Antibodies against staphylococcus aureus isdb and uses thereof

Abstract

The application discloses an antibody against Staphylococcus aureus IsdB and application thereof. The antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown as SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11. In addition, the application further discloses a product for detecting Staphylococcus aureus IsdB and application of the antibody in preparation of a diagnosis, prevention and / or treatment of diseases related to Staphylococcus aureus infection. The application provides a new idea for detection of Staphylococcus aureus IsdB and treatment of related diseases, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of drug development, antibody engineering and biomedicine, specifically relating to antibodies against Staphylococcus aureus IsdB and their applications. Background Technology

[0002] Staphylococcus aureus (SA) is a highly pathogenic bacterium widely found in nature. It can infect various parts of the host, causing infections such as skin and soft tissue infections, nipple inflammation, pneumonia, and bacteremia; severe infections can lead to death.

[0003] Iron is an essential metallic element for the growth and metabolism of various cells and bacteria. However, only free iron can participate in biochemical reactions. In vertebrates, iron is usually found in heme. Only after separating iron from heme into iron ions can it be used in various biochemical reactions. Staphylococcus aureus possesses an iron-regulated surface determinant system that can separate heme into free iron within the host. This free iron serves as a nutrient for the growth and proliferation of Staphylococcus aureus. Iron-responsive surface determinant B (IsdB) is a crucial virulence factor for Staphylococcus aureus infecting the host and proliferating within the body. IsdB binds to the host's hemoglobin and extracts iron, helping Staphylococcus aureus survive in low-iron environments.

[0004] Immunotherapy agents based on the Staphylococcus aureus IsdB antigen have shown protective efficacy in both active and passive immunization in animal models. The IsdB antigen has become a vaccine target for preventing Staphylococcus aureus infection, and clinical trials using it as a vaccine have been reported. Currently, several biological products for the prevention or treatment of Staphylococcus aureus are undergoing or have undergone clinical trials both domestically and internationally, but none have yet been successfully marketed. Therefore, developing anti-IsdB antibodies with high specificity, low toxicity, and excellent clinical efficacy is urgent and necessary, providing patients with more treatment options. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention aims to provide an antibody that specifically binds to Staphylococcus aureus IsdB and its application.

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

[0007] The first aspect of the present invention provides an antibody or antigen-binding fragment thereof against Staphylococcus aureus IsdB.

[0008] Furthermore, the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and the light chain variable region comprises light chain CDR1, light chain CDR2, and light chain CDR3; wherein:

[0009] The amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; the amino acid sequences of the light chain CDR1, light chain CDR2 and light chain CDR3 are shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively.

[0010] Furthermore, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8, or an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:8, or a sequence having one or more amino acid substitutions, deletions, or insertions, or any combination thereof, compared to the amino acid sequence shown in SEQ ID NO:8.

[0011] Furthermore, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:16, or an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:16, or a sequence having one or more amino acid substitutions, deletions, or insertions, or any combination thereof, compared to the amino acid sequence shown in SEQ ID NO:16.

[0012] In this invention, the term "antibody" refers to any form of antibody that exhibits the desired biological activity (e.g., inhibiting the binding of a ligand to its receptor or by inhibiting receptor signal transduction induced by the ligand). Therefore, "antibody" is used in its broadest sense and is explicitly included, but not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and multispecific antibodies (e.g., bispecific antibodies), fully human, humanized, primate-derived, chimeric antibodies, single-chain antibodies, etc.

[0013] In this invention, the term "antigen-binding fragment" refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of its structure, the antibody fragment binds to the same antigen recognized by the intact antibody. The term "antigen-binding fragment" includes aptamers, mirror isoforms, and bivalent antibodies. The term "antigen-binding fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by forming a complex with a specific antigen.

[0014] The "complementarity-determining region" (CDR) or "hypervariant region" is a region within the variable domain of an antibody that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes.

[0015] Based on the variable region amino acid sequence contained in the given antibody or its fragment according to the present invention, those skilled in the art can routinely determine the CDR contained therein. For example, the Kabat, AbM, Chothia, or Contact protocols can be used to define the CDR in the variable region amino acid sequence.

[0016] When referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0017] The boundaries of the CDR of the antibody of the present invention can be determined artificially according to any method or combination thereof in the art. Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers the CDR sequence determined in any of the foregoing methods.

[0018] In this invention, the term "identity" refers to sequence similarity to the amino acid sequence used in this invention. To determine sequence identity, sequence alignment can be performed, which can be done in various ways known to those skilled in the art, such as using BLAST, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment, including any algorithm required to achieve optimal alignment across the full-length sequences being compared. Therefore, amino acid sequences that are at least 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequences of this invention are within the scope of protection of this invention.

[0019] In this invention, the modified antibody sequences also fall within the scope of protection. The term "modification" refers to any chemical modification of an amino acid sequence, such as substitution, deletion, insertion, and / or addition of amino acids. The term "substitution" refers to replacing one or more amino acids with different amino acids. "Deletion" refers to the reduction of one or more amino acids in the amino acid sequence. "Insertion" or "addition" refers to a change in the amino acid sequence resulting in an increase of one or more amino acids compared to a naturally occurring molecule. It should be noted that in the modified antibodies provided by this invention, the modification preferably occurs in regions other than the variable region, such as the frame region or constant region of the antibody, and the modified antibody still retains the desired functional properties of the antibody or its antigen-binding fragment of this invention, or has improved antigen-binding properties.

[0020] A second aspect of the present invention provides a polynucleotide that encodes the antibody or antigen-binding fragment thereof described in the first aspect of the present invention.

[0021] Furthermore, the polynucleotide sequence encoding the heavy chain variable region of the antibody or its antigen-binding fragment according to the first aspect of the present invention is shown in SEQ ID NO:24; the polynucleotide sequence encoding the light chain variable region of the antibody or its antigen-binding fragment according to the first aspect of the present invention is shown in SEQ ID NO:32.

[0022] In this invention, the term "polynucleotide" includes sequences of ribonucleotides and deoxyribonucleotides, such as modified or unmodified RNA or DNA, each in a linear or circular form in single-stranded and / or double-stranded form, or mixtures thereof (including hybrid molecules). Therefore, nucleic acids according to the invention include DNA (e.g., dsDNA, ssDNA, cDNA), RNA (e.g., dsRNA, ssRNA, mRNA, ivtRNA), combinations thereof, or derivatives thereof (e.g., PNA). Preferably, the nucleic acid is DNA or RNA.

[0023] A third aspect of the present invention provides a carrier comprising the polynucleotide described in the second aspect of the present invention.

[0024] Furthermore, the vector includes plasmids and viral vectors.

[0025] In this invention, a vector refers to an artificial construct capable of delivering and preferably expressing one or more target genes or sequences in a host cell. The vector of this invention is not limited and can be an expression vector, viral vector, etc. In some embodiments, the vector contains a target gene encoding an antibody or its precursor of this invention, a promoter, a terminator, or optionally a marker gene. The vector can be a known vector or a self-constructed vector. Known vectors include plasmid vectors, lentiviral vectors, adenovirus vectors, AAV viral vectors, etc.

[0026] A fourth aspect of the present invention provides a host cell comprising the polynucleotide described in the second aspect of the present invention or the vector described in the third aspect of the present invention.

[0027] Furthermore, the host cells include prokaryotic cells and eukaryotic cells.

[0028] In this invention, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli, fungal cells such as yeast cells, or cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0029] The fifth aspect of this invention provides an antibody derivative.

[0030] Furthermore, the antibody derivative is a complex obtained by modifying the antibody or its antigen-binding fragment as described in the first aspect of the present invention; the modification includes conjugation modification using a detectable marker, conjugation modification using a therapeutic agent, or conjugation modification using an imaging agent.

[0031] Furthermore, the detectable markers include fluorescent dyes, avidin, paramagnetic atoms, radioactive isotopes, enzyme markers, or colloidal gold.

[0032] Furthermore, the fluorescent pigments include fluorescein, rhodamine, Texas red, phycoerythrin, phycocyanin, allophycocyanin, and polydinoflavin-chlorophyll protein.

[0033] Furthermore, the avidin includes biotin, egg white avidin, streptavidin, egg yolk avidin, and avidin-like substances.

[0034] Furthermore, the radioactive isotopes include radioactive iodine, radioactive cesium, radioactive iridium, and radioactive cobalt.

[0035] Furthermore, the enzyme markers include horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, lysozyme, and malate dehydrogenase.

[0036] Furthermore, the therapeutic agents include cytotoxic agents, hormonal preparations, targeted small molecule preparations, proteasome inhibitors, chemotherapeutic agents, oncolytic drugs, cytokines, activators of co-stimulatory molecules, or inhibitors of inhibitory molecules.

[0037] The term "detectable marker" refers to a reagent that is detectable, for example, by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Useful detectable markers include, but are not limited to, fluorescent dyes, chemiluminescent compounds, radioactive isotopes, electron-dense reagents, enzymes, colored particles, biotin, or digoxigenin. Detectable markers often produce measurable signals, such as radioactivity, fluorescence, color, or enzyme activity. Antibodies conjugated to detectable reagents can be used for diagnostic or therapeutic purposes. Examples of detectable reagents include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals using various positron emission tomography techniques, and non-radioactive paramagnetic metal ions. Detectable substances can be directly linked to or conjugated with antibodies, or indirectly through intermediates such as linkers known in the art, using techniques known in the art. See U.S. Patent No. 4,741,900, which describes the conjugation of metal ions with antibodies for diagnostic purposes. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; examples of suitable cofactor complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriazinylamine, dansyl chloride, and phycoerythrin; one example of a luminescent material is luminescent ammonia; examples of bioluminescent materials include insect luciferin and luminescent proteins.

[0038] The sixth aspect of this invention provides a product for detecting Staphylococcus aureus IsdB.

[0039] Furthermore, the product includes the antibody or its antigen-binding fragment as described in the first aspect of the present invention, the polynucleotide as described in the second aspect of the present invention, the vector as described in the third aspect of the present invention, the host cell as described in the fourth aspect of the present invention, or the antibody derivative as described in the fifth aspect of the present invention.

[0040] Furthermore, the products include reagent kits and test strips.

[0041] A seventh aspect of the present invention provides a pharmaceutical composition.

[0042] Furthermore, the pharmaceutical composition comprises the antibody or its antigen-binding fragment as described in the first aspect of the present invention, the polynucleotide as described in the second aspect of the present invention, the carrier as described in the third aspect of the present invention, the host cell as described in the fourth aspect of the present invention, or the antibody derivative as described in the fifth aspect of the present invention.

[0043] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier and / or excipients.

[0044] In this invention, pharmaceutically acceptable carriers may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous mediators, non-aqueous mediators, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, clamping or chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0045] The pharmaceutical composition may be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained-release formulation, or powder. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0046] The eighth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the polynucleotide described in the second aspect of the present invention, the vector described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, or the antibody derivative described in the fifth aspect of the present invention in the preparation of products for diagnosing diseases related to Staphylococcus aureus infection.

[0047] The ninth aspect of this invention provides the use of the antibody or antigen-binding fragment thereof described in the first aspect of this invention, the polynucleotide described in the second aspect of this invention, the carrier described in the third aspect of this invention, the host cell described in the fourth aspect of this invention, the antibody derivative described in the fifth aspect of this invention, or the pharmaceutical composition described in the seventh aspect of this invention in the preparation of a medicament for the prevention and / or treatment of diseases related to Staphylococcus aureus infection.

[0048] Furthermore, the diseases associated with Staphylococcus aureus infection include pneumonia, systemic infection, osteomyelitis, necrotizing fasciitis, endocarditis, peritonitis, septic arthritis, sepsis, bacteremia, septicemia, abscess, and toxic shock syndrome.

[0049] Furthermore, the Staphylococcus aureus infection-related diseases are selected from pneumonia or systemic infections.

[0050] In this invention, Staphylococcus aureus infection, including MRSA, typically begins as a small, red bump resembling a papule, boil, or spider bite. These bumps or scars can rapidly develop into painful, deep abscesses requiring surgical drainage. Sometimes, the bacteria remain confined to the skin. Other times, they penetrate deep into the body, causing potentially life-threatening infections in a wide range of tissues, including the skin, soft tissues, bones, joints, surgical wounds, bloodstream, heart valves, lungs, or other organs. Therefore, Staphylococcus aureus infection can cause associated conditions that can be fatal, such as osteomyelitis, necrotizing fasciitis, endocarditis, peritonitis, septic arthritis, sepsis, bacteremia, septicemia, abscesses, and toxic shock syndrome, as well as various forms of pneumonia, including necrotizing pneumonia, and toxin production in furuncles and carbuncles.

[0051] The present invention also provides any one of the following methods, the method comprising:

[0052] (1) A method for producing the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, the method comprising the following steps: culturing the host cell described in the fourth aspect of the present invention to obtain a culture product, and separating and purifying the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention from the culture product;

[0053] (2) A method for detecting Staphylococcus aureus IsdB in a test sample for non-diagnostic and non-therapeutic purposes, the method comprising the following steps: contacting the test sample with the antibody or its antigen-binding fragment as described in the first aspect of the present invention, and detecting the formation of a complex of the antibody or its antigen-binding fragment with Staphylococcus aureus IsdB;

[0054] (3) A method for preparing the host cell according to the fourth aspect of the present invention, the method comprising the following steps: introducing the polynucleotide according to the second aspect of the present invention or a vector containing the polynucleotide into the host cell;

[0055] (4) A method for inhibiting the growth of Staphylococcus aureus and / or enhancing the killing ability of phagocytes against Staphylococcus aureus for non-therapeutic purposes, said method comprising applying the antibody or antigen-binding fragment thereof described in the first aspect of the present invention.

[0056] Furthermore, the methods of introduction include calcium phosphate transfection, DEAE, dextran-mediated transfection, electroporation, and phage infection.

[0057] Advantages and beneficial effects of the present invention:

[0058] The antibody provided by this invention can specifically bind to the Staphylococcus aureus IsdB antigen, effectively inhibit the growth of Staphylococcus aureus, and promote the killing ability of phagocytes against Staphylococcus aureus. It provides a new approach for the detection of Staphylococcus aureus IsdB and the treatment of related diseases, and has broad application prospects. Attached Figure Description

[0059] Figure 1 IsdB- N2 Image of protein SDS-PAGE purity test results;

[0060] Figure 2 The image shows the expression and purification results of the IsdB-36 monoclonal antibody.

[0061] Figure 3 The graph shows the results of the binding activity assay for the IsdB-36 monoclonal antibody.

[0062] Figure 4 The epitope type identification results for the IsdB-36 monoclonal antibody are shown in the figure.

[0063] Figure 5 Figure showing the results of IsdB-36 monoclonal antibody inhibiting the growth of Staphylococcus aureus in a low-iron environment;

[0064] Figure 6 The image shows the opsonization and killing of Staphylococcus aureus by the IsdB-36 monoclonal antibody; among them, Figure 6 In the diagram, A represents the phagocytosis result. Figure 6 B in the graph represents the kill rate results;

[0065] Figure 7 The results of the IsdB-36 monoclonal antibody in a Staphylococcus aureus systemic infection model are shown in the figure.

[0066] Figure 8 The image shows the results of using the IsdB-36 monoclonal antibody in a Staphylococcus aureus pneumonia model. Detailed Implementation

[0067] The present invention is further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. Reagents or instruments used that do not specify the manufacturer are all commercially available conventional products.

[0068] Example 1 Recombinant Staphylococcus aureus IsdB- N2 Antigen expression and purification

[0069] The full-length sequence of the IsdB gene was synthesized (Shanghai Jierui) and cloned into the expression vector pGEX-6P-2 using the restriction endonuclease recognition sites BamHI and SacI to construct the IsdB- gene. N2 protein prokaryotic expression vector pGEX-IsdB N2 / XL-1, after transfection and induction, makes IsdB- N2 The protein was expressed in E. coli, and IsdB- N2 The recombinant bacteria were inoculated into LB medium containing ampicillin and cultured overnight (37°C, 100 rpm). The next day, the culture was scaled up to OD at a ratio of 1:100 (V / V). 600 The pH value was 0.6-0.8. IPTG was added overnight to induce expression (4℃, 200 rpm). The cells were collected by centrifugation and lysed by sonication. The lysate was added to GST-4FF affinity packing material and vertically mixed overnight at 4℃. Glutathione was used as elution to obtain IsdB-. N2 -GST fusion protein, then Prescission protease is added to cleave the GST tag, and IsdB- is obtained by SPHP chromatography. N2 The protein was purified by replacing the buffer with PBS and then further concentrating and purifying it using ultrafiltration. The purity was assessed by SDS-PAGE gel electrophoresis. N2 The molecular weight and purity test results are as follows: Figure 1 As shown, the results indicate IsdB- N2 The protein purity is 100.0%.

[0070] Example 2: Isolation of PBMC cells

[0071] IsdB- based on Staphylococcus aureus vaccine N2 The protective antigen was approved by the ethics review committee of Jiangsu Provincial Center for Disease Control and Prevention, and peripheral blood lymphocyte samples of Phase Ia subjects were obtained from the Phase Ia clinical trial center of recombinant Staphylococcus aureus vaccine (Escherichia coli) and frozen in liquid nitrogen.

[0072] Example 3: Establishment of a human-specific anti-Staphylococcus aureus antibody library

[0073] 1. cDNA preparation:

[0074] Total RNA was extracted from peripheral blood lymphocytes isolated in Example 2, and then cDNA was synthesized by reverse transcription using the total RNA as a template.

[0075] 2. Construction of a human-specific anti-Staphylococcus aureus Fab antibody library:

[0076] The inverted cDNA was mixed and used as a gene template. Human light chain (VL+CL) Kappa / Lambda and heavy chain Fd fragment gene sequences were amplified by PCR using Ig primers. The PCR product of the VL+CL light chain gene was digested with SacI-HF and XbaI-HF enzymes and ligated into the pComb3XSS phage display vector. After successful construction of the VL+CL (Kappa) and VL+CL (Lambda) light chain libraries, the heavy chain Fd fragment was cloned into the pComb3XSS vector containing the light chain gene fragment using XhoI-HF and SpeI-HF restriction enzyme sites, respectively, to construct Fab phage particles. The ligation product was transformed into TG1 competent cells and cultured overnight at 37°C on ampicillin-containing plates. Cells were collected to calculate the library size, and 10 single colonies were selected 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 the PCR product was analyzed by 2% agarose gel electrophoresis.

[0077] 3. Positive rate identification:

[0078] Screening results showed that the positive clone rates of both the Fab (Kappa light chain) and Fab (Lambda light chain) libraries exceeded 80%, and the library sizes exceeded 10. 8 .

[0079] Example 4: Screening of Human-Specific Anti-Staphylococcus aureus Fab Antibody Library

[0080] 1. Bacterial purification and antibody phage preparation:

[0081] The frozen bacterial culture was inoculated into LB-ampicillin medium at a ratio of 1:100 for resuscitation and grown to OD. 600When the pH value is 0.5-0.6, remove the bacterial culture and add helper phage M13 at a ratio of 1:1000. Incubate at 37°C for 30 min, then continue shaking for another 30 min. Centrifuge to collect the bacterial cells and incubate overnight at 30°C in LB medium containing ampicillin and kanamycin on a shaker. Centrifuge the bacterial culture at 4°C at 3000 g for 10 min to collect the supernatant phage. Add PEG / NaCl solution and mix thoroughly. Incubate on ice for 30 min, then centrifuge at 4°C at 3000 g for 20 min. Remove the supernatant, add PEG / NaCl solution to the precipitate and mix thoroughly. Incubate on ice for 30 min, then centrifuge at 4°C at 12000 g for 2 min. Remove the supernatant, resuspend the precipitate in 1 mL PBS, add glycerol, and store at -80°C for later use.

[0082] 2. Phage library selection:

[0083] Add antibody-phage to the antigen-coated ELISA plate and incubate at 37°C for 20 min. Wash 10 times with PBST and PBS solution to remove unbound phage. Digest the bound phage with trypsin and then add the plate to culture until OD200. 600 TG1 bacteria with a pH of 0.5–0.6 were infected for 30 min, and the bacterial cells were collected by centrifugation and plated onto LB agar plates containing ampicillin, and incubated overnight at 37°C. The next day, the titers of the panned phage sub-libraries were determined by serial dilution, and the bacterial cells were collected. The phage sub-libraries were then repackaged using the method described above as input phage libraries for the next round of panning. The panning process was repeated three times to maximize the enrichment of high-affinity antibodies.

[0084] 3. Phage library screening:

[0085] Enriched phages were plated on LB agar plates containing ampicillin to generate bacterial single clones. Single clones were picked and cultured in 96-well plates for phage ELISA screening. Antibody-coated phages were added to antigen-coated ELISA plates and incubated at 37°C for 20 min. The plates were washed three times with PBST to remove unbound phages. Fluorescently labeled antibodies were added and incubated at 37°C for 20 min, followed by three washes with PBST. Specific antibodies were screened by detecting the ELISA fluorescence signal intensity. Corresponding positive well antibody-coated phages were amplified and sequenced.

[0086] Example 5: Cloning, expression, and purification of the IsdB-36 fully human antibody

[0087] 1. Experimental Methods

[0088] Using the positive clone display vector selected in Example 3 as a template, human Ig VH and VK / L were amplified by PCR. The PCR products were identified by agarose gel electrophoresis. Antibody genes that were identified as positive and whose light and heavy chains could be paired were purified using the Qiagen PCR product purification kit. The purified products were sequenced in both forward and reverse directions, and the antibody gene family, mutation rate, and CDR region were analyzed using the IMGT online server (https: / / www.imgt.org / IMGT_vquest / input).

[0089] Using the TA cloning method, the PCR product of the antibody variable region gene, which was positive by gel electrophoresis and whose heavy and light chains could be paired, was ligated into the pcDNA3.4 vector containing either the heavy chain constant region or the light chain constant region to construct an expression vector for the fully human IsdB-36 antibody. This expression vector was then transformed into DH5α competent cells, plated on LB agar plates containing ampicillin, and incubated overnight at 37°C. The next day, 10 single colonies were picked and identified by PCR 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 100 s, 28 cycles; 72°C extension for 5 min. 5 μL of the PCR product was detected by 1% agarose gel electrophoresis.

[0090] The PCR product identified as positive vector plasmid was transformed into DH5α for large-scale amplification. After rapid extraction of the recombinant plasmid, it was incubated with PEI transfection reagent at 37℃ for 15-20 min and then transfected into HEK 293F cells. The cells were cultured in a 37℃, 5% CO2 incubator. After 5 days of cell culture, the cell supernatant was collected by centrifugation at 3000 g, 4℃ for 30 min. The supernatant was purified by protein A affinity chromatography, and the expression and purification status of the antibody were detected by SDS-PAGE gel electrophoresis.

[0091] 2. Experimental Results

[0092] This approach successfully constructed a series of antibody heavy and light chain expression vectors. One of the antibody clones was named the fully human IsdB monoclonal antibody IsdB-36 (abbreviated as IsdB-36 antibody or IsdB-36 monoclonal antibody).

[0093] SDS-PAGE test results are as follows: Figure 2 As shown, the relative molecular weight of the IsdB-36 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-36 antibody sequence is shown in Table 1.

[0094] Table 1 IsdB-36 antibody sequence

[0095]

[0096]

[0097] Example 6: IsdB-36 monoclonal antibody and Staphylococcus aureus iron-regulated surface determinant B (IsdB- N2 ) binding activity detection

[0098] 1. Experimental Methods

[0099] 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-36 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. To exclude false positives caused by non-specific components in the serum sample, a negative serum group (1:100 dilution) was set up with 100 μL per well. To exclude false positives caused by the universal structure of the antibody, an unrelated control antibody IgG1 (0.5 μg / mL) was set up with 100 μL per well. 100 μL of PBST was added to the blank, and the plates were incubated at 37°C for 1 h.

[0100] 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 control-irrelevant antibody IgG1, determine the threshold (3 times the mean value), and calculate the EC50 for antibodies exceeding the threshold.

[0101] 2. Experimental Results

[0102] Test results as follows Figure 3 As shown, the results indicate that the fully human IsdB monoclonal antibody IsdB-36 can bind to Staphylococcus aureus iron-regulated surface determinant B (IsdB-36). N2 ) combined. IsdB- N2 The calculated EC50 value was 0.09 μg / mL.

[0103] Example 7: Determination of Epitope Type of IsdB-36 Monoclonal Antibody

[0104] 1. Experimental Methods

[0105] Western blotting (WB) was used to detect antibody-antigen binding reactions. IsdB-36 protein was subjected to SDS-PAGE (reduced) gel electrophoresis at 140 V for 1–2 hours. Electrophoresis was stopped when 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 sealed 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.

[0106] 2. Experimental Results

[0107] Test results as follows Figure 4 As shown, the results indicate that the IsdB-36 antibody can bind to IsdB- N2 Antigen binding indicates that the epitope of the IsdB-36 antibody is a linear epitope.

[0108] Example 8: Inhibitory effect of anti-IsdB antibody on the growth of recombinant Staphylococcus aureus in a low-iron environment.

[0109] 1. Experimental Methods

[0110] 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.

[0111] First, USA300 cells were cultured using TSB as a base. Then, the activated bacterial culture was cultured overnight in RPMI 1640 (containing 2,2′-bipyridine and casein hydrolysates). The next day, the cells were collected by centrifugation and washed twice with RPMI 1640 (containing 2,2′-bipyridine and casein hydrolysates). Finally, the cells were resuspended in RPMI 1640 (containing 2,2′-bipyridine and / or human hemoglobin). For the antibody group, 1 μM and 100 μM IsdB-36 antibody were mixed with an equal volume of resuspended bacterial culture (resuspended in RPMI 1640 (containing 2,2′-bipyridine and human hemoglobin)) and added to 96-well cell culture plates. For the control group, PBS buffer was used to mix an equal volume of resuspended bacterial culture (resuspended in RPMI 1640). The bacterial suspension was resuspended in RPMI 1640 (containing 2,2′-bipyridine and human hemoglobin) and added to a 96-well cell culture plate. For the control group, an equal volume of the resuspended bacterial culture (resuspended in RPMI 1640 (containing 2,2′-bipyridine)) was added to a 96-well cell culture plate using PBS buffer. The 96-well cell culture plates were incubated at 37°C for 24 hours. The OD values ​​of each well were periodically measured using a microplate reader. 600 The values ​​were used to monitor bacterial growth, plot bacterial growth curves, and further calculate the inhibition rate of antibody on bacterial growth.

[0112] 2. Experimental Results

[0113] The results are as follows Figure 5 The absorbance (OD) of Staphylococcus aureus cultured in RPMI 1640 (containing 2,2′-bipyridine and human hemoglobin) for 24 hours was shown. 600 For a 100% calculation, Staphylococcus aureus cultured in RPMI 1640 (containing 2,2′-bipyridine and human hemoglobin) with 1 μM antibody for 24 hours showed an inhibition rate of 21%; Staphylococcus aureus cultured in RPMI 1640 (containing 2,2′-bipyridine and human hemoglobin) with 100 μM antibody for 24 hours showed an inhibition rate of 56%.

[0114] Example 9: IsdB-36 monoclonal antibody opsonizes and kills Staphylococcus aureus.

[0115] 1. Experimental Methods

[0116] Antibodies can promote the phagocytosis and killing of Staphylococcus aureus by immune cells, but Staphylococcus aureus possesses various virulence factors that prevent phagocytosis. To evaluate the ability of the IsdB-36 monoclonal antibody to promote the killing of Staphylococcus aureus by phagocytic cells, an opsonization phagocytosis-killing assay (OPK) was designed to assess the ability of the monoclonal antibody of this invention to promote the killing of Staphylococcus aureus USA300 in the presence of complement. The procedure involved serially diluting the IsdB-36 monoclonal antibody, positive serum, negative serum, and control antibody seven times in a 96-well plate; culturing Staphylococcus aureus USA300 in TSB and then resuspending it in OBB buffer to a concentration of 1.0 × 10⁻⁶. 5 CFU / mL was added to microplates (10 μL / well), and incubated with shaking at room temperature for 1 hour. Leukocytes were then isolated from fresh rabbit whole blood, washed, and the concentration adjusted to 2.0 × 10⁻⁶ using buffer. 6 Cells / mL, after being mixed with complement in a certain ratio, were incubated in an inverted incubator at 37°C for half an hour. The cell-complement mixture was then added to a microplate (50 μL / well) and phagocytosed at 37°C with 5% CO2 for 60 min. After phagocytosis, the microplate was placed on ice for 20 min, and 10 μL of the mixture was seeded onto MHA for counting.

[0117] 2. Experimental Results

[0118] The results are as follows Figure 6 As shown, the control antibody did not promote cell killing of Staphylococcus aureus, while the IsdB-36 monoclonal antibody did promote cell killing of Staphylococcus aureus, with a maximum killing rate of 69%.

[0119] Example 10: The role of IsdB-36 monoclonal antibody in a systemic MRSA infection model

[0120] 1. Experimental Methods

[0121] The protective efficacy of the IsdB-36 monoclonal antibody against Staphylococcus aureus USA300 was evaluated in a systemic infection model. Staphylococcus aureus USA300 was cultured in TSB, passaged, and grown to mid-log phase (OD). 600 =1.0). The culture was then washed twice in physiological saline and measured with OD. 600The bacterial suspension was resuspended in physiological saline at an optical density of 1.6. Mice were injected via tail vein with 100 μL of the bacterial suspension. One day prior to infection, mice in the experimental group were treated with 100 mg / kg of IsdB-36 monoclonal antibody, the control group was treated with 100 mg / kg of hIgG1 (PcrV-A039), and the blank group was treated with physiological saline. After tail vein challenge with 100 μL of the bacteria, the mice's health was monitored until 7 days post-infection, with the number of surviving mice recorded every 12 hours to calculate the survival rate.

[0122] 2. Experimental Results

[0123] The results are as follows Figure 7 As shown, the protection rate of the monoclonal antibody containing 100 mg / kg IsdB-36 was 60%, while the protection rate of the monoclonal antibody containing 100 mg / kg hIgG1 (PcrV-A039) was 10%.

[0124] Example 11: In vivo prophylactic effect of IsdB-36 monoclonal antibody on an acute pneumonia model of MRSA

[0125] 1. Experimental Methods

[0126] The protective efficacy of the IsdB-36 monoclonal antibody against Staphylococcus aureus USA300 was evaluated in a pneumonia model. Staphylococcus aureus USA300 was cultured in BHI broth and passaged to OD. 600 The value was 1.0. The culture was then washed twice in physiological saline and diluted with 2 × 10⁻⁶ solution. 10 The bacterial suspension was resuspended in physiological saline at a density of CFU / mL. Mice were challenged with 20 μL of the bacterial suspension via endotracheal intubation. One day before infection, mice in the experimental group were treated with monoclonal antibody containing 100 mg / kg IsdB-36, mice in the control group were treated with hIgG1 (PcrV-A039) containing 100 mg / kg, and mice in the blank group were treated with physiological saline. After endotracheal intubation and challenge with 20 μL of the bacterial suspension, the survival status of the mice was monitored until 7 days post-infection. The number of surviving mice was recorded every 12 hours, and the survival rate was calculated.

[0127] 2. Experimental Results

[0128] The protection rate of monoclonal antibody containing 100 mg / kg IsdB-36 was 70%, while the protection rate of monoclonal antibody containing 100 mg / kg hIgG1 (PcrV-A039) was 20%. Figure 8 ).

[0129] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof directed against S. aureus IsdB, characterized in that, The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, the light chain variable region comprising a light chain CDR1, a light chain CDR2 and a light chain CDR3; wherein: the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are respectively as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; the amino acid sequences of the light chain CDR1 and the light chain CDR3 are respectively as shown in SEQ ID NO: 9, SEQ ID NO: 11; and the amino acid sequence of the light chain CDR2 is DNN.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 8; and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

16.

3. A polynucleotide, comprising, The polynucleotide encodes the antibody or antigen-binding fragment thereof of any one of claims 1-2.

4. A vector, characterized by, The vector comprises the polynucleotide of claim 3.

5. A host cell, characterized in that, The host cell comprises the polynucleotide of claim 3 or the vector of claim 4.

6. An antibody derivative, characterized in that The antibody derivative is a complex obtained by modification of the antibody or antigen-binding fragment thereof of any one of claims 1-2; the modification is a conjugation modification using a detectable label, an imaging agent conjugation modification.

7. A product for detecting S. aureus IsdB, characterized in that, The product comprises the antibody or antigen-binding fragment thereof of any one of claims 1-2, the polynucleotide of claim 3, the vector of claim 4, the host cell of claim 5 or the antibody derivative of claim 6.

8. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the antibody or antigen-binding fragment thereof of any one of claims 1-2, the polynucleotide of claim 3, the vector of claim 4, the host cell of claim 5 or the antibody derivative of claim 6.

9. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-2, the polynucleotide of claim 3, the vector of claim 4, the host cell of claim 5 or the antibody derivative of claim 6 in the preparation of a product for diagnosing a disease associated with Staphylococcus aureus infection.

10. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-2, the polynucleotide of claim 3, the vector of claim 4, the host cell of claim 5, the antibody derivative of claim 6 or the pharmaceutical composition of claim 8 in the preparation of a medicament for preventing and / or treating a disease associated with Staphylococcus aureus infection.

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