Production and use of anti-staphylococcus aureus isdb antibodies

Abstract

The application discloses preparation and application of anti-Staphylococcus aureus IsdB antibody, which comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 in the heavy chain variable region shown as SEQ ID NO: 8; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 in the light chain variable region shown as SEQ ID NO: 16. The application also provides nucleic acid encoding the antibody, a carrier comprising the nucleic acid and a cell comprising the carrier. In addition, experiments prove that the antibody can inhibit systemic infection of MRSA and resist invasion of MRSA on pneumonia, and has a good application prospect in clinic.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to preparation and application of anti-Staphylococcus aureus IsdB antibody. BACKGROUND

[0002] Staphylococcus aureus (S. aureus) as a representative of gram-positive bacteria is a kind of pathogenic coccus widely existing in nature, and is also an important pathogenic bacterium causing hospital infection and community infection. S. aureus infection is characterized by acute and pyogenic, and can cause pyogenic infection of skin and soft tissue, etc., and is difficult to heal; and can cause serious infections such as osteomyelitis, septic arthritis, endocarditis, pneumonia, sepsis and complications, and the mortality rate is as high as 20%. Meanwhile, the exotoxin of S. aureus can also cause food poisoning, scalded skin syndrome and toxic shock syndrome and other systemic fatal infections.

[0003] Iron regulated surface determinant B (IsdB) is distributed on the cell surface of S. aureus, is a receptor of hemoglobin, maintains the growth of S. aureus, and is closely related to the pathogenesis of MRSA infective endocarditis.

[0004] At present, for the treatment of S. aureus infection, antibiotics such as erythromycin, new penicillin, gentamicin, vancomycin or cephalothin are usually selected. However, due to the emergence of multi-drug resistant S. aureus, it is more and more difficult to treat S. aureus infection with a single antibiotic. Clinical data shows that S. aureus with multi-drug resistance accounts for 60% in community S. aureus infection, and accounts for 80% in hospital. Among them, methicillin-resistant S. aureus (MRSA) is particularly prominent. The infection caused by MRSA is difficult to cure by antibiotic therapy and has a high mortality rate. The sepsis caused by invasive infection of MRSA has a mortality rate of more than 50% within 90 days. Therefore, since the current main antibiotic therapy has no effect on controlling MRSA infection, antibody development is imminent. SUMMARY

[0005] In order to make up for the deficiency of the prior art, the purpose of the present application is to provide an antibody specifically binding to S. aureus iron regulated surface determinant B and application thereof in preparation of a product for diagnosing or treating diseases related to S. aureus infection.

[0006] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0007] The present application provides an antibody against IsdB of Staphylococcus aureus in a first aspect.

[0008] Further, the antibody comprises CDR regions in a heavy chain variable region as shown in SEQ ID NO: 8; and CDR regions in a light chain variable region as shown in SEQ ID NO: 16.

[0009] Further, the determination scheme of the CDR regions is any one of IMGT, Chothia, Kabat, AbM, Contact scheme.

[0010] Further, the antibody includes a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, or a murine antibody.

[0011] In the present application, the term "antibody" means an immunoglobulin molecule that recognizes and specifically binds to an antigen through at least one antigen recognition site within the variable region of the immunoglobulin molecule. The target can be a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing. As used herein, the term "antibody" encompasses intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins incorporating an antibody, and any other modified configuration of the immunoglobulin molecule that presents the desired biological activity. In particular embodiments of the present application, the antibody is a monoclonal antibody. Antibodies can be of any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, based on the identity of their constant domains (a, d, e, g, and m, respectively). The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, and the like.

[0012] A complete antibody is typically composed of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CHI, CH2, and CH3) regions, and each light chain contains one N-terminal variable (VL(Kappa, Lambda)) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen binding site of the antibody. The VH and VL regions have the same general structure, with each region comprising four framework regions whose sequences are relatively conserved. As used herein, the term "framework region" refers to the relatively conserved amino acid sequences within the variable region that are located between the hypervariable regions or complementarity determining regions (CDRs). There are four framework regions in each variable domain, designated FR1, FR2, FR3, and FR4. The framework regions form a beta sheet that provides the structural framework of the variable region (see, e.g., C. A. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001)). The three CDRs, designated CDR1, CDR2, and CDR3, form the "hypervariable region" of the antibody that is responsible for antigen binding.

[0013] In the present application, the terms "VL" and "VL domain" are used interchangeably to refer to the variable region of the light chain of an antibody. The terms "VH" and "VH domain" are used interchangeably to refer to the variable region of the heavy chain of an antibody.

[0014] In the present application, CDR regions are defined in the art in a variety of ways, including but not limited to, Kabat, Chothia, Abm, Contact, and IMGT. The IMGT definition used in the present application is from IMGT® (the international Im Muno Gene Tics database, imgt.org, creator and manager: Marie-Paule Lefranc, Montpellier, France, see, e.g., Lefranc, M.-P., 1999, The Immunologist, 7:132-136 and Lefranc, M.-P. et al., 1999, Nucleic Res., 27:209-212, both of which are incorporated by reference herein in their entireties). For the IMGT numbering system, (i) HCDR1 is generally at heavy chain amino acid positions 26-33, (ii) HCDR2 is generally at heavy chain amino acid positions 51-58, (iii) HCDR3 is generally at heavy chain amino acid positions 97-111. For the IMGT numbering system, (i) LCDR1 is generally at light chain amino acid positions 27-32, (ii) LCDR2 is generally at light chain amino acid positions 50-52, (iii) LCDR3 is generally at light chain amino acid positions 89-97.

[0015] In a specific embodiment of the present application, the CDR regions of the heavy chain variable region are designated as HCDR1, HCDR2 and HCDR3, respectively, the sequences of which are set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and the CDR regions of the light chain variable region are designated as LCDR1, LCDR2 and LCDR3, respectively, the sequences of which are set forth in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively.

[0016] In the present application, the terms "constant region" or "constant domain" are interchangeable and have their common meaning in the art. The constant region is a portion of an antibody, e.g., a carboxy-terminal portion of the light and / or heavy chains that is not directly involved in binding of the antibody to an antigen but which can exhibit various effector functions (e.g., interaction with the Fc receptor). The amino acid sequence of the constant region of an immunoglobulin molecule is generally more conserved than the variable domain of the immunoglobulin.

[0017] In the present application, the term "heavy chain" when used in reference to an antibody, based on the amino acid sequence of the constant domain, can refer to any of the different classes, e.g., alpha (a), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which give rise to IgA, IgD, IgE, IgG, and IgM classes, respectively, of antibodies, including subclasses of IgG, e.g., IgGl, IgG2, IgG3, and IgG4. The heavy chain amino acid sequences are well known in the art.

[0018] In the present application, the term "light chain" when used in reference to an antibody, based on the amino acid sequence of the constant domain, can refer to any of the different classes, e.g., kappa (κ) or lambda (λ). The light chain amino acid sequences are well known in the art.

[0019] Further, the antibody has a heavy chain variable region sequence set forth in SEQ ID NO: 8 and a light chain variable region sequence set forth in SEQ ID NO: 16.

[0020] In some embodiments, the antibody includes an amino acid sequence having at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 16, and having the same function.

[0021] In some embodiments, the antibody further includes an amino acid sequence having one or more amino acids modified, substituted, deleted or added to the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 16, and having the same function.

[0022] In the present application, the terms "identity", "homology" mean a sequence identity over the entire length of the nucleotide sequence or a sequence identity over the entire length of the amino acid sequence of at least 60%, preferably of at least 70%, more preferably of at least 80%, particularly of at least 90%, further more preferably of at least 95%, 96%, 97%, 98%, 99% and most preferably of higher. The present application thus also includes all these modifications of all nucleic acid or amino acid sequences according to the present application having the above-mentioned percentage identities as described herein.

[0023] The second aspect of the present application provides an antibody derivative.

[0024] Further, the antibody derivative is a complex obtained by modifying the antibody according to the first aspect of the present application; the modification comprises a detectable label conjugation modification, a therapeutic agent conjugation modification, an imaging agent conjugation modification.

[0025] Further, the detectable label comprises a fluorescent dye, an avidin, a paramagnetic atom, a radioisotope, an enzymatic label or a colloidal gold.

[0026] Further, the therapeutic agent comprises a cytotoxic agent, a hormonal agent, a targeted small molecule agent, a proteasome inhibitor, a chemotherapeutic agent, an oncolytic drug, a cytokine, an activator of a costimulatory molecule or an inhibitor of an inhibitory molecule.

[0027] In the present application, the term "detectable label" refers to a molecule or a compound or a set of molecules or a set of compounds associated with a probe that is used to identify the probe hybridized to a genomic nucleic acid or a reference nucleic acid. In some cases, the detectable label can be detected directly. In other cases, the detectable label can be part of a binding pair that can then be followed by detection. The signal from the detectable label can be detected in a variety of ways and will depend on the nature of the detectable label. Examples of ways to detect a detectable label include, but are not limited to, spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as a fluorescent dye, colloidal gold, a chemiluminescent catalyst or a chemiluminescent marker, or any other suitable means. The term "chemiluminescent marker" relates to a marker that is capable of emitting light (luminescence) with limited heat as a result of a chemical reaction. Chemiluminescent markers include, but are not limited to, luminol and its derivatives, isoluminol and its derivatives, cya lume, pyrogallol, oxalyl chloride, lucigenin, TDAE (tetra(dimethylamino)ethylene), acridinum ester and its derivatives, adamantane, rare earth elements, a ruthenium complex of dipyridyl, acridinum ester or dioxetane. Chemiluminescent catalysts include, but are not limited to, horseradish peroxidase, alkaline phosphatase.

[0028] The term "therapeutic agent" as used herein refers to a compound that when present in an effective amount produces a desired therapeutic effect on a subject in need thereof. Therapeutic agents include, but are not limited to, radionuclides, cytokines, gold nanoparticles, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes, chemotherapeutic agents. The cytokines include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-14, IFN-gamma, TNF-beta, TNF-alpha, G-CSF, M-CSF; the chemotherapeutic agents include, but are not limited to, cisplatin, paclitaxel, vincristine, asparaginase, oxaliplatin, platinous oxalate, oxaliplatin.

[0029] The third aspect of the present application provides a nucleic acid molecule encoding the antibody of the first aspect of the present application.

[0030] Further, the nucleotide sequence encoding the heavy chain variable region of the antibody of the first aspect of the present application is shown in SEQ ID NO: 24.

[0031] Further, the nucleotide sequence encoding the light chain variable region of the antibody of the first aspect of the present application is shown in SEQ ID NO: 32.

[0032] In the present application, the term "nucleic acid molecule" is intended to encompass polymers of DNA or RNA, i.e., a polynucleotide, which can be single-stranded or double-stranded and which can contain non-natural or altered nucleotides. The terms "nucleic acid" and "polynucleotide" as used herein refer to polymeric forms of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecules and thus include double- and single-stranded DNA, as well as double- and single-stranded RNA. The terms include RNA or DNA analogs, as equivalents. Although many other linkages are known in the art (e.g., phosphorothioates, phosphoroboranoates, etc.), nucleic acids are typically linked via phosphodiester bonds to form nucleic acid sequences or polynucleotides.

[0033] The fourth aspect of the present application provides an expression vector comprising the nucleic acid molecule of the third aspect of the present application.

[0034] Further, the expression vector comprises a plasmid or a viral vector.

[0035] The term "expression vector" refers to a recombinant DNA construct designed for proper insertion of a selected gene of interest into the expression vector to express the gene (typically a protein). Non-limiting examples of expression vectors include plasmids or viral vectors. Suitable expression vectors can include regulatory elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals, and enhancers, and can be prepared in various ways depending on the intended use. The promoter of the vector can be constitutive or inducible. In particular, the expression vector can be prepared by using a plasmid vector.

[0036] The fifth aspect of the present application provides a host cell comprising the nucleic acid molecule of the third aspect of the present application or the expression vector of the fourth aspect of the present application.

[0037] Further, the host cell comprises a prokaryotic cell or a eukaryotic cell.

[0038] In the present application, the host cell includes any suitable prokaryotic or eukaryotic cell. Cells that can be used include cells that can be easily and reliably grown, have a reasonably fast growth rate, have a well-characterized expression system, and can be easily and efficiently transformed or transfected. Eukaryotic cells are known in the art and include, but are not limited to, for example, yeast cells, insect cells, and mammalian cells. In one embodiment, the vector is expressed in a mammalian cell. Many suitable mammalian host cells are known in the art. Examples of suitable mammalian cells include, but are not limited to, Chinese hamster ovary cells, CHO DHFR cells, human embryonic kidney (HEK) 293, HEK293E, HEK293-6E, HEK293F, or 293T cells (ATCC No. CRL 1573), and 3T3 cells (ATCC No. CCL92). Other suitable mammalian cell lines are the monkey COS-1 (ATCC No. CRL 1650) and COS-7 cell lines (ATCC No. CRL 1651), per.C6 cells, CV-1 cell line (ATCC No. CCL70), myeloma cells, hybridoma cells, and NSO cells. Desirably, the mammalian cell is a human cell. In a particular embodiment of the present application, the host cell is a HEK293F cell. The nucleic acid sequences encoding the amino acids of any of the antibodies described herein can be introduced into the cell by transfection, transformation, or transduction.

[0039] The sixth aspect of the present application provides a pharmaceutical composition.

[0040] Further, the pharmaceutical composition comprises the antibody according to the first aspect of the present application, the antibody derivative according to the second aspect of the present application, the nucleic acid molecule according to the third aspect of the present application, the expression vector according to the fourth aspect of the present application or the host cell according to the fifth aspect of the present application.

[0041] In the present application, composition refers to any mixture of two or more products, substances or compounds (including cells). It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous or any combination thereof. The term "pharmaceutical composition" as used in the present application refers to a preparation which is effective in the form in which it is administered in order to exert the biological activity of one or more active ingredients, and which does not contain other components which are unacceptable in terms of toxicity to the subject to which the formulation is administered. It is thus a composition suitable for medical use in a mammalian subject, typically a human being. A pharmaceutical composition typically comprises an effective amount of an active agent and a carrier, excipient or diluent. The carrier, excipient or diluent is typically a pharmaceutically acceptable carrier, excipient or diluent, respectively. Such formulations can be sterile.

[0042] The seventh aspect of the present application provides a product for detecting Staphylococcus aureus IsdB.

[0043] Further, the product comprises the antibody according to the first aspect of the present application, the antibody derivative according to the second aspect of the present application, the nucleic acid molecule according to the third aspect of the present application, the expression vector according to the fourth aspect of the present application or the host cell according to the fifth aspect of the present application.

[0044] Further, the product comprises a kit, a test strip.

[0045] The eighth aspect of the present application provides use of the antibody according to the first aspect of the present application, the antibody derivative according to the second aspect of the present application, the nucleic acid molecule according to the third aspect of the present application, the expression vector according to the fourth aspect of the present application, the host cell according to the fifth aspect of the present application or the pharmaceutical composition according to the sixth aspect of the present application for the manufacture of a product for diagnosing or treating a disease caused by Staphylococcus aureus infection.

[0046] Further, the disease caused by Staphylococcus aureus infection comprises pneumonia, systemic infection, skin abscess, folliculitis, myocardial endocarditis, meningitis, otitis media, osteomyelitis, toxic shock syndrome, septicemia or sepsis.

[0047] Further, the disease caused by Staphylococcus aureus infection is pneumonia, systemic infection.

[0048] In the present application, the term "treatment" refers to (1) preventing the onset of symptoms or disease in a subject susceptible to or otherwise at risk of developing a disorder; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or disorder. As understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of the present technology, beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or remission of the condition (including disease), state and / or improvement no matter how small, whether detectable or undetectable.

[0049] The ninth aspect of the present application provides use of the antibody of the first aspect of the present application in the manufacture of the antibody derivative of the second aspect of the present application, the nucleic acid molecule of the third aspect of the present application, the expression vector of the fourth aspect of the present application, the host cell of the fifth aspect of the present application, the pharmaceutical composition of the sixth aspect of the present application or the product for detecting Staphylococcus aureus IsdB of the seventh aspect of the present application.

[0050] The tenth aspect of the present application provides use of the antibody of the first aspect of the present application, the antibody derivative of the second aspect of the present application, the nucleic acid molecule of the third aspect of the present application, the expression vector of the fourth aspect of the present application, the host cell of the fifth aspect of the present application or the pharmaceutical composition of the sixth aspect of the present application in the manufacture of a product for enhancing the killing ability of phagocytes against Staphylococcus aureus.

[0051] In addition, the present application also provides any one of the following methods:

[0052] (1) A method for detecting Staphylococcus aureus IsdB in a sample for non-diagnostic purposes, the method comprising contacting the sample with the antibody of the first aspect of the present application, and detecting the immunoreaction of Staphylococcus aureus IsdB with the antibody;

[0053] (2) A method for preparing the host cell of the fifth aspect of the present application, the method comprising introducing the nucleic acid molecule of the third aspect of the present application or the expression vector of the fourth aspect of the present application into a cell;

[0054] (3) A method for producing the antibody of the first aspect of the present application, the method comprising culturing the host cell of the fifth aspect of the present application, and isolating the antibody from the culture;

[0055] (4) A method for enhancing the killing ability of phagocytes against Staphylococcus aureus for non-therapeutic purposes, the method comprising administering the antibody of the first aspect of the present application;

[0056] (5) A method of specifically inhibiting the activity of S. aureus IsdB, the method comprising inhibiting the activity of S. aureus IsdB using the antibody of the first aspect of the application or introducing the nucleic acid molecule of the third aspect of the application into a cell of an organism to inhibit the activity of S. aureus IsdB by expressing the antibody of the first aspect of the application.

[0057] In the present application, the term "introducing" refers to providing a vector to a cell so that the vector becomes internalized in the cell. For example, the vector can be introduced into a cell using transfection, transformation, and / or injection, and can also be introduced into a cell using other methods known to one of ordinary skill in the art. Transfection reagents include, but are not limited to, liposome transfection reagents such as Lipofectamine 2000, Lipofectamine 3000, Turbiofect, PEI, etc. or chemical transfection reagents or transfection by electroporation.

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

[0059] The present application provides an antibody (named IsdB-H6) that specifically binds to S. aureus IsdB, the IsdB-H6 antibody can inhibit the growth of S. aureus in a low iron ion environment, and can resist systemic invasion of MRSA, resist invasion of MRSA on pneumonia, and has good application prospect in clinic. BRIEF DESCRIPTION OF DRAWINGS

[0060] Figure 1 Figure 1 is a result graph of SDS-PAGE purity detection of IsdB-H6 monoclonal antibody; N2 Figure 1 is a result graph of SDS-PAGE purity detection of IsdB-H6 monoclonal antibody;

[0061] Figure 2 Figure 1 is a result graph of SDS-PAGE purity detection of IsdB-H6 monoclonal antibody;

[0062] Figure 3 Figure 1 is a result graph of SDS-PAGE purity detection of IsdB-H6 monoclonal antibody;

[0063] Figure 4 Figure 1 is a result graph of SDS-PAGE purity detection of IsdB-H6 monoclonal antibody;

[0064] Figure 5 Figure 1 is a result graph of SDS-PAGE purity detection of IsdB-H6 monoclonal antibody;

[0065] Figure 6 Figure 1 is a result graph of SDS-PAGE purity detection of IsdB-H6 monoclonal antibody; Figure 6 Figure 1 is a result graph of SDS-PAGE purity detection of IsdB-H6 monoclonal antibody; Figure 6 Figure 1 is a result graph of SDS-PAGE purity detection of IsdB-H6 monoclonal antibody;

[0066] Figure 7 Figure 4 is a graph showing the results of IsdB-H6 monoclonal antibody in a systemic Staphylococcus aureus infection model;

[0067] Figure 8 Figure 5 is a graph showing the results of IsdB-H6 monoclonal antibody in a Staphylococcus aureus pneumonia model. DETAILED DESCRIPTION

[0068] The present application is further described in the following Examples. It should be understood that the particular implementations described herein are meant to be illustrative only and not limiting of the application. The main features of the application can be used in various embodiments without departing from the scope of the application.

[0069] The experimental methods used in the following examples are routine unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0070] Example 1 Recombinant Staphylococcus aureus IsdB- N2 Expression and purification of antigen

[0071] The full-length sequence of IsdB gene was synthesized (Jierui, Shanghai) and cloned into the expression vector pGEX-6P-2 using the restriction endonuclease recognition sites BamHI and SacI to construct the IsdB- N2 Prokaryotic expression vector pGEX-IsdB N2 XL-1, and the IsdB- N2 protein was expressed in E. coli. The recombinant bacteria were inoculated in LB medium containing ampicillin and cultured overnight (37°C, 100 rpm), and the next day the culture was expanded to an OD N2 value of 0.6-0.8 at a ratio of 1:100 (V / V), and IPTG was added for overnight induction of expression (4°C, 200 rpm). The bacteria were centrifuged and the bacterial cells were lysed by sonication. The lysate supernatant was added to GST-4FF affinity filler and mixed vertically at 4°C overnight, and glutathione was added to elute the IsdB- 600 -GST fusion protein. Prescission protease was then added to remove the GST tag, and IsdB- N2 protein was obtained by SPHP chromatography. The buffer was replaced with PBS, and the purified IsdB- N2 protein was further concentrated using an ultrafiltration tube. The purity of the IsdB- N2 protein was detected by SDS-PAGE gel electrophoresis. The results of the molecular weight and purity detection are shown in Figure 1 Figure 1, which shows that the purity of the IsdB- N2 protein was 100.0%.

[0072] Isolation of PBMC cells

[0073] IsdB-based S. aureus vaccine N2 Protective antigens, ethical review approval was obtained from Jiangsu Provincial Center for Disease Control and Prevention, and peripheral blood lymphocyte samples from phase Ia subjects stored in liquid nitrogen were obtained from the recombinant S. aureus vaccine (E. coli) phase Ia clinical trial center.

[0074] Example 3 Establishment of human-specific anti-S. aureus antibody library

[0075] 1. Preparation of cDNA:

[0076] Total RNA was extracted from the peripheral blood lymphocytes isolated in Example 2, and reverse transcription was performed to synthesize cDNA using the total RNA as a template.

[0077] 2. Construction of human-specific anti-S. aureus Fab antibody library:

[0078] The reverse-transcribed cDNA was mixed and used as a gene template, and Ig primers were used to amplify the human light chain (VL+CL) Kappa / Lambda and heavy chain Fd gene sequences by PCR. The light chain VL+CL gene PCR recovery product was double-digested by SacI-HF and XbaI-HF enzymes, and then ligated into 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 with the light chain gene fragment through XhoI-HF and SpeI-HF restriction enzyme cleavage sites, respectively, to form the Fab phagemid. The ligation product was transformed into TG1 competent cells, which were cultured on plates containing ampicillin at 37°C overnight. 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 as follows: 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 the PCR product was detected by 2% agarose gel electrophoresis.

[0079] 3. Positive rate identification:

[0080] The screening results showed that the positive clone rates of the Fab (Kappa light chain) library and the Fab (Lambda light chain) library were both more than 80%, and the library capacity was more than 10 8 .

[0081] Example 4 Screening of human-specific anti-S. aureus Fab antibody library

[0082] 1. Bacterial purification and antibody phage preparation

[0083] The frozen library bacterial solution was inoculated into LB-ampicillin culture medium at a ratio of 1:100 for recovery, and grown to OD 600 When the OD value was 0.5-0.6, the bacterial solution was removed, and helper phage M13 was added at a ratio of 1:1000. After standing at 37°C for 30 min, the shaking culture was continued for 30 min, the bacterial cells were collected by centrifugation, and the LB medium containing ampicillin and kanamycin was used for overnight culture at 30°C on a constant temperature shaker. The phage supernatant was collected by centrifugation of the bacterial solution at 3000 g for 10 min at 4°C, PEG / NaCl solution was added for thorough mixing, and the mixture was placed on ice for 30 min, then centrifuged at 3000 g for 20 min at 4°C, the supernatant was removed, the precipitate was mixed with PEG / NaCl solution, and the mixture was placed on ice for 30 min, then centrifuged at 12000 g for 2 min at 4°C, the supernatant was removed, the precipitate was resuspended with 1 mL PBS, and glycerol was added for storage at -80°C.

[0084] 2. Phage library panning

[0085] The antibody phage was added to the ELISA plate coated with antigen, incubated at 37°C for 20 min, and washed 10 times with PBST and PBS solution to remove unbound phage; after trypsin digestion of the bound phage, TG1 bacteria infected with OD 600 The OD value was 0.5-0.6, the bacterial solution was removed, and helper phage M13 was added at a ratio of 1:1000. After standing at 37°C for 30 min, the shaking culture was continued for 30 min, the bacterial cells were collected by centrifugation, and the LB medium containing ampicillin and kanamycin was used for overnight culture at 30°C on a constant temperature shaker. The phage supernatant was collected by centrifugation of the bacterial solution at 3000 g for 10 min at 4°C, PEG / NaCl solution was added for thorough mixing, and the mixture was placed on ice for 30 min, then centrifuged at 3000 g for 20 min at 4°C, the supernatant was removed, the precipitate was mixed with PEG / NaCl solution, and the mixture was placed on ice for 30 min, then centrifuged at 12000 g for 2 min at 4°C, the supernatant was removed, the precipitate was resuspended with 1 mL PBS, and glycerol was added for storage at -80°C.

[0086] 3. Phage library screening

[0087] The phage enriched by panning was plated on LB plates containing ampicillin to produce bacterial monoclonals, which were picked into 96-well culture plates for culture and phage packaging for phage ELISA screening. The antibody phage was added to the ELISA plate coated with antigen, incubated at 37°C for 20 min, washed 3 times with PBST solution to remove unbound phage; fluorescently labeled antibody was added, incubated at 37°C for 20 min, and washed 3 times with PBST; the specific antibody was screened by detecting the ELISA fluorescence signal intensity, the corresponding positive well antibody phage was amplified, and sequencing analysis was performed.

[0088] Example 5 Cloning, expression and purification of anti-IsdB-H6 fully human antibody Example 5 Cloning, expression and purification of anti-IsdB-H6 fully human antibody

[0089] Example 5 Cloning, expression and purification of anti-IsdB-H6 fully human antibody1. Experimental method Example 5 Cloning, expression and purification of anti-IsdB-H6 fully human antibody

[0090] Example 5 Cloning, expression and purification of anti-IsdB-H6 fully human antibodyThe positive clone display vector screened in Example 3 was used as a template to amplify human Ig VH and VK / L by PCR. The PCR products were identified by agarose gel electrophoresis. The antibody genes identified as positive and matched pairs of light and heavy chains were purified by Qiagen PCR product purification kit. The purified products were subjected to forward and reverse sequence determination. The antibody gene family, mutation rate and CDR region were analyzed by IMGT online server (https: / / www.imgt.org / IMGT_vquest / input). Example 5 Cloning, expression and purification of anti-IsdB-H6 fully human antibody

[0091] Example 5 Cloning, expression and purification of anti-IsdB-H6 fully human antibodyThe PCR products of the antibody variable region genes identified as positive and matched pairs of heavy and light chains by gel electrophoresis were ligated to pcDNA3.4 vectors containing heavy chain constant region or light chain constant region by TA cloning method to construct expression vectors of anti-IsdB-H6 fully human antibodies. Then the expression vectors were transformed into DH5a competent cells, which were plated on LB plates containing ampicillin and incubated in a 37°C incubator overnight. The next day, 10 single colonies were picked and subjected to PCR identification with specific primers. The reaction conditions were as follows: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 100 s, 28 cycles; 72°C extension for 5 min. 5 μL of the PCR products were detected by 1% agarose gel electrophoresis. Example 5 Cloning, expression and purification of anti-IsdB-H6 fully human antibody

[0092] Example 5 Cloning, expression and purification of anti-IsdB-H6 fully human antibodyThe vector plasmids with positive PCR products were transformed into DH5a for mass amplification. The recombinant plasmids were quickly extracted and incubated with transfection reagent PEI at 37°C for 15-20 min, then transfected into HEK 293F cells, which were cultured in a 37°C, 5% CO2 incubator. After 5 days of cell culture, the cell supernatant was collected by centrifugation at 3000 g, 4°C for 30 min. The antibody was purified by protein A affinity chromatography. The expression and purification of the antibody were detected by SDS-PAGE gel electrophoresis. Example 5 Cloning, expression and purification of anti-IsdB-H6 fully human antibody

[0093] Example 5 Cloning, expression and purification of anti-IsdB-H6 fully human antibody2. Experimental results Example 5 Cloning, expression and purification of anti-IsdB-H6 fully human antibody

[0094] Example 5 Cloning, expression and purification of anti-IsdB-H6 fully human antibodyThis scheme successfully constructed a series of heavy chain and light chain expression vectors of antibodies. One of the antibodies was named fully human IsdB monoclonal antibody IsdB-H6 (abbreviated as IsdB-H6 antibody or IsdB-H6 monoclonal antibody). Example 5 Cloning, expression and purification of anti-IsdB-H6 fully human antibody

[0095] Example 5 Cloning, expression and purification of anti-IsdB-H6 fully human antibodyThe SDS-PAGE detection results are as follows: Figure 2 Example 5 Cloning, expression and purification of anti-IsdB-H6 fully human antibodyAs shown, the relative molecular weight of the IsdB-H6 antibody in the transfected cells is approximately 150 kDa, with the heavy chain at approximately 50 kDa and the light chain at approximately 25 kDa, indicating that the transfected cells can successfully express the antibody. The IsdB-H6 antibody sequence is shown in Table 1.

[0096] Table 1 IsdB-H6 antibody sequence

[0097]

[0098]

[0099] Example 6: IsdB-H6 monoclonal antibody and Staphylococcus aureus iron-regulated surface determinant B (IsdB- N2 ) binding activity detection

[0100] 1. Experimental Methods

[0101] Recombinant expression of IsdB- N2 100 μL of 2 μg / mL protein was coated onto each well of a 96-well ELISA plate and incubated overnight at 4°C. The plate was washed the next day, and then blocked at 37°C for 2 h with 3% BSA blocking buffer. The IsdB-H6 antibody transfected and expressed in Example 5 was diluted to 100 μg / mL and then serially diluted with 1×PBST. 100 μL of each antibody was added to each well of the ELISA plate, with three replicates for each concentration. The positive control was 100 μL of vaccine serum (1:2000 dilution) per well, and the negative control was 100 μL of negative serum (1:100 dilution) and negative control-irrelevant antibody IgG1 (0.5 μg / mL) per well. 100 μL of PBST was added to the blank control, and the plates were incubated at 37°C for 1 h.

[0102] Wash the plate three times with PBST buffer. Add 100 μL of Goat-Anti-Human-IgG-Fab-HRP (secondary antibody) diluted 1:5000 with PBST to each well and incubate at 37°C for 1 h. Wash the plate three times with PBST buffer. Add 100 μL of TMB chromogenic solution to each well and incubate at 37°C in the dark for 5–10 min. Immediately stop the reaction by adding 50 μL of 2 M H₂SO₄. Detect the OD value at dual wavelengths of 450 / 650 nm. Calculate the mean value of the negative control unrelated antibody IgG1, determine the threshold (3 times the mean value), and calculate the EC value. Antibody values ​​greater than the threshold are considered positive antibodies. 50 .

[0103] 2. Experimental Results

[0104] Test results as follows Figure 3 As shown, the results indicate that the fully human IsdB monoclonal antibody IsdB-H6 can bind to Staphylococcus aureus iron-regulated surface determinant B (IsdB-H6).N2 ) combined. IsdB- N2 EC 50 The calculated result is 0.27 μg / mL.

[0105] Example 7: Determination of Epitope Type of IsdB-H6 Monoclonal Antibody

[0106] 1. Experimental Methods

[0107] The antibody-antigen binding reaction was detected using Western blotting (WB). The IsdB-H6 antibody was subjected to SDS-PAGE (reduced) gel electrophoresis at 140 V for 1–2 hours. Electrophoresis was stopped when the bromophenol blue just appeared. After electrophoresis, the PVDF membrane was transferred using a semi-dry transfer apparatus at 23 V for 20 minutes. The transferred membrane was then completely covered with TBST solution containing 5% skim milk powder and blocked, then placed on a shaker at room temperature for 1 hour. The PVDF membrane was washed three times with TBST solution for 5 min each time. Then, a solution containing 1% skim milk powder was prepared with TBST, and anti-human IgG-AP was added at a ratio of 1:7500. The PVDF membrane was placed in the solution and incubated at room temperature on a horizontal shaker for 1 h. Then, it was washed three times with TBST solution for 5 min each time. The PVDF membrane was placed in a clean Petri dish, and about 1 mL of AP colorimetric solution was added to each membrane in the dark. The color development of the bands was observed. When the bands were clear, water was added to stop the reaction.

[0108] 2. Experimental Results

[0109] Test results as follows Figure 4 As shown, the results indicate that the IsdB-H6 antibody can bind to IsdB- N2 Antigen binding indicates that the epitope of the IsdB-H6 antibody is a linear epitope.

[0110] Example 8: Inhibitory effect of anti-IsdB antibody on the growth of recombinant Staphylococcus aureus in a low-iron environment.

[0111] 1. Experimental Methods

[0112] By using anti-IsdB antibody under low iron conditions, we observed its effect on the iron uptake pathway of recombinant Staphylococcus aureus and evaluated the potential effect of the antibody in inhibiting bacterial growth.

[0113] First, USA300 was cultured based on TSB, second, activated bacteria solution was cultured overnight using RPMI 1640 (containing 2,2'-dipyridyl and casein hydrolyzed amino acid), the next day, bacteria were collected by centrifugation, then washed twice using RPMI 1640 (containing 2,2'-dipyridyl and casein hydrolyzed amino acid), finally, bacteria were resuspended using RPMI 1640 (containing 2,2'-dipyridyl and / or human hemoglobin); antibody group, 1 μM and 100 μM IsdB-H6 antibody was mixed with equal volume of resuspended bacteria solution (resuspended using RPMI 1640 (containing 2,2'-dipyridyl and human hemoglobin)) and added into 96-well cell culture plate; control group, PBS buffer was mixed with equal volume of resuspended bacteria solution (resuspended using RPMI 1640 (containing 2,2'-dipyridyl and human hemoglobin)) and added into 96-well cell culture plate; blank group, PBS buffer was mixed with equal volume of resuspended bacteria solution (resuspended using RPMI 1640 (containing 2,2'-dipyridyl)) and added into 96-well cell culture plate; 96-well cell culture plate was placed in 37°C incubator and cultured for 24 hours. Enzyme marker was used to measure OD value of each well regularly to monitor bacterial growth and draw bacterial growth curve, and further calculate inhibition rate of antibody on bacterial growth. 600

[0114] 2、Experimental results

[0115] Results are shown in Figure 5 OD6 00 100% for Staphylococcus aureus cultured in RPMI 1640 (containing 2,2'-dipyridyl and human hemoglobin) for 24 hours; inhibition rate was 12% for Staphylococcus aureus cultured in RPMI 1640 (containing 2,2'-dipyridyl and human hemoglobin) containing 1 μM antibody for 24 hours; inhibition rate was 58% for Staphylococcus aureus cultured in RPMI 1640 (containing 2,2'-dipyridyl and human hemoglobin) containing 100 μM antibody for 24 hours.

[0116] Example 9 IsdB-H6 monoclonal antibody opsonizes and kills Staphylococcus aureus

[0117] 1、Experimental method

[0118] ​Antibodies can promote phagocytic killing of S. aureus by immune cells, but S. aureus has multiple virulence factors to prevent being killed by phagocytes; to assess the ability of IsdB-H6 monoclonal antibody to promote the killing of S. aureus by phagocytes, the IsdB-H6 monoclonal antibody was tested in an opsonophagocytic killing assay (OPK) to assess the ability of the monoclonal antibody of the present application to promote the killing of S. aureus USA300 by phagocytes in the presence of complement. The procedure was to use 7 dilutions of IsdB-H6 monoclonal antibody, positive serum, negative serum, and control antibody in a 96-well plate using a 2-fold dilution; S. aureus USA300 was cultured in TSB, and then suspended in OBB buffer to a concentration of 1.0 x 10 5 CFU / mL, added to the microplate (10 μL / well), and incubated at room temperature for 1 hour, and then washed with buffer to a concentration of 2.0 x 10 6

[0119] 2. Experimental results

[0120] The results are shown in Table 1, and the control antibody did not promote the killing of S. aureus by the cells, but the IsdB-H6 monoclonal antibody promoted the killing of S. aureus by the cells, and the maximum killing rate was 73%. Figure 6

[0121] Example 10. Role of IsdB-H6 monoclonal antibody in a model of systemic infection with MRSA

[0122] 1. Experimental method

[0123] The present application assessed the protective efficacy of IsdB-H6 monoclonal antibody against S. aureus USA300 in a model of systemic infection. S. aureus USA300 was cultured in TSB, subcultured, and grown to mid-log phase (OD 600 = 1.0). The culture was then washed twice in physiological saline, and 1 x 10 600 ​​The bacteria were resuspended in physiological saline at an optical density of 1.6. The mice were injected with 100 μL of the bacterial suspension via the tail vein. One day before infection, the experimental group was treated with 100 mg / kg of the IsdB-H6 monoclonal antibody, the control group was treated with 100 mg / kg of hlgGl (PcrV-A039), and the blank group was treated with physiological saline. After the mice were infected with 100 μL of the challenge bacteria via the tail vein, the mice were monitored for 7 days after infection, and the number of surviving mice was recorded every 12 h to calculate the survival rate.

[0124] 2. Experimental results

[0125] The protection rate of the IsdB-H6 monoclonal antibody containing 100 mg / kg was 60%, and the protection rate of the hlgGl (PcrV-A039) containing 100 mg / kg was 10% ( Figure 7 ).

[0126] Example 11 In vivo prophylactic effect of the IsdB-H6 monoclonal antibody on an MRSA acute pneumonia model

[0127] 1. Experimental method

[0128] The IsdB-H6 monoclonal antibody was evaluated for its protective efficacy against S. aureus USA300 in a pneumonia model. S. aureus USA300 was cultured in BHI broth and subcultured to an OD 600 of 1.0. The culture was then washed twice in physiological saline and resuspended in physiological saline at a density of 2 x 10 10 cfu / mL. The mice were infected with 20 μL of the bacterial suspension via tracheal intubation. One day before infection, the experimental group was treated with 100 mg / kg of the IsdB-H6 monoclonal antibody, the control group was treated with 100 mg / kg of hlgGl (PcrV-A039), and the blank group was treated with physiological saline. After the mice were infected with 20 μL, the mice were monitored for 7 days after infection, and the number of surviving mice was recorded every 12 h to calculate the survival rate.

[0129] 2. Experimental results

[0130] The protection rate of the IsdB-H6 monoclonal antibody containing 100 mg / kg was 70%, and the protection rate of the hlgGl (PcrV-A039) containing 100 mg / kg was 10% ( Figure 8 ).

[0131] The above description of the embodiments is only for understanding the method of the present application and its core idea. It should be noted that, for those skilled in the art, without departing from the principles of the present application, some improvements and modifications can be made to the present application, and these improvements and modifications will also fall within the protection scope of the claims of the present application.

Claims

1. An antibody against Staphylococcus aureus IsdB, characterized in that, The antibody comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region includes HCDR1, HCDR2 and HCDR3, whose sequences are shown as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively. The light chain variable region includes LCDR1, LCDR2 and LCDR3, whose sequences are shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively.

2. The antibody according to claim 1, characterized in that, The antibody has the heavy chain variable region sequence shown in SEQ ID NO: 8 and the light chain variable region sequence shown in SEQ ID NO:

16.

3. An antibody derivative, characterized in that, The antibody derivative is a complex obtained by modifying the antibody according to any one of claims 1-2; the modification is a conjugation modification using a detectable marker or an imaging agent.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody according to any one of claims 1-2.

5. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule of claim 4.

6. A host cell, characterized in that, The host cell comprises the nucleic acid molecule of claim 4 or the expression vector of claim 5.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibody according to any one of claims 1-2, the antibody derivative according to claim 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, or the host cell according to claim 6.

8. A product for detecting Staphylococcus aureus IsdB, characterized in that, The product comprises the antibody according to any one of claims 1-2, the antibody derivative according to claim 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, or the host cell according to claim 6.

9. The use of the antibody according to any one of claims 1-2, the antibody derivative according to claim 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, the host cell according to claim 6, or the pharmaceutical composition according to claim 7 in the preparation of products for diagnosing or treating diseases caused by Staphylococcus aureus infection.

10. The use of the antibody according to any one of claims 1-2 in the preparation of the antibody derivative according to claim 3, the pharmaceutical composition according to claim 7, or the product for detecting Staphylococcus aureus IsdB according to claim 8.

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