A monoclonal antibody specifically binding to Staphylococcus aureus manganese ion transporter C and its application

Abstract

The present invention discloses a monoclonal antibody specifically binding to Staphylococcus aureus manganese ion transporter C and its application. The antibody MntC-15 provided by the present invention can specifically bind to the MntC protein, can effectively inhibit the growth of Staphylococcus aureus, can resist sepsis caused by MRSA infection, and inhibit the release of mouse spleen lymphocyte cytokines induced by MntC, and has broad application prospects in clinical practice.

Description

Technical Field

[0001] The invention belongs to the field of medical immunity, and in particular relates to a monoclonal antibody specifically binding to Staphylococcus aureus manganese ion transporter protein C and an application thereof. Background Art

[0002] Staphylococcus aureus is an important pathogen that causes hospital and community infections. The infection is characterized by acute and suppurative characteristics. It can cause severe infections and complications such as sepsis, acute pneumonia, endocarditis, septic arthritis, and osteomyelitis throughout the body, with an infection mortality rate of up to 20%; it can cause suppurative infections of the skin and soft tissues locally, which are long-lasting. At the same time, the exotoxins of Staphylococcus aureus can also cause systemic fatal infections such as food poisoning, scalded skin syndrome, and toxic shock syndrome. With the long-term and widespread use of antibiotics, the problem of bacterial resistance has become increasingly prominent. As a typical representative, methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most common nosocomial pathogens in the world since it was first discovered in 1961. In addition, due to its strong pathogenicity, wide transmission routes, easy outbreaks, and the development of multiple drug resistance, it has become a difficult point in clinical treatment.

[0003] So far, there is no effective antibody drug or vaccine product in the world to treat or prevent Staphylococcus aureus infection. Staphylococcus aureus manganese ion binding protein C (MntC) is a highly conserved cell surface protein and an important virulence factor for systemic infection of Staphylococcus aureus. It plays an important role in Staphylococcus aureus infection by promoting the acquisition of manganese ions from the host environment. The loss or reduction of manganese ion transporter activity will make Staphylococcus aureus sensitive to oxidative stress, making the bacteria vulnerable to attack by immune cells and death. At the same time, MntC has adhesin activity, which may be a feature of the pathogenesis of Staphylococcus aureus.

[0004] Therefore, developing antibodies against S. aureus manganese-binding protein C is crucial for the treatment of S. aureus infections. Summary of the invention

[0005] To make up for the deficiencies of the prior art, the present invention provides a monoclonal antibody specifically binding to Staphylococcus aureus manganese ion transporter protein C and application thereof.

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

[0007] The first aspect of the present invention provides a monoclonal antibody that specifically binds to MntC, the monoclonal antibody comprising three CDRs of a heavy chain variable region complementary determining region and three CDRs of a light chain variable region complementary determining region, wherein the amino acid sequences of the heavy chain variable region complementary determining regions CDR1, CDR2, and CDR3 have 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: 1, 2, and 3, respectively, and the amino acid sequences of the light chain variable region complementary determining regions CDR1, CDR2, and CDR3 have 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, respectively.

[0008] Furthermore, the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NOs: 1, 2, and 3, respectively, and the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NOs: 9, 10, and 11, respectively.

[0009] Further, the heavy chain variable region also includes a heavy chain variable region framework region of four FRs, and the light chain variable region also includes a light chain variable region framework region of four FRs, wherein the amino acid sequences of the heavy chain variable region framework regions FR1, FR2, FR3 and FR4 have 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 amino acid sequences shown in SEQ ID NOs: 4, 5, 6, and 7, respectively, and the amino acid sequences of the light chain variable region framework regions FR1, FR2, FR3 and FR4 have 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 amino acid sequences shown in SEQ ID NOs: 12, 13, 14, and 15, respectively.

[0010] Furthermore, the amino acid sequences of the heavy chain variable region framework regions FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs: 4, 5, 6, and 7, respectively, and the amino acid sequences of the light chain variable region framework regions FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs: 12, 13, 14, and 15, respectively.

[0011] Further, the amino acid sequence of the heavy chain variable region 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 amino acid sequence shown in SEQ ID NO:8,

[0012] The amino acid sequence of the light chain variable region 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 with the amino acid sequence shown in SEQ ID NO:16.

[0013] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:16.

[0014] Further, the monoclonal antibody binds to a linear epitope.

[0015] Further, the CDRs are defined according to the IMGT numbering system.

[0016] Furthermore, the monoclonal antibody is afucosylated.

[0017] The second aspect of the present invention provides an antigenic epitope polypeptide, which comprises a polypeptide fragment of MntC or a variant thereof.

[0018] Furthermore, the antigenic epitope polypeptide has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO:33.

[0019] Furthermore, the sequence of the antigen epitope polypeptide is shown in SEQ ID NO:33.

[0020] The third aspect of the present invention provides a nucleic acid molecule, which encodes the monoclonal antibody described in the first aspect of the present invention or the antigen epitope polypeptide described in the second aspect of the present invention.

[0021] Furthermore, the nucleotide sequences of the nucleic acid molecules encoding the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the monoclonal antibody 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.

[0022] The nucleotide sequences of the nucleic acid molecules encoding the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the monoclonal antibody 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.

[0023] Furthermore, the nucleotide sequences of the nucleic acid molecules encoding the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the monoclonal antibody are shown in SEQ ID NOs: 17, 18, and 19, respectively.

[0024] The nucleotide sequences of the nucleic acid molecules encoding the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the monoclonal antibody are shown in SEQ ID NOs: 25, 26, and 27, respectively.

[0025] Further, the nucleotide sequences of the nucleic acid molecules encoding the framework regions FR1, FR2, FR3 and FR4 of the heavy chain variable region of the monoclonal antibody have 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 sequences shown in SEQ ID NOs: 20, 21, 22, and 23, respectively.

[0026] The nucleotide sequences of the nucleic acid molecules encoding the monoclonal antibody light chain variable region framework regions FR1, FR2, FR3 and FR4 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.

[0027] Further, the nucleotide sequences of the nucleic acid molecules encoding the framework regions FR1, FR2, FR3 and FR4 of the heavy chain variable region of the monoclonal antibody are shown in SEQ ID NOs: 20, 21, 22 and 23, respectively.

[0028] The nucleotide sequences of the nucleic acid molecules encoding the monoclonal antibody light chain variable region framework regions FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs: 28, 29, 30 and 31, respectively.

[0029] Furthermore, the amino acid sequence encoding the heavy chain variable region of the monoclonal antibody 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 amino acid sequence shown in SEQ ID NO: 24,

[0030] The amino acid sequence encoding the light chain variable region of the monoclonal antibody 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:32.

[0031] Furthermore, the amino acid sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:24, and the amino acid sequence encoding the light chain variable region is shown in SEQ ID NO:32.

[0032] The fourth aspect of the present invention provides a recombinant expression vector, which comprises the nucleic acid molecule described in the third aspect of the present invention.

[0033] Furthermore, the recombinant expression vector also includes a promoter.

[0034] Furthermore, the recombinant expression vector includes a plasmid vector, a viral vector or a phage vector.

[0035] The fifth aspect of the present invention provides a host cell, wherein the host cell comprises the nucleic acid molecule described in the third aspect of the present invention or the recombinant expression vector described in the fourth aspect of the present invention.

[0036] Furthermore, the host cells include prokaryotic cells and eukaryotic cells.

[0037] Furthermore, the eukaryotic cells include lower eukaryotic cells and higher eukaryotic cells.

[0038] Furthermore, the higher eukaryotic cells include mammalian cells.

[0039] The sixth aspect of the present invention provides a pharmaceutical composition, which comprises the monoclonal antibody described in the first aspect of the present invention, the antigenic epitope described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the recombinant expression vector described in the fourth aspect of the present invention, or the host cell described in the fifth aspect of the present invention.

[0040] Furthermore, the pharmaceutical composition also includes a pharmaceutically compatible carrier.

[0041] Furthermore, the pharmaceutical composition also includes a buffer.

[0042] The seventh aspect of the present invention provides a kit for detecting MntC protein, wherein the kit comprises the monoclonal antibody described in the first aspect of the present invention.

[0043] Furthermore, the kit also includes a detectable label conjugated to the monoclonal antibody.

[0044] Furthermore, the detectable label includes a fluorescent label, a radioactive isotope, a chemiluminescent molecule, a paramagnetic ion or a spin-trapping agent.

[0045] The eighth aspect of the present invention provides a method for detecting MntC protein in a sample, the method comprising contacting the monoclonal antibody described in the first aspect of the present invention with the sample to be tested, thereby detecting the level of MntC protein in the sample to be tested.

[0046] Furthermore, the method is a method for non-diagnostic purposes.

[0047] The ninth aspect of the present invention provides a method for preparing the monoclonal antibody described in the first aspect of the present invention, the method comprising culturing the host cell described in the fifth aspect of the present invention and recovering the monoclonal antibody.

[0048] The tenth aspect of the present invention provides any of the following applications:

[0049] (1) Use of the monoclonal antibody described in the first aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the recombinant expression vector described in the fourth aspect of the present invention, and the host cell described in the fifth aspect of the present invention in detecting the content of MntC protein;

[0050] (2) Use of the monoclonal antibody described in the first aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the recombinant expression vector described in the fourth aspect of the present invention, and the host cell described in the fifth aspect of the present invention in the preparation of a product for detecting MntC protein;

[0051] (3) Use of the monoclonal antibody of the first aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention, the recombinant expression vector of the fourth aspect of the present invention, or the host cell of the fifth aspect of the present invention in inhibiting Staphylococcus aureus or in preparing a pharmaceutical composition for preventing and / or treating diseases related to Staphylococcus aureus infection;

[0052] 4) Use of the antigen epitope polypeptide according to the second aspect of the present invention in the preparation of anti-MntC antibodies;

[0053] 5) Use of the antigenic epitope polypeptide according to the second aspect of the present invention in the preparation of a vaccine for preventing Staphylococcus aureus infection;

[0054] 6) Use of the antigen epitope polypeptide described in the second aspect of the present invention in the preparation of a product for detecting anti-MntC antibodies.

[0055] Furthermore, the diseases associated with Staphylococcus aureus infection include osteomyelitis, necrotizing fasciitis, endocarditis, peritonitis, septic arthritis, sepsis, bacteremia, sepsis, pneumonia, abscess and toxic shock syndrome.

[0056] Furthermore, the disease related to Staphylococcus aureus infection is selected from sepsis.

[0057] Furthermore, the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus.

[0058] Advantages and beneficial effects of the present invention:

[0059] The antibody MntC-15 provided by the present invention can specifically bind to the MntC protein, can effectively inhibit the growth of Staphylococcus aureus, can resist sepsis caused by MRSA infection, and inhibit the release of cytokines in mouse spleen lymphocytes induced by MntC, and has broad application prospects in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is a diagram showing the results of SDS-PAGE detection of the recombinant MntC protein;

[0061] Figure 2 2A is a diagram of Fab (Kappa light chain) library, and 2B is a diagram of Fab (Lambda light chain) library;

[0062] Figure 3 This is the result of SDS-PAGE detection of expression and purification of the fully human monoclonal antibody MntC-15;

[0063] Figure 4 This is a graph showing the binding activity test results of the fully human monoclonal antibody MntC-15;

[0064] Figure 5 This is the WB test result of MntC-15;

[0065] Figure 6 This is the Elisa result of the fully human monoclonal antibody MntC-15 against the MntC Mapping peptide;

[0066] Figure 7 This is the result of the fully human monoclonal antibody MntC-15 interfering with the oxidative stress of Staphylococcus aureus;

[0067] Figure 8 The results of the fully human monoclonal antibody MntC-15 specifically inhibiting the MntC protein of Staphylococcus aureus are shown in Figure 8A, where 8A is the result of 20 mM methyl viologen, and 8B is the result of 45 mM methyl viologen;

[0068] Fig. 9 The figures are the evaluation results of the fully human monoclonal antibody MntC-15 on the animal model of sepsis, wherein 9A is the infection dose exploration figure of the MRSA sepsis model, and 9B is the survival rate analysis figure of the MntC-15 treatment of the MRSA sepsis model;

[0069] Fig.10 These are the results of the human monoclonal antibody MntC-15 inhibiting renal abscesses and bacterial loads caused by MRSA infection, wherein 10A is the result of the number of renal abscesses, and 10B is the result of the number of renal bacterial loads. DETAILED DESCRIPTION

[0070] 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.

[0071] The present invention provides a monoclonal antibody specifically binding to MntC, wherein the monoclonal antibody comprises three CDRs of the heavy chain variable region complementary determining regions and three CDRs of the light chain variable region complementary determining regions.

[0072] In one embodiment of the present invention, a monoclonal antibody (mAB) refers to an antibody molecule with a single molecular composition obtained from a group of substantially identical antibodies. An antibody comprises two heavy (H) chains and two light (L) chains. A 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); a 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 comprises a variable region and a first constant region of a single heavy chain bound to a variable region and a 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.

[0073] The precise amino acid sequence boundaries of the complementarity determining regions (CDRs) of antibodies can be defined according to well-known methods, such as Chothia (Chothia et al., Nature, 342:877-883, 1989; Al-Lazikani et al., Journal of Molecular Biology, 273:927-948, 1997), which is based on the three-dimensional structure of antibodies and the topology of the CDR loops; or Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health, 1987), AbM (University of Bath), Contact (University College London), and IMGT (the international ImMunoGeneTics database, 1999 Nucleic Acids Research, 27, 209-212), which are based on antibody sequence variability; or the North CDR definition based on affinity propagation clustering using a large number of crystal structures. The boundaries of the CDR of the antibody in the present application can be determined by a person skilled in the art according to any scheme in the art (such as the above optional definition method).

[0074] It should be noted that the boundaries of the CDRs of the same antibody obtained based on different definition methods may be different, that is, the CDR sequences of the variable regions of the same antibody obtained under different definition methods are different. Therefore, when the specific CDR sequences defined in this application are used to define antibodies, the antibodies also include antibodies whose complementary determining region sequences contain the CDR sequences described in this application, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in this application due to the use of different CDR boundary definition methods.

[0075] In a specific embodiment of the invention, the CDRs are defined according to the IMGT numbering system.

[0076] The monoclonal antibody is afucosylated.

[0077] 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.

[0078] The present invention provides a recombinant expression vector, which comprises the above nucleic acid molecule.

[0079] In one embodiment of the present invention, when a prokaryotic cell is used as a host, the recombinant expression vector generally comprises a strong promoter that enables transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter), a ribosome binding site for starting translation and a transcription / translation end sequence. When using Escherichia coli strains (E. coli) (for example, HB101, BL21, DH5α, Top10, JM109, etc.) as host cells, the promoter and operator site of the E. coli tryptophan biosynthesis pathway (Yanofsky, C., J. Bacteriol., (1984) 158: 1018-1024) and the leftward promoter of bacteriophage λ (pLλ promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., (1980) 14: 399-445) can be used as regulatory sites. When Bacillus is used as a host cell, the promoter of the toxin protein gene of Bacillus thuringiensis (Appl. Environ. Microbiol. (1998) 64: 3932-3938; Mol. Gen. Genet. (1996) 250: 734-741) or any promoter that can be expressed in Bacillus can be used as a regulatory site.

[0080] When eukaryotic cells are used as hosts, the recombinant expression vector can utilize promoters derived from the genome of mammalian cells (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter), and usually has a polyadenylation sequence as a transcription termination sequence.

[0081] In addition, the recombinant expression vectors of the present invention also include plasmids (for example, pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pcDNA3.4, pET series and pUC19, etc.), bacteriophages (for example, λgt4.λB, λ-Charon, λΔz1 and M13, etc.) or viruses (for example, SV40, etc.).

[0082] The present invention provides a host cell, which comprises the above nucleic acid molecule or the above recombinant expression vector.

[0083] In one embodiment of the present invention, the host cell can be a prokaryotic cell, such as E. coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus sp. In addition, the host cell can be a fungal cell, such as Aspergillus sp., a yeast cell such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp. and Neurospora crassa, a lower eukaryotic cell and a higher eukaryotic cell such as an insect cell. In addition, the host cell can be from a plant and / or a mammal. Preferred examples of host cells include, but are not limited to, PER.C6 cells, monkey kidney cells 7 (COS7, particularly simian COS cells), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, Madin-Darby canine kidney (MDCK) cells, myeloma cell lines, HuT78 cells, 293T cells, 293F cells and other mammalian host cells that produce antibody proteins according to the present invention.

[0084] In the present invention, the method for transformation into host cells includes any method for introducing nucleic acid into organisms, cells, tissues or organs, which can be carried out using standard techniques selected according to the type of host cells as known in the art. The method includes but is not limited to electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, agitation using silicon carbide fibers, Agrobacterium-mediated transformation, and PEG, dextran sulfate, liposomes (lipofectamine) or transformation mediated by drying / inhibition.

[0085] The present invention provides a pharmaceutical composition, which comprises the above-mentioned antibody, the above-mentioned nucleic acid molecule, the above-mentioned recombinant expression vector or the above-mentioned host cell.

[0086] The pharmaceutical composition also includes a pharmaceutically compatible carrier.

[0087] In the present invention, pharmaceutical compatibility refers to non-toxic materials that do not interact with the action of the active ingredient of the pharmaceutical composition. The pharmaceutically compatible carrier refers to a natural or synthetic, organic or inorganic component that is used in conjunction with the active ingredient to facilitate application. According to the present invention, the pharmaceutically compatible carrier includes one or more compatible solid or liquid fillers, diluents or encapsulating materials, and the carrier is suitable for administration to a patient. The components of the pharmaceutical composition of the present invention generally do not interact with each other in a manner that significantly affects the desired drug efficacy.

[0088] The present invention provides a kit for detecting MntC protein, and the kit comprises the above-mentioned monoclonal antibody.

[0089] The kit also includes a detectable label conjugated to the monoclonal antibody.

[0090] In one embodiment of the invention, the detectable label includes, but is not limited to, a fluorescent label, a radioisotope, a chemiluminescent molecule, a paramagnetic ion, or a spin-trapping agent.

[0091] Among them, fluorescent labels include but are not limited to 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 and / or Texas Red.

[0092] 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) and / or yttrium 90 .

[0093] 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).

[0094] The present invention provides the use of the monoclonal antibody, the nucleic acid molecule, the recombinant expression vector and the host cell in inhibiting Staphylococcus aureus or in preparing a pharmaceutical composition for preventing and / or treating diseases related to Staphylococcus aureus infection.

[0095] In one embodiment of the invention, S. aureus infections, including MRSA, usually begin as small red bumps similar to papules, boils or spider bites. These bumps or scars can quickly turn into painful deep abscesses that require surgical drainage. Sometimes, the bacteria stay confined to the skin. Sometimes, they penetrate deep into the body and cause potentially life-threatening infections in a wide range of human tissues, including skin, soft tissue, bones, joints, surgical wounds, blood flow, heart valves, lungs or other organs. Therefore, S. aureus infections can cause conditions associated with them, which can be fatal diseases, such as osteomyelitis, necrotizing fasciitis, endocarditis, peritonitis, septic arthritis, sepsis, bacteremia, sepsis, abscesses and toxic shock syndrome, and various forms of pneumonia, including necrotizing pneumonia, and toxin production in furunculosis and carbuncle.

[0096] In a preferred embodiment of the present invention, the disease associated with Staphylococcus aureus infection is selected from sepsis.

[0097] In a specific embodiment of the present invention, the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus.

[0098] 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.

[0099] Example 1 Expression and purification of Staphylococcus aureus manganese ion transporter C (MntC)

[0100] 1. Expression and purification of Staphylococcus aureus manganese transporter C (MntC)

[0101] MntC was selected from the mature peptide of MRSA252 (BX571856.1GI:49240382) SAR0641, and the PMNTCBamHI and PMNTCNotI primer pairs were designed to PCR amplify the mature peptide gene from the 252 genome. The MntC PCR product was digested with BamHI+NotI, and the target gene was connected to the pGEX-6P-2 vector using a DNA ligation kit to complete the construction of the gene expression vector pGEX-MntC, which was expressed in XL1-Blue Escherichia coli. The MntC engineered bacteria pGEX-MntC / XL1 was cultured in LB medium containing ampicillin at 37°C overnight, and the next day, the culture was expanded and IPTG-induced expression was performed, the bacteria were centrifuged and the lysis buffer was added, and the bacteria were lysed by a high-pressure homogenizer. The MntC-GST fusion protein was purified using glutathione sepharose 4B (GE Healthcare, USA), and then the GST tag was removed by enzyme cleavage and elution using PP enzyme (Prescission protease, GE Healthcare, USA). The MntC protein solution after initial purification was finely purified using MMC filler, and linear gradient elution was performed with eluent to collect the target protein recombinant MntC protein.

[0102] 2. Experimental results

[0103] The molecular weight of the purified MntC protein was about 33.0 kDa, and the purity was 100% as determined by SDS-PAGE. Figure 1 ).

[0104] Example 2 Establishment of a Human Specific Anti-Staphylococcus aureus Fab Antibody Library

[0105] 1. Source of PBMC cells

[0106] Based on the MntC protective antigen of the recombinant Staphylococcus aureus vaccine (Escherichia coli), the present invention unit obtained an ethics review approval through the Ethics Review Committee of the Jiangsu Provincial Center for Disease Control and Prevention, and obtained peripheral blood lymphocyte samples of Phase Ia subjects frozen in liquid nitrogen from the Phase Ia clinical trial center of the recombinant Staphylococcus aureus vaccine (Escherichia coli).

[0107] 2. Construction of human-specific anti-Staphylococcus aureus Fab antibody library using phage surface display technology

[0108] Based on the results of humoral immunogenicity (Luminex test using MntC protein (HPLC purity > 95%) as antigen) and efficacy (OPK test), PBMCs of 9 subjects whose Luminex and OPK 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. 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.

[0109] 3. Experimental results

[0110] 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 10 8 .

[0111] Example 3 Screening of human specific anti-Staphylococcus aureus Fab antibody library

[0112] 1. Bacterial purification and antibody phage preparation

[0113] The frozen library bacterial solution was inoculated into LB-penicillin medium at a ratio of 1:100 for recovery. 600nmWhen the reading is between 0.5 and 0.6, remove the bacterial solution and add the helper phage M13 at a ratio of 1:1000. After standing at 37°C for 30 minutes, continue to shake and culture for 30 minutes, collect the bacteria by centrifugation and culture them overnight in a constant temperature shaker at 30°C with LB medium containing ampicillin and kanamycin. Centrifuge the bacterial solution at 4°C and 3000g for 10 minutes to collect the supernatant phage, add PEG / NaCl solution and mix thoroughly, place it on ice for 30 minutes, centrifuge it at 4°C and 3000g for 20 minutes, remove the supernatant, add PEG / NaCl solution to the precipitate and mix thoroughly, place it on ice for 30 minutes, centrifuge it at 4°C and 11000g for 2 minutes, remove the supernatant, resuspend the precipitate with 1mL PBS and add glycerol to store it at -80°C for later use.

[0114] 2. Antigen coating

[0115] MntC protein was added at 100 μL with 5×10 11 μg / mL coated overnight at 4℃; after removing the supernatant, wash once with PBS, pat dry, block with 3% skim milk powder, incubate at 37℃ for 2h, remove the blocking solution, wash 3 times with PBST, and store at 4℃ for use.

[0116] 3. Phage library panning

[0117] The antibody phage was added to the antigen-coated ELISA plate and incubated at 37°C for 20 min. The unbound phage was removed by washing with PBST and PBS solution 10 times in sequence. The bound phage was digested with trypsin and then added to the plate until the OD 600nm Infect bacteria at TG1 of 0.5-0.6 for 30 minutes. Collect the cells by centrifugation and spread them on LB plates containing ampicillin and culture them overnight at 37°C. The next day, the titer of the selected phage sub-library was determined by gradient dilution and the cells 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.

[0118] 4. Phage library screening

[0119] 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.

[0120] Example 4 Cloning, expression and purification of anti-MntC fully human antibodies

[0121] 1. Experimental methods

[0122] 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.

[0123] 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 / ).

[0124] The PCR products of the antibody variable region genes that were positive by gel electrophoresis and the heavy and light chains could be matched were connected to the pcDNA3.4 vector containing the heavy chain constant region or the light chain constant region by TA cloning to construct the expression vector of the fully human anti-MntC antibody, and then the expression vector was transformed into DH5α competent bacteria, cultured at 37°C on a plate containing ampicillin overnight, 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. After the reaction was completed, 5μL of PCR product was taken and detected by 1% agarose gel electrophoresis.

[0125] The vector plasmid in the positive transformants detected by gel electrophoresis was transformed into DH5α for large-scale amplification. After rapid extraction of the recombinant plasmid, it was incubated with the transfection reagent PEI at 37°C for 15-20 minutes and then transfected using the concentration method. Before transfection, the concentration of HEK293F cells was adjusted to 2×10 7 cells / mL for 4h. During transfection, the cell volume was controlled to be less than 60mL. After 6h of transfection, a certain amount of OPM serum-free medium was added. After shaking and culturing at 125rpm / min in a 37°C shaker and 5% CO2 incubator for 108h, the expression supernatant was collected by centrifugation at 3000g and 4°C for 30min, and purified using protein A affinity filler. The expression and purification of the antibody were verified by SDS-PAGE.

[0126] 2. Experimental results

[0127] The results showed that the heavy chain and light chain expression vectors of the antibody were successfully constructed and the transfected cells successfully expressed the antibody. The antibody clone was named the fully human MntC monoclonal antibody MntC-15 (abbreviated as MntC-15 antibody or MntC-15 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, with a heavy chain of about 55kDa and a light chain of about 25kDa ( Figure 3 ).

[0128] Table 1 MntC-15 antibody sequence

[0129]

[0130]

[0131]

[0132] Example 5 Detection of Binding Activity of MntC-15 Antibody

[0133] 1. Experimental methods

[0134] The recombinantly expressed MntC (4 μg / mL) protein was used as an antigen to coat the ELISA plate, 100 μL per well, coated overnight at 4°C, and blocked for 2h at 37°C with 3% BSA blocking solution, 250 μL per well. The transfected and expressed MntC-15 antibody (adjusted to 10 μg / mL) in Example 4 was diluted in multiples, 200 μL / well was added to the first column of the ELISA plate, 100 μL of sample was taken out from the first column and added to the second column until the last well was diluted, 100 μL per well for each dilution; the positive control was vaccine serum (1:2000 times dilution), 100 μL per well, the negative control was negative serum (1:50 times dilution) and negative control irrelevant antibody IgG1 (10 μg / mL) 100 μL per well, and the blank control was 100 μL diluent (PBST), all of which were 3 replicates, incubated at 37°C for 60min.

[0135] After washing the plate three times with a plate washer, dilute Anti-human HRP-IgG with diluent at 1:5000, add 100 μL to each well of the ELISA plate, and incubate in a 37°C incubator for 45 min. Wash the plate once with PBST buffer (3 cycles), protect from light, add 100 μL of TMB colorimetric solution to each well, place at 37°C for 15 min, and add 50 μL of 2M sulfuric acid stop solution to each well. Detect OD with an ELISA reader 450 nm Calculate the mean of the negative control irrelevant antibody IgG1 and calculate the threshold (3 times the mean, see Figure 4 If the expression level is greater than the threshold, it is considered a positive well, and the EC50 is calculated.

[0136] 2. Experimental results

[0137] The results showed that when the concentration of MntC-15 was 0.078-10 μg / mL, the EC50 of MntC-15 binding to MntC was 0.021 μg / mL, and the control antibody did not bind to the recombinant MntC protein, indicating that the binding was specific ( Figure 4 ).

[0138] Example 6 Determination of epitope type of MntC-15 antibody

[0139] 1. Experimental methods

[0140] 1) Protein electrophoresis: Add 16 μL of reduced loading buffer (containing β-mercaptoethanol) to 4 μg of recombinant MntC protein sample and boil in a metal bath for 5 min. During this time, install the prepared 10% electrophoresis gel into the electrophoresis tank, pour in 1× electrophoresis solution, remove the comb and load the sample, protein product 2 μg / well. Set the electrophoresis instrument voltage to 80V and the electrophoresis time to 15-20min. After the band is flattened, increase the electrophoresis voltage to 200V and run for 35-40min. Stop the electrophoresis when bromophenol blue just runs out of the inner tank.

[0141] 2) Electrophoresis gel transfer: Take out the filter paper soaked in the electrophoresis transfer solution and place it flat on the transfer instrument, place the methanol-activated PVDF membrane on the filter paper, then place the electrophoresis gel with the concentrated gel removed on the membrane, cover the filter paper on the top, assemble and place it in the semi-dry transfer instrument, adjust the working voltage to 21V, transfer the membrane for 20 minutes, take 2L / pack of TBS powder, dissolve 2L with ultrapure water, add 1mL of Tween-20, mix and use. The PVDF membrane after transfer is blocked with TBST solution containing 3% BSA at room temperature for 2 hours. The PVDF membrane was washed 3 times with TBST solution, each time for 6 minutes; the fully human monoclonal antibody MntC-15 obtained by expression and purification in Example 4 was diluted to a final concentration of 1μg / mL, and the PVDF membrane was incubated overnight at 4°C, and washed 3 times with TBST solution, each time for 6 minutes; then the goat anti-human IgG-HRP was diluted with blocking solution at 1:5000, and the PVDF membrane was placed in the above solution and placed on a horizontal shaker for incubation at room temperature for 1h.

[0142] 3) Color development: Wash the PVDF membrane with TBST solution for 3 times, 5 min each time; put the PVDF membrane into a disposable dish, prepare DAB color development solution according to the instructions of the kit, add about 10 drops of color development solution to each membrane in the dark, observe whether the bands are displayed and the color development position, and when the bands are obvious, put the PVDF membrane into a prepared level dish.

[0143] like Figure 5As shown, the MntC-15 antibody can bind to the denatured MntC, thus judging that the epitope of the MntC-15 antibody is a linear epitope.

[0144] Example 7 Determination of antigenic epitopes

[0145] 1. Experimental methods

[0146] The amino acid sequence of MntC was truncated to synthesize a polypeptide with a length of 18 amino acids. The 6 amino acids between two adjacent polypeptides overlapped, and 45 polypeptides were synthesized in this way.

[0147] The synthesized MntC polypeptide was dissolved in DMSO. The polypeptide was diluted with carbonate coating solution to a concentration of 80 μg / mL, and 100 μL / well was used to coat the ELISA plate, overnight at 4°C, washed 3 times with PBST buffer, and blocked with 2% BSA at 37°C for 60 min. The plate was washed three times with PBST buffer, and 100 μL of MntC-15 monoclonal antibody at a concentration of 5 μg / mL was added, and 3 replicates were made. The positive control was the recombinant MntC protein immune positive rabbit serum (1:200), and the negative control was the negative serum (1:200) and the negative control irrelevant antibody IgG15 μg / mL, 100 μL per well, incubated at 37°C for 60 min.

[0148] After washing the plate three times with a plate washer, dilute Anti-human HRP-IgG with diluent at 1:5000, add 100 μL to each well of the ELISA plate, and incubate in a 37°C incubator for 45 min. Wash the plate once with PBST buffer (3 cycles), protect from light, add 100 μL of TMB colorimetric solution to each well, place at 37°C for 15 min, and add 50 μL of 2M sulfuric acid stop solution to each well. Detect OD with an ELISA reader 450nm Calculate the mean of the negative control irrelevant antibody IgG1, calculate the threshold (3 times the mean), and the wells with a value greater than the threshold are positive. Figure 6 It shows that polypeptide No. 21 can bind to MntC-15 monoclonal antibody. The sequence of polypeptide No. 21 is shown in Table 2. Therefore, the antigenic site recognized by MntC-15 monoclonal antibody at least includes the entire amino acid sequence of LDNGIKYVKTIQQTFIDN or a partial fragment and some amino acids.

[0149] Table 2 Peptide sequences binding to MntC-15 monoclonal antibody

[0150] Sequence number describe sequence SEQ ID NO:33 Peptide No. 21 LDNGIKYVKTIQQTFIDN

[0151] Example 8 Effect of MntC-15 antibody on the oxidative stress of Staphylococcus aureus

[0152] 1. Experimental methods

[0153] Pick single clones of MRSA strains cultured overnight at 37°C on blood agar solid medium, add them to 20mL of TSB (TSB-C) liquid culture treated with resin, and culture them overnight at 37°C and 220rpm. Take 200μL of overnight cultured bacterial solution and add it to fresh 20mL TSB-C liquid medium, and culture it at 37°C and 220rpm for about 5h. Collect the bacterial solution into a centrifuge tube, centrifuge at 4000rpm for 5min. Resuspend it with physiological saline and adjust it to OD 600nm The absorbance value at 600nm =1, using the method of serial dilution, dilute the bacterial solution to 2×10 4 CFU / mL. Then 100μL / well was added to a 96-well cell culture plate. MntC-15 antibody was added to the experimental wells; an equal volume of saline was added to the blank control wells; human IgG1 was added to the irrelevant control wells; 3 replicates were added to each concentration gradient, incubated at 37°C for 45 minutes, and then methyl viologen was added at a final concentration of 45mM, and incubated overnight for 16-20h. Serial multiple dilutions were applied to the plate to count the number of residual bacteria on the plate.

[0154] 2. Experimental results

[0155] The growth of Staphylococcus aureus requires a certain amount of iron, manganese, zinc and other trace elements. 2+ It plays an important role in bacterial metabolism, cell wall synthesis and virulence enhancement. Staphylococcus aureus encodes two manganese ion transport systems: ATP-binding cassette transporter (MntABC) and proton-dependent NRAMP transporter (MntH). MntA encodes the nucleotide binding domain, MntB encodes the transmembrane domain, and MntC encodes the substrate-binding lipoprotein. The main function of MntC is to obtain Mn from the host environment. 2+ , Mn 2+ The acquisition of Mn is crucial to the virulence of S. aureus because it is a cofactor of superoxide dismutase, which can neutralize the reactive oxygen species produced during the host's oxidative burst. Methyl viologen is a generator of reactive oxygen species in vitro. The MRSA strain containing only culture medium maintains normal proliferation. After adding reactive oxygen releasers (methyl viologen, MV) to the culture medium, the growth of MRSA strains is inhibited, and the MntABC transporter expressed on its membrane can function to obtain superoxide dismutase cofactor-Mn from the environment to neutralize reactive oxygen species. 2+ , and still survive against reactive oxygen species. When MV and MntC-15 monoclonal antibody (100 μg) were added at the same time, the bacterial sensitivity to reactive oxygen species produced by MV increased and the number of bacteria decreased, indicating that targeted blocking of the MntABC transporter would make S. aureus sensitive to oxidative stress.

[0156] Therefore, MntC-15 can play an immune protective role against MRSA. Figure 7 As shown, MntC-15 monoclonal antibody effectively inhibited the growth of Staphylococcus aureus compared with the control group.

[0157] Example 9 Determination of the specificity of the MntC-15 antibody in inhibiting Staphylococcus aureus

[0158] 1. Experimental methods

[0159] Single clones of MntC knockout MRSA and wild-type MRSA strains cultured overnight at 37°C on blood agar solid medium were picked and added to 20 mL of TSB (TSB-C) liquid culture treated with resin, and cultured overnight at 37°C and 220 rpm. Take 200 μL of overnight cultured bacterial solution into fresh 20 mL TSB-C liquid medium and culture for about 5 hours at 37°C and 220 rpm. Collect the bacterial solution into a centrifuge tube and centrifuge at 4000 rpm for 5 minutes. Resuspend with physiological saline and adjust it to OD 600nm The absorbance value at 600nm =1, using the method of serial dilution, dilute the bacterial solution to 2×10 4 CFU / mL. Then 100μL / well was added to a 96-well cell culture plate. MntC-15 antibody was added to the experimental wells; an equal volume of saline was added to the blank control wells; human IgG1 was added to the irrelevant control wells; 3 replicates were added to each concentration gradient, and incubated at 37°C for 45 minutes. For the manganese ion remediation experiment, manganese sulfate was added at a final concentration of 5μM, incubated for 30min, and finally methyl viologen was added at a final concentration of 45mM, and incubated overnight for 16-20h. Serial dilutions were plated to count the number of residual bacteria on the plate.

[0160] 2. Experimental results

[0161] When MntC was knocked out and the knockout strain with MntC-15 antibody was added, no further increase in bacterial resistance to oxidative stress was observed, e.g. Figure 8 As shown, the specificity of the fully human monoclonal antibody MntC-15 was demonstrated; under the blocking of MntC-15 antibody, the addition of an appropriate amount of manganese ions could partially restore the growth of MRSA, proving that the blocking target of the fully human monoclonal antibody MntC-15 is MntC, and also confirming that the antibacterial mechanism of the MntC-15 antibody is the targeted blocking of the transport of manganese ions by the MntABC transporter, thereby exerting a good antibacterial effect.

[0162] Example 10 Establishment of MRSA lethal model and protective evaluation

[0163] 1. Exploration of infection dose for MRSA sepsis model

[0164] The experimental animals were BALB / c mice, 7-8 weeks old, female 16-18g; source: Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. There were 10 groups of mice, each with 10 experimental animals, and 100 μL MRSA liquid was injected into the tail vein at a concentration of CUF / mL, and the control group was 100 μL normal saline.

[0165] The mice were observed for 7 consecutive days after infection, and the survival status of the mice was recorded every 12 h. The mortality rate was calculated after the experiment.

[0166] From the experimental results Fig. 9 A, 9×10 9 The lethality rate of MRSA dose at CFU / mL reaches 90%, which can be used to establish a sepsis model caused by MRSA infection.

[0167] 2. Survival analysis of MntC-15 treatment of MRSA-infected sepsis model

[0168] Female BALB / c mice aged 7-8 weeks, weighing 16-18g, were selected and divided into four groups, with 10 mice in each group. The fully human monoclonal antibody MntC-15 expressed and purified in Example 4 was diluted to high and low concentrations. Each mouse in the high concentration group was injected with 1.5 mg, 100 μL; each mouse in the low concentration group was injected with 0.75 mg, 100 μL; each mouse in the non-specific antibody control group was injected with 1.5 mg human IgG1; each mouse in the negative control group was injected with normal saline, 100 μL. The implementation time was 24 hours before the establishment of the sepsis model.

[0169] The MRSA activated by blood plate was inoculated into TSB medium for bacterial growth and the OD was adjusted. 600 nm The absorbance value was set to 9 × 10 9 CFU / mL, and then 100 μL of bacterial solution was injected into the tail vein of mice. The survival of mice was observed for 7 consecutive days and recorded every 12 hours. Fig. 9 As shown in B, the survival rate of mice in the 1.5 mg MntC-15 group was 90%, and the survival rate of mice in the 0.75 mg MntC-15 group was 70%. The survival rates of mice in the two groups were significantly higher than those in the negative control group (0%), and the difference was statistically significant (p<0.05). The survival rate of mice in the human IgG1 control group was 20%. This shows that the fully human anti-MntC antibody MntC-15 can resist sepsis caused by MRSA infection to a certain extent.

[0170] Example 11 Therapeutic evaluation of MntC-15 antibody in animals

[0171] 1. Experimental methods

[0172] 45 6-8 week old Balb / C mice were divided into three groups according to body weight: experimental, irrelevant antibody, and saline, with 15 mice in each group. 18 hours before the challenge, mice in the experimental group were injected with 100 μg / mouse MntC-15 antibody, mice in the irrelevant antibody group were injected with 100 μg / mouse hIgG1 antibody, and mice in the saline group were injected with an equal volume of saline.

[0173] The MRSA activated by blood plate was inoculated into TSB medium for bacterial growth. The cultured bacterial liquid was collected and washed three times with saline. The OD was adjusted. 600 nm The absorbance value was set to 8 × 10 7 CFU / mL. Mice were anesthetized by intraperitoneal injection of 80 mg / kg ketamine + 8 mg / kg xylazine, and the resuspended bacteria were injected into the periorbital venous plexus at 100 μL / mouse. After 15 days, the mice were killed by CO2 inhalation and the kidneys were removed. Surface abscesses were visible on intact kidneys. Kidneys were weighed, homogenized, serially diluted and plated on MHA agar plates to count the number of bacterial loads in kidney tissue.

[0174] 2. Experimental results

[0175] The results are as follows Fig.10 As shown, Fig.10 A is the number of renal abscesses. Fig.10 B is the result of the number of bacterial loads in the kidney. As can be seen from the results, compared with the human IgG1 and saline groups, the number of renal abscess lesions and bacterial loads in the mice treated with the fully human monoclonal antibody MntC-15 were less than those in the human IgG1 and saline control groups.

[0176] 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 that specifically binds to MntC, characterized in that: The monoclonal antibody comprises three heavy chain variable region complementary determining regions and three light chain variable region complementary determining regions, wherein the amino acid sequences of the heavy chain variable region complementary determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 1, 2, and 3, respectively, and the amino acid sequences of the light chain variable region complementary determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 9, 10, and 11, respectively, and the sequence of SEQ ID NO: 10 is WAS.

2. The monoclonal antibody according to claim 1, characterized in that The heavy chain variable region also includes four heavy chain variable region framework regions, and the light chain variable region also includes four light chain variable region framework regions, wherein the amino acid sequences of the heavy chain variable region framework regions FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs: 4, 5, 6, and 7, respectively, and the amino acid sequences of the light chain variable region framework regions FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs: 12, 13, 14, and 15, respectively.

3. The monoclonal antibody according to claim 2, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

16.

4. The monoclonal antibody according to claim 3, characterized in that The monoclonal antibody binds to a linear epitope.

5. The monoclonal antibody according to claim 1, characterized in that The CDRs are defined according to the IMGT numbering system.

6. The monoclonal antibody according to claim 4, characterized in that The monoclonal antibody is afucosylated.

7. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the monoclonal antibody according to any one of claims 1 to 6.

8. The nucleic acid molecule according to claim 7, characterized in that The nucleotide sequences of the nucleic acid molecules encoding the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the monoclonal antibody are shown in SEQ ID NOs: 17, 18, and 19, respectively. The nucleotide sequences of the nucleic acid molecules encoding the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the monoclonal antibody are shown in SEQ ID NOs: 25, 26, and 27, respectively.

9. The nucleic acid molecule according to claim 7, characterized in that The nucleotide sequences of the nucleic acid molecules encoding the framework regions FR1, FR2, FR3 and FR4 of the heavy chain variable region of the monoclonal antibody are shown in SEQ ID NOs: 20, 21, 22 and 23, respectively. The nucleotide sequences of the nucleic acid molecules encoding the monoclonal antibody light chain variable region framework regions FR1, FR2, FR3 and FR4 are shown in SEQ ID NOs: 28, 29, 30 and 31, respectively.

10. The nucleic acid molecule according to claim 9, characterized in that The amino acid sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:24, and the amino acid sequence encoding the light chain variable region is shown in SEQ ID NO:

32.

11. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid molecule according to any one of claims 7 to 10.

12. The recombinant expression vector according to claim 11, characterized in that: The recombinant expression vector also includes a promoter.

13. The recombinant expression vector according to claim 11, characterized in that: The recombinant expression vector includes a plasmid vector, a virus vector or a phage vector.

14. A host cell, characterized in that The host cell comprises the nucleic acid molecule according to any one of claims 7 to 10 or the recombinant expression vector according to any one of claims 11 to 13.

15. The host cell according to claim 14, characterized in that The host cells include prokaryotic cells and eukaryotic cells.

16. The host cell according to claim 15, characterized in that The eukaryotic cells include mammalian cells.

17. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the monoclonal antibody according to any one of claims 1 to 6, the nucleic acid molecule according to any one of claims 7 to 10, the recombinant expression vector according to any one of claims 11 to 13, or the host cell according to any one of claims 14 to 16.

18. The pharmaceutical composition according to claim 17, characterized in that The pharmaceutical composition also includes a pharmaceutically compatible carrier.

19. The pharmaceutical composition according to claim 17, characterized in that The pharmaceutical composition also includes a buffer.

20. A kit for detecting MntC protein, characterized in that: The kit comprises the monoclonal antibody according to any one of claims 1 to 6.

21. The kit according to claim 20, characterized in that The kit also includes a detectable label conjugated to the monoclonal antibody.

22. The kit according to claim 21, characterized in that The detectable labels include fluorescent labels, radioisotopes, chemiluminescent molecules, paramagnetic ions or spin-trapping agents.

23. A method for detecting MntC protein in a sample for non-diagnostic purposes, characterized in that: The method comprises contacting the monoclonal antibody according to any one of claims 1 to 6 with a sample to be tested, thereby detecting the level of MntC protein in the sample to be tested.

24. A method for preparing the monoclonal antibody according to any one of claims 1 to 6, characterized in that: The method comprises culturing the host cell according to any one of claims 14 to 16 and recovering the monoclonal antibody.

25. Any of the following applications: (1) Use of the monoclonal antibody according to any one of claims 1 to 6, the nucleic acid molecule according to any one of claims 7 to 10, the recombinant expression vector according to any one of claims 11 to 13, or the host cell according to any one of claims 14 to 16 in detecting the content of MntC protein for non-diagnostic purposes; (2) Use of the monoclonal antibody according to any one of claims 1 to 6, the nucleic acid molecule according to any one of claims 7 to 10, the recombinant expression vector according to any one of claims 11 to 13, or the host cell according to any one of claims 14 to 16 in the preparation of a product for detecting MntC protein; (3) Use of the monoclonal antibody according to any one of claims 1 to 6, the nucleic acid molecule according to any one of claims 7 to 10, the recombinant expression vector according to any one of claims 11 to 13, or the host cell according to any one of claims 14 to 16 for non-therapeutic inhibition of Staphylococcus aureus or in the preparation of a pharmaceutical composition for the prevention and / or treatment of diseases related to Staphylococcus aureus infection; The diseases related to Staphylococcus aureus infection are peritonitis, bacteremia, sepsis, and renal abscess.

26. The use according to claim 25, characterized in that The Staphylococcus aureus is methicillin-resistant Staphylococcus aureus.

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