Antibody for resisting OXA-23 type carbapenemase and application thereof

Abstract

The invention discloses an antibody for resisting OXA-23 type carbapenemase and an application of the antibody. The meropenem derivative has good binding activity with OXA-23 type carbapenem enzyme, and can reduce the carbapenem drug resistance level of the acinetobacter baumannii, so that the clinical use concentration of meropenem is reduced, and the effect of meropenem on the acinetobacter baumannii is improved. In addition, the antibody disclosed by the invention can also inhibit OXA-23 protein secreted by bacteria, prevent the bacteria from hydrolyzing carbapenem antibiotics, increase the concentration of antibiotics in a medication part, and facilitate the removal of mixed infection with acinetobacter baumannii.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to an antibody against OXA-23 type carbapenemase and its application. Background Art

[0002] Among β-lactam antibiotics, carbapenems possess the broadest antimicrobial activity, resisting the hydrolytic activity of most β-lactamases and extended-spectrum β-lactamases and attenuating the action of chromosome-mediated AmpC enzymes. Acinetobacter exhibits some resistance to many antibiotics, including β-lactams, aminoglycosides, and fluoroquinolones. Carbapenems have consistently demonstrated good antimicrobial activity against Acinetobacter. However, with the widespread clinical use of carbapenems, the prevalence of resistance in Acinetobacter baumannii has been increasing annually. In recent years, there have been numerous reports of nosocomial infections caused by Acinetobacter carrying blaOXA-23. Literature reports indicate that 80% of carbapenem-resistant Acinetobacter baumannii in China produce OXA-23 carbapenemases, making the clinical treatment of carbapenem-resistant Acinetobacter extremely challenging. According to the data from the Ministry of Health's Antimicrobial Resistance Monitoring Network CHINET in 2011, the resistance rates of Acinetobacter baumannii to the carbapenems imipenem and meropenem in my country were 56.8% and 58.7%, respectively, indicating that carbapenem-resistant Acinetobacter baumannii poses a severe challenge to clinical anti-infection treatment.

[0003] Therefore, providing an antibody that can neutralize the active site of OXA-23, thereby blocking the enzyme's hydrolysis of carbapenem antibiotics, restoring the effectiveness of carbapenem antibiotics or reducing the clinical dosage of carbapenem antibiotics is of great significance for the treatment of Acinetobacter baumannii. Summary of the Invention

[0004] In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide an antibody that can reduce the carbapenem resistance level of Acinetobacter baumannii and enhance the therapeutic effect of carbapenem antibiotics on Acinetobacter baumannii.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides an anti-OXA-23 type carbapenemase antibody or an antigen-binding fragment thereof.

[0007] Further, 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 HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13 or SEQ ID NO:17; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14 or SEQ ID NO:18; the CDR sequence is determined by any one of the IMGT, Chothia, Kabat, AbM, and Contact schemes.

[0008] Furthermore, the CDR sequence determination scheme is the IMGT scheme.

[0009] "Complementarity determining regions" or "CDR regions" or "CDRs" or "hypervariable regions" are regions of an antibody variable domain that are highly variable in sequence and form structurally defined loops ("hypervariable loops") and / or contain antigen-contacting residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes.

[0010] Based on the variable region amino acid sequence of a given antibody or fragment thereof of the present invention, one skilled in the art can routinely determine the CDRs contained therein. For example, the CDRs in the variable region amino acid sequence can be defined using the IMGT scheme, the Kabat scheme, the AbM scheme, the Chothia scheme, or the Contact scheme.

[0011] When referring to antibodies defined by specific CDR sequences defined herein, the scope of said antibodies also encompasses antibodies whose variable region sequences comprise said specific CDR sequences, but whose declared CDR boundaries differ from the specific CDR boundaries defined herein due to the application of a different scheme (e.g., a different assignment system rule or combination).

[0012] The CDRs of the antibodies of the present invention can be manually assessed and determined according to any protocol or combination thereof in the art. Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above manners. In a specific embodiment of the present invention, the CDR sequence determination protocol is the IMGT protocol.

[0013] Further, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13 or SEQ ID NO:17, or an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13 or SEQ ID NO:17, 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:1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13 or SEQ ID NO:17.

[0014] Further, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14 or SEQ ID NO:18, or an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14 or SEQ ID NO:18, 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:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14 or SEQ ID NO:18.

[0015] In the present invention, term " identity " refers to the sequence similarity with amino acid sequence used in the present invention.In order to determine sequence identity, can carry out sequence alignment, it can be carried out in the various ways that those skilled in the art understand, for example, use BLAST, ALIGN, NEEDLE or Megalign (DNASTAR) software etc. Those skilled in the art can determine the appropriate parameters for comparison, be included in and realize any algorithm required for optimal comparison in the full-length sequence compared.Therefore, with the amino acid sequence of the present invention at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical all within protection scope of the present invention.

[0016] In the present invention, the antibody sequences obtained by modification also fall within the scope of protection of the present invention. 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 the replacement of one or more amino acids by different amino acids. "Deletion" refers to the reduction of one or more amino acids in an amino acid sequence. "Insertion" or "addition" refers to a change in an amino acid sequence that results 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 the present invention, the modification preferably occurs in a region outside the variable region, such as in the framework region or constant region of the antibody, and the modified antibody still retains the desired functional properties of the antibody of the present invention or its antigen-binding fragment, or has improved antigen-binding properties.

[0017] A second aspect of the present invention provides a biomaterial.

[0018] Furthermore, the biomaterial comprises:

[0019] 1) a polynucleotide encoding the antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0020] 2) a vector comprising the polynucleotide described in 1);

[0021] 3) A host cell comprising the polynucleotide described in 1) or the vector described in 2).

[0022] Furthermore, the polynucleotide sequence encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention is shown as SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15 or SEQ ID NO: 19; the polynucleotide sequence encoding the light chain variable region of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention is shown as SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16 or SEQ ID NO: 20.

[0023] Furthermore, the vector includes plasmid and viral vector.

[0024] Furthermore, the viral vector includes lentivirus, adenovirus, and adeno-associated virus vector.

[0025] Furthermore, the host cells include prokaryotic cells and eukaryotic cells.

[0026] As used herein, the term "polynucleotide" includes sequences of ribonucleotides and deoxyribonucleotides, such as modified or unmodified RNA or DNA, each in single-stranded and / or double-stranded linear or circular form, or mixtures thereof (including hybrid molecules). Thus, nucleic acids according to the present 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.

[0027] In some embodiments, the nucleic acid molecule is isolated or purified. The sequence of the nucleic acid molecule can be obtained using conventional techniques or hybridoma technology. Once the relevant sequence is obtained, it can be obtained in large quantities using recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells using conventional methods. In addition, artificial synthesis methods can also be used to synthesize the relevant sequence, especially when the fragment length is relatively short. Generally, a longer sequence fragment is obtained by first synthesizing multiple small fragments and then ligating them.

[0028] In the present invention, a vector refers to an artificial construct that can deliver and preferably express one or more target genes or sequences in a host cell. The vector of the present invention is not limited and can be an expression vector, a viral vector, etc. In certain embodiments, the vector comprises a target gene, a promoter, a terminator encoding an antibody of the present invention or a precursor thereof, or optionally further comprises a marker gene. The vector can use a known vector or a self-constructed vector. Known vectors include plasmid vectors, lentiviral vectors, adenoviral vectors, AAV viral vectors, etc.

[0029] In the present 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 fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells or HEK293 cells.

[0030] A third aspect of the present invention provides a composition.

[0031] Furthermore, the composition is a complex obtained by modifying the antibody or antigen-binding fragment thereof described in the first aspect of the present invention; the modification includes modification by conjugation with a detectable marker, modification by conjugation with a therapeutic agent, and modification by conjugation with an imaging agent.

[0032] Furthermore, the detectable markers include fluorescent dyes, avidin, paramagnetic atoms, radioactive isotopes, enzyme markers, and colloidal gold.

[0033] Furthermore, the fluorescent pigments include fluorescein, rhodamine, Texas red, phycoerythrin, phycocyanin, allophycocyanin, and peridinin-chlorophyll protein.

[0034] Furthermore, the avidin includes biotin, egg albumin avidin, streptavidin, egg yolk avidin, and avidin-like.

[0035] Furthermore, the radioactive isotopes include radioactive iodine, radioactive cesium, radioactive iridium, and radioactive cobalt.

[0036] Furthermore, the enzyme markers include horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, lysozyme, and malate dehydrogenase.

[0037] Furthermore, the therapeutic agents include cytotoxic agents, hormone preparations, targeted small molecule preparations, proteasome inhibitors, chemotherapeutic agents, oncolytic drugs, cytokines, activators of costimulatory molecules, and inhibitors of inhibitory molecules.

[0038] The term "detectable label" refers to an agent that is detectable, for example, by spectroscopy, photochemistry, biochemistry, immunochemistry, or chemical means. Useful detectable labels include, but are not limited to, fluorescent dyes, chemiluminescent compounds, radioisotopes, electron-dense reagents, enzymes, colored particles, biotin, or digoxigenin. Detectable labels often produce a measurable signal, such as radioactivity, fluorescence, color, or enzymatic activity. Antibodies coupled to detectable agents can be used for diagnostic or therapeutic purposes. Examples of detectable agents 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 or coupled to antibodies using techniques known in the art, or indirectly through intermediates such as linkers known in the art. See, U.S. Patent No. 4,741,900, which describes the coupling of metal ions to antibodies for diagnosis. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferin and photoprotein.

[0039] A fourth aspect of the present invention provides a product.

[0040] Furthermore, the product includes the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the biomaterial described in the second aspect of the present invention, or the composition described in the third aspect of the present invention.

[0041] Furthermore, the product includes a test kit, a test paper, a nucleic acid membrane strip, a chip, a system, or a device.

[0042] Preferably, the product is a kit.

[0043] A fifth aspect of the present invention provides any one of the following methods, comprising:

[0044] (1) A method for producing the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the method comprising the following steps: culturing the host cell according to the second aspect of the present invention to obtain a culture product, and isolating and purifying the antibody or antigen-binding fragment thereof according to the first aspect of the present invention from the culture product;

[0045] (2) A method for detecting OXA-23 carbapenemase 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 antigen-binding fragment thereof described in the first aspect of the present invention or contacting the test sample with the composition described in the third aspect of the present invention, and detecting the formation of an immune complex between the OXA-23 carbapenemase and the antibody;

[0046] (3) A method for preparing the host cell according to the second aspect of the present invention, the method comprising the following steps: introducing the polynucleotide molecule according to the second aspect of the present invention or a vector containing the same into the host cell; preferably, the introduction method includes calcium phosphate transfection, DEAE, dextrose-mediated transfection, electroporation, and phage infection;

[0047] In some embodiments, the method of introduction includes physical methods, chemical methods, and biological methods. In some embodiments, the physical method includes calcium phosphate precipitation, lipofection, particle bombardment, microinjection, and electroporation; the chemical method includes colloidal dispersion systems and lipid-based systems; the colloidal dispersion system includes macromolecular complexes, nanocapsules, microspheres, and beads; the lipid-based system includes oil-in-water emulsions, micelles, mixed micelles, and liposomes; and the biological method includes DNA vectors and RNA vectors.

[0048] (4) An in vitro method for inhibiting the activity of OXA-23 carbapenemase and / or inactivating OXA-23 carbapenemase for non-diagnostic and non-therapeutic purposes, the method comprising the following steps: contacting a target sample with the antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0049] The present invention has no particular limitation on the sample type.

[0050] A sixth aspect of the present invention provides any one of the following applications, comprising:

[0051] (1) Use of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the biological material according to the second aspect of the present invention, and / or the composition according to the third aspect of the present invention in the preparation of a product for detecting OXA-23 type carbapenemases;

[0052] (2) Use of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the biomaterial according to the second aspect of the present invention, and / or the composition according to the third aspect of the present invention in the preparation of a product for inhibiting the activity of OXA-23 type carbapenemase;

[0053] (3) Use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the biomaterial described in the second aspect of the present invention, and / or the composition described in the third aspect of the present invention in the preparation of a medicament for treating diseases caused by Acinetobacter baumannii.

[0054] In some embodiments, the related diseases caused by Acinetobacter baumannii include but are not limited to: bacteremia, pneumonia, meningitis, peritonitis, endocarditis, urinary tract and skin infections. Various symptoms or diseases caused by Acinetobacter baumannii infection are within the scope of protection of the present invention.

[0055] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0056] The present invention provides a novel anti-OXA-23 carbapenemases antibody with excellent binding activity to OXA-23 carbapenemases, capable of reducing the carbapenem resistance level of Acinetobacter baumannii, thereby lowering the clinical concentration of meropenem and improving its efficacy against Acinetobacter baumannii. Furthermore, the antibody can inhibit bacterially secreted OXA-23 proteins, preventing their hydrolysis of carbapenem antibiotics, increasing antibiotic concentration at the site of administration, and facilitating the elimination of mixed infections with Acinetobacter baumannii. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is a graph showing the effect of the antibodies provided by the present invention on drug resistance of Acinetobacter baumannii;

[0058] Figure 2 This is a graph showing the effect of the antibodies provided by the present invention on the MIC of Acinetobacter baumannii. DETAILED DESCRIPTION

[0059] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solutions and inventive concepts of the present invention, should be covered by the scope of protection of the present invention.

[0060] Unless otherwise specified, the materials and reagents used in the following examples are all commercially available. Experimental methods in the following examples where specific conditions are not specified were generally performed under conventional conditions or conditions recommended by the manufacturer.

[0061] Example 1 Preparation of Anti-OXA-23 Carbapenemase Antibodies

[0062] 1. Sequencing of Acinetobacter baumannii expressing OXA-23 carbapenemase was performed to obtain the DNA sequence of OXA-23. This sequence was cloned into the high-expression vector pET28a and transformed into the Escherichia coli BL21 (DE3) strain to induce expression and purify the OXA-23 protein.

[0063] 2. Use purified OXA-23 protein to immunize mice to obtain OXA-23 monoclonal antibodies.

[0064] The present invention obtained a total of 5 monoclonal antibodies, named No. 3, No. 4, No. 7, No. 11 and No. 22 antibodies, respectively. The amino acid sequences and base sequences of their heavy chain variable regions and light chain variable regions are shown below, respectively.

[0065] The amino acid sequence of the heavy chain variable region of antibody No. 3 is DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYNWHWIRQFPGNKLEWMGYIKYSGITNYNPSLKSRISITQDTSKNQFFLQLNSVTTDDTATYCCTRGWDWFPYWGQGALVTVSA (SEQ ID NO: 1); the amino acid sequence of the light chain variable region of antibody No. 3 is DIVMSQSPSSLAVSVGEKVTMTCKSSQSLFFSSNQKNCLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVEAEDLAVYYCQQYYIYTYTFGGGTTLEIK (SEQ ID NO: 2); The base sequence of the variable region of the heavy chain of antibody No. 3 is GATGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTCACTGGCTACTCCATCACCAGTGGTTATAACTGGCACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATAAAATACAGT GGTATCACTAACTACAACCCATCTCTCAAAAGTCGAATTTCTATCACTCAAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGACGACACAGCCACATATTGCTGTACAAGAGGGTGGGACTGGTTTCCTTACTGGGGCCAAGGGGCTCTGGTCACTGTCTCTGCA (SEQ ID NO:3);The base sequence of the light chain variable region of antibody No. 3 is GACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGACCTGCAAGTCCAGTCAGAGCCTTTTCTTTAGTAGCAATCAAAAGAACTGCTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGTTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGGAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATATCTATACGTACACGTTCGGAGGGGGGACCACGCTGGAAATAAAA (SEQ ID NO:4).;

[0066] The amino acid sequence of the heavy chain variable region of antibody No. 4 is EVQLQQSGPELVKPGASVKISCKASGYTIIDYYINWVKQSHGKSLEWIGDINPNNGITTYNQKFKGKATLTVDMSSSTAYMDLRSLTSEDSAVYYCASSPDTGNYFAMDYWGQGTAVTVSS (SEQ ID NO: 5); the amino acid sequence of the light chain variable region of antibody No. 4 is DIQMTQSPASQSASLGESVTITCLASQTIGTWLAWYQQKPGKSPQLLIYAATRLADGVPSRFSGSGSGTKFSFKISSLQAEDFVSYYCQQLYINPRTFGGGTKLEIK (SEQ ID NO: 6); The base sequence of the variable region of the heavy chain of antibody No. 4 is GAGGTCCAGCTGCAACAATCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGTAAGGCTTCTGGATACACGATCATTGACTACTACATAAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTAATAATGGTATTAC TACCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACATGTCCTCCAGCACAGCCTACATGGACCTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGTAGTCCGGACACTGGTAACTACTTTGCTATGGACTACTGGGGTCAGGGAACCGCAGTCACCGTCTCCTCA (SEQ ID NO:7);The base sequence of the light chain variable region of antibody No. 4 is GACATTCAGATGACCCAGTCTCCTGCCTCCCAGTCTGCATCTCTGGGAGAAAGTGTCACCATCACATGCCTGGCAAGTCAGACCATTGGTACATGGTTAGCATGGTATCAGCAGAAACCAGGGAAATCTCCTCAGCTCCTGATTTATGCTGCAACCAGGTTGGCAGATGGGGTCCCATCAAGGTTCAGTGGGAGTGGATCTGGCACAAAATTTTCTTTCAAGATCAGCAGCCTACAGGCTGAAGATTTTGTAAGTTATTACTGTCAACAACTTTACATTAATCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO:8).;

[0067] The amino acid sequence of the heavy chain variable region of antibody No. 7 is QVQLKQSGPSLVQPSQSLSITCTVSGFSLSSYGVHWVRQSPGKALEWLGVIWRGGNTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTATYYCAIGPITTVAAYWGQGTTLTVSS (SEQ ID NO: 9); the amino acid sequence of the light chain variable region of antibody No. 7 is DIVMTQSHKFMSTSVGDRVSITCKASQDVRTAVAWYQQKPGQPPKLLIYWTSTRHTGVPDRFTGSGSGTDYSLIISIVQAEDLALYYCQQHFSTPLTFGAGTKLELK (SEQ ID NO: 10); The base sequence of the variable region of the heavy chain of antibody No. 7 is CAGGTGCAGCTGAAGCAGTCAGGACCTAGCCTAGTGCAGCCCTCACAGAGCCTGTCCATAACCTGCACAGTCTCTGGTTTCTCATTGTCTAGCTATGGTGTACACTGGGTTCGCCAGTCTCCAGGAAAGGCTCTGGAGTGGCTGGGAGTGATATGGAGAGGTGGAA ACACAGACTACAATGCAGCTTTCATGTCCAGACTGAGCATCACCAGGACAATTCCAAGAGCCAAGTTTTCTTAAATGAACAGTCTACAAGCTGATGACACTGCCACATACTACTGTGCCATAGGACCCATTACTACGGTAGCAGCCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:11);The base sequence of the light chain variable region of antibody No. 7 is GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGAGGACTGCTGTAGCCTGGTATCAACAAAAACCAGGGCAACCTCCTAAACTTCTGATTTACTGGACATCCACCCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTATAGTCTCATCATCAGCATTGTGCAGGCTGAAGACCTGGCACTTTATTACTGTCAGCAACATTTTAGCACTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO:12).;

[0068] The amino acid sequence of the heavy chain variable region of antibody No. 11 is QVQLKQSGPSLVQPSQSLSITCTVSGFSLSSYGVHWVRQSPGKALEWLGVIWRGGNTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTATYYCAIGPITTVAAYWGQGTTLTVSS (SEQ ID NO: 13); the amino acid sequence of the light chain variable region of antibody No. 11 is DIVMTQSHKFMSTSVGDRVSITCKASQDVRTAVAWYQQKPGQPPKLLIYWTSTRHTGVPDRFTGSGSGTDYSLIISIVQAEDLALYYCQQHFSTPLTFGAGTKLELK (SEQ ID NO: 14); The base sequence of the variable region of the heavy chain of antibody No. 11 is CAGGTGCAGCTGAAGCAGTCAGGACCTAGCCTAGTGCAGCCCTCACAGAGCCTGTCCATAACCTGCACAGTCTCTGGTTTCTCATTGTCTAGCTATGGTGTACACTGGGTTCGCCAGTCCAGGAAAGGCTCTGGAGTGGCTGGGAGTGATATGGAGAGGTGGAA ACACAGACTACAATGCAGCTTTCATGTCCAGACTGAGCATCACCAGGACAATTCCAAGAGCCAAGTTTTCTTAAATGAACAGTCTACAAGCTGATGACACTGCCACATACTACTGTGCCATAGGACCCATTACTACGGTAGCAGCCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:15);The base sequence of the light chain variable region of antibody No. 11 is GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGAGGACTGCTGTAGCCTGGTATCAACAAAAACCAGGGCAACCTCCTAAACTTCTGATTTACTGGACATCCACCCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTATAGTCTCATCATCAGCATTGTGCAGGCTGAAGACCTGGCACTTTATTACTGTCAGCAACATTTTAGCACTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO:16).;

[0069] The amino acid sequence of the heavy chain variable region of antibody No. 22 is QIQFVQSGPELKKPGETVKISCKASVYSFTEAPVHWVKQAPGKGFKWMGWINTYSGRPSYADDFTERFAFSLETSASTAYLQINNLKNEDTATYFCARRDGNLCFVYWGQGTLVTVSA (SEQ ID NO: 17); the amino acid sequence of the light chain variable region of antibody No. 22 is DIVMTQSQKFMSTSIGDRVSVTCKASQSVRTNVAWYQKKRGQSPKPLIHSASYRYSGVPDRFTGSGSGTEFTLSISNVQSEDLAEYFCHQYNTYPLTFGAGTNL (SEQ ID NO: 18); The base sequence of the variable region of the heavy chain of antibody No. 22 is CAGATCCAGTTCGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGTGTATCCTTCACAGAAGCTCCAGTGCACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTCAAGTGGATGGGCTGGATAAACACCTACTCTGG AAGGCCATCATTGCTGACGACTTCACGGAACGGTTTGCCTTTTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAGGAGGGATGGTAATCTTTGCTTTGTTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO:19);The base sequence of the variable region of the light chain of antibody No. 22 is GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAATAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAGTGTGAGAACTAATGTAGCCTGGTATCAAAAGAAACGAGGGCAATCTCCTAAACCACTGATTCACTCGGCAT CCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGAATTCACTCTCAGCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCACCAATATAACACCTATCCGCTCACGTTCGGCGCTGGGACCAACCTGGAGCTGAAA (SEQ ID NO:20). ;

[0070] The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of antibody No. 3 are GYSITSGYN (SEQ ID NO: 21), IKYSGIT (SEQ ID NO: 22) and TRGWDWFPY (SEQ ID NO: 23), respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region of antibody No. 3 are QSLFFSSNQKNC (SEQ ID NO: 24), WAS (SEQ ID NO: 25) and QQYYIYTYT (SEQ ID NO: 26), respectively.

[0071] The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of antibody No. 4 are GYTIIDYY (SEQ ID NO: 27), INPNNGIT (SEQ ID NO: 28) and ASSPDTGNYFAMDY (SEQ ID NO: 29), respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region of antibody No. 4 are QTIGTW (SEQ ID NO: 30), AAT (SEQ ID NO: 31) and QQLYINPRT (SEQ ID NO: 32), respectively.

[0072] The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of antibody No. 7 are GFSLSSYG (SEQ ID NO: 33), IWRGGNT (SEQ ID NO: 34) and AIGPITTVAAY (SEQ ID NO: 35), respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region of antibody No. 7 are QDVRTA (SEQ ID NO: 36), WTS (SEQ ID NO: 37) and QQHFSTPLT (SEQ ID NO: 38), respectively.

[0073] The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of antibody No. 11 are GFSLSSYG (SEQ ID NO: 39), IWRGGNT (SEQ ID NO: 40) and AIGPITTVAAY (SEQ ID NO: 41), respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region of antibody No. 11 are QDVRTA (SEQ ID NO: 42), WTS (SEQ ID NO: 43) and QQHFSTPLT (SEQ ID NO: 44), respectively.

[0074] The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of antibody No. 11 are VYSFTEAP (SEQ ID NO: 45), INTYSGRP (SEQ ID NO: 46) and ARRDGNLCFVY (SEQ ID NO: 47), respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region of antibody No. 11 are QSVRTN (SEQ ID NO: 48), SAS (SEQ ID NO: 49) and HQYNTYPLT (SEQ ID NO: 50), respectively.

[0075] Example 2 Neutralizing Effect of Anti-OXA-23 Carbapenemase Antibodies

[0076] 1. Experimental Materials

[0077] 1. Consumables: 96-well plate, 12 mL shake tube, 1.5 mL Eppenem tube, LB liquid medium, MH solid medium, meropenem aqueous solution, meropenem E-test strips, sterile PBS.

[0078] 2. Instruments: microplate reader, 37°C constant temperature incubator, Nanodrop micro-spectrophotometer.

[0079] 3. Bacterial species: clinical strain of carbapenem-resistant Acinetobacter baumannii; Antibody: monoclonal antibody against OXA-23 provided in Example 1 of the present invention.

[0080] 2. Experimental Methods

[0081] 1. Bacterial culture: Pick a single colony of Acinetobacter baumannii strain 057 and culture it in 1 mL of LB liquid medium at 37°C overnight.

[0082] 2. System Preparation: Add the meropenem aqueous solution to LB liquid medium to prepare meropenem solutions at concentrations of 0 and 24 μg / mL. Transfer 188 μL of meropenem-added LB to a 96-well plate. Add 2 μL of the antibody solution or PBS solution to the 96-well plate, following the procedure in Table 1.

[0083] Table 1 Anti-OXA-23 carbapenemase antibodies and Acinetobacter baumannii incubation experiments

[0084] Meropenem 0 + PBS Meropenem 24 ug / mL + PBS Meropenem 0 + Antibody 3 Meropenem 24 ug / mL + Antibody No. 3 Meropenem 0 + Antibody 4 Meropenem 24 ug / mL + Antibody No. 4 Meropenem 0 + Antibody 7 Meropenem 24 ug / mL + Antibody 7 Meropenem 0 + Antibody 11 Meropenem 24 ug / mL + Antibody No. 11 Meropenem 0 + Antibody 22 Meropenem 24 ug / mL + Antibody 22 Meropenem 0 + PBS (NC well, no sterile) Meropenem 24 ug / mL + PBS (NC well)

[0085] 3. Bacterial inoculation: Dilute the overnight bacterial solution to OD = 0.2, take 10ul and add it to a 96-well plate using a dispenser. Incubate at 37℃ for 24h and then measure the OD600 value using a microplate reader.

[0086] 4. E-test: Dilute the overnight bacterial solution to OD = 0.15, mix with Antibody No. 3 at a dilution of 1:100, incubate for 10 minutes, spread evenly on a MH plate, place an E-test strip, and incubate at 37°C overnight. Observe the MIC value of meropenem the next day.

[0087] 3. Experimental Results

[0088] The results of the effect of antibodies on bacterial resistance growth are as follows Figure 1 As shown. Determine OD in 96-well plate 600 , relative growth rate = value of the well with meropenem added / value of the well without meropenem added. It can be seen that antibodies 3, 4, 7, 11, and 22 can inhibit the growth ability of carbapenem-resistant Acinetobacter baumannii under the pressure of meropenem antibiotics, indicating that antibodies 3, 4, 7, 11, and 22 can bind to OXA-23 type carbapenemase and reduce the level of carbapenem resistance of Acinetobacter baumannii. We then incubated carbapenem-resistant Acinetobacter baumannii with antibody 3 and performed an E-test. Its MIC for meropenem decreased from 24ug / ml to 16ug / ml ( Figure 2 ).

[0089] These results demonstrate that the antibodies provided herein can bind to OXA-23 carbapenemases, reducing the level of carbapenem resistance in Acinetobacter baumannii, thereby lowering the clinical concentration of meropenem and enhancing its efficacy against Acinetobacter baumannii. Furthermore, the antibodies provided herein can inhibit secreted OXA-23 proteins, preventing their hydrolysis of carbapenem antibiotics, increasing antibiotic concentrations at the site of administration, and facilitating the elimination of mixed infections with Acinetobacter baumannii.

[0090] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. An anti-OXA-23 carbapenemase antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, or SEQ ID NO: 17; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, or SEQ ID NO: 18; and the CDR sequences are determined by any one of the IMGT, Chothia, Kabat, AbM, and Contact schemes. Preferably, the CDR sequence determination scheme is the IMGT scheme.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13 or SEQ ID NO: 17, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13 or SEQ ID NO: 17, or a sequence having one or more amino acid substitutions, deletions or insertions, or any combination thereof, compared to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13 or SEQ ID NO: 17; Preferably, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14 or SEQ ID NO: 18, or an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14 or SEQ ID NO: 18, or a sequence that has one or more amino acid substitutions, deletions or insertions, or any combination thereof, compared to the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14 or SEQ ID NO:

18.

3. A biomaterial, characterized in that The biological material includes: 1) A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2; 2) a vector comprising the polynucleotide described in 1); 3) A host cell comprising the polynucleotide described in 1) or the vector described in 2).

4. The biomaterial according to claim 3, characterized in that The polynucleotide sequence encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 is shown in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15 or SEQ ID NO: 19; the polynucleotide sequence encoding the light chain variable region of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 is shown in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16 or SEQ ID NO: 20; Preferably, the vector comprises a plasmid or a viral vector; Preferably, the viral vector comprises a lentivirus, an adenovirus, or an adeno-associated virus vector; Preferably, the host cells include prokaryotic cells and eukaryotic cells.

5. A composition, characterized in that The composition is a complex obtained by modifying the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2; the modification includes modification by conjugation with a detectable marker, modification by conjugation with a therapeutic agent, and modification by conjugation with an imaging agent.

6. The composition according to claim 5, characterized in that The detectable markers include fluorescent pigments, avidin, paramagnetic atoms, radioisotopes, enzyme markers, and colloidal gold; Preferably, 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 co-stimulatory molecule, or an inhibitor of an inhibitory molecule.

7. A product, characterized in that The product comprises the antibody or antigen-binding fragment thereof according to any one of claims 1-2, the biomaterial according to any one of claims 3-4, or the composition according to any one of claims 5-6.

8. The product according to claim 7, characterized in that The products include test kits, test strips, nucleic acid membrane strips, chips, systems, or devices.

9. Any of the following methods, comprising: (1) A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, characterized in that the method comprises the following steps: culturing the host cell according to any one of claims 3 to 4 to obtain a culture product, and isolating and purifying the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 from the culture product; (2) A method for detecting OXA-23 carbapenemase in a test sample for non-diagnostic and non-therapeutic purposes, characterized in that the method comprises the following steps: contacting the test sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 or contacting the test sample with the composition according to any one of claims 5 to 6, and detecting the formation of an immune complex between the OXA-23 carbapenemase and the antibody; (3) A method for preparing the host cell according to any one of claims 3 to 4, characterized in that the method comprises the following steps: introducing the polynucleotide molecule according to any one of claims 3 to 4 or a vector containing the same into the host cell; preferably, the introduction method comprises calcium phosphate transfection, DEAE, dextrose-mediated transfection, electroporation, or phage infection; (4) An in vitro method for inhibiting the activity of OXA-23 carbapenemase and / or inactivating OXA-23 carbapenemase for non-diagnostic and non-therapeutic purposes, characterized in that the method comprises the following steps: contacting a target sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2.

10. Any of the following applications, comprising: (1) Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-2, the biological material according to any one of claims 3-4, and / or the composition according to any one of claims 5-6 in the preparation of a product for detecting OXA-23 carbapenemases; (2) Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-2, the biomaterial according to any one of claims 3-4, and / or the composition according to any one of claims 5-6 in the preparation of a product for inhibiting the activity of OXA-23 type carbapenemase; (3) Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-2, the biomaterial according to any one of claims 3-4, and / or the composition according to any one of claims 5-6 in the preparation of a medicament for treating diseases caused by Acinetobacter baumannii.

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