An antibody, a method of preparation and use thereof

Abstract

The application discloses an antibody, a preparation method and application thereof. The antibody has good binding activity with OXA-23 type carbapenamase, can reduce the carbapenam antibiotic resistance level of acinetobacter baumannii, thereby reducing the use concentration of meropenem in clinic, and improving the treatment effect of meropenem on acinetobacter baumannii. In addition, the antibody can also inhibit the OXA-23 protein secreted by bacteria, prevent the hydrolysis of carbapenam antibiotics by bacteria, increase the antibiotic concentration at a drug application site, and is favorable for clearing mixed infections with acinetobacter baumannii. The application provides a new strategy for treating acinetobacter baumannii infections, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to an antibody against OXA-23 type carbapenemase, its preparation method and its application. Background Technology

[0002] In β Among β-lactam antibiotics, carbapenems have the broadest antibacterial activity, capable of resisting the vast majority of β-lactam antibiotics. Lactamase, extended-spectrum β It exhibits hydrolytic activity due to lactamases and can attenuate the activity of chromosome-mediated AmpC enzymes. Acinetobacter is resistant to many antibiotics, such as β-lactamases. Lactams, aminoglycosides, and fluoroquinolones all exhibit some degree of resistance, while carbapenems have consistently shown good antibacterial activity against Acinetobacter baumannii. However, with the widespread clinical use of carbapenems, the resistance rate of Acinetobacter baumannii to them is increasing year by year. In recent years, there have been increasing reports of bacteria carrying blaOXA... There have been reports of Acinetobacter baumannii causing hospital-acquired infections. Literature reports that 80% of carbapenem-resistant Acinetobacter baumannii in China are OXA-producing bacteria. Carbapenemase type 23 makes the treatment of carbapenem-resistant Acinetobacter baumannii extremely difficult in clinical practice. According to data from the Ministry of Health's CHINET drug resistance monitoring network 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 serious challenge to clinical anti-infective therapy.

[0003] Therefore, providing an antibody that can neutralize the active site of OXA-23, thereby blocking the hydrolysis of carbapenem antibiotics by this enzyme, 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] To overcome the shortcomings of the prior art, the purpose of this invention is to provide an antibody that can reduce the carbapenem resistance level of Acinetobacter baumannii and improve the therapeutic effect of carbapenem antibiotics on Acinetobacter baumannii.

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

[0006] The first aspect of the present invention provides an antibody against OXA-23 type carbapenemase or an antigen-binding fragment thereof.

[0007] Further, 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 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; and 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 sequence is determined by any one of the IMGT, Chothia, Kabat, AbM, and Contact schemes.

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

[0009] 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.

[0010] 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 CDR in the variable region amino acid sequence can be defined using the IMGT protocol, Kabat protocol, AbM protocol, Chothia protocol, or Contact protocol.

[0011] 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).

[0012] The boundaries of the CDR of the antibody of the present invention can be determined manually according to any scheme or combination thereof in the art. Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the above-described ways. In a specific embodiment of the present invention, the scheme for determining the CDR sequence is the IMGT scheme.

[0013] Furthermore, 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 having at least 90% identity with 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] Furthermore, 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 having at least 90% identity with 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 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequences of this invention are within the scope of protection of this invention.

[0016] 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.

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

[0018] Furthermore, the biomaterial includes:

[0019] 1) A polynucleotide encoding the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention;

[0020] 2) A carrier comprising the polynucleotide described in 1);

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

[0022] Furthermore, the polynucleotide sequences encoding the heavy chain variable region of the antibody or its antigen-binding fragment according to the first aspect of the present invention are 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 sequences encoding the light chain variable region of the antibody or its antigen-binding fragment according to the first aspect of the present invention are shown in 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 plasmids and viral vectors.

[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] 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.

[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 techniques. Once the relevant sequence is obtained, it can be obtained in large quantities using recombinant methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods. Alternatively, the relevant sequence can be synthesized artificially, especially when the fragment length is short. Generally, longer fragments can be obtained by first synthesizing multiple small fragments and then ligating them.

[0028] 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.

[0029] 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.

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

[0031] Furthermore, the composition 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.

[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 polydinoflavin-chlorophyll protein.

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

[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, hormonal preparations, targeted small molecule preparations, proteasome inhibitors, chemotherapeutic agents, oncolytic drugs, cytokines, activators of co-stimulatory molecules, and inhibitors of inhibitory molecules.

[0038] 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, cofactors, 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, and β-phosphatase. 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.

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

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

[0041] Furthermore, the products include reagent kits, test strips, nucleic acid membrane strips, chips, systems, or devices.

[0042] Preferably, the product is a reagent kit.

[0043] The fifth aspect of the present invention provides any one of the following methods, the method 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 separating 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 its antigen-binding fragment as described in the first aspect of the present invention or contacting the test sample with the composition as described in the third aspect of the present invention, and detecting the formation of OXA-23 carbapenemase-antibody immune complex;

[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 polynucleotide molecule into the host cell; preferably, the introduction method includes calcium phosphate transfection, DEAE, dextran-mediated transfection, electroporation, and phage infection;

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

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

[0049] This invention does not impose any particular restrictions on the type of sample.

[0050] The sixth aspect of the present invention provides any of the following applications, the applications including:

[0051] (1) The use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the biological material 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 product for detecting OXA-23 type carbapenemase;

[0052] (2) The use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the biological material 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 product for inhibiting the activity of OXA-23 type carbapenemase;

[0053] (3) The use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the biological material 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 diseases caused by Acinetobacter baumannii include, but are not limited to: bacteremia, pneumonia, meningitis, peritonitis, endocarditis, urinary tract and skin infections. All symptoms or diseases caused by Acinetobacter baumannii infection are within the scope of protection of this invention.

[0055] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0056] This invention provides a novel antibody against OXA-23 carbapenemase, exhibiting good binding activity to OXA-23 carbapenemase. This antibody can reduce carbapenem resistance levels in *Acinetobacter baumannii*, thereby lowering the clinical concentration of meropenem and enhancing its efficacy against *Acinetobacter baumannii*. Furthermore, the antibody can inhibit the secreted OXA-23 protein, preventing its hydrolysis of carbapenem antibiotics and increasing antibiotic concentration at the application site, which is beneficial for clearing mixed infections with *Acinetobacter baumannii*. Attached Figure Description

[0057] Figure 1 The figure shows the effect of the antibody provided by this invention on drug resistance in Acinetobacter baumannii.

[0058] Figure 2 The figure shows the effect of the antibody provided by this invention on the MIC of Acinetobacter baumannii. Detailed Implementation

[0059] The present invention will be further described below with reference to specific embodiments. However, the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

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

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

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

[0063] 2. Mice were immunized with purified OXA-23 protein to obtain OXA-23 monoclonal antibodies.

[0064] This invention yielded five monoclonal antibodies, named antibodies 3, 4, 7, 11, and 22, respectively. The amino acid and base sequences of their heavy chain variable regions and light chain variable regions are shown below.

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

[0066] The amino acid sequence of the heavy chain variable region of antibody 4 is EVQLQQSGPELVKPGASVKISCKASGYTIIDYYINWVKQSHGKSLEWIGDINPNNGITTYNQKFKGKATLTVDMSSSTAYMDLRSLTSEDSAVYYCASSPDTGNYFAMDYWGQGTAVTVSS (SEQ ID NO:5); the amino acid sequence of the light chain variable region of antibody 4 is DIQMTQSPASQSASLGESVTITCLASQTIGTWLAWYQQKPGKSPQLLIYAATRLADGVPSRFSGSGSGTKFSFKISSLQAEDFVSYYCQQLYINPRTFGGGTKLEIK (SEQ ID NO:5). 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 7 is QVQLKQSGPSLVQPSQSLSITCTVSGFSLSSYGVHWVRQSPGKALEWLGVIWRGGNTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTATYYCAIGPITTVAAYWGQGTTLTVSS (SEQ ID NO: 9); the amino acid sequence of the light chain variable region of antibody 7 is DIVMTQSHKFMSTSVGDRVSITCKASQDVRTAVAWYQQKPGQPPKLLIYWTSTRHTGVPDRFTGSGSGTDYSLIISIVQAEDLALYYCQQHFSTPLTFGAGTKLELK (SEQ ID NO: 9). 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 11 is QVQLKQSGPSLVQPSQSLSITCTVSGFSLSSYGVHWVRQSPGKALEWLGVIWRGGNTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTATYYCAIGPITTVAAYWGQGTTLTVSS (SEQ ID NO:13); the amino acid sequence of the light chain variable region of antibody 11 is DIVMTQSHKFMSTSVGDRVSITCKASQDVRTAVAWYQQKPGQPPKLLIYWTSTRHTGVPDRFTGSGSGTDYSLIISIVQAEDLALYYCQQHFSTPLTFGAGTKLELK (SEQ ID NO:13). 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 22 is QIQFVQSGPELKKPGETVKISCKASVYSFTEAPVHWVKQAPGKGFKWMGWINTYSGRPSYADDFTERFAFSLETSASTAYLQINNLKNEDTATYFCARRDGNLCFVYWGQGTLVTVSA (SEQ ID NO:17); the amino acid sequence of the light chain variable region of antibody 22 is DIVMTQSQKFMSTSIGDRVSVTCKASQSVRTNVAWYQKKRGQSPKPLIHSASYRYSGVPDRFTGSGSGTEFTLSISNVQSEDLAEYFCHQYNTYPLTFGAGTNL (SEQ ID NO:17). NO: 18); The base sequence of the heavy chain variable region of antibody No. 22 is CAGATCCAGTTCGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGTGTATCCTTCACAGAAGCTCCAGTGCACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTCAAGTGGATGGGCTGGATAAACACCTACTCTGG AAGGCCATCATATGCTGACGACTTCACGGAACGGTTTGCCTTTTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAGGAGGGATGGTAATCTTTGCTTTGTTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (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 in the heavy chain variable region of antibody 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 in the light chain variable region of antibody 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 in the heavy chain variable region of antibody 4 are GYTIIDYY (SEQ ID NO:27), INPNGIT (SEQ ID NO:28) and ASSPDTGNYFAMDY (SEQ ID NO:29), respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region of antibody 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 in the heavy chain variable region of antibody 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 in the light chain variable region of antibody 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 in the heavy chain variable region of antibody 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 in the light chain variable region of antibody 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 in the heavy chain variable region of antibody 22 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 in the light chain variable region of antibody 22 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 antibody

[0076] I. Experimental Materials

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

[0078] 2. Instruments: Microplate reader, 37℃ constant temperature incubator, Nanodrop micro spectrophotometer.

[0079] 3. Bacterial strain: Carbapenem-resistant Acinetobacter baumannii clinical strain; Antibody: Monoclonal antibody against OXA-23 provided in Example 1 of this invention.

[0080] II. Experimental Methods

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

[0082] 2. System preparation: Add meropenem aqueous solution to LB liquid medium to prepare LB solutions with a meropenem concentration of 0.24 ug / mL. Add 188 μL of LB medium containing meropenem to a 96-well plate, and add 2 μL of antibody solution or PBS solution to the 96-well plate as shown in Table 1.

[0083] Table 1. Anti-OXA-23 carbapenemase antibody and Acinetobacter baumannii incubation experiment.

[0084]

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

[0086] 4. E-test: Dilute the overnight bacterial culture to OD=0.15, mix with antibody No. 3 at a ratio of 1:100 and incubate for 10 minutes. Spread evenly on MH plate, place E-test strip on it, and incubate at 37℃ overnight. Observe the MIC value of meropenem the next day.

[0087] III. Experimental Results

[0088] The effect of antibodies on the growth of antibiotic-resistant bacteria is as follows: Figure 1 As shown. OD of a 96-well plate was measured. 600 The relative growth rate was calculated as the ratio of the value of wells with meropenem to the value of wells without meropenem. This indicates that antibodies 3, 4, 7, 11, and 22 all inhibited the growth of carbapenem-resistant Acinetobacter baumannii under meropenem antibiotic stress, demonstrating that antibodies 3, 4, 7, 11, and 22 can bind to OXA-23 carbapenemase and reduce the carbapenem resistance level of Acinetobacter baumannii. Subsequently, after co-incubating carbapenem-resistant Acinetobacter baumannii with antibody 3, an E-test was performed, and the MIC of meropenem decreased from 24 ug / ml to 16 ug / ml. Figure 2 ).

[0089] These results indicate that the antibody provided by this invention can bind to OXA-23 carbapenemase, reducing carbapenem resistance levels in Acinetobacter baumannii, thereby lowering the clinically required concentration of meropenem and enhancing its efficacy against Acinetobacter baumannii. Furthermore, the antibody of this invention can also inhibit secreted OXA-23 protein, preventing its hydrolysis of carbapenem antibiotics, increasing antibiotic concentration at the application site, and facilitating the clearance of mixed infections with Acinetobacter baumannii.

[0090] 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 against an OXA-23 type carbapenemase, characterized in that, The antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2 and HCDR3, and the light chain variable region includes LCDR1, LCDR2 and LCDR3. The sequence of HCDR1 is shown in SEQ ID NO:27; the sequence of HCDR2 is shown in SEQ ID NO:28; and the sequence of HCDR3 is shown in SEQ ID NO:

29. The sequence of LCDR1 is shown in SEQ ID NO:30; the sequence of LCDR2 is shown in SEQ ID NO:31; and the sequence of LCDR3 is shown in SEQ ID NO:

32.

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

5.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein, The light chain variable region contains the amino acid sequence shown in SEQ ID NO:

6.

4. A biomaterial, characterized in that, The biomaterials include: 1) A polynucleotide encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1-3; 2) A carrier comprising the polynucleotide described in 1); 3) A host cell containing the polynucleotide described in 1) or the vector described in 2).

5. The biomaterial according to claim 4, characterized in that, The polynucleotide sequence encoding the heavy chain variable region of the antibody or antigen-binding fragment of any one of claims 1-3 is shown in SEQ ID NO:7; the polynucleotide sequence encoding the light chain variable region of the antibody or antigen-binding fragment of any one of claims 1-3 is shown in SEQ ID NO:

8.

6. The biomaterial according to claim 4, characterized in that, The vectors include plasmids and viral vectors.

7. The biomaterial according to claim 6, characterized in that, The viral vectors include lentiviruses, adenoviruses, and adeno-associated virus vectors.

8. The biomaterial according to claim 4, characterized in that, The host cells include prokaryotic cells and eukaryotic cells.

9. An antibody derivative, characterized in that, The antibody derivative is a complex obtained by modifying the antibody or its antigen-binding fragment as described in any one of claims 1-3; the modification includes conjugation modification using a detectable marker and conjugation modification using an imaging agent.

10. The antibody derivative according to claim 9, characterized in that, The detectable markers include fluorescent dyes, avidin, paramagnetic atoms, radioactive isotopes, enzyme markers, and colloidal gold.

11. A product characterized in that, The product includes the antibody or antigen-binding fragment thereof as described in any one of claims 1-3, the biomaterial as described in any one of claims 4-8, or the antibody derivative as described in any one of claims 9-10.

12. The product according to claim 11, characterized in that, The products include reagent kits, test strips, nucleic acid membrane strips, chips, or devices.

13. The method comprising: (1) A method for producing the antibody or antigen-binding fragment of any one of claims 1-3, characterized in that the method comprises the following steps: culturing the host cell of any one of claims 4-8 to obtain a culture product, and separating and purifying the antibody or antigen-binding fragment of any one of claims 1-3 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 its antigen-binding fragment as described in any one of claims 1-3 or contacting the test sample with the antibody derivative as described in any one of claims 9-10, and detecting the formation of OXA-23 carbapenemase-antibody immune complex; (3) A method for preparing a host cell according to any one of claims 4-8, characterized in that the method comprises the following steps: introducing a polynucleotide molecule according to any one of claims 4-8 or a vector containing the polynucleotide molecule into a host cell; (4) A method for inhibiting and / or inactivating OXA-23 carbapenemase activity in vitro 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 as described in any one of claims 1-3.

14. The method according to claim 13, characterized in that, The methods of introduction include calcium phosphate transfection, DEAE, dextran-mediated transfection, electroporation, and phage infection.

15. Any of the following applications, the application including: (1) The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-3, the biological material according to any one of claims 4-8, and / or the antibody derivative according to any one of claims 9-10 in the preparation of a product for detecting OXA-23 type carbapenemase; (2) The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-3, the biological material according to any one of claims 4-8, and / or the antibody derivative according to any one of claims 9-10 in the preparation of a product for inhibiting the activity of OXA-23 type carbapenemase; (3) The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-3, the biological material according to any one of claims 4-8, and / or the antibody derivative according to any one of claims 9-10 in the preparation of a medicament for treating Acinetobacter baumannii infection.

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