Antibody against Staphylococcus aureus manganese ion transporter C and its application

Abstract

The present invention discloses an antibody against Staphylococcus aureus manganese ion transporter C and its application. The antibody provided by the present invention can specifically bind to MntC protein, can effectively inhibit the growth of Staphylococcus aureus, provides a new idea for the detection of MntC protein and the treatment of related diseases, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to an antibody against Staphylococcus aureus manganese ion transporter protein C and an application thereof. Background Art

[0002] Staphylococcus aureus is the main pathogen causing hospital-acquired cross-infection, with high morbidity and mortality. It mainly causes osteomyelitis, endocarditis, septic arthritis, pneumonia, abscesses and toxic shock syndrome. Staphylococcus aureus can survive on dry surfaces, increasing the chance of bacterial infection. Infection with any type of Staphylococcus aureus can cause staphylococcal scalded skin syndrome, mainly because the skin tissue overreacts to toxins after bacterial endotoxins enter human blood samples. Staphylococcus aureus can also cause bacteremia / sepsis, endangering human life. Among them, methicillin-resistant Staphylococcus aureus (MRSA) can secrete a variety of toxins to destroy normal humoral immunity and cellular immunity and reshape the host's immune environment and develop multidrug resistance. At present, MRSA has become the Gram-positive bacteria with the highest infection rate in ICU wards, postoperative infections, burns, etc. It has become a difficult problem in clinical treatment, a serious threat to human health, and a major pathogen of hospital cross-infection. At present, infections caused by Staphylococcus aureus are usually treated with antibiotics such as penicillin, and vancomycin is used as an alternative therapy for penicillin-resistant strains. Vancomycin, as the drug of choice for the clinical treatment of Gram-positive bacterial infections, was once called the "last line" of clinical anti-infection. However, with the emergence of vancomycin-resistant Staphylococcus aureus in recent years, there is an urgent need to develop new anti-infection drugs. Therefore, the search for anti-MRSA drugs has become an important direction in the field of MRSA treatment research in recent years.

[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 die; at the same time, MntC has adhesin activity. In the early stage of infection, Staphylococcus aureus invades and colonizes host cells through surface adhesins. In recent years, Staphylococcus aureus vaccines have gone through several stages, including capsular polysaccharide vaccines, DNA vaccines, and inactivated vaccines, but none of them have been successful. Passive immunotherapy based on antibody drugs for MRSA infection can, on the one hand, quickly establish specific immune protection for the infected person and quickly and efficiently eliminate pathogens. On the other hand, it can also help those MRSA susceptible people with low immunity or even immunodeficiency to obtain immediate and effective immune protection. However, no extensive humanized monoclonal antibody against MntC has been found so far, so it is of great importance in this field to develop an antibody against S. aureus Mn-binding protein C. Summary of the invention

[0004] To make up for the deficiencies of the prior art, the present invention provides an antibody against Staphylococcus aureus manganese ion transporter protein C and its application.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides an antibody against Staphylococcus aureus manganese ion transporter C, the antibody comprising:

[0007] The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region 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,

[0008] The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region 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.

[0009] 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.

[0010] 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.

[0011] Furthermore, the antibody further comprises:

[0012] The framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region have amino acid sequences that are at least 90%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the amino acid sequences shown in SEQ ID NOs: 4, 5, 6, and 7, respectively.

[0013] The amino acid sequences of the framework regions FR1, FR2, FR3, and FR4 of the light chain variable region 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 amino acid sequences shown in SEQ ID NOs: 12, 13, 14, and 15, respectively.

[0014] Furthermore, the amino acid sequences of the framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region are shown in SEQ ID NOs: 4, 5, 6, and 7, respectively.

[0015] The amino acid sequences of the framework regions FR1, FR2, FR3, and FR4 of the light chain variable region are shown in SEQ ID NOs: 12, 13, 14, and 15, respectively.

[0016] 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,

[0017] 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.

[0018] 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.

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

[0020] Further, the antibody is afucosylated.

[0021] Further, the antibody lacks fucose at Asn297.

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

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

[0024] Further, the antibody is linked to one or more conjugate moieties.

[0025] Further, the conjugate moieties include covalent linkages directly or through a linker.

[0026] Further, the conjugate moiety includes a therapeutic agent, a radioisotope, a detectable label, a pharmacokinetic modulating moiety, or a purification moiety.

[0027] The second aspect of the present invention provides an antigenic epitope, which comprises a polypeptide fragment of Staphylococcus aureus manganese ion transporter C or a variant thereof.

[0028] Furthermore, the antigenic epitope 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 sequence shown in SEQ ID NO:33, 34, 35.

[0029] Furthermore, the sequence of the antigen epitope is shown in SEQ ID NO:33, 34, 35.

[0030] The third aspect of the present invention provides an isolated polynucleotide encoding the antibody according to the first aspect of the present invention or the antigenic epitope according to the second aspect of the present invention.

[0031] Furthermore, the nucleic acid sequence of the polynucleotide encoding the complementary determining regions CDR1, CDR2, and CDR3 of the antibody heavy chain variable region has at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with the nucleic acid sequences shown in SEQ ID NOs: 17, 18, and 19, respectively.

[0032] The nucleic acid sequences of the polynucleotides encoding the complementary determining regions CDR1, CDR2, and CDR3 of the antibody light chain variable region have at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with the nucleic acid sequences shown in SEQ ID NOs: 25, 26, and 27, respectively.

[0033] Furthermore, the nucleic acid sequences of the polynucleotides encoding the complementary determining regions CDR1, CDR2, and CDR3 of the antibody heavy chain variable region are shown in SEQ ID NOs: 17, 18, and 19, respectively.

[0034] The nucleic acid sequences of the polynucleotides encoding the complementary determining regions CDR1, CDR2, and CDR3 of the antibody light chain variable region are shown in SEQ ID NOs: 25, 26, and 27, respectively.

[0035] Furthermore, the nucleic acid sequences of the polynucleotides encoding the framework regions FR1, FR2, FR3, and FR4 of the antibody heavy chain variable region 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 nucleic acid sequences shown in SEQ ID NOs: 20, 21, 22, and 23, respectively.

[0036] The nucleic acid sequences of the polynucleotides encoding the framework regions FR1, FR2, FR3, and FR4 of the antibody light chain variable region 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 nucleic acid sequences shown in SEQ ID NOs: 28, 29, 30, and 31, respectively.

[0037] Furthermore, the nucleic acid sequences of the polynucleotides encoding the framework regions FR1, FR2, FR3, and FR4 of the antibody heavy chain variable region are shown in SEQ ID NOs: 20, 21, 22, and 23, respectively.

[0038] The nucleic acid sequences of the polynucleotides encoding the framework regions FR1, FR2, FR3, and FR4 of the antibody light chain variable region are shown in SEQ ID NOs: 28, 29, 30, and 31, respectively.

[0039] Further, the nucleic acid sequence of the polynucleotide encoding the antibody 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%, 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 24,

[0040] The nucleic acid sequence of the polynucleotide encoding the antibody 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%, 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO:32.

[0041] Furthermore, the nucleic acid sequence of the polynucleotide encoding the antibody heavy chain variable region is shown in SEQ ID NO: 24,

[0042] The nucleic acid sequence of the polynucleotide encoding the antibody light chain variable region is shown in SEQ ID NO:32.

[0043] The fourth aspect of the present invention provides a vector, which comprises the polynucleotide described in the third aspect of the present invention.

[0044] Furthermore, the vector also includes at least one promoter operably linked to the nucleic acid sequence.

[0045] Furthermore, the vector includes plasmid, bacteriophage, and animal virus.

[0046] The fifth aspect of the present invention provides a host cell, wherein the host cell comprises the polynucleotide described in the third aspect of the present invention or the vector described in the fourth aspect of the present invention.

[0047] Furthermore, the host cell includes prokaryotes, yeast or higher eukaryotic cells.

[0048] Furthermore, the higher eukaryotic cells include animal cells, plant cells or fungal cells.

[0049] Furthermore, the animal cells include mammalian cells, bird cells, and insect cells.

[0050] Furthermore, the mammalian cells include CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK 293T cells, and 293F cells.

[0051] Furthermore, the mammalian cells are selected from HEK 293F cells.

[0052] The sixth aspect of the present invention provides a product for detecting Staphylococcus aureus manganese ion transporter C, wherein the product comprises the antibody described in the first aspect of the present invention.

[0053] Furthermore, the product includes a test kit and a test paper.

[0054] The seventh aspect of the present invention provides a pharmaceutical composition, which comprises the antibody described in the first aspect of the present invention, the antigen epitope described in the second aspect of the present invention, the polynucleotide described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention or the host cell described in the fifth aspect of the present invention.

[0055] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0056] An eighth aspect of the present invention provides any of the following methods:

[0057] 1) A method for producing the antibody according to the first aspect of the present invention, the method comprising culturing the host cell according to the fifth aspect of the present invention and recovering the antibody;

[0058] 2) A method for detecting Staphylococcus aureus manganese ion transporter C in a sample, the method comprising contacting the sample with the antibody according to the first aspect of the present invention, thereby detecting the level of Staphylococcus aureus manganese ion transporter C in the sample;

[0059] 3) A method for opsonizing phagocytic bactericidal activity, the method comprising administering the antibody of the first aspect of the present invention;

[0060] 4) A method for regulating cytokines produced by Staphylococcus aureus stimulation, the method comprising administering the antibody according to the first aspect of the present invention;

[0061] 5) A method for detecting antibodies to Staphylococcus aureus manganese ion transporter C, the method comprising using the antigen epitope described in the second aspect of the present invention.

[0062] Furthermore, the cytokines in 4) include one or more of IFN-γ, IL-17A or IL-5.

[0063] The ninth aspect of the present invention provides a vaccine comprising the antigenic epitope described in the second aspect of the present invention.

[0064] Furthermore, the epitope is a linear epitope.

[0065] Further, the method described in 5) comprises the following steps:

[0066] Furthermore, the method is not for diagnostic or therapeutic purposes.

[0067] 1) obtaining a sample containing antibodies against Staphylococcus aureus manganese ion transporter C;

[0068] 2) contacting the antigen epitope described in the second aspect of the present invention with the sample of step 1);

[0069] 3) Detect antigen-antibody reaction.

[0070] The tenth aspect of the present invention provides any of the following applications:

[0071] 1) Use of the antibody according to the first aspect of the present invention, the polynucleotide according to the third aspect of the present invention, the vector according to the fourth aspect of the present invention, or the host cell according to the fifth aspect of the present invention in detecting Staphylococcus aureus manganese ion transporter C or in preparing a product for diagnosing diseases related to Staphylococcus aureus infection;

[0072] 2) Use of the antibody according to the first aspect of the present invention, the polynucleotide according to the third aspect of the present invention, the vector according to the fourth aspect of the present invention, or the host cell according to the fifth aspect of the present invention in inhibiting Staphylococcus aureus manganese ion transporter C or in preparing a pharmaceutical composition for preventing and / or treating diseases related to Staphylococcus aureus infection;

[0073] 3) Use of the antibody according to the first aspect of the present invention in regulating the bactericidal activity of opsonized phagocytes;

[0074] 4) Use of the antigen epitope described in the second aspect of the present invention in preparing antibodies;

[0075] 5) Use of the antigenic epitope described in the second aspect of the present invention in the preparation of a vaccine for preventing Staphylococcus aureus infection;

[0076] 6) Use of the antigenic epitope described in the second aspect of the present invention in the preparation of a product for detecting antibodies against Staphylococcus aureus manganese ion transporter C.

[0077] 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.

[0078] Furthermore, the disease related to Staphylococcus aureus infection is selected from sepsis.

[0079] Advantages and beneficial effects of the present invention:

[0080] The antibody provided by the present invention can specifically bind to the MntC protein, can effectively inhibit the growth of Staphylococcus aureus, provides a new idea for the detection of the MntC protein and the treatment of related diseases, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 This is a diagram showing the results of SDS-PAGE detection of the recombinant MntC protein;

[0082] Figure 2 2A is a graph of the screening results of the Fab library, wherein 2B is a graph of the screening results of the Fab library (Lambda light chain), and 2B is a graph of the screening results of the Fab library (Lambda light chain);

[0083] Figure 3 This is the result of SDS-PAGE detection of the expression and purification of the fully human monoclonal antibody MntC-C12;

[0084] Figure 4 This is a graph showing the binding activity test results of the fully human monoclonal antibody MntC-C12;

[0085] Figure 5 This is the WB test result of the fully human monoclonal antibody MntC-C12;

[0086] Figure 6 This is the Elisa result of the fully human monoclonal antibody MntC-C12 against the MntC Mapping peptide;

[0087] Figure 7 7A is the A600 graph and 7B is the inhibition rate graph;

[0088] Figure 8 8A is a graph showing the phagocytosis result of HL60 cells after the fully human monoclonal antibody MntC-C12 opsonized MRSA, and 8B is a graph showing the phagocytosis result statistics;

[0089] Fig. 9 The figures are the evaluation results of the fully human monoclonal antibody MntC-C12 on the animal model of sepsis, wherein 9A is the figure for exploring the lethal dose, and 9B is the figure for analyzing the survival rate of the sepsis model;

[0090] Fig.10 These are the results of the inhibition test of cytokines produced by MntC stimulation by the fully human monoclonal antibody MntC-C12, wherein 10A is the result of the IL-5 test, 10B is the result of the IFN-γ test, and 10C is the result of the IL-17A test. DETAILED DESCRIPTION

[0091] 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.

[0092] The invention provides an antibody against Staphylococcus aureus manganese ion transporter C, which comprises: complementary determining regions CDR1, CDR2 and CDR3 of heavy chain and light chain variable regions, and framework regions FR1, FR2, FR3 and FR4 of heavy chain and light chain variable regions.

[0093] In the present invention, antibody refers to an immunoglobulin-derived molecule that can specifically bind to a target antigen, and the immunoglobulin-derived molecule binds to the target antigen through at least one antigen binding site located in its variable region. When referring to the term antibody, unless the context clearly indicates, it includes not only complete antibodies, but also antigen-binding fragments that can specifically bind to a target antigen.

[0094] The precise amino acid sequence boundaries of the complementary determining regions (CDRs) of antibodies can be defined according to known methods, such as Chothia based on the three-dimensional structure of antibodies and the topology of CDR loops, or Kabat based on antibody sequence variability, and IMGT, AbM or Contact definitions. The CDRs of antibodies in this application can be defined by those skilled in the art according to any scheme in the art (e.g., the optional definition method described above).

[0095] 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 include the sequences of the CDRs 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.

[0096] In a specific embodiment of the present invention, the complementarity determining regions (CDRs) of an antibody are determined according to the IMGT method.

[0097] In the present invention, epitope shall particularly refer to a molecular structure that completely constitutes the specific binding partner of the antibody binding site or a part of the specific binding partner. The epitope may be composed of carbohydrates, peptide structures, fatty acids, organic substances, biochemical substances or inorganic substances or their derivatives and any combination thereof. The epitope may be a linear or conformational epitope. A linear epitope consists of a single segment of the primary sequence of a polypeptide or sugar chain. Linear epitopes may be continuous or overlapping.

[0098] In a specific embodiment of the invention, said antibody binds to a linear epitope.

[0099] In certain embodiments, the Staphylococcus aureus manganese transporter C antibody provided by the present invention may contain one or more modifications or substitutions in one or more variable region sequences provided by the present invention, and still maintain specific binding affinity to Staphylococcus aureus manganese transporter C. In certain embodiments, at least one (or all) of the substitutions in the CDR sequence, FR sequence, or variable region sequence include conservative substitutions.

[0100] The antibody is afucosylated.

[0101] In the present invention, afucosylated (i.e., does not contain fucose). In some embodiments, the afucosylated antibodies provided by the present invention do not have fucose at asparagine 297 (Asn297) of the heavy chain. Asn297 is a conserved N-linked glycosylation site present in each CH2 domain of the Fc region of the antibody IgG1 isotype.

[0102] The antibody is linked to one or more conjugate moieties.

[0103] The conjugate moiety can be a therapeutic agent (e.g., a cytotoxic agent), a radioisotope, a detectable label (e.g., a lanthanide, a luminescent label, a fluorescent label, or an enzyme-substrate label), a pharmacokinetic modulating moiety, or a purification moiety (e.g., a magnetic bead or nanoparticle).

[0104] Examples of detectable labels may include fluorescent labels for detection (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme-substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, saccharide oxidase, or β-D-galactosidase), radioisotopes, luminescent labels, chromogenic moieties, digoxigenin, biotin / avidin, DNA molecules, or gold. Examples of radioisotopes may include 123 I. 124 I. 125 I. 131 I. 35 S. 3 H. 111 In, 112 In, 14 C. 64 Cu, 67 Cu, 86 Y. 88 Y. 90 Y. 177 Lu, 211 At 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P and other lanthanides. Antibodies labeled with radioisotopes can be used in receptor-targeted imaging experiments.

[0105] In certain embodiments, the pharmacokinetic regulating part can be a clearance regulator that helps increase the half-life of the antibody. Illustrative examples include water-soluble polymers such as PEG, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, ethylene glycol / propylene glycol copolymers, etc. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers connected to the antibody can vary, and if multiple polymers are connected, they can be the same or different molecules.

[0106] The present invention provides an isolated polynucleotide encoding the above antibody.

[0107] In the present invention, polynucleotide refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and its polymer in single-stranded or double-stranded form.Unless explicitly limited, the term covers polynucleotides containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, specific polynucleotide sequences also implicitly cover conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, as well as sequences explicitly indicated.

[0108] The present invention provides an expression vector, which comprises the above-mentioned polynucleotide.

[0109] In the present invention, the vector includes a cloning vector or an expression vector, which contains the nucleic acid sequence provided by the present invention encoding the antibody, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker. Examples of vectors include, but are not limited to, plasmids; phagemids; cosmids; and artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages, such as lambda phages or M13 phages; and animal viruses. The types of animal viruses used as expression vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Exemplary plasmids include pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMA L, 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, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0110] The present invention provides a host cell, which comprises the above polynucleotide or the above vector.

[0111] The vector comprising the polynucleotide sequence encoding the antibody or antigen-binding fragment can be introduced into the host cell for cloning or gene expression. The host cell suitable for cloning or expressing the DNA of the vector provided by the present invention is the above-mentioned prokaryotic organism, yeast or higher eukaryotic cell. Suitable prokaryotes for this purpose include 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. marcescans); and Shigella; and Bacilli, such as B. subtilis and B. licheniformis; Pseudomonas, such as Pseudomonas aeruginosa; and Streptomyces.

[0112] Higher eukaryotic cells include, but are not limited to, protist cells, animal cells, plant cells, or fungal cells, wherein the animal cells include mammalian cells, avian cells, and insect cells. Among them, mammalian cells include, but are not limited to, CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK 293T cells, and 293F cells.

[0113] The present invention provides a pharmaceutical composition, which comprises the above-mentioned antibody, the above-mentioned polynucleotide, the above-mentioned vector or the above-mentioned host cell.

[0114] In the present invention, the pharmaceutically acceptable carrier may include, for example, a pharmaceutically acceptable liquid, gel or solid carrier, an aqueous vehicle, a non-aqueous vehicle, an antimicrobial agent, an isotonic agent, a buffer, an antioxidant, an anesthetic, a suspending / dispersing agent, a chelating agent or a chelating agent, a diluent, an adjuvant, an excipient or a non-toxic auxiliary substance, other components known in the art, or various combinations thereof.

[0115] The pharmaceutical composition can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation or powder. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0116] The present invention provides use of the above-mentioned antibody, the above-mentioned polynucleotide, the above-mentioned vector or the above-mentioned host cell in inhibiting Staphylococcus aureus manganese ion transporter C or in preparing a pharmaceutical composition for preventing and / or treating diseases related to Staphylococcus aureus infection.

[0117] In the present invention, S. aureus infections, including MRSA, usually begin with 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 may 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.

[0118] 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.

[0119] Example 1 Expression and purification of Staphylococcus aureus manganese ion transporter C (MntC)

[0120] 1. Expression and purification of Staphylococcus aureus manganese transporter C (MntC)

[0121] 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 recombinant MntC protein.

[0122] 2. Experimental results

[0123] 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 ).

[0124] Example 2 Establishment of a Human Specific Anti-Staphylococcus aureus Fab Antibody Library

[0125] 1. Source of PBMC cells

[0126] 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).

[0127] 2. Construction of human-specific anti-Staphylococcus aureus Fab antibody library using phage surface display technology

[0128] 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, and the human light chain (VL+CL) Kappa / Lambda and heavy chain Fd segment gene sequences were amplified by PCR using the Ig primer set. The light chain VL+CL gene PCR recovery product was double-digested with SacI-HF enzyme and XbaI-HF enzyme, and then connected to the pComb3XSS phage display vector. After the VL+CL (Kappa) and VL+CL (Lambda) light chain libraries were successfully constructed, the heavy chain Fd segment was cloned into the pComb3XSS vector already connected with the light chain gene fragment through the XhoI-HF and SpeI-HF restriction enzyme sites to form the Fab phagemid. The ligation product was transformed into TG1 competent cells and cultured overnight at 37°C on a plate containing ampicillin. The bacterial cells were collected to calculate the library capacity, and 10 single colonies were picked for PCR identification using specific primers. The reaction conditions were: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, 30 cycles; 72°C extension for 10 min. 5 μL of PCR product was detected by 2% agarose gel electrophoresis.

[0129] 3. Experimental results

[0130] The results showed that the positive clone rates of Fab (Kappa light chain) library and Fab (Lambda light chain) library were both over 80%, and the library capacity exceeded 10 8 ( Figure 2 ).

[0131] Example 3 Screening of human specific anti-Staphylococcus aureus Fab antibody library

[0132] 1. Bacterial purification and antibody phage preparation

[0133] The frozen library bacterial solution was inoculated into LB-penicillin medium at a ratio of 1:100 for recovery. 600 nmWhen 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.

[0134] 2. Antigen coating

[0135] MntC protein was coated at 100 μL per well at 4°C overnight; after removing the supernatant, the cells were washed once with PBS, patted dry, blocked with 3% skimmed milk powder, incubated at 37°C for 2 h, the blocking solution was removed, washed three times with PBST, and stored at 4°C for later use.

[0136] 3. Phage library panning

[0137] 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 600 nm 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.

[0138] 4. Phage library screening

[0139] 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 and incubated at 37°C for 20 minutes.

[0140] The solution was washed three times to remove unbound phages; fluorescent-labeled antibodies were added, incubated at 37°C for 20 min, and washed three times with PBST; the ELISA fluorescence signal intensity was detected to screen specific antibodies, the antibody phagemids corresponding to the positive wells were amplified, and sequencing analysis was performed.

[0141] Example 4 Cloning, expression and purification of anti-MntC fully human antibodies

[0142] 1. Experimental methods

[0143] 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.

[0144] 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 / ).

[0145] 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.

[0146] 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.

[0147] 2. Experimental results

[0148] 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-C12 (abbreviated as MntC-C12 antibody or MntC-C12 monoclonal antibody). Figure 3 As shown, the transfected cells successfully expressed the antibody, and the relative molecular weight of the antibody was about 160-180 kDa, the heavy chain was about 55 kDa, and the light chain was about 25 kDa. Its sequence is shown in Table 1.

[0149] Table 1 MntC-C12 antibody sequence

[0150]

[0151]

[0152]

[0153] Example 5 Binding Activity Detection of MntC-C12 Antibody

[0154] 1. Experimental methods

[0155] 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-C12 antibody (adjusted to 100 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 (100 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.

[0156] 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 4If the expression level is greater than the threshold, it is considered a positive well, and the EC50 is calculated.

[0157] 2. Experimental results

[0158] The results showed that when the concentration of MntC-C12 was 0.078-100 μg / mL, the EC50 of MntC-C12 binding to MntC was 0.024 μg / mL, and the control antibody did not bind to the recombinant MntC protein, indicating that the binding was specific ( Figure 4 ).

[0159] Example 6 Determination of epitope type of MntC-C12 antibody

[0160] 1. Experimental methods

[0161] 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 period, 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.

[0162] 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 put it into the semi-dry transfer instrument, adjust the working voltage to 21V, transfer the membrane for 20min, 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 6min; the fully human monoclonal antibody MntC-C12 expressed and purified 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 6min; 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.

[0163] 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.

[0164] 2. Experimental results

[0165] like Figure 5 As shown, the MntC-C12 antibody can bind to the denatured MntC, thereby judging that the epitope of the MntC-C12 antibody is a linear epitope.

[0166] Example 7 Determination of antigenic epitopes

[0167] 1. Experimental methods

[0168] 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.

[0169] 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-C12 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.

[0170] 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, calculate the threshold (3 times the mean), and the wells with a value greater than the threshold are positive. Figure 6 It shows that polypeptides 2 and 3 can bind to the MntC-C12 monoclonal antibody. The sequences of polypeptides 1-3 are shown in Table 2. Therefore, the antigenic site recognized by the MntC-C12 monoclonal antibody includes at least the entire amino acid sequence of SSDKSNGKLKVVTTNSILYDMAKN (SEQID NO: 33) or a partial fragment and some amino acids.

[0171] Table 2 Peptide sequences binding to MntC-C12 monoclonal antibody

[0172] Sequence number describe sequence SEQ ID NO:33 Peptide No. 1 SSDKSNGKLKVVTTNSILYDMAKN SEQ ID NO:34 Peptide No. 2 SSDKSNGKLKVVTTNSIL SEQ ID NO:35 Peptide No. 3 GKLKVVTTNSILYDMAKN

[0173] Example 8 Antagonistic effect of MntC-C12 antibody on Staphylococcus aureus manganese ion transporter C

[0174] 1. Experimental methods

[0175] Pick a single clone of the MRSA strain cultured overnight at 37°C on blood agar solid medium, add it to 20mL TSB liquid culture, and culture it overnight at 37°C and 220rpm. Take 200μL of the overnight cultured MRSA and add it to fresh 20mL TSB liquid culture medium, and culture it at 37°C and 220rpm for about 5h. Collect the MRSA bacterial liquid into a centrifuge tube, centrifuge at 4000rpm for 5min. Resuspend it with physiological saline and adjust it to OD 600 nm The absorbance value at 600 nm =1, using the method of serial dilution, dilute the bacterial solution to 1×10 5 CFU / mL. Then 100μL / well was added to a 96-well cell culture plate. 10μL of 30μg / μL MntC-C12 was added to the experimental wells, and 10μL of saline was added to the control wells. Each concentration gradient was repeated 3 times. The OD was detected every 1h using a multifunctional microplate reader. 600 nm The absorbance value at .

[0176] 2. Experimental results

[0177] 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 MntC is essential for the virulence of S. aureus because it is a cofactor of superoxide dismutase, which can neutralize reactive oxygen species produced during the host oxidative burst. Therefore, antibodies targeting MntC will interfere with the production of Mn 2+ Nutritional acquisition to play an immune protection role. Figure 7 As shown, MntC-C12 monoclonal antibody effectively inhibited the growth of Staphylococcus aureus compared with the control group.

[0178] Example 9 Effect of MntC-C12 antibody on opsonization of Staphylococcus aureus and promotion of phagocytosis of bacteria by HL60 cells

[0179] 1. Experimental methods

[0180] Cell recovery: HL60 main cells purchased from the Chinese Academy of Sciences were taken out of liquid nitrogen and recovered and cultured in 1640 medium containing 20% ​​FBS for about 3 weeks. Then, the medium was replaced with 1640 medium containing 10% FBS to continue proliferation. After 3 weeks of culture, the culture system was expanded to maintain the continuous proliferation of CM1. Each time, a portion of cells was taken from the medium and centrifuged at 400g for 10 minutes to collect the cell precipitate. The cells were resuspended in CM2 medium (10% FBS) containing 1% DMF, and the cells were counted with trypan blue to adjust the cell concentration to 5×10 5 cells / mL, differentiate into 100 mL, and culture at 37°C, 5% CO2 for 5 days.

[0181] Antibody dilution process: Add 20 μL of prepared OBB (containing 1% gelatin, 1× HBSS with Ca 2+ Mg 2+ Sterile water was added). In addition to adding 20 μL of prepared OBB in the first and second columns of row H, different concentrations of MntC-C12 were added to the remaining columns of row H, and serial 2-fold dilutions were performed in sequence.

[0182] Bacterial treatment: Take out the MRSA working strain from the -80℃ refrigerator, dissolve it in a 37℃ water bath, and centrifuge it at 12000g for 2min. Discard the supernatant, add 1mL OBB to resuspend, and then centrifuge it at 12000g for 2min. Use OBB to adjust to the original volume, take a certain volume of MRSA and add it to 10mL OBB, and add 10μL / well to the corresponding wells of the 96-well plate with the diluted fully human monoclonal antibody MntC-C12. Place the cell culture plate on a shaker and shake it at room temperature for 50min.

[0183] Cell treatment: Take the differentiated HL-60 cells out of the incubator and centrifuge at 400g for 10 min. Resuspend the cells in 50 mL 1× HBSS (without Ca 2+ Mg 2+ ), centrifuge at 400g for 10 min at room temperature. Resuspend the cells in 50 mL 1× HBSS (with Ca 2+ Mg 2+ ), centrifuged at 400 g for 10 min at room temperature. The cells were adjusted to 1×10 7cells / mL, trypan blue was used to detect cell activity, the number of live cells must be ≥90%, complement was taken out from the -80℃ refrigerator, thawed at room temperature, and placed on ice. After the shaking reaction was completed, the cells and complement were mixed at a ratio of 4:1, and 50μL / well was added to the 96-well plate. The cell culture plate was placed on a shaker and incubated for 70min at 37℃, 5% CO2, and 700rpm. After 70min, the 96-well culture plate was placed on ice for 20min to terminate the phagocytic reaction. According to the dilution concentration of the fully human antibody MntC-C12, 10μL of liquid was taken out from each well, the bacterial solution was applied to the MHA plate, and then the plate was immediately tilted to make the liquid flow into a line. By analogy, H and G are one plate, and by analogy, F, E, D, C, B, and A are applied on another plate. Place in a 37℃ constant temperature incubator and culture overnight. Count the number of bacteria on the plate. The OI value of the antibody is defined as the antibody dilution multiple with a 50% bactericidal rate. The maximum bacterial well condition of this experiment is the number of surviving colonies after co-incubation of MRSA, complement, HL-60 cells, and OBB; the number of colonies in the experimental group is the number of surviving colonies after co-incubation of MRSA, complement, HL-60 cells, and MntC-C12; the control group is the number of surviving colonies after co-incubation of MRSA, complement, HL-60 cells, and saline. The nonspecific bactericidal value is calculated using the maximum bacterial well ratio in the presence of inactivated / live complement.

[0184] 2. Experimental results

[0185] The opsonophagocytic bactericidal activity of MntC-C12 was detected by co-incubating 15-30 μg / μL MntC-C12 with HL-60 phagocytes. Figure 8 As shown, the opsonization index of 30μg / μL MntC-C12 on bacteria reached 133, and the opsonization index of 15μg / μL MntC-C12 on bacteria was 24, both of which were significantly higher than the control group and had an obvious dose-dependent relationship, indicating that MntC-C12 has strong opsonization and phagocytic bactericidal activity.

[0186] Example 10 Establishment of MRSA lethal model and protective evaluation

[0187] 1. Exploration of infection dose for MRSA sepsis model

[0188] 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 each group was injected with 100 μL MRSA liquid at a concentration of 3-9×10 9 CUF / mL, and the control group was 100 μL normal saline.

[0189] The mice were observed for 7 consecutive days after infection. The survival status of the mice was observed and recorded every 12 h, and the number of deaths of the mice was recorded. The mortality rate was calculated after the experiment.

[0190] From the experimental results Fig. 9 A look, 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.

[0191] 2. Survival analysis of MntC-C12 in the treatment of MRSA-infected sepsis model

[0192] 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-C12 expressed and purified in Example 4 was diluted to high and low concentrations. Each mouse in the high concentration group was injected with 1.5mg, 100μL; each mouse in the low concentration group was injected with 0.75mg, 100μL; each mouse in the non-specific antibody control group was injected with 1.5mg human IgG1; each mouse in the negative control group was injected with normal saline, 100μL. The implementation time was 24h before the establishment of the sepsis model.

[0193] 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-C12 group was 90%, and the survival rate of mice in the 0.75 mg MntC-C12 group was 60%, and the survival rates of mice in the two groups were significantly higher than those in the normal saline group (0%) and the human IgG1 control group (10%), and the difference was statistically significant (p<0.05). This shows that the fully human anti-MntC antibody MntC-C12 can resist sepsis caused by MRSA infection to a certain extent.

[0194] Example 11 MntC-C12 inhibits cytokine production stimulated by MntC

[0195] 1. Experimental methods

[0196] Twenty Balb / C mice were taken, and each mouse was intramuscularly injected with 30 μg MntC and 50 μL aluminum adjuvant. The immunization schedule was 0, 14, and 21 days. On the 7th day after the last immunization, the mouse spleen cells were obtained by cervical dislocation, ground with a grinder, and the grinding liquid was filtered with a mesh to obtain a single cell suspension. After adding 2 times the volume of red blood cell lysis solution, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and 5 mL RPMI 1640 medium was added to the precipitate. The precipitate was washed by blowing and centrifuged at 1000 rpm for 5 minutes. The cell concentration was adjusted to 5×10 7 cells / mL, 100μL / well was added to a 96-well cell culture plate, MntC-C12 was diluted to a concentration of 40μg / mL, 20μg / mL, 40μg / mL with RPMI1640 complete medium, and 8 concentration gradients were diluted twice, MntC was diluted to a concentration of 40μg / mL with RPMI1640 complete medium, MntC protein was mixed with the diluted MntC-C12 at a volume ratio of 1:1, and the mixture was incubated in a 37°C incubator for 30min. 100μL of the above MntC-C12 / MntC mixture was added to the upper layer of cells, the negative control was RPMI1640 liquid medium, and the positive control was MntC. Three replicate wells were used for each experiment. The cell culture plate was placed in a 5% CO2, 37°C incubator for 72 h, and the cytokine concentrations of IFN-γ, IL-17A and IL-5 in the cell supernatant were detected according to the instructions of the Dakoway ELISA kit.

[0197] 2. Experimental results

[0198] The inhibitory effect of the fully human antibody MntC-C12 on the production of cytokines (IFN-γ, IL-5, IL-17A) in mouse spleen lymphocytes induced by MntC Fig.10 As shown, according to the calculation formula cytokine inhibition rate (%) = (cytokine concentration of positive control group - cytokine concentration of antibody group) / (cytokine concentration of positive control group - cytokine concentration of negative control group), when the concentration of MntC-C12 is 10 μg / mL, the inhibition rates of IFN-γ, IL-17A and IL-5 are all greater than 95%, indicating that MntC-C12 can inhibit the release of cytokines from mouse spleen lymphocytes induced by MntC.

[0199] 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 manganese ion transporter C, characterized in that: The antibodies include: 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. 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, and the sequence of SEQ ID NO: 10 is DDS.

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

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

4. The antibody according to claim 1, characterized in that 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, 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%, at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

16.

5. The antibody according to claim 4, 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.

6. The antibody according to claim 1, characterized in that The antibody binds to a linear epitope.

7. The antibody according to claim 1, characterized in that The antibody is afucosylated.

8. The antibody according to claim 7, characterized in that The antibody lacks fucose at Asn297.

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

10. The antibody according to claim 1, characterized in that The antibody is linked to one or more conjugate moieties, which are detectable labels.

11. An isolated polynucleotide, characterized in that The polynucleotide encodes the antibody according to any one of claims 1 to 10.

12. The polynucleotide according to claim 11, characterized in that The nucleic acid sequences of the polynucleotides encoding the complementary determining regions CDR1, CDR2, and CDR3 of the antibody heavy chain variable region are shown in SEQ ID NOs: 17, 18, and 19, respectively. The nucleic acid sequences of the polynucleotides encoding the complementary determining regions CDR1, CDR2 and CDR3 of the antibody light chain variable region are shown in SEQ ID NOs: 25, 26 and 27, respectively. The sequence of SEQ ID NO: 26 is GATGATAGC.

13. The polynucleotide according to claim 11, characterized in that The nucleic acid sequence of the polynucleotide encoding the framework regions FR1, FR2, FR3, and FR4 of the antibody heavy chain variable region has 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 nucleic acid sequences shown in SEQ ID NOs: 20, 21, 22, and 23, respectively. The nucleic acid sequences of the polynucleotides encoding the framework regions FR1, FR2, FR3, and FR4 of the antibody light chain variable region 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 nucleic acid sequences shown in SEQ ID NOs: 28, 29, 30, and 31, respectively.

14. The polynucleotide according to claim 13, characterized in that The nucleic acid sequences of the polynucleotides encoding the framework regions FR1, FR2, FR3, and FR4 of the antibody heavy chain variable region are shown in SEQ ID NOs: 20, 21, 22, and 23, respectively. The nucleic acid sequences of the polynucleotides encoding the framework regions FR1, FR2, FR3, and FR4 of the antibody light chain variable region are shown in SEQ ID NOs: 28, 29, 30, and 31, respectively.

15. The polynucleotide according to claim 11, characterized in that The nucleic acid sequence of the polynucleotide encoding the antibody 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%, 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 24, The nucleic acid sequence of the polynucleotide encoding the antibody 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%, 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO:

32.

16. The polynucleotide according to claim 15, characterized in that The nucleic acid sequence of the polynucleotide encoding the antibody heavy chain variable region is shown in SEQ ID NO: 24, The nucleic acid sequence of the polynucleotide encoding the antibody light chain variable region is shown in SEQ ID NO:

32.

17. A carrier, characterized in that The vector comprises the polynucleotide according to any one of claims 11 to 16.

18. The carrier according to claim 17, characterized in that The vector also includes at least one promoter operably linked to the nucleic acid sequence.

19. The carrier according to claim 18, characterized in that The vectors include plasmids, bacteriophages, and animal viruses.

20. A host cell, characterized in that The host cell comprises the polynucleotide according to any one of claims 11 to 16 or the vector according to any one of claims 17 to 19.

21. The host cell according to claim 20, characterized in that The host cells include prokaryotic cells, yeast or eukaryotic cells.

22. The host cell according to claim 21, characterized in that The eukaryotic cell includes an animal cell, a plant cell or a fungal cell.

23. The host cell according to claim 22, characterized in that The animal cells include mammalian cells, avian cells, and insect cells.

24. The host cell according to claim 23, characterized in that The mammalian cells include CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, AT1080 cells, A549 cells, HEK293T cells, and 293F cells.

25. The host cell according to claim 24, characterized in that The mammalian cells are selected from CHO cells and HEK 293F cells.

26. A product for detecting Staphylococcus aureus manganese ion transporter C, characterized in that: The product comprises the antibody according to any one of claims 1-10.

27. The product according to claim 26, characterized in that The products include test kits and test strips.

28. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the antibody according to any one of claims 1 to 10, the polynucleotide according to any one of claims 11 to 16, the vector according to any one of claims 17 to 19, or the host cell according to any one of claims 20 to 25.

29. The pharmaceutical composition according to claim 28, characterized in that The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

30. Any of the following methods: 1) A method for producing the antibody according to any one of claims 1 to 10, characterized in that: The method comprises culturing the host cell of any one of claims 20 to 25, and recovering the antibody; 2) A method for detecting Staphylococcus aureus manganese ion transporter C in a sample for non-diagnostic purposes, characterized in that the method comprises contacting the sample with the antibody according to any one of claims 1 to 10, thereby detecting the level of Staphylococcus aureus manganese ion transporter C in the sample; 3) A method for regulating the bactericidal activity of phagocytes for non-therapeutic purposes, characterized in that the method comprises administering the antibody according to any one of claims 1 to 10, and the bacterium is Staphylococcus aureus; 4) A method for regulating cytokines produced by Staphylococcus aureus for non-therapeutic purposes, characterized in that the method comprises administering the antibody according to any one of claims 1 to 10.

31. The method according to claim 30, characterized in that 4) The cytokines include one or more of IFN-γ, IL-17A or IL-5.

32. Any of the following applications: 1) Use of the antibody according to any one of claims 1 to 10, the polynucleotide according to any one of claims 11 to 16, the vector according to any one of claims 17 to 19, or the host cell according to any one of claims 20 to 25 in vitro detection of Staphylococcus aureus manganese transporter C for non-diagnostic purposes or in the preparation of a product for detecting Staphylococcus aureus; 2) Use of the antibody according to any one of claims 1 to 10, the polynucleotide according to any one of claims 11 to 16, the vector according to any one of claims 17 to 19, or the host cell according to any one of claims 20 to 25 in the preparation of a pharmaceutical composition for preventing and / or treating diseases related to Staphylococcus aureus infection; 3) Use of the antibody according to any one of claims 1 to 10 for the non-therapeutic purpose of opsonizing phagocyte bactericidal activity, wherein the bacterium is Staphylococcus aureus; The disease associated with Staphylococcus aureus infection is selected from sepsis.

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