A monoclonal antibody against Staphylococcus aureus protein A and its application

Abstract

The present invention discloses a monoclonal antibody against Staphylococcus aureus protein A and its application. The SpA-A6 comprises HCDR1, HCDR2, HCDR3 of the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 1, 2, 3; and LCDR1, LCDR2, LCDR3 of the light chain variable region of the amino acid sequence shown in SEQ ID NO: 9, 10, 11. The present invention also discloses a method for generating the Staphylococcus aureus protein A antibody SpA-A6 and its application in preparing a product for diagnosing or treating Staphylococcus aureus protein A infection. This antibody and combination drug are suitable for symptoms related to Staphylococcus aureus infection. The antibody can prevent the severity and / or duration of the disease.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a monoclonal antibody against Staphylococcus aureus protein A and an application thereof. Background Art

[0002] Staphylococcus aureus is a gram-positive coccus belonging to the genus Staphylococcus, and is an important pathogen causing hospital and community infections. Staphylococcus aureus is present in the nose and skin of 25-30% of healthy adults, but for people with weakened immune systems, if it manages to break through the periphery, it can cause minor infections such as papules, boils and other soft tissue infections, or even serious infections that are fatal.

[0003] Administration of antibiotics is the standard treatment for S. aureus infections, but antibiotic-resistant infections may be more prevalent depending on the type of infection (e.g., skin infections are disproportionately antibiotic-resistant) and region. Fatal infections are often associated with bacteremia, which had a mortality rate of 65-70% before widespread antibiotic use. Today, even with optimal therapy and the latest antibiotics, mortality from bacteremia within 30 days is still as high as 20-40% due to antibiotic-resistant strains.

[0004] The current misuse of antibiotics has led to the evolution of Staphylococcus aureus' ability to resist β-lactam antibiotics (such as penicillin and cephalosporin), and Staphylococcus aureus resistant to vancomycin and linezolid is often encountered. However, considering the continued increase in antimicrobial resistance and the slowdown in the development of new antibiotics, new methods for preventing and treating Staphylococcus aureus infections need to be developed. Summary of the invention

[0005] To make up for the deficiencies of the prior art, the present invention provides a monoclonal antibody against Staphylococcus aureus protein A and application thereof.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides an antibody against Staphylococcus aureus protein A, the antibody comprising heavy chain variable regions HCDR1, HCDR2, HCDR3 having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs: 1, 2, 3,

[0008] And light chain variable regions LCDR1, LCDR2, and LCDR3 having at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with the amino acid sequences shown in SEQ ID NOs: 9, 10, and 11.

[0009] Furthermore, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 1, 2 and 3, respectively.

[0010] The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 9, 10 and 11, respectively.

[0011] Further, the antibody also includes heavy chain variable region framework regions HFR1, HFR2, HFR3, and HFR4 having 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% sequence identity with the amino acid sequences shown in SEQ ID NOs: 4, 5, 6, and 7,

[0012] And light chain variable region framework regions LFR1, LFR2, LFR3, and LFR4 having 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% sequence identity with the amino acid sequences shown in SEQ ID NO:12, 13, 14, and 15.

[0013] Furthermore, the amino acid sequences of the heavy chain variable region framework regions HFR1, HFR2, HFR3, and HFR4 are shown in SEQ ID NOs: 4, 5, 6, and 7, respectively.

[0014] The amino acid sequences of the light chain variable region framework regions LFR1, LFR2, LFR3, and LFR4 are shown in SEQ ID NOs: 12, 13, 14, and 15, respectively.

[0015] Further, the antibody heavy chain variable region has a VH that has 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% sequence identity to the amino acid sequence shown in SEQ ID NO:8,

[0016] The light chain variable region has a VL that 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 VH of the antibody has the amino acid sequence shown in SEQ ID NO:8, and the VL of the antibody has the amino acid sequence 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] Further, the CDRs are defined according to the IMGT numbering system.

[0021] Further, the antibody is afucosylated.

[0022] A second aspect of the present invention provides any of the following substances:

[0023] (1) a nucleic acid molecule encoding the antibody according to the first aspect of the present invention;

[0024] (2) a vector, wherein the vector comprises the nucleic acid molecule described in (1);

[0025] (3) a host cell, wherein the host cell comprises the nucleic acid molecule described in (1) or the vector described in (2);

[0026] (4) A product for detecting Staphylococcus aureus protein A, comprising the antibody described in the first aspect of the present invention.

[0027] Furthermore, the nucleic acid molecules encoding HCDR1, HCDR2, and HCDR3 in (1) have at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 17, 18, and 19, respectively.

[0028] The nucleic acid molecules encoding LCDR1, LCDR2, and LCDR3 have at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 25, 26, and 27, respectively.

[0029] Furthermore, the nucleotide sequences of the nucleic acid molecules encoding HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 17, 18, and 19, respectively, and the nucleotide sequences of the nucleic acid molecules encoding LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 25, 26, and 27, respectively.

[0030] Furthermore, the nucleic acid molecules encoding HFR1, HFR2, HFR3, and HFR4 have 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% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 20, 21, 22, and 23, respectively.

[0031] The nucleic acid molecules encoding LFR1, LFR2, LFR3, and LFR4 have 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% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 28, 29, 30, and 31, respectively.

[0032] Furthermore, the nucleotide sequences of the nucleic acid molecules encoding HFR1, HFR2, HFR3, and HFR4 are shown in SEQ ID NOs: 20, 21, 22, and 23, respectively, and the nucleotide sequences of the nucleic acid molecules encoding LFR1, LFR2, LFR3, and LFR4 are shown in SEQ ID NOs: 28, 29, 30, and 31, respectively.

[0033] Further, the nucleic acid molecule encoding VH has 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% sequence identity to the nucleotide sequence shown in SEQ ID NO: 24,

[0034] The nucleic acid molecule encoding VL has 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% sequence identity with the nucleotide sequence shown in SEQ ID NO:32.

[0035] Furthermore, the nucleotide sequence of the nucleic acid molecule encoding VH is shown in SEQ ID NO:24, and the nucleotide sequence of the nucleic acid molecule encoding VL is shown in SEQ ID NO:32.

[0036] Furthermore, the vector described in (2) has a signal peptide operably linked to the antibody.

[0037] Furthermore, the vector also includes elements for controlling expression.

[0038] Furthermore, the vector may also include materials that facilitate its entry into cells.

[0039] Furthermore, the host cells described in (3) include prokaryotic cells and eukaryotic cells.

[0040] Furthermore, the product described in (4) includes a test kit and a test strip.

[0041] Further, the kit includes a conjugate of an antibody linked to a signal generating compound.

[0042] The third aspect of the present invention provides a derivative, wherein the derivative comprises a diagnostic agent or a therapeutic agent connected to the antibody described in the first aspect of the present invention or the nucleic acid molecule described in the second aspect of the present invention.

[0043] Furthermore, the diagnostic reagent includes a fluorescent label, a radioactive isotope, a chemiluminescent molecule, a paramagnetic ion or a spin-trapping reagent.

[0044] Furthermore, the therapeutic agent includes a cytokine, a chemotherapeutic agent, a radiotherapeutic agent, an anti-inflammatory agent, a hormone, an antibody Fc fragment, a TLR agonist, a CpG-containing molecule or an immune co-stimulatory molecule.

[0045] A fourth aspect of the present invention provides any of the following methods:

[0046] (1) A method for preparing the antibody according to the first aspect of the present invention, the method comprising: culturing the host cell according to the second aspect of the present invention, and recovering the antibody;

[0047] (2) A method for detecting Staphylococcus aureus protein A in a sample, the method comprising: contacting the anti-Staphylococcus aureus protein A antibody described in the first aspect of the present invention with a sample to be tested, thereby detecting the level of Staphylococcus aureus protein A in the sample to be tested.

[0048] Furthermore, the method described in (1) further comprises purifying the antibody.

[0049] Furthermore, the method is a method for non-diagnostic purposes.

[0050] The fifth aspect of the present invention provides a pharmaceutical composition, which comprises the antibody described in the first aspect of the present invention, the substance described in the second aspect of the present invention, or the derivative described in the third aspect of the present invention.

[0051] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0052] A sixth aspect of the present invention provides any of the following applications:

[0053] (1) Use of the antibody according to the first aspect of the present invention, the substance according to the second aspect of the present invention, or the derivative according to the third aspect of the present invention in detecting Staphylococcus aureus protein A;

[0054] (2) Use of the antibody according to the first aspect of the present invention, the substance according to the second aspect of the present invention, or the derivative according to the third aspect of the present invention in the preparation of a product for detecting Staphylococcus aureus protein A;

[0055] (3) Use of the antibody described in the first aspect of the present invention, the substance described in the second aspect of the present invention, the derivative described in the third aspect of the present invention, or the pharmaceutical composition described in the fifth aspect of the present invention in the preparation of a drug for preventing and / or treating diseases related to Staphylococcus aureus.

[0056] Furthermore, diseases associated with Staphylococcus aureus include purulent infections or toxic diseases.

[0057] Furthermore, the purulent infection includes purulent infection of the skin, purulent infection of various organs, disseminated infection or systemic infection.

[0058] Furthermore, the suppurative skin infection includes folliculitis, furuncle, carbuncle, suppuration of wound or abscess.

[0059] Furthermore, the suppurative infections of various organs include tracheitis, pneumonia, empyema, otitis media or osteomyelitis.

[0060] Furthermore, the systemic infection includes sepsis and septicemia.

[0061] Furthermore, the toxic diseases include food poisoning, scalded skin syndrome or toxic shock syndrome.

[0062] Furthermore, the Staphylococcus aureus-related disease is selected from disseminated infection and / or systemic infection.

[0063] Advantages and beneficial effects of the present invention:

[0064] The target antigen of the present application is derived from antibiotic-sensitive and multi-drug-resistant Staphylococcus aureus protein A. Antibodies against SpA are isolated from vaccine-immunized human PBMCs. The antibodies and combined drugs are suitable for symptoms related to Staphylococcus aureus infection. The antibodies can prevent the severity and / or duration of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 This is the SDS-PAGE test result of wSpA and mSpA proteins;

[0066] Figure 2 2A is the colony map of Fab (Kappa light chain) library, and 2B is the colony map of Fab (Lambda light chain) library;

[0067] Figure 3 This is the result of SDS-PAGE detection of SpA-A6 expression and purification;

[0068] Figure 4 This is the result of enzyme-linked immunosorbent assay to detect the binding specificity of SpA-A6 monoclonal antibody to wSpA and mSpA;

[0069] Figure 5 5A is a graph showing the killing of Staphylococcus aureus by SpA-A6 monoclonal antibody, wherein 5A is a graph showing the phagocytosis result, and 5B is a graph showing the killing rate;

[0070] Figure 6 It is a graph showing the in vivo protective effect of SpA-A6 monoclonal antibody in a disseminated Staphylococcus aureus infection model;

[0071] Figure 7 It is a graph showing the in vivo protective effect of SpA-A6 monoclonal antibody in the Staphylococcus aureus pneumonia model;

[0072] Figure 8 It is a graph showing the in vivo protective effect of SpA-A6 monoclonal antibody on the Staphylococcus aureus systemic infection model. DETAILED DESCRIPTION

[0073] 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.

[0074] The present invention provides an antibody against Staphylococcus aureus protein A, wherein the antibody comprises heavy chain variable regions HCDR1, HCDR2, HCDR3, and light chain variable regions LCDR1, LCDR2, LCDR3.

[0075] In one embodiment of the present invention, antibody or monoclonal antibody refers to an immunoglobulin-derived molecule capable of specifically binding to a target antigen, wherein the immunoglobulin-derived molecule binds to the target antigen through at least one antigen binding site located in its variable region. When referring to the term antibody, unless the context clearly indicates, it includes not only complete antibodies, but also antigen binding fragments capable of specifically binding to a target antigen.

[0076] Antibodies contain two heavy (H) chains and two light (L) chains. The mammalian heavy chain consists of a variable region (VH) and a first, second, third and optionally a fourth constant region (CH1, CH2, CH3, CH4, respectively); the mammalian light chain consists of a variable region (VL) and a constant region. The antibody is Y-shaped, wherein the stem of the Y consists of the second and third constant regions of two heavy chains bound together by disulfide bonds. Each arm of the Y includes the variable region and the first constant region of a single heavy chain combined with the variable region and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in the two chains generally contain three highly variable loops, called complementarity determining regions (CDRs), the light chain CDRs include LCDR1, LCDR2 and LCDR3, and the heavy chain CDRs include HCDR1, HCDR2, and HCDR3. The variable regions of the light and heavy chains also include framework regions (FRs), the light chain FRs include LFR1, LFR2, LFR3, and LFR4, and the heavy chain FRs include HFR1, HFR2, HFR3, and HFR4. The constant regions of the heavy and light chains do not participate in antigen binding, but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of the constant region of the antibody heavy chain.

[0077] In one embodiment of the present invention, the antibody has any variant with one or more conservative amino acid substitutions, and the variant refers to a molecule that is different from the amino acid sequence of the parent anti-Staphylococcus aureus protein A antibody due to the addition, deletion and / or substitution of one or more amino acid residues in the parent antibody sequence and retains at least one desired activity of the parent anti-Staphylococcus aureus protein A antibody. The desired activity may include the ability to specifically bind to the antigen, the ability to reduce, inhibit or neutralize the activity of Staphylococcus aureus protein A in animals, and the ability to inhibit Staphylococcus aureus protein A-mediated signal transduction in cell-based assays. In one embodiment, the variant comprises one or more amino acid substitutions in one or more hypervariable regions and / or framework regions of the parent antibody. For example, the variant may comprise at least one, or about one to about ten, or about two to about five substitutions in one or more hypervariable regions and / or framework regions of the parent antibody. Variants retain the ability to bind to S. aureus Protein A variants and may have stronger binding affinity, enhanced ability to reduce, inhibit or neutralize S. aureus Protein A activity in animals, and / or enhanced ability to inhibit S. aureus Protein A-mediated signaling in cell-based assays.

[0078] For the determination or definition of antibody CDR, the deterministic description of CDR and the identification of residues comprising the binding site of the antibody can be completed by resolving the structure of the antibody and / or resolving the structure of the antibody-ligand complex. This can be achieved by any of the various techniques known to those skilled in the art, such as X-ray crystallography. A variety of analytical methods can be used to identify CDR, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, and conformational definitions.

[0079] In a specific embodiment of the invention, the CDRs are defined according to the IMGT numbering system.

[0080] The antibody is afucosylated.

[0081] In one embodiment of the present invention, afucosylation refers to an antibody of the IgG1 or IgG3 isotype (preferably of the IgG1 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. Antibodies recombinantly expressed in non-sugar-modified CHO host cells are usually fucosylated at Asn297 in an amount of at least 85%. It should be understood that as used in the present invention, afucosylated antibodies include antibodies without fucose in their glycosylation pattern. It is generally known that the typical glycosylation residue position in antibodies is asparagine at position 297 (Asn297) according to the EU numbering system.

[0082] The present invention provides any of the following substances:

[0083] (1) a nucleic acid molecule encoding the above-mentioned antibody;

[0084] (2) a vector, wherein the vector comprises the nucleic acid molecule described in (1);

[0085] (3) a host cell, wherein the host cell comprises the nucleic acid molecule described in (1) or the vector described in (2);

[0086] (4) A product for detecting Staphylococcus aureus protein A, comprising the above-mentioned antibody.

[0087] In one embodiment of the present invention, vector refers to a medium into which a polynucleotide encoding a protein can be operably inserted to cause expression of the protein. The vector can be used for transforming, transducing or transfecting a host cell so that it expresses the genetic elements carried in the host cell. The vector can contain a variety of elements for controlling expression, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element and a reporter gene. In addition, the vector can contain a replication origin. The replication origin refers to a sequence that initiates replication when present in a vector. The replication origin can be recognized by a replication initiation factor or alternatively by a DNA helicase. The vector can also include materials that help it enter the cell, including but not limited to virus particles, liposomes or protein envelopes.

[0088] The vector can be a recombinant expression vector or a cloning vector. The present invention provides a vector (e.g., an expression vector) containing a nucleic acid sequence provided by the present invention encoding an anti-Staphylococcus aureus protein A antibody, at least one promoter operably connected to the nucleic acid sequence and / or at least one selective marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phages and M13 phages, plasmids, including but not limited to pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUS E. pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV -SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pCDNA3.4, pRc / RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos.

[0089] In one embodiment of the present invention, the host cell can be virtually any cell for which the expression vector can be used. It includes prokaryotic cells, eukaryotic cells, and the prokaryotic cells include, but are not limited to, true bacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, such as Escherichia coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, such as Salmonella typhimurium; Serratia, such as Serratia marcescens; marcescens); and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis; Pseudomonas, such as P. aeruginosa; and Streptomyces.

[0090] Eukaryotic cells include but are not limited to protist cells, animal cells, plant cells or fungal cells, wherein the animal cells include mammalian cells, bird cells, and insect cells; wherein mammalian cells include but are not limited to Chinese hamster ovary cells, SP2 / 0 cells, HeLa cells, baby hamster kidney cells, and cancer cells.

[0091] The present invention provides a derivative, wherein the derivative comprises a diagnostic agent or a therapeutic agent connected to the above antibody or the above nucleic acid molecule.

[0092] In one embodiment of the present invention, the diagnostic reagent is known in the art, and methods for linking it to proteins (including antibodies) are also known, including but not limited to fluorescent labels, radioactive isotopes, chemiluminescent molecules, paramagnetic ions or spin capture agents.

[0093] Among them, the fluorescent label includes but is not limited to one or more of Alexa 350, Alexa 430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5, 6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, OregonGreen 514, Pacific Blue, REG, rhodamine green, rhodamine red, Renographin, ROX, TAMRA, TET, tetramethylrhodamine or Texas Red.

[0094] Radioactive isotopes include but are not limited to astatine 211 , 14 carbon, 51 chromium, 36 chlorine, 57 cobalt, 58 Cobalt, copper 67 , 152 Eu, Gallium 67 , 3 Hydrogen, iodine 123 ,iodine 125 ,iodine 131 ,indium 111 , 59 iron, 32 Phosphorus, Rhenium 186 ,rhenium 188 , 75 selenium, 35 Sulfur, technetium 99m (technicium) or yttrium 90 One or more of the .

[0095] Paramagnetic ions include, but are not limited to, ions of chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and / or erbium (III).

[0096] The therapeutic agents include, but are not limited to, cytokines, chemotherapeutic agents, radiotherapeutic agents, anti-inflammatory agents, hormones, antibody Fc fragments, TLR agonists, CpG-containing molecules, or immune co-stimulatory molecules.

[0097] Among them, cytokines include but are not limited to IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL -14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, TNFα, GM-CSF, INFα, IFNβ and IFNγ.

[0098] Chemotherapeutic agents include, but are not limited to, cisplatin (CDDP), carboplatin, procarbazine, nitrogen mustard, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, paclitaxel, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine, and methotrexate.

[0099] Another class of therapeutic agents are toxins, including but not limited to cholera toxin, botulinum toxin, pertussis toxin, ricin A and B chains, and other natural and synthetic toxins.

[0100] Anti-inflammatory agents include, but are not limited to, NSAIDs, steroids, rapamycin, infliximab, and denileukin (ontak).

[0101] TLR agonists are compounds that stimulate or turn on the immune system. The natural agonists of TLR9 are components of DNA commonly found in bacteria and viruses. The natural agonists of TLR7 and TLR8 are RNA patterns found in viruses. After recognizing their natural DNA and RNA agonists, TLR7, 8, and 9 each trigger a different cascade of protective immune responses. TLR agonists include oligodeoxynucleotides, hyaluronic acid fragments, imiquimod, lavandin C, lipid A, loroxibine, LPS, monophosphoryl lipid A (monophosphoryl lipda A), myristyl ether, resiquimod, S. typhimurium flagellin, HKLM, PAM3CSK4, and polyinosinic acid (polyI: C).

[0102] The present invention provides use of the above antibody, the above substance, the above derivative or the above pharmaceutical composition in preparing a drug for preventing and / or treating diseases related to Staphylococcus aureus.

[0103] In one embodiment of the present invention, diseases associated with Staphylococcus aureus include but are not limited to: 1) suppurative infection (invasive disease); 2) toxic disease: a toxic disease caused by exotoxin.

[0104] Among them, suppurative infection includes but is not limited to: 1) suppurative infection of the skin: such as folliculitis, furuncle, carbuncle, suppuration of wound and abscess, etc. It can also invade the respiratory tract or bloodstream to cause infection. Clinical manifestations: pus is golden and viscous, the lesion boundary is clear, and it is 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 focus of the skin is squeezed by external force or the body's resistance is reduced, it will cause sepsis and septicemia.

[0105] 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 1-6 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 1-2 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: It is more common in infants and adults with low immunity. At first, erythema appears on the skin, and the epidermis wrinkles in 1-2 days, followed by the appearance of bullae containing sterile, clear fluid, 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 have heart and kidney failure, and may even suffer from shock.

[0106] In a specific embodiment of the present invention, the Staphylococcus aureus-related disease is selected from disseminated infection and / or systemic infection.

[0107] 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.

[0108] Example 1 Expression and purification of wild-type (wSpA) and mutant (mSpA) proteins of Staphylococcus aureus protein A

[0109] 1. Expression and purification of wild-type (wSpA) and mutant (mSpA) proteins of Staphylococcus aureus protein A

[0110] The SAR0114 gene (Staphylococcus aureus protein A) of the genome (BX571856.1GI:49240382) of Staphylococcus aureus (MRSA 252) was used as a template to design corresponding point mutations in the sequence of this segment. The amino acids at the corresponding positions of wSpA are (DEABC(7Q8Q, 34D35D, 68Q69Q, 95D96D, 126Q127Q, 153D154D, 184Q185Q, 211D212D, 242Q243Q, 269D270D)), which are mutated to mSpA amino acids (DEABC(7K8K, 34A35A, 68K69K, 95A96A, 126K127K, 153A154A, 184K185K, 211A212A, 242K243K, 269A270A)), and the codons are optimized, restriction sites are added to the gene sequence, a BamHⅠ site is added to the N-terminus, and a Not I site is added to the C-terminus. . The target gene sequences 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 working strains were cultured overnight at 37°C in LB medium containing ampicillin. The next day, the bacterial solution was expanded and IPTG-induced expression was performed. The bacterial cells were collected by centrifugation and homogenized. The fusion proteins wSpA-GST and mSpA-GST were obtained by SP FF cation chromatography (Borgron (Shanghai) Biotechnology Co., Ltd.), and the GST tag was specifically removed by 3C protease of human rhinovirus type 14 (Prescission Protease enzyme, PP enzyme, GE Healthcare, USA), and then filtered with a sterilizing filter. The filtrate was desalted by SP HP cation chromatography, Phenyl HP hydrophobic chromatography, G25 (Borgron (Shanghai) Biotechnology Co., Ltd.), and Q HP anion exchange chromatography (GE Healthcare, USA) to obtain wild-type wSpA and mutant mSpA proteins.

[0111] 2. Experimental results

[0112] The molecular weight of the purified wSpA protein was about 30.7 kDa, and the purity was 100.0% 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 ).

[0113] Example 2 Establishment of a Human Specific Anti-Staphylococcus aureus Fab Antibody Library

[0114] 1. Source of human PBMC cells

[0115] 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 subjects stored in liquid nitrogen with the recombinant Staphylococcus aureus vaccine (Escherichia coli) were obtained from the Phase Ia clinical trial center.

[0116] 2. Construction of human-specific anti-Staphylococcus aureus Fab antibody library using phage surface display technology

[0117] The immunogenicity (Luminex test with mSpA protein (HPLC purity > 95%) as antigen) and efficacy (OPKA test) evaluation results of active immune sera were selected, and the Luminex and OPKA test results were significantly higher than the average values ​​of the placebo group and the PBMC of the 9 subjects before vaccine injection. Total RNA was extracted (QIAGEN, RNeasy Plus Mini Kit (250)), and ezDNase was used to TM Enzyme SuperScript TM IV First-Strand Synthesis System (Invitrogen, containing ezDNase TM Enzyme SuperScript TM IV first-chain synthesis system) to synthesize cDNA, the above-mentioned reverse cDNA library was mixed as a gene template, and the forward primer and reverse primer set of the heavy chain and light chain were used to amplify the light chain (VL+CL) Kappa / Lambda and heavy chain Fd segment gene sequences by PCR. The PCR recovery product of the light chain VL+CL gene was double-digested with SacI-HF enzyme and XbaI-HF enzyme, and then cloned into the pComb3XSS phage display vector. Similarly, the heavy chain Fd segment was cloned into the pComb3XSS vector connected to the light chain gene fragment through the XhoI-HF and SpeI-HF restriction enzyme sites to form a Fab phage antibody library. The ligation product was electroporated into TG1 electrocompetent cells to generate a phage library, 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.

[0118] 3. Experimental results

[0119] The results showed that the Fab (Kappa light chain) library ( Figure 2 A) and Fab (Lambda light chain) library ( Figure 2B) The positive clone rate was more than 80%, and the library capacity was more than 10 8 .

[0120] Example 3 Screening of human specific anti-Staphylococcus aureus Fab antibody library

[0121] 1. Bacterial purification and antibody phage preparation

[0122] Using Dynabeads TM The phage library was bio-panned for 7 consecutive rounds without phage amplification by coupling biotinylated mSpA protein with M-280 streptavidin. The method was to incubate the phage library with mSpA-coated magnetic beads for 1 hour. After the tube was placed in a magnetic stand for adsorption, the supernatant in the tube was carefully removed; the magnetic beads were then washed 10-15 times with 0.5% PBST; the bound phage particles were then eluted with glycine-HCl (pH 2.2), and the eluate was neutralized to pH 7.0 with 1M Tris-HCl (pH 9.0) for the next round of panning. After 7 rounds of panning, the phage particles were collected and mixed with TG1 cells, plated and colonies were picked. All clones were analyzed using phage Elisa and the DNA was sequenced.

[0123] 2. Purification of phage

[0124] Take the bacterial clone carrying the recombinant phagemid and grow it to OD600 of 0.5-0.6 in 1 mL SB medium (containing 50 μg / mL carbenicillin), and infect the culture with helper phage at a ratio of 1:20 between cells and helper phage; incubate at room temperature for 30 min, then place in a shaker at 200 rpm / min, incubate at 37°C for 2 h, then add 2 mL SB medium (containing 50 μg / mL carbenicillin, 15 μg / mL tetracycline, 37.5 μg / mL kanamycin), and culture at 37°C overnight. The next day, the culture supernatant was collected by centrifugation at 2300×g for 5 minutes at room temperature; 4 times the volume of the supernatant was added with 25mM PEG-8000 and 2.5M NaCl to precipitate the phage particles, and the mixture was placed on ice for 1 hour and centrifuged for 15 minutes. The obtained phage precipitate was resuspended in sterile PBS buffer; the absorbance values ​​at 269nm and 320nm were used to calculate the phage physical titer.

[0125] 3. Bacteriophage Elisa

[0126] The physical titer of the resuspended phage was adjusted to 3-5×10 12virus / mL, 30 μL of phage resuspension was added to the Elisa plate, incubated overnight at 4°C, and washed 3 times with 0.1% PBST buffer; 3% BSA blocking solution was added for blocking for 1 hour, and then washed 6 times with washing solution; 10 μg / mL biotinylated mSpA protein was added for incubation for 1 hour, and then washed 6 times with washing solution; finally, Streptavidin HRP was diluted 1:1000 with blocking solution, and then tested, and 8 highly positive clones were selected for further analysis.

[0127] Example 4 Cloning, expression and purification of SpA-A6 monoclonal antibody

[0128] 1. Experimental methods

[0129] The positive Fab display vector screened in Example 3 was used as a template, and the heavy chain and light chain were cloned into pCDNA3.4 using a forward primer containing Kozak and Lonza specific leader sequences and homologous to the antibody sequence, and a reverse primer homologous to the 3' segment of the heavy chain and light chain variable regions to construct an expression vector for a fully human anti-SpA antibody. The expression vector was then transformed into DH5α competent bacteria, cultured overnight at 37°C on a plate containing ampicillin, and 10 single colonies were picked for PCR 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. 5μL of PCR product was detected by 1% agarose gel electrophoresis.

[0130] 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 / ).

[0131] 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.

[0132] 2. Experimental results

[0133] The results showed that a series of antibody heavy chain (H435R and Y436F mutations in hIgG1 Fc) and light chain expression vectors were successfully constructed. The antibody clone was named the fully human SpA monoclonal antibody SpA-A6 (abbreviated as SpA-A6 antibody or SpA-A6 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 ), whose sequence is shown in Table 1.

[0134] Table 1 SpA-A6 antibody sequences

[0135]

[0136]

[0137]

[0138] Example 5 Specificity of the binding of SpA-A6 monoclonal antibody to Staphylococcus aureus protein A

[0139] 1. Experimental methods

[0140] 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-A6 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.

[0141] Wash 3 times with 0.1% PBST buffer, add 100μL of 5000-fold diluted Chicken anti-HumanIgG Fc Secondary Antibody, HRP to each well, and incubate at 37℃ for 1h. Wash 5 times with 0.1% PBST buffer, protect from light, add 100μL of TMB to each well, place at 37℃ for 5-10min, and then add 50μL of 2M H2SO4 to terminate. Detect OD value at dual wavelength 450 / 650nm.

[0142] 2. Experimental results

[0143] The results are as follows Figure 4As 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).

[0144] Due to the presence of H435R and / or Y436F mutations in the Fc of SpA-A6 and hIgG1 (PcrV-A039) Fc monoclonal antibodies, SpA-A6 monoclonal antibodies specifically bind to mSpA with an EC50 of 2.50×10 -10 ; SpA-A6 monoclonal antibody can specifically bind to wSpA with an EC50 of 3.01×10 -10 .

[0145] Table 2 Binding results of SpA-A6 and Staphylococcus aureus protein A (wild-type wSpA and mutant mSpA)

[0146]

[0147] Example 6 SpA-A6 monoclonal antibody opsonizing and killing Staphylococcus aureus

[0148] 1. Experimental methods

[0149] The antibody can promote the phagocytic killing of Staphylococcus aureus by HL60 cells, but Staphylococcus aureus has a variety of virulence factors to prevent killing by phagocytes; in order to evaluate the ability of SpA-A6 monoclonal antibody to promote the killing of Staphylococcus aureus by phagocytes, SpA-A6 monoclonal antibody was tested in the opsonized phagocytic killing test (OPK) to evaluate the ability of the monoclonal antibody of the present invention to promote the killing of Staphylococcus aureus Newman in the presence of complement. The process steps are: SpA-A6 monoclonal antibody, positive serum, negative serum, and control antibody are diluted in 7 gradients using a microtiter plate; Staphylococcus aureus Newman is cultured in TSB and suspended in a buffer solution to a concentration of 1.2×10 5 CFU / mL, add 10 μL to the microplate and shake and incubate; add 10 μL complement and shake and incubate again; wash the HL-60 cells differentiated for 3.5 days and use buffer to adjust the concentration to 1.0×10 7Cells / mL, add 40 μL to each well, and incubate the microplate for phagocytosis at 37°C, 5% CO2 for 75 min. After the phagocytosis is completed, place the microplate on ice for 20 min, and take 10 μL of the mixture and inoculate it on MHA for counting.

[0150] 2. Experimental results

[0151] The results are as follows Figure 5 As shown, the control antibody could not promote cell killing of Staphylococcus aureus, while the SpA-A6 monoclonal antibody could promote cell killing of Staphylococcus aureus, and the maximum killing rate was 63%.

[0152] Example 7 Evaluation of the effect of SpA-A6 monoclonal antibody in a disseminated Staphylococcus aureus infection model

[0153] 1. Experimental methods

[0154] S. aureus causes disseminated infection in mice when injected intraperitoneally with high levels of bacterial replication in the kidney. In this experiment, SpA-A6 monoclonal antibodies were tested for their ability to reduce kidney burden when administered 1 day after S. aureus infection.

[0155] The SpA-A6 monoclonal antibodies of the present invention were 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 8 cfu / 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 10 mg / kg of SpA-A6 monoclonal antibody, the control group was treated with 10 mg / kg of hIgG1 (PcrV-A039), 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 X-100 and homogenized using a Cmax dissociation tube. Bacteria were counted by colony forming units via serial dilution and inoculated onto TSA.

[0156] 2. Experimental results

[0157] The SpA-A6 monoclonal antibody reduced the S. aureus kidney burden by approximately 2 logs, whereas hIgG1(PcrV-A039) was ineffective in reducing the S. aureus burden compared with the control antibody ( Figure 6 ).

[0158] Example 8: Evaluation of the effect of SpA-A6 monoclonal antibody in the Staphylococcus aureus pneumonia model

[0159] 1. Experimental methods

[0160] The SpA-A6 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-A6 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.

[0161] 2. Experimental results

[0162] The protection rate of SpA-A6 monoclonal antibody containing 100 mg / kg was 70%, and the protection rate of hIgG1 (PcrV-A039) containing 100 mg / kg was 10% ( Figure 7 ).

[0163] Example 9 Evaluation of the effect of SpA-A6 monoclonal antibody in the Staphylococcus aureus systemic infection model

[0164] The SpA-A6 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-A6 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 the tail vein was challenged with 100 μL of the challenge bacteria, 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.

[0165] The protection rates of SpA-A6 monoclonal antibody at 100 mg / kg and hIgG1 (PcrV-A039) at 100 mg / kg were 60% and 10% respectively. Figure 8 ).

[0166] 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. An antibody against Staphylococcus aureus protein A, characterized in that The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the antibody are shown in SEQ ID NOs: 1, 2 and 3, respectively. The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 9, 10 and 11, respectively, and the CDRs are defined according to the IMGT numbering system.

2. The antibody according to claim 1, characterized in that The antibody further comprises heavy chain variable region framework regions HFR1, HFR2, HFR3, HFR4 having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 4, 5, 6, 7, And light chain variable region framework regions LFR1, LFR2, LFR3, and LFR4 having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs: 12, 13, 14, and 15.

3. The antibody according to claim 2, characterized in that The amino acid sequences of the heavy chain variable region framework regions HFR1, HFR2, HFR3, and HFR4 are shown in SEQ ID NOs: 4, 5, 6, and 7, respectively. The amino acid sequences of the light chain variable region framework regions LFR1, LFR2, LFR3, and LFR4 are shown in SEQ ID NOs: 12, 13, 14, and 15, respectively.

4. The antibody according to claim 3, characterized in that The antibody heavy chain variable region has a VH having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 8, The light chain variable region has a VL that has at least 70% sequence identity to the amino acid sequence shown in SEQ ID NO:

16.

5. The antibody according to claim 4, characterized in that The VH of the antibody has the amino acid sequence shown in SEQ ID NO:8, and the VL of the antibody has the amino acid sequence shown in SEQ ID NO:

16.

6. The antibody according to claim 5, characterized in that The hIgG1 Fc of the antibody has H435R and / or Y436F mutations.

7. The antibody according to claim 5, characterized in that The antibody binds to a linear epitope.

8. The antibody according to claim 5, characterized in that The antibody is afucosylated.

9. Any of the following substances: (1) A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the antibody according to any one of claims 1 to 8; (2) A vector, characterized in that the vector comprises the nucleic acid molecule described in (1); or (3) a host cell, characterized in that the host cell comprises the nucleic acid molecule described in (1) or the vector described in (2).

10. The substance according to claim 9, characterized in that The nucleic acid molecules encoding HCDR1, HCDR2, and HCDR3 in (1) have at least 95% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 17, 18, and 19, respectively, The nucleic acid molecules encoding LCDR1, LCDR2, and LCDR3 have at least 95% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 25, 26, and 27, respectively.

11. The substance according to claim 10, characterized in that The nucleotide sequences of the nucleic acid molecules encoding HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 17, 18, and 19, respectively, and the nucleotide sequences of the nucleic acid molecules encoding LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 25, 26, and 27, respectively.

12. The substance according to claim 9, characterized in that The nucleic acid molecules encoding HFR1, HFR2, HFR3, and HFR4 have at least 90% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 20, 21, 22, and 23, respectively, The nucleic acid molecules encoding LFR1, LFR2, LFR3, and LFR4 have at least 90% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 28, 29, 30, and 31, respectively.

13. The substance according to claim 12, characterized in that The nucleotide sequences of the nucleic acid molecules encoding HFR1, HFR2, HFR3, and HFR4 are shown in SEQ ID NOs: 20, 21, 22, and 23, respectively, and the nucleotide sequences of the nucleic acid molecules encoding LFR1, LFR2, LFR3, and LFR4 are shown in SEQ ID NOs: 28, 29, 30, and 31, respectively.

14. The substance according to claim 9, characterized in that The nucleic acid molecule encoding VH has at least 70% sequence identity with the nucleotide sequence shown in SEQ ID NO: 24, The nucleic acid molecule encoding VL has at least 70% sequence identity with the nucleotide sequence shown in SEQ ID NO:

32.

15. The substance according to claim 14, characterized in that The nucleotide sequence of the nucleic acid molecule encoding VH is shown in SEQ ID NO:24, and the nucleotide sequence of the nucleic acid molecule encoding VL is shown in SEQ ID NO:

32.

16. The substance according to claim 9, characterized in that The vector described in (2) has a signal peptide operably linked to the antibody.

17. The substance according to claim 9, characterized in that The vector also includes elements for controlling expression.

18. The substance according to claim 9, characterized in that The vector may also include materials that facilitate its entry into cells.

19. The substance according to claim 9, characterized in that The host cells mentioned in (3) include prokaryotic cells and eukaryotic cells.

20. A product for detecting Staphylococcus aureus protein A, characterized in that: The product comprises the antibody according to any one of claims 1-8.

21. The product according to claim 20, characterized in that The products include test kits and test strips.

22. The product according to claim 21, characterized in that The kit includes a conjugate of an antibody linked to a signal generating compound.

23. A derivative, characterized in that The derivative is a diagnostic agent or a therapeutic agent connected to the antibody according to any one of claims 1 to 8 or the nucleic acid molecule according to any one of claims 9 to 15.

24. The derivative according to claim 23, characterized in that The diagnostic reagents include fluorescent labels, radioisotopes, chemiluminescent molecules, paramagnetic ions or spin-trapping reagents.

25. The derivative according to claim 23, characterized in that The therapeutic agents include cytokines, hormones, antibody Fc fragments, TLR agonists, CpG-containing molecules or immune co-stimulatory molecules.

26. The derivative according to claim 23, characterized in that The therapeutic agent includes a chemotherapeutic agent or a radiotherapeutic agent.

27. The derivative according to claim 23, characterized in that The therapeutic agents include anti-inflammatory agents.

28. Any of the following methods: (1) A method for preparing the antibody according to any one of claims 1 to 8, characterized in that: The method comprises: culturing the host cell of claim 9 or 19, and recovering the antibody; (2) A method for detecting Staphylococcus aureus protein A in a sample for non-diagnostic purposes, characterized in that the method comprises: contacting the anti-Staphylococcus aureus protein A antibody according to any one of claims 1 to 8 with a sample to be tested, thereby detecting the level of Staphylococcus aureus protein A in the sample to be tested.

29. The method according to claim 28, characterized in that The method described in (1) further comprises purifying the antibody.

30. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the antibody according to any one of claims 1-8, the substance according to any one of claims 9-19, the product according to any one of claims 20-22 or the derivative according to any one of claims 23-27.

31. The pharmaceutical composition according to claim 30, characterized in that The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

32. Any of the following applications: (1) Use of the antibody according to any one of claims 1 to 8, the substance according to any one of claims 9 to 19, the product according to any one of claims 20 to 22, or the derivative according to any one of claims 23 to 27 for the detection of Staphylococcus aureus protein A for non-diagnostic purposes; (2) Use of the antibody according to any one of claims 1 to 8, the substance according to any one of claims 9 to 19, the product according to any one of claims 20 to 22, or the derivative according to any one of claims 23 to 27 in the preparation of a product for detecting Staphylococcus aureus protein A; (3) Use of the antibody according to any one of claims 1 to 8, the substance according to any one of claims 9 to 19, the product according to any one of claims 20 to 22, the derivative according to any one of claims 23 to 27, or the pharmaceutical composition according to any one of claims 30 to 31 in the preparation of a medicament for preventing and / or treating diseases associated with Staphylococcus aureus.

33. The use according to claim 32, characterized in that Diseases associated with Staphylococcus aureus include suppurative infections or toxic diseases.

34. The use according to claim 33, characterized in that The purulent infection includes purulent infection of the skin, purulent infection of various organs, disseminated infection or systemic infection.

35. The use according to claim 34, characterized in that The skin suppurative infection includes folliculitis, furuncle, carbuncle, wound suppuration or abscess.

36. The use according to claim 34, characterized in that The suppurative infections of the various organs include tracheitis, pneumonia, empyema, otitis media or osteomyelitis.

37. The use according to claim 34, characterized in that The systemic infection includes sepsis and septicemia.

38. The use according to claim 33, characterized in that The toxic diseases include food poisoning, scalded skin syndrome or toxic shock syndrome.

39. The use according to claim 34, characterized in that The Staphylococcus aureus-related disease is selected from disseminated infection and / or systemic infection.

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