyr Tyr Gly Leu Asp Val Trp Gly
100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 18
<211> 111
<212> PRT
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Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly
20 25 30
Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
35 40 45
Leu Ile Phe Gly Asn Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu
65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser
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Leu Ser Gly Ser Ile Phe Gly Gly Gly Thr Met Leu Thr Val Leu
100 105 110
<210> 19
<211> 363
<212> PRT
<213> homo sapiens
<400> 19
Gly Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Gly Thr Gly Gly
1 5 10 15
Ala Gly Thr Cys Thr Gly Gly Ala Gly Gly Ala Gly Gly Cys Thr Thr
20 25 30
Gly Ala Thr Cys Cys Ala Gly Cys Cys Thr Gly Gly Gly Gly Gly Gly
35 40 45
Thr Cys Cys Cys Thr Gly Ala Gly Ala Cys Thr Cys Thr Cys Cys Thr
50 55 60
Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly Gly Gly Cys Thr
65 70 75 80
Cys Ala Cys Cys Gly Thr Cys Ala Gly Thr Ala Gly Cys Ala Ala Cys
85 90 95
Thr Ala Cys Ala Thr Gly Ala Gly Cys Thr Gly Gly Gly Thr Cys Cys
100 105 110
Gly Cys Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Gly Gly Ala Ala
115 120 125
Gly Gly Gly Gly Cys Thr Gly Gly Ala Gly Thr Gly Gly Gly Thr Cys
130 135 140
Thr Cys Ala Gly Thr Thr Ala Thr Thr Thr Ala Thr Ala Gly Cys Gly
145 150 155 160
Gly Thr Gly Gly Thr Ala Gly Cys Ala Cys Ala Thr Ala Cys Thr Ala
165 170 175
Cys Gly Cys Ala Gly Ala Cys Thr Cys Cys Gly Thr Gly Ala Ala Gly
180 185 190
Gly Gly Cys Cys Gly Ala Thr Thr Cys Ala Cys Cys Ala Thr Cys Thr
195 200 205
Cys Cys Ala Gly Ala Gly Ala Cys Ala Ala Thr Thr Cys Cys Ala Ala
210 215 220
Gly Ala Ala Cys Ala Cys Gly Cys Thr Gly Thr Thr Thr Cys Thr Thr
225 230 235 240
Cys Ala Ala Ala Thr Gly Ala Ala Cys Ala Gly Cys Cys Thr Gly Ala
245 250 255
Gly Ala Gly Cys Cys Gly Ala Gly Gly Ala Cys Ala Cys Gly Gly Cys
260 265 270
Cys Gly Thr Gly Thr Ala Thr Thr Ala Cys Thr Gly Thr Gly Cys Gly
275 280 285
Ala Gly Gly Gly Gly Gly Gly Ala Cys Ala Gly Cys Thr Ala Thr Gly
290 295 300
Ala Cys Ala Ala Cys Thr Ala Cys Thr Ala Cys Thr Ala Cys Gly Gly
305 310 315 320
Thr Thr Thr Gly Gly Ala Cys Gly Thr Cys Thr Gly Gly Gly Gly Cys
325 330 335
Cys Ala Ala Gly Gly Gly Ala Cys Cys Ala Cys Gly Gly Thr Cys Ala
340 345 350
Cys Cys Gly Thr Cys Thr Cys Cys Thr Cys Ala
355 360
<210> 20
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<213> homo sapiens
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Cys Ala Gly Thr Cys Thr Gly Thr Gly Cys Thr Gly Ala Cys Gly Cys
1 5 10 15
Ala Gly Cys Cys Gly Cys Cys Cys Thr Cys Ala Gly Thr Gly Thr Cys
20 25 30
Thr Gly Gly Gly Gly Cys Cys Cys Cys Ala Gly Gly Gly Cys Ala Gly
35 40 45
Ala Gly Gly Gly Thr Cys Ala Cys Cys Ala Thr Cys Thr Cys Cys Thr
50 55 60
Gly Cys Ala Cys Thr Gly Gly Gly Ala Gly Cys Ala Gly Cys Thr Cys
65 70 75 80
Cys Ala Ala Cys Ala Thr Cys Gly Gly Gly Gly Cys Ala Gly Gly Thr
85 90 95
Thr Ala Thr Gly Ala Thr Gly Thr Ala Cys Ala Cys Thr Gly Gly Thr
100 105 110
Ala Cys Cys Ala Gly Cys Ala Gly Cys Thr Thr Cys Cys Ala Gly Gly
115 120 125
Ala Ala Cys Ala Gly Cys Cys Cys Cys Cys Ala Ala Ala Cys Thr Cys
130 135 140
Cys Thr Cys Ala Thr Cys Thr Thr Thr Gly Gly Thr Ala Ala Thr Ala
145 150 155 160
Cys Cys Ala Ala Thr Cys Gly Gly Cys Cys Cys Thr Cys Ala Gly Gly
165 170 175
Gly Gly Thr Cys Cys Cys Thr Gly Ala Cys Cys Gly Ala Thr Thr Cys
180 185 190
Thr Cys Thr Gly Gly Cys Thr Cys Cys Ala Ala Gly Thr Cys Thr Gly
195 200 205
Gly Cys Ala Cys Cys Thr Cys Ala Gly Cys Cys Thr Cys Cys Cys Thr
210 215 220
Gly Gly Cys Cys Ala Thr Cys Ala Cys Thr Gly Gly Gly Cys Thr Cys
225 230 235 240
Cys Ala Gly Gly Cys Thr Gly Ala Gly Gly Ala Thr Gly Ala Gly Gly
245 250 255
Cys Thr Gly Ala Thr Thr Ala Thr Thr Ala Cys Thr Gly Cys Cys Ala
260 265 270
Gly Thr Cys Cys Thr Ala Thr Gly Ala Cys Ala Gly Cys Ala Gly Cys
275 280 285
Cys Thr Gly Ala Gly Thr Gly Gly Thr Thr Cys Gly Ala Thr Ala Thr
290 295 300
Thr Cys Gly Gly Cys Gly Gly Ala Gly Gly Gly Ala Cys Cys Ala Thr
305 310 315 320
Gly Cys Thr Gly Ala Cys Cys Gly Thr Cys Cys Thr Ala
325 330

Claims (13)

1. An isolated antibody or fragment thereof that specifically binds to a human novel coronavirus (SARS-CoV-2) RBD protein, the antibody or fragment thereof comprising a VH CDR1-3 as set forth in SEQ ID nos. 1-3 and a VL CDR1-3 as set forth in SEQ ID nos. 4-6.
2. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.
3. The antibody or fragment thereof of claim 2, wherein the antibody or fragment thereof comprises a light chain variable region consisting of SEQ ID NO: 7-8; or with SEQ ID NO: 7-8 amino acid sequences having at least 70% homology.
4. The antibody or fragment thereof of any one of claims 1 to 3, wherein the antibody or fragment thereof is of one of the isotypes IgG, IgM, IgA, IgE or IgD.
5. The antibody or fragment thereof of any one of claims 1 to 3, wherein the antibody or fragment thereof is a chimeric, humanized or fully human antibody.
6. The antibody or fragment thereof of any one of claims 1 to 3, wherein the antibody or fragment thereof is a Fab, Fab 'and F (ab') 2, Fvs or single chain antibody.
7. The antibody or fragment thereof of any one of claims 1-3, wherein the antibody or fragment thereof further comprises amino acid sequence modifications including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modifications.
8. A polynucleotide encoding the antibody or fragment thereof of any one of claims 1-7.
9. A composition/complex comprising the antibody or fragment thereof of any one of claims 1-7.
10. A composition/complex as claimed in claim 9, further comprising a pharmaceutically acceptable carrier.
11. An isolated cell comprising one or more polynucleotides encoding the antibody or fragment thereof of any one of claims 1-7.
12. A method of screening, identifying or detecting comprising the step of contacting the antibody or fragment thereof of any one of claims 1 to 7 with a further solution, the screening, identifying or detecting being based on the contacting step; the method is a method for non-disease diagnostic purposes.
13. Use of the antibody or fragment thereof according to any one of claims 1 to 7 in the manufacture of a reagent or medicament for the diagnosis or treatment of a disease caused by a novel coronavirus.
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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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