Antibody specifically binding to Staphylococcus aureus protein A and its application

Abstract

The present invention discloses an antibody specifically binding to Staphylococcus aureus protein A and an application thereof. The antibody provided by the present invention can specifically bind to Staphylococcus aureus protein A through a Fab segment, and the anti-SpA antibody of the present application can regulate and kill Staphylococcus aureus, has a good therapeutic effect on disseminated Staphylococcus aureus infection, Staphylococcus aureus pneumonia and systemic infection, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to an antibody specifically binding to Staphylococcus aureus protein A and an application thereof. Background Art

[0002] Staphylococcus aureus (SA) is an aerobic Gram-positive coccus that usually colonizes the nose and skin of healthy humans and is an important pathogen that causes nosocomial and community infections. Hospital-acquired S. aureus infections are common and are the most common cause of hospital-acquired surgical site infections and pneumonia. S. aureus infections are also the second most common cause of cardiovascular and bloodstream infections. Antibiotic administration is the standard treatment for S. aureus infections, but antibiotic-resistant infections may be more common depending on the type of infection (e.g., skin infections are proportionally antibiotic-resistant) and region. Currently, methicillin-resistant S. aureus (MRSA) has evolved the ability to resist β-lactam antibiotics (such as penicillin and cephalosporin), and S. aureus resistant to vancomycin and linezolid is often encountered. New methods for preventing and treating S. aureus infections are urgently needed. Therefore, screening highly sensitive and specific antibodies against Staphylococcus aureus is of great significance for the early detection, early treatment and improved prognosis of related diseases. Summary of the invention

[0003] To make up for the deficiencies of the prior art, the present invention provides an antibody specifically binding to Staphylococcus aureus protein A and application thereof.

[0004] To achieve the above object, the present invention adopts the following technical solution:

[0005] The first aspect of the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to the SpA protein, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region complementarity determining region VH CDR and a light chain variable region complementarity determining region VLCDR,

[0006] wherein the amino acid sequences of VH CDR1, VH CDR2 and VH CDR3 are at least 95%, at least 96%, at least 97%, at least 98% and at least 99% identical to the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively;

[0007] The amino acid sequences of VL CDR1, VL CDR2 and VL CDR3 are at least 95%, at least 96%, at least 97%, at least 98% and at least 99% identical to the amino acid sequences shown in SEQ ID NOs: 9, 10 and 11, respectively.

[0008] Furthermore, the amino acid sequences of VH CDR1, VH CDR2 and VH CDR3 are shown in SEQ ID NOs: 1, 2 and 3, respectively.

[0009] The amino acid sequences of VL CDR1, VL CDR2 and VL CDR3 are shown in SEQ ID NOs: 9, 10 and 11, respectively.

[0010] Furthermore, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region framework region VH FR and a light chain variable region framework region VL FR,

[0011] wherein the amino acid sequences of VH FR1, VH FR2, VH FR3 and VH FR4 are at least 90%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99% identical to the amino acid sequences shown in SEQ ID NOs: 4, 5, 6 and 7, respectively;

[0012] The amino acid sequences of VL FR1, VL FR2, VL FR3 and VL FR4 are at least 90%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99% identical to the amino acid sequences shown in SEQ ID NOs: 12, 13, 14 and 15, respectively.

[0013] Furthermore, the amino acid sequences of VH FR1, VH FR2, VH FR3 and VH FR4 are shown in SEQ ID NOs: 4, 5, 6 and 7, respectively.

[0014] The amino acid sequences of VL FR1, VL FR2, VL FR3 and VL FR4 are shown in SEQ ID NOs: 12, 13, 14 and 15, respectively.

[0015] Furthermore, the amino acid sequence of the heavy chain variable region VH of the antibody or antigen-binding fragment thereof is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence shown in SEQ ID NO: 8,

[0016] The amino acid sequence of the light chain variable region VL is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO:16.

[0017] Furthermore, the amino acid sequence of the heavy chain variable region VH of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO:16.

[0018] Furthermore, the hIgG1 Fc of the antibody has H435R and / or Y436F mutations.

[0019] Further, the antibody binds to a linear epitope.

[0020] Furthermore, the antibody or antigen-binding fragment thereof is linked to a marker.

[0021] Furthermore, the label includes an enzyme, a radionuclide, a fluorescent dye, a luminescent substance or biotin.

[0022] Further, the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems.

[0023] Further, the CDRs are defined according to the IMGT numbering system.

[0024] Furthermore, the antibody or antigen-binding fragment thereof is afucosylated.

[0025] The second aspect of the present invention provides any of the following products:

[0026] (1) an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0027] (2) a vector comprising the isolated nucleic acid molecule described in (1);

[0028] (3) a host cell comprising the isolated nucleic acid molecule described in (1) or the vector described in (2);

[0029] (4) A product for detecting SpA protein, the product comprising the antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0030] (5) A pharmaceutical composition, comprising the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the isolated nucleic acid molecule according to (1), the vector according to (2), and the host cell according to (3).

[0031] Furthermore, the nucleotide sequences of the nucleic acid molecules encoding VH CDR1, VH CDR2, and VH CDR3 in (1) are at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the nucleotide sequences shown in SEQ ID NOs: 17, 18, and 19, respectively.

[0032] The nucleotide sequences of the nucleic acid molecules encoding VL CDR1, VL CDR2, and VL CDR3 are at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the nucleotide sequences shown in SEQ ID NOs: 25, 26, and 27, respectively.

[0033] Furthermore, the nucleotide sequences of the nucleic acid molecules encoding VH CDR1, VH CDR2, and VH CDR3 in (1) are shown in SEQ ID NOs: 17, 18, and 19, respectively.

[0034] The nucleotide sequences of the nucleic acid molecules encoding VL CDR1, VL CDR2, and VL CDR3 are shown in SEQ ID NOs: 25, 26, and 27, respectively.

[0035] Furthermore, the nucleotide sequences of the nucleic acid molecules encoding VH FR1, VH FR2, VH FR3 and VH FR4 in (1) are at least 90%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99% identical to the nucleotide sequences shown in SEQ ID NOs: 20, 21, 22 and 23, respectively.

[0036] The nucleotide sequences of the nucleic acid molecules encoding VL FR1, VL FR2, VL FR3 and VL FR4 are at least 90%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99% identical to the nucleotide sequences shown in SEQ ID NOs: 28, 29, 30 and 31, respectively.

[0037] Furthermore, the nucleotide sequences of the nucleic acid molecules encoding VH FR1, VH FR2, VH FR3 and VH FR4 in (1) are shown in SEQ ID NOs: 20, 21, 22 and 23, respectively.

[0038] The nucleotide sequences of the nucleic acid molecules encoding VL FR1, VL FR2, VL FR3 and VL FR4 are shown in SEQ ID NOs: 28, 29, 30 and 31, respectively.

[0039] Furthermore, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region in (1) is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 24, respectively.

[0040] The nucleotide sequence of the nucleic acid molecule encoding the light chain variable region is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the nucleotide sequence shown in SEQ ID NO:32.

[0041] Furthermore, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region in (1) is shown as SEQ ID NO:24, and the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region is shown as SEQ ID NO:32.

[0042] Furthermore, the vector described in (2) includes plasmid, bacteriophage, and artificial chromosome.

[0043] Furthermore, the host cells described in (3) include prokaryotic cells, insect cells, and animal cells.

[0044] Furthermore, the product described in (4) includes a test kit and a test paper.

[0045] Furthermore, the pharmaceutical composition described in (5) further comprises a pharmaceutically acceptable carrier.

[0046] The third aspect of the present invention provides a method for preparing the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the method comprising culturing the host cell according to (3) in the second aspect of the present invention and recovering the antibody.

[0047] Furthermore, the method is a non-therapeutic method.

[0048] The fourth aspect of the present invention provides a method for detecting the presence or amount of SpA protein in a sample, the method comprising:

[0049] (1) contacting the sample with the antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0050] (2) Detecting the formation of a complex between the antibody or antigen-binding fragment thereof and the SpA protein or detecting the amount of the complex.

[0051] Furthermore, the method is a method for non-diagnostic purposes.

[0052] The fifth aspect of the present invention provides use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention or the product described in the second aspect of the present invention in detecting SpA protein or in preparing a product for detecting SpA protein.

[0053] The sixth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention or the product described in the second aspect of the present invention in inhibiting Staphylococcus aureus infection or in preparing a pharmaceutical composition for preventing / treating Staphylococcus aureus-related diseases.

[0054] Furthermore, diseases associated with Staphylococcus aureus include purulent infections or toxic diseases.

[0055] Furthermore, the purulent infection includes purulent infection of the skin, purulent infection of various organs, disseminated infection or systemic infection.

[0056] Furthermore, the suppurative skin infection includes folliculitis, furuncle, carbuncle, suppuration of wound or abscess.

[0057] Furthermore, the suppurative infections of various organs include tracheitis, pneumonia, empyema, otitis media or osteomyelitis.

[0058] Furthermore, the systemic infection includes sepsis and septicemia.

[0059] Furthermore, the toxic diseases include food poisoning, scalded skin syndrome or toxic shock syndrome.

[0060] Furthermore, the Staphylococcus aureus-related disease is selected from disseminated infection or pneumonia infection.

[0061] Advantages and beneficial effects of the present invention:

[0062] The present invention provides an antibody that specifically binds to SpA protein and has good binding activity with SpA. The SpA protein antibody of the present application can regulate and kill Staphylococcus aureus, has a good therapeutic effect on disseminated Staphylococcus aureus infection and Staphylococcus aureus pneumonia infection, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 This is the SDS-PAGE test result of wSpA and mSpA proteins;

[0064] Figure 2 2A is a graph of the Fab library screening results, wherein 2B is a graph of the Fab (Kappa light chain) library results, and 2A is a graph of the Fab (Lambda light chain) library results;

[0065] Figure 3This is the result of SDS-PAGE detection of SpA-16 monoclonal antibody expression and purification;

[0066] Figure 4 4A is a result diagram of the binding specificity of the SpA-16 monoclonal antibody, wherein 4A is a result diagram with wSpA, and 4B is a result diagram with mSpA;

[0067] Figure 5 5A is a graph showing the killing of Staphylococcus aureus by SpA-16 monoclonal antibody, wherein 5A is a graph showing the phagocytosis result, and 5B is a graph showing the killing rate;

[0068] Figure 6 It is a graph showing the in vivo protective effect of SpA-16 monoclonal antibody in a disseminated Staphylococcus aureus infection model;

[0069] Figure 7 It is a graph showing the in vivo protective effect of SpA-16 monoclonal antibody in the Staphylococcus aureus pneumonia model;

[0070] Figure 8 It is a graph showing the in vivo protective effect of SpA-16 monoclonal antibody on the Staphylococcus aureus systemic infection model. DETAILED DESCRIPTION

[0071] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0072] The present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to SpA protein. The antibody or the antigen-binding fragment thereof comprises a heavy chain variable region complementarity determining region VH CDR and a light chain variable region complementarity determining region VL CDR.

[0073] In the present invention, an antibody refers to an immunoglobulin-derived molecule that can specifically bind to a target antigen, and the immunoglobulin-derived molecule binds to the target antigen through at least one antigen binding site located in its variable region. When the term antibody is mentioned, unless the context clearly indicates, it includes not only complete antibodies, but also antigen binding fragments that can specifically bind to a target antigen. A complete antibody is typically composed of two pairs of polypeptide chains (each pair has a light chain and a heavy chain). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light chain and the heavy chain, the variable region and the constant region are connected by a J region of about 12 or more amino acids, and the heavy chain also includes a D region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domain is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions with high variability (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form antigen binding sites, respectively.

[0074] Complementarity determining region or CDR refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs in each of the variable regions of the heavy chain and light chain, named CDR1, CDR2 and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883), AbM, Contact or IMGT numbering systems (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, a person skilled in the art will easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (eg, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0075] The CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention can be identified according to various numbering systems known in the art. In a specific embodiment of the present invention, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention are identified by the IMGT numbering system.

[0076] The antibody or antigen-binding fragment thereof is afucosylated.

[0077] In the present invention, the term afucosylated antibody refers to an antibody of the IgG1 or IgG3 isotype having an altered glycosylation pattern at Asn297 in the Fc region and having a reduced level of fucosyl residues. Glycosylation of human IgG1 or IgG3 occurs at Asn297 as a core fucosylated biantennary complex oligosaccharide glycosylation with up to two Gal residues at the end. Depending on the amount of the terminal Gal residue, these structures are referred to as G0, G1 (α1,6 or α1,3) or G2 glycan residues (Raju, TS, BioProcess Int.1 (2003) 44-53). CHO-type glycosylation of the Fc portion of an antibody is described, for example, by Routier, FH, Glycoconjugate J.14 (1997) 201-207. It should be understood that, as used in the present invention, the term afucosylated antibody includes antibodies having no fucose in their glycosylation pattern. It is generally known that the typical glycosylation residue position in antibodies is asparagine (Asn297) at position 297 according to the EU numbering system. The EU numbering system or EU index is generally used when referring to residues in the constant region of an immunoglobulin heavy chain (e.g., the EU index reported in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), which is expressly incorporated herein by reference).

[0078] The present invention provides any of the following products:

[0079] (1) an isolated nucleic acid molecule encoding the above-mentioned antibody or antigen-binding fragment thereof;

[0080] (2) a vector comprising the isolated nucleic acid molecule described in (1);

[0081] (3) a host cell comprising the isolated nucleic acid molecule described in (1) or the vector described in (2);

[0082] (4) A product for detecting SpA protein, the product comprising the above-mentioned antibody or antigen-binding fragment thereof;

[0083] (5) A pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment thereof, the isolated nucleic acid molecule described in (1), the vector described in (2), and the host cell described in (3).

[0084] In the present invention, vector refers to a nucleic acid carrier into which a polynucleotide can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; bacteriophages; artificial chromosomes. Artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage, pComb3XSS phage or M13 phage and animal viruses; plasmids such as pGEX-6p-2, pCMV-myc, pCMV-HA, pIRES-puro3, pIRES-neo3, pCAGGS, etc. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication initiation site.

[0085] In the present invention, host cells refer to cells that can be used to introduce vectors, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0086] The pharmaceutical composition described in (5) further comprises a pharmaceutically acceptable carrier.

[0087] In the present invention, a pharmaceutically acceptable carrier will generally be any type of non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary.

[0088] The term carrier refers to a diluent, adjuvant, auxiliary material or excipient used together with the therapeutic agent. Such a pharmaceutical carrier can be a sterile liquid, such as water and oil, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous glucose solutions and glycerol solutions can also be used as liquid carriers, particularly for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum, sodium chloride, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If necessary, the composition can also contain a small amount of wetting agent or emulsifier, or pH buffer, such as acetate, citrate or phosphate. Antimicrobial agents such as benzyl alcohol or methyl paraben are also contemplated; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for adjusting tonicity such as sodium chloride or dextrose. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The composition can be formulated into a suppository together with conventional binders and carriers (e.g., triglycerides). Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such a composition will contain a therapeutically effective amount of an antigen-binding polypeptide, preferably in a purified form, together with an appropriate amount of a carrier to provide a form for appropriate administration to a patient. The formulation should be suitable for the mode of administration. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0089] The present invention provides the use of the above-mentioned antibody or antigen-binding fragment thereof, and the above-mentioned product in inhibiting Staphylococcus aureus infection or in preparing a pharmaceutical composition for preventing / treating Staphylococcus aureus-related diseases.

[0090] In the present invention, diseases associated with Staphylococcus aureus include but are not limited to: 1) suppurative infection (invasive disease); 2) toxin disease: a toxic disease caused by exotoxin.

[0091] Among them, suppurative infection includes but is not limited to: ① suppurative infection of the skin: such as folliculitis, furuncle, carbuncle, suppuration of wounds and abscesses, etc. It can also invade the respiratory tract or bloodstream to cause infection. Clinical manifestations: pus is golden and viscous, the lesions are clearly defined, and are mostly localized; ② suppurative infection of various organs: such as tracheitis, pneumonia, empyema, otitis media, osteomyelitis, etc.; ③ disseminated infection; ④ systemic infection: if the primary suppurative lesions of the skin are squeezed by external force or the body's resistance is reduced, it will cause sepsis, septicemia, etc.

[0092] Toxic diseases include but are not limited to: ① Food poisoning: After ingesting food contaminated by enterotoxin-producing Staphylococcus aureus, after an incubation period of 1 to 6 hours, symptoms of acute gastroenteritis such as nausea, vomiting, and diarrhea may occur, i.e. food poisoning. It is not accompanied by fever and generally recovers quickly within 1 to 2 days. In a few severe cases, collapse or shock may occur. Food poisoning caused by this bacterium is a common gastrointestinal disease in summer and autumn; ② Scalded skin syndrome: more common in infants and adults with low immunity. At first, erythema appears on the skin, and the epidermis wrinkles in 1 to 2 days, followed by large bullae containing sterile, clear liquid, which may rupture with a slight touch and finally fall off the epidermis. If not treated in time, the mortality rate can reach 20%; ③ Toxic shock syndrome (TSS): patients show sudden high fever, vomiting, diarrhea, diffuse rash, followed by peeling (especially on the palms and soles), hypotension, mucosal lesions (oropharynx, vagina, etc.), and severe patients also experience heart and kidney failure, and may even suffer from shock.

[0093] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0094] Example 1: Expression and purification of wild-type Staphylococcus aureus protein A (wSpA) and mutant Staphylococcus aureus protein A (mSpA)

[0095] 1. Expression and purification of wild-type Staphylococcus aureus protein A (wSpA) and mutant Staphylococcus aureus protein A (mSpA)

[0096] The SAR0114 gene (protein A) of the Staphylococcus aureus MRSA 252 genome (BX571856.1GI:49240382) was used as a template, and corresponding point mutations were designed according to the sequence of this segment. The amino acids at the corresponding sites of wSpA (DEABC) are (7Q8Q, 34D35D, 68Q69Q, 95D96D, 126Q127Q, 153D154D, 184Q185Q, 211D212D, 242Q243Q, 269D270D), and the amino acids of the mutated mSpA are (7K8K, 34A35A, 68K69K, 95A96A, 126K127K, 153A154A, 184K185K, 211A212A, 242K243K, 269A270A). Codon optimization was performed according to the codon preference of Escherichia coli, and a BamHⅠ site was added to the N-terminus and a NotI site was added to the C-terminus. The target genes of wSpA and mSpA were synthesized by Shanghai Jierui Biotechnology Co., Ltd., and were constructed on the pGEX-6p-2 vector and expressed in XL1 Escherichia coli. The wSpA and mSpA engineering bacteria were cultured in LB medium containing ampicillin at 37°C overnight, expanded and IPTG-induced expression was performed the next day, and the bacteria were collected and lysed by centrifugation. The wSpA-GST and mSpA-GST fusion proteins were purified using SP FF cationic chromatography column (Borgron (Shanghai) Biotechnology Co., Ltd.), and the GST tag was specifically cut off by 3C protease of human rhinovirus type 14 (Prescission Protease enzyme, PP enzyme, GE Healthcare, USA). After filtering and sterilization with a sterilizing filter, the wild-type wSpA and mutant mSpA proteins were obtained by SP HP cationic chromatography column, Phenyl HP hydrophobic chromatography column, G25 (Borgron (Shanghai) Biotechnology Co., Ltd.) desalting, and Q HP (GE Healthcare, USA) anion exchange chromatography.

[0097] 2. Experimental results

[0098] The molecular weight of the purified wSpA protein was about 30.7 kDa, and the purity was 100% by SDS-PAGE. The molecular weight of the wSpA protein was about 30.5 kDa, and the purity was 100% by SDS-PAGE. Figure 1 ).

[0099] Example 2: Establishment of a human-specific anti-Staphylococcus aureus Fab antibody library

[0100] 1. Source of PBMC cells

[0101] Based on the SpA mutant protective antigen of the recombinant Staphylococcus aureus vaccine (Escherichia coli), the ethical review approval was obtained with the approval of the Ethics Review Committee of the Jiangsu Provincial Center for Disease Control and Prevention, and peripheral blood lymphocyte samples of Phase Ia subjects frozen in liquid nitrogen were obtained from the Phase Ia clinical trial center of the recombinant Staphylococcus aureus vaccine (Escherichia coli).

[0102] 2. Construction of human-specific anti-Staphylococcus aureus Fab antibody library using phage surface display technology

[0103] Based on the results of humoral immunogenicity (Luminex test using mSpA protein (HPLC purity > 95%) as antigen) and efficacy (OPKA test), PBMCs of 9 subjects whose Luminex and OPKA results were significantly higher than the mean values ​​of the placebo group and before vaccine injection were selected, and total RNA was extracted (QIAGEN, RNeasy Plus Mini Kit (250)) and SuperScript TM III reverse transcriptase (Invitrogen, SuperScript TM III Reverse Transcriptase) and random primers were used to synthesize cDNA, and the reverse cDNA was mixed as a gene template, and the human light chain (VL+CL) Kappa / Lambda and heavy chain Fd segment gene sequences were amplified by PCR using the Ig primer set. The light chain VL+CL gene PCR recovery product was double-digested with SacI-HF enzyme and XbaI-HF enzyme, and then connected to the pComb3XSS phage display vector. After the VL+CL (Kappa) and VL+CL (Lambda) light chain libraries were successfully constructed, the heavy chain Fd segment was cloned into the pComb3XSS vector already connected with the light chain gene fragment through the XhoI-HF and SpeI-HF restriction enzyme sites to form the Fab phagemid. The ligation product was transformed into TG1 competent cells and cultured overnight at 37°C on a plate containing ampicillin. The bacterial cells were collected to calculate the library capacity, and 10 single colonies were picked for PCR identification using specific primers. The reaction conditions were: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, 30 cycles; 72°C extension for 10 min. 5 μL of PCR product was detected by 2% agarose gel electrophoresis.

[0104] 3. Experimental results

[0105] The results showed that the Fab (Kappa light chain) library ( Figure 2 A) and Fab (Lambda light chain) library ( Figure 2 B) The positive clone rate was more than 80%, and the library capacity was more than 108 .

[0106] Example 3: Screening of human specific anti-Staphylococcus aureus Fab antibody library

[0107] 1. Bacterial purification and antibody phage preparation

[0108] The frozen library bacterial solution was inoculated into LB-penicillin medium at a ratio of 1:100 for recovery. When the OD600 reading was between 0.5 and 0.6, the bacterial solution was removed and the helper phage M13 was added at a ratio of 1:1000. After standing at 37°C for 30 minutes, the shaking culture was continued for 30 minutes. The bacteria were collected by centrifugation and cultured overnight in a constant temperature shaker at 30°C with LB medium containing ampicillin and kanamycin. The bacterial solution was centrifuged at 4°C and 3000g for 10 minutes to collect the supernatant phage, and the PEG / NaCl solution was added to mix thoroughly, placed on ice for 30 minutes, centrifuged at 4°C and 3000g for 20 minutes, the supernatant was removed, the precipitate was added with PEG / NaCl solution and mixed thoroughly, placed on ice for 30 minutes, centrifuged at 4°C and 11000g for 2 minutes, the supernatant was removed, the precipitate was resuspended with 1mL PBS and glycerol was added to -80°C for storage.

[0109] 2. Antigen coating

[0110] mSpA protein was coated at 100 μL per well at 4°C overnight; after removing the supernatant, the cells were washed once with PBS, patted dry, blocked with 3% skimmed milk powder, incubated at 37°C for 2 h, the blocking solution was removed, washed three times with PBST, and stored at 4°C for later use.

[0111] 3. Phage library panning

[0112] The antibody phage was added to the antigen-coated ELISA plate, incubated at 37°C for 20 minutes, and washed 10 times with PBST and PBS solution to remove the unbound phage; after digesting the bound phage with trypsin, TG1 bacteria cultured to OD600 of 0.5-0.6 were added for infection for 30 minutes. The bacteria were collected by centrifugation and spread on LB plates containing ampicillin and cultured overnight at 37°C. The next day, the titer of the selected phage sub-library was determined by gradient dilution and the bacteria were collected. The phage sub-library was re-packaged as the input phage library for the next round of panning using the above method. The panning process was repeated three times to maximize the enrichment of high-affinity antibodies.

[0113] 4. Phage library screening

[0114] After panning, the enriched phages were plated on LB plates containing ampicillin to produce bacterial monoclones. The monoclones were picked and cultured in 96-well culture plates and packaged for phage ELISA screening. The antibody phages were added to the antigen-coated ELISA plates, incubated at 37°C for 20 minutes, and washed 3 times with PBST solution to remove the unbound phages; fluorescent labeled antibodies were added, incubated at 37°C for 20 minutes, and washed 3 times with PBST; the ELISA fluorescence signal intensity was detected to screen specific antibodies, the antibody phagemids in the corresponding positive wells were amplified, and sequencing analysis was performed.

[0115] Example 4: Cloning, expression and purification of SpA-16 monoclonal antibody

[0116] 1. Experimental methods

[0117] The positive Fab display vector screened in Example 3 was used as a template, and human Ig VH and VK / L were amplified by PCR using vector primers. The products were identified by 1.2% agarose gel electrophoresis.

[0118] Antibody gene sequence determination and bioinformatics analysis: Antibody gene PCR products that were positive by gel electrophoresis and whose heavy and light chains could be matched were purified using the Qiagen PCR product purification kit and sequenced from the forward and reverse directions, respectively. The antibody gene family, mutation rate and CDR region were analyzed using the IMGT online server (http: / / imgt.cines.fr / ).

[0119] The PCR products of antibody variable region genes that were positive by gel electrophoresis and that could match the heavy chain and light chain were connected to the pcDNA3.4 vector containing the heavy chain constant region (H435R and Y436F mutations in hIgG1 Fc) or the light chain constant region by TA cloning to construct the expression vector of the fully human anti-SpA antibody. The expression vector was then transformed into DH5α competent bacteria and cultured overnight at 37°C on a plate containing ampicillin. Ten single colonies were picked and PCR was performed using specific primers. The reaction conditions were: 94°C pre-denaturation for 3min; 94°C denaturation for 30s, 55°C annealing for 30s, 72°C extension for 100s, 28 cycles; 72°C extension for 5min. Take 5μL of PCR product and detect it by 1% agarose gel electrophoresis.

[0120] The vector plasmid in the obtained positive transformant was transformed into DH5α for large-scale amplification. After rapid extraction of the recombinant plasmid, it was co-incubated with the transfection reagent PEI at 37°C for 15-20 minutes and then transfected into HEK 293F cells, and cultured in a 37°C shaker with 125rpm / min in a 5% CO2 incubator. After 5 days, the cell supernatant was collected by centrifugation at 3000g and 4°C for 30 minutes, and purified by protein G affinity chromatography; the expression and purification of the antibody were tested by SDS-PAGE.

[0121] 2. Experimental results

[0122] The results showed that a series of heavy chain and light chain expression vectors of antibodies were successfully constructed. The antibody clone was named the fully human SpA monoclonal antibody SpA-16 (abbreviated as SpA-16 antibody or SpA-16 monoclonal antibody). SDS-PAGE test results showed that the transfected cells successfully expressed the antibody, and the relative molecular weight of the antibody was about 160-180kDa, the heavy chain was about 55kDa, and the light chain was about 25kDa ( Figure 3 ), and the antibody sequences are shown in Table 1.

[0123] Table 1 SpA monoclonal antibody SpA-16 antibody sequence

[0124]

[0125]

[0126]

[0127]

[0128] Example 5: Specificity of SpA-16 monoclonal antibody binding to Staphylococcus aureus protein A

[0129] 1. Experimental methods

[0130] A 96-well ELISA plate was coated with wild-type wSpA and mutant mSpA (1 μg / mL) proteins as antigens, 100 μL per well, coated overnight at 4°C, and blocked with blocking solution at room temperature for 2 h; the SpA-16 antibody (100 μg / mL) transfected and expressed in Example 4 was diluted 3-fold, and 100 μL per well was added for each dilution; hIgG1 (PcrV-A039, a specific fully human IgG1 monoclonal antibody against Pseudomonas aeruginosa antigen PcrV, as an isotype control antibody for the specific fully human IgG1 monoclonal antibody for SpA) and hIgG1 (PcrV-A039) Fc (100 μg / mL) were diluted 3-fold, and 100 μL per well was added for each dilution; both were repeated in 2 replicates and incubated at 37°C for 1 h.

[0131] Wash the plate once with PBST buffer (3 cycles), add 100 μL Chicken anti-Human IgG Fc Secondary Antibody, HRP diluted 1:5000 with blocking buffer to each well, and incubate at 37℃ for 1 hour. Wash the plate once with PBST buffer (5 cycles), protect from light, add 100 μL TMB to each well, place at 37℃ for 5-10 minutes, and immediately terminate with 50 μL 2M H2SO4. Detect OD value at dual wavelength 450 / 650nm.

[0132] 2. Experimental results

[0133] The results are as follows Figure 4 As shown in A, hIgG1(PcrV-A039) can bind to wild-type wSpA non-specifically. The H435R and / or Y436F mutations of hIgG1(PcrV-A039) Fc significantly reduced the non-specific binding ability to wild-type wSpA, with an EC50 of 6.74×10 -8 ; Figure 4 WSpA was mutated into mSpA as shown in B, and hIgG1(PcrV-A039) did not bind to the mutant mSpA (Table 2).

[0134] Due to the presence of H435R and / or Y436F mutations in the Fc of SpA-16 and hIgG1 (PcrV-A039) Fc monoclonal antibodies, SpA-16 monoclonal antibodies specifically bind to mSpA with an EC50 of 2.82×10 -10 ; SpA-16 monoclonal antibody can specifically bind to wSpA with an EC50 of 4.35×10 -11 .

[0135] Table 2 Binding results of SpA-16 and Staphylococcus aureus protein A (wild-type wSpA and mutant mSpA)

[0136]

[0137] Example 6: SpA-16 monoclonal antibody opsonizing and killing Staphylococcus aureus

[0138] 1. Experimental methods

[0139] Staphylococcus aureus evades complement-dependent killing by interfering with the expression of complement activation virulence factors before the formation of the attack complex. The ability of the anti-protein A antibody of the present invention to promote killing of Staphylococcus aureus Newman in the presence of complement is evaluated by testing the ability of the antibody to overcome complement escape and induce antibody-induced serum killing. Briefly, 40 μL of SpA-16 antibody, positive serum, negative serum, and control antibody were added to a microtiter plate, and the antibody was diluted 7 gradients using a buffer; Staphylococcus aureus Newman was cultured in TSB and then suspended with a buffer to a concentration of 1.2×10 5 Colony forming units (CFU) / mL, add 10 μL to each well, incubate with shaking; then add 10 μL complement for co-incubation; wash the HL-60 cells differentiated for 3.5 days and adjust the concentration to 1×10 using buffer 7 Cells / mL, 40 μL was added to each well, and phagocytosis was conditioned at 37°C, 5% CO2 for 75 min. After phagocytosis, the microplate was placed on ice for 20 min, and 10 μL of the mixture was inoculated on MHA for counting.

[0140] 2. Experimental results

[0141] The results are as follows Figure 5 As shown, the control antibody could not promote cell killing of Staphylococcus aureus, while the SpA-16 monoclonal antibody could promote cell killing of Staphylococcus aureus, and the maximum killing rate was 71%.

[0142] Example 7: Evaluation of the effect of SpA-16 monoclonal antibody in a disseminated Staphylococcus aureus infection model

[0143] 1. Experimental methods

[0144] S. aureus causes disseminated infection in mice when injected intraperitoneally with high levels of bacterial replication in the kidney. In this experiment, anti-Protein A antibodies were tested for their ability to reduce kidney burden when administered 1 day after S. aureus infection.

[0145] The anti-protein A monoclonal antibody SpA-16 of the present invention was evaluated for therapeutic efficacy against S. aureus Newman in a disseminated infection model. Briefly, cultures of S. aureus Newman were grown overnight in TSB, subcultured and grown to mid-log phase (OD 600 =1.0). The cultures were then washed twice in PBS and plated at OD 600 =3(7.5×10 8cfu / mL) were resuspended in PBS. Mice were injected intraperitoneally with 200 μL of bacterial suspension. One day after infection, 100 μL of the drug was administered subcutaneously, and the experimental group was treated with SpA-16 monoclonal antibody containing 10 mg / kg, the control group was treated with hIgG1 (PcrV-A039) containing 10 mg / kg, and the blank group was treated with normal saline. The weight loss and physical condition of the mice were monitored until 4 days after infection, when they were euthanized. The kidneys were removed and resuspended in PBS containing 0.1% Triton X100 and homogenized using Cmax dissociation tubes. Bacteria were counted by colony forming units via serial dilution and inoculated onto TSA.

[0146] 2. Experimental results

[0147] SpA-16 monoclonal antibody reduced S. aureus kidney burden by 3 logs, whereas IgG1 (PcrV-A039) was ineffective in reducing S. aureus burden compared with control antibody ( Figure 6 ).

[0148] Example 8: Evaluation of the effect of SpA-16 monoclonal antibody in the Staphylococcus aureus pneumonia model

[0149] 1. Experimental methods

[0150] The SpA-16 monoclonal antibody of the present invention was evaluated for its protective efficacy against Staphylococcus aureus USA300 in a pneumonia model. Staphylococcus aureus USA300 was cultured in BHI broth and subcultured until OD 600 The culture was then washed twice in saline and plated at 2 × 10 10 The density of cfu / mL was resuspended in normal saline. The mice were intubated with 20 μL of bacterial suspension. One day before infection, the experimental group treated mice with 100 mg / kg of SpA-16 monoclonal antibody, the control group treated mice with 100 mg / kg of hIgG1 (PcrV-A039), and the blank group treated mice with normal saline; after endotracheal intubation and infection with 20 μL, the survival status of the mice was monitored until 7 days after infection, the number of surviving mice was recorded every 12 hours, and the survival rate was calculated.

[0151] 2. Experimental results

[0152] The protection rate of SpA-16 monoclonal antibody containing 100 mg / kg was 75%, and the protection rate of hIgG1 (PcrV-A039) containing 100 mg / kg was 10% ( Figure 7 ).

[0153] Example 9 Evaluation of the effect of SpA-16 monoclonal antibody in the Staphylococcus aureus systemic infection model

[0154] The SpA-16 monoclonal antibody of the present invention was evaluated for protective efficacy against Staphylococcus aureus USA300 in a systemic infection model. Staphylococcus aureus USA300 was cultured in TSB, subcultured and grown to mid-log phase (OD 600 =1.0). The cultures were then washed twice in saline and the OD 600 The optical density was 1.6 and resuspended in normal saline. 100 μL of bacterial suspension was injected into the tail vein of mice. One day before infection, the experimental group treated mice with 100 mg / kg of SpA-16 monoclonal antibody, the control group treated mice with 100 mg / kg of hIgG1 (PcrV-A039), and the blank group treated mice with normal saline. After 100 μL of attack bacteria were injected into the tail vein, the physical condition of the mice was monitored until 7 days after infection, and the number of surviving mice was recorded every 12 hours to calculate the survival rate.

[0155] The protection rate of SpA-16 monoclonal antibody containing 100 mg / kg was 65%, and the protection rate of hIgG1 (PcrV-A039) containing 100 mg / kg was 10% ( Figure 8 ).

[0156] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof that specifically binds to SpA protein, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region complementarity determining region VH CDR and a light chain variable region complementarity determining region VLCDR, The amino acid sequences of VH CDR1, VH CDR2 and VH CDR3 are shown in SEQ ID NOs: 1, 2 and 3, respectively. The amino acid sequences of VL CDR1, VL CDR2 and VL CDR3 are shown in SEQ ID NOs: 9, 10 and 11, respectively.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The monoclonal antibody or antigen-binding fragment thereof further comprises a heavy chain variable region framework region VH FR and a light chain variable region framework region VL FR, The amino acid sequences of VH FR1, VH FR2, VH FR3 and VH FR4 are shown in SEQ ID NOs: 4, 5, 6 and 7, respectively. The amino acid sequences of VL FR1, VL FR2, VL FR3 and VL FR4 are shown in SEQ ID NOs: 12, 13, 14 and 15, respectively.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The amino acid sequence of the heavy chain variable region VH of the monoclonal antibody or its antigen-binding fragment is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO:

16.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The hIgG1 Fc of the monoclonal antibody has H435R and / or Y436F mutations.

5. The monoclonal antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The monoclonal antibody binds to a linear epitope.

6. The monoclonal antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The monoclonal antibody or antigen-binding fragment thereof is linked to a label.

7. The monoclonal antibody or antigen-binding fragment thereof according to claim 6, characterized in that: The label includes enzymes, radionuclides, fluorescent dyes, luminescent substances or biotin.

8. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The CDRs are defined according to the IMGT numbering system.

9. The monoclonal antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The monoclonal antibody or antigen-binding fragment thereof is afucosylated.

10. Any of the following products: (1) An isolated nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9; (2) a vector comprising the isolated nucleic acid molecule described in (1); (3) a host cell comprising the isolated nucleic acid molecule described in (1) or the vector described in (2); (4) A product for detecting SpA protein, characterized in that: The product comprises the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9; (5) A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the isolated nucleic acid molecule according to (1), the vector according to (2), and the host cell according to (3).

11. The product according to claim 10, characterized in that The nucleotide sequences of the nucleic acid molecules encoding VH CDR1, VH CDR2, and VH CDR3 in (1) are at least 95% identical to the nucleotide sequences shown in SEQ ID NOs: 17, 18, and 19, respectively; The nucleotide sequences of the nucleic acid molecules encoding VL CDR1, VL CDR2, and VL CDR3 are at least 95% identical to the nucleotide sequences shown in SEQ ID NOs: 25, 26, and 27, respectively.

12. The product according to claim 11, characterized in that The nucleotide sequences of the nucleic acid molecules encoding VH CDR1, VH CDR2, and VH CDR3 in (1) are shown in SEQ ID NOs: 17, 18, and 19, respectively. The nucleotide sequences of the nucleic acid molecules encoding VL CDR1, VL CDR2, and VL CDR3 are shown in SEQ ID NOs: 25, 26, and 27, respectively.

13. The product according to claim 10, characterized in that The nucleotide sequences of the nucleic acid molecules encoding VH FR1, VH FR2, VH FR3 and VH FR4 in (1) are at least 90% identical to the nucleotide sequences shown in SEQ ID NOs: 20, 21, 22 and 23, respectively; The nucleotide sequences of the nucleic acid molecules encoding VL FR1, VL FR2, VL FR3 and VL FR4 are at least 90% identical to the nucleotide sequences shown in SEQ ID NOs: 28, 29, 30 and 31, respectively.

14. The product according to claim 13, characterized in that The nucleotide sequences of the nucleic acid molecules encoding VH FR1, VH FR2, VH FR3 and VH FR4 in (1) are shown in SEQ ID NOs: 20, 21, 22 and 23, respectively. The nucleotide sequences of the nucleic acid molecules encoding VL FR1, VL FR2, VL FR3 and VL FR4 are shown in SEQ ID NOs: 28, 29, 30 and 31, respectively.

15. The product according to claim 10, characterized in that The nucleotide sequences of the nucleic acid molecules encoding the heavy chain variable regions in (1) are at least 70% identical to the nucleotide sequences shown in SEQ ID NO: 24, The nucleotide sequences of the nucleic acid molecules encoding the light chain variable regions are at least 70% identical to the nucleotide sequences shown in SEQ ID NO:

32.

16. The product according to claim 15, characterized in that The nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region in (1) is shown in SEQ ID NO:24, and the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region is shown in SEQ ID NO:

32.

17. The product according to claim 10, characterized in that The vectors mentioned in (2) include plasmids, bacteriophages, and artificial chromosomes.

18. The product according to claim 10, characterized in that The host cells mentioned in (3) include prokaryotic cells, insect cells, and animal cells.

19. The product according to claim 10, characterized in that The products described in (4) include test kits and test strips.

20. The product according to claim 10, characterized in that The pharmaceutical composition described in (5) further includes a pharmaceutically acceptable carrier.

21. A method for preparing the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, characterized in that: The method comprises culturing the host cell described in claim 10 (3) and recovering the monoclonal antibody.

22. A method for detecting the presence or amount of SpA protein in a sample for non-diagnostic purposes, characterized in that: The method comprises: (1) contacting the sample with the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9; (2) Detecting the formation of a complex between the monoclonal antibody or the antigen-binding fragment thereof and the SpA protein or detecting the amount of the complex.

23. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the product according to any one of claims 10 to 20, in detecting SpA protein for non-diagnostic purposes or in preparing a product for detecting SpA protein.

24. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the product according to any one of claims 10 to 20, in inhibiting Staphylococcus aureus for non-therapeutic purposes or in preparing a pharmaceutical composition for preventing / treating diseases related to Staphylococcus aureus.

25. The use according to claim 24, characterized in that Diseases associated with Staphylococcus aureus include suppurative infections or toxic diseases.

26. The use according to claim 25, characterized in that The purulent infection includes purulent infection of the skin, purulent infection of various organs, disseminated infection or systemic infection.

27. The use according to claim 26, characterized in that The skin suppurative infection includes folliculitis, furuncle, carbuncle, wound suppuration or abscess.

28. The use according to claim 26, characterized in that The suppurative infections of the various organs include tracheitis, pneumonia, empyema, otitis media or osteomyelitis.

29. The use according to claim 26, characterized in that The systemic infection includes sepsis and septicemia.

30. The use according to claim 25, characterized in that The toxic diseases include food poisoning, scalded skin syndrome or toxic shock syndrome.

31. The use according to claim 26, characterized in that The Staphylococcus aureus-related disease is selected from a disseminated infection or a systemic infection.

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