An antibody binding to helicobacter pylori antigen protein urease and use thereof

Abstract

The embodiment of the present specification provides an antibody or antigen binding fragment binding to Helicobacter pylori antigen protein urease, the antibody or antigen binding fragment comprising a heavy chain variable region and a light chain variable region. Wherein, the heavy chain variable region comprises CDRH1 of an amino acid sequence as shown in SEQ ID NO: 2, CDRH2 of an amino acid sequence as shown in SEQ ID NO: 3 and CDRH of an amino acid sequence as shown in SEQ ID NO: 4. The light chain variable region comprises CDRL1 of an amino acid sequence as shown in SEQ ID NO: 6, CDRL2 of an amino acid sequence of DTS and CDRL3 of an amino acid sequence as shown in SEQ ID NO: 7. The antibody or antigen binding fragment has the characteristics of high purity, high specificity and sensitivity. The embodiment of the present specification also provides related application of the antibody or antigen binding fragment binding to Helicobacter pylori antigen protein urease.

Description

Technical Field

[0001] This specification relates to the field of biomedical technology, and in particular to an antibody that binds to the Helicobacter pylori antigen protein urease and its applications. Background Technology

[0002] Helicobacter pylori (HP) is a Gram-negative microaerophilic bacterium that can survive in highly acidic environments (e.g., stomach acid). HP infection is closely related to various digestive tract diseases; for example, it is a significant pathogenic factor for chronic gastritis and peptic ulcers. Furthermore, HP infection is closely associated with the development of gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma.

[0003] Helicobacter pylori does not react to most classic biochemical tests commonly used in clinical microbiology to identify intestinal bacteria. Urease is one of the main bases for biochemical identification of Helicobacter pylori, and clinically, Helicobacter pylori infection can be diagnosed through invasive (e.g., rapid urease test) and non-invasive (e.g., urea breath test, serological immunological test) urease-based assays. For example, serological immunological assays targeting urease detect Helicobacter pylori infection by measuring the Helicobacter pylori antigen protein urease in serum. Compared to histological sections and histochemical staining microscopy, which are the gold standard for Helicobacter pylori detection, serological immunological assays targeting urease have room for improvement in specificity and sensitivity. Furthermore, serological assays targeting urease may not react in infected individuals with existing symptoms. Therefore, it is necessary to provide an antibody that can specifically bind to the Helicobacter pylori antigen protein urease. Summary of the Invention

[0004] According to a first aspect of this specification, an antibody or antigen-binding fragment that binds to the Helicobacter pylori antigen protein urease is provided. The antibody or antigen-binding fragment includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes the amino acid sequences CDRH1 (as shown in SEQ ID NO:2), CDRH2 (as shown in SEQ ID NO:3), and CDRH3 (as shown in SEQ ID NO:4). The light chain variable region includes the amino acid sequence CDRL1 (as shown in SEQ ID NO:6), CDRL2 (with the amino acid sequence DTS), and CDRL3 (as shown in SEQ ID NO:7).

[0005] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:5.

[0006] In some embodiments, the antibody is a mouse antibody, a rabbit antibody, a chimeric antibody, or a humanized antibody.

[0007] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0008] In some embodiments, the antigen-binding fragment is selected from the group consisting of: Fab, Fab', F(ab')2, Fv, scFv, dsFv, and dAb.

[0009] According to a second aspect of this specification, a nucleic acid molecule is provided. The nucleic acid molecule comprises a nucleotide sequence encoding an antibody or antigen-binding fragment as described above.

[0010] In some embodiments, the nucleic acid molecule includes nucleotide sequences encoding the heavy chain variable region and nucleotide sequences encoding the light chain variable region. The nucleotide sequences encoding the heavy chain variable region include the coding sequences for CDRH1 as shown in SEQ ID NO:9, CDRH2 as shown in SEQ ID NO:10, and CDRH3 as shown in SEQ ID NO:11. The nucleotide sequences encoding the light chain variable region include the coding sequences for CDRL1 as shown in SEQ ID NO:13, CDRL2 with the sequence GACACATCC, and CDRL3 as shown in SEQ ID NO:14.

[0011] In some embodiments, the nucleotide sequence encoding VH is shown in SEQ ID NO:8; the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO:12.

[0012] According to a third aspect of this specification, a recombinant vector is provided. The recombinant vector comprises the nucleic acid molecules as described above.

[0013] According to a fourth aspect of this specification, a host cell is provided. The host cell comprises a nucleic acid molecule or a recombinant vector as described above.

[0014] According to a fifth aspect of this specification, a pharmaceutical composition is provided. The pharmaceutical composition comprises an antibody or antigen-binding fragment as described above.

[0015] In some embodiments, the antibody or antigen-binding fragment is conjugated to a drug compound or peptide.

[0016] According to aspect six of this specification, the use of antibodies or antigen-binding fragments, nucleic acid molecules, recombinant vectors, host cells, or pharmaceutical compositions as described above in the preparation of medicaments for the treatment of Helicobacter pylori infection is provided.

[0017] According to aspect seven of this specification, a kit for detecting Helicobacter pylori is provided. The kit comprises an antibody or antigen-binding fragment as described above.

[0018] According to aspect eight of this specification, the use of antibodies or antigen-binding fragments, nucleic acid molecules, recombinant vectors or host cells as described above in the preparation of kits for the detection of Helicobacter pylori is provided. Detailed Implementation

[0019] It should be understood that although the terms "first," "second," "third," etc., may be used herein to describe various elements, these elements should not be limited by these terms. These terms are used only to distinguish one element from another. For example, a first product may be referred to as a second product, and similarly, a second product may be referred to as a first product without departing from the scope of exemplary embodiments described herein.

[0020] As indicated in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" do not specifically refer to the singular and may also include the plural. Generally speaking, the terms "comprising" and "including" only indicate the inclusion of expressly identified steps and elements, which do not constitute an exclusive list, and the method or apparatus may also include other steps or elements.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0022] This specification provides an antibody or antigen-binding fragment that binds to Helicobacter pylori antigen protein urease. Specifically, Balb / c mice can be immunized with Helicobacter pylori antigen protein urease. Highly specific hybridoma cells are obtained through specific high-throughput screening. Large amounts of mouse ascites are obtained through culture and re-immunization. High-purity, high-sensitivity, and high-specificity monoclonal antibodies against Helicobacter pylori urease are then obtained through multi-step separation and purification. On the one hand, the antibody or antigen-binding fragment provided in this specification has excellent specific binding ability to Helicobacter pylori antigen protein urease. Its application in various immunological detection methods can improve the sensitivity and specificity of the corresponding detection methods. The antibody or antigen-binding fragment provided in this specification can provide the necessary raw materials for developing immunological detection methods or products for detecting Helicobacter pylori urease. On the other hand, the antibody or antigen-binding fragment provided in this specification can support the development of new drugs for treating Helicobacter pylori infection. For example, the antibody or antigen-binding fragment provided in this specification can be used to develop and prepare antibody-drug conjugates (ADCs), providing the possibility of reducing or delaying drug resistance.

[0023] Some embodiments of this specification provide an antibody or antigen-binding fragment capable of binding to Helicobacter pylori antigen protein urease. The antibody or antigen-binding fragment includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes three complementarity-determining regions, namely CDRH1, CDRH2, and CDRH3; the light chain variable region includes three complementarity-determining regions, namely CDRL1, CDRL2, and CDRL3.

[0024] As used herein, the term "antibody" or "Ab" refers to a molecule comprising at least one immunoglobulin heavy chain (HC) and at least one immunoglobulin light chain (LC). Each heavy chain may include a heavy chain variable region (VH) and a heavy chain constant region (CH), the heavy chain variable region having three complementarity-determining regions (CDRs) and four frame regions (FRs). Each light chain may include a light chain variable region (VL) and a light chain constant region (CL), the light chain variable region having three complementarity-determining regions and four frame regions. Antibodies can be classified as IgM, IgG, IgA, IgD, and IgE based on the antigenicity of the VH. In some embodiments, antibodies include, but are not limited to, monoclonal antibodies, bispecific antibodies, multispecific antibodies, dimeric antibodies, trimeric antibodies, and polymeric antibodies.

[0025] The term "antigen-binding fragment" refers to a polypeptide fragment containing a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen. Non-limiting examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fd, Fv, CDR fragments, single-chain antibodies (e.g., scFv or dsFv), bispecific antibody fragments (dAb), single-domain antibodies (sdAb), and nanobodies (Nb).

[0026] The term "CDR," or "complementarity-determining region," refers to the region in which an antibody specifically recognizes an antigen.

[0027] In some embodiments, the amino acid sequence of CDRH1 is DYTFTNSW (SEQ ID NO:2). The amino acid sequence of CDRH2 is INPSTGST (SEQ ID NO:3). The amino acid sequence of CDRH3 is ASEEYDGFDY (SEQ ID NO:4).

[0028] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment is PGASVKMSCRASDYTFTNSWMHWVKQRPGQGLEWVGYINPSTGSTDYNQKFRDKATLTADKSSST AYMQLSSLTSEDSAVYYCASEEYDGFDYWGQGTTLTVSS (SEQ ID NO:1).

[0029] In some embodiments, the heavy chain variable region may contain no more than two amino acids or no more than one conserved substitution. As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the fundamental properties of a protein / peptide comprising an amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue having a similar side chain, such as substitutions of physically or functionally similar residues (e.g., having similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (e.g., threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, the corresponding amino acid residue is preferably substituted by another amino acid residue from the same side chain family.

[0030] In some embodiments, the heavy chain variable region has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity in the frame region compared to the amino acid sequence shown in SEQ ID NO:1. As used herein, the term “identity” refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules determined by alignment and comparison of sequences. “Percentage identity” refers to the percentage of identical residues among amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest molecule being compared. In some embodiments, the heavy chain variable region has an amino acid sequence having one or more added, deleted, and / or substituted amino acids in the frame region compared to the amino acid sequence shown in SEQ ID NO:1.

[0031] In some embodiments, the amino acid sequence of CDRL1 is SSIIYM (SEQ ID NO:6). The amino acid sequence of CDRL2 is DTS. The amino acid sequence of CDRL3 is QQWSSSPYT (SEQ ID NO:7).

[0032] In some embodiments, the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment is DIVITQSP AIMSASPGEKVTMTCSANSSIIYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTS YSLTISSMEAEDAATYYCQQWSSSPYTFGGGTKLEIK (SEQ ID NO:5).

[0033] In some embodiments, the light chain variable region may contain conservative substitutions of no more than two amino acids or no more than one amino acid. In some embodiments, the light chain variable region has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity in the frame region compared to the amino acid sequence shown in SEQ ID NO:5. In some embodiments, the light chain variable region has an amino acid sequence with one or more added, deleted, and / or substituted amino acids in the frame region compared to the amino acid sequence shown in SEQ ID NO:5.

[0034] The antibodies provided in the embodiments of this specification may be derived from the same species. In some embodiments, the antibodies may be mouse-derived antibodies or rabbit-derived antibodies. The antibodies provided in the embodiments of this specification may also be derived from different species. In some embodiments, the antibodies may be chimeric antibodies or humanized antibodies.

[0035] As used herein, a "chimeric antibody" is an antibody formed by fusing the variable region of an antibody from one species with the constant region of an antibody from another species. Chimeric antibodies are characterized by reducing the immunogenicity of heterologous antibodies. In some embodiments, the variable region of a chimeric antibody may be rabbit-derived or mouse-derived, and the constant region of a chimeric antibody may be human-derived. The term "humanized antibody" refers to a genetically engineered antibody form containing human antibody sequences and non-human antibody sequences. In some embodiments, all or part of the CDR region of a humanized antibody is derived from a non-human (e.g., mouse or rabbit) antibody, and all or part of the non-CDR region (e.g., the constant region and variable region framework) of a humanized antibody is derived from a human antibody.

[0036] In some embodiments, the antibody may be a monoclonal antibody, a bispecific antibody, or a multispecific antibody. Those skilled in the art can determine the number and type of antigenic epitopes that the antibody can recognize based on application requirements.

[0037] In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, dsFv, and dAb.

[0038] As used herein, the term "Fab" refers to an antibody fragment consisting of the VL, VH, CL, and CH1 regions. The term "F(a b')2" refers to an antibody fragment containing two Fab fragments linked by disulfide bridges on their hinge regions. The term "Fab'" refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light and heavy chain Fd fragment (composed of the VH and CH1 regions). The term "Fv" refers to an antibody fragment consisting of the VL and VH regions of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (e.g., an Fd fragment containing only three antigen-specific CDRs) can recognize and bind to antigens, although its affinity may be lower than that of a complete binding site. The term "scFv" refers to an antibody fragment consisting of the VH and VL regions linked by short linkers, which does not contain an Fc fragment. The term "dsFv" refers to an antibody fragment in which Fv is linked by interchain disulfide bonds; it is also known as a disulfide-stabilized antibody. The term "dAb" refers to an antibody fragment composed of VH regions.

[0039] Methods for preparing antibodies should be known to those skilled in the art. For example, hybridoma techniques, or the in vitro production of antibodies using recombinant DNA technology with an antibody-encoding gene as the starting material. Methods for preparing antigen-binding fragments should also be known to those skilled in the art. For example, antigen-binding fragments of antibodies can be generated via recombinant DNA technology or through enzymatic or chemical fragmentation of intact antibodies.

[0040] Some embodiments of this specification also provide an isolated nucleic acid molecule. The nucleic acid molecule may include a nucleotide sequence encoding an antibody or antigen-binding fragment that binds to the Helicobacter pylori antigen protein urease described above. In some embodiments, the nucleic acid molecule may include a nucleotide sequence encoding a heavy chain variable region of the antibody or antigen-binding fragment, and a nucleotide sequence encoding a light chain variable region of the antibody or antigen-binding fragment.

[0041] In some embodiments, the nucleotide sequences encoding the heavy chain variable regions may include the coding sequences for the heavy chain variable regions CDRH1, CDRH2, and CDRH3. Specifically, the coding sequence for CDRH1 is GACTACACCTTTACTAACTCCTGG (SEQ ID NO:9). The coding sequence for CDRH2 is ATTAATCCTAGCACTGGTTCTACT (SEQ ID NO:10). The coding sequence for CDRH3 is GCAAGCGAGGAGTACGACGGCTTTGACTAC (SEQ ID NO:11).

[0042] In some embodiments, the nucleotide sequence encoding the heavy chain variable region is CCTGGGGCCTCAGTGAAGATG TCCTGCAGGGCTTCTGACTACACCTTTACTAACTCCTGGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGGTTGGATACATTAATCCTAGCACTGGTTCTACTGACTACAATCAGAAGTTCAGGGACAAGGCCACTTTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGCGAGGAGTACGACGGCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:8). In other embodiments, the nucleotide sequence encoding the variable region of the heavy chain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the frame region coding sequence compared to the sequence shown in SEQ ID NO:8.

[0043] In some embodiments, the nucleotide sequence encoding the light chain variable region may include the coding sequences for the light chain variable regions CDRL1, CDRL2, and CDRL3. Specifically, the coding sequence for CDRL1 is TCAAGTATAATTTAC (SEQ ID NO:13). The coding sequence for CDRL2 is GACACATCC. The coding sequence for CDRL1 is CAGCAGTGGAGTAGT AGCCCGTACACG (SEQ ID NO:14).

[0044] In some embodiments, the nucleotide sequence encoding the light chain variable region is GATATTGTGATAACCCAGTCT CCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAACTCAAGTATAATTTACATGCATTGGTACCAGCAGAAGTCAGGCACGTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGTTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAGCCCGTACACGTTCGGAGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO:12). In other embodiments, the nucleotide sequence encoding the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the frame region coding sequence compared to the sequence shown in SEQ ID NO:12.

[0045] Methods for achieving the isolation of nucleic acid molecules should be known to those skilled in the art; for example, they can be prepared using recombinant DNA technology or isolated from suitable other sources.

[0046] Some embodiments of this specification also provide a recombinant vector comprising the aforementioned nucleic acid molecules. In some embodiments, the recombinant vector is an expression vector.

[0047] Methods for preparing recombinant vectors should be known to those skilled in the art. For example, recombinant DNA technology and gene transfection methods can be used to insert the aforementioned nucleic acid molecules into one or more expression vectors, so that the nucleic acid molecules are operatively linked to transcriptional and translational regulatory sequences.

[0048] Some embodiments of this specification also provide a host cell comprising the nucleic acid molecules or recombinant vectors described above, enabling the host cell to produce the antibody or antigen-binding fragments provided in this specification.

[0049] As used herein, the term "host cell" refers to a cell capable of introducing and stably maintaining a foreign gene. In some embodiments, host cells include prokaryotic cells and eukaryotic cells.

[0050] In some embodiments, the host cell is a eukaryotic cell or a prokaryotic cell. In some embodiments, eukaryotic cells that can serve as host cells include, but are not limited to, rodent cells, primate cells, plant cells, insect cells, and yeast cells. In some embodiments, prokaryotic cells that can serve as host cells include, but are not limited to, Escherichia coli, Bacillus, and Salmonella.

[0051] Methods for preparing host cells should be known to those skilled in the art. For example, the aforementioned recombinant vector can be introduced into the host cell to integrate the aforementioned nucleic acid molecules into the host cell's genome. The transcription and translation of the encoding gene of the aforementioned antibody or antigen-binding fragment can be achieved through the host cell's expression system, thereby producing the antibody or antigen-binding fragment provided in this specification.

[0052] Some embodiments of this specification also provide a pharmaceutical composition comprising the aforementioned antibody or antigen-binding fragment.

[0053] In some embodiments, the pharmaceutical composition includes an immunoconjugate, which is prepared by conjugating the aforementioned antibody or antigen-binding fragment with a pharmaceutical compound or peptide. Methods for preparing immunoconjugates should be known to those skilled in the art; for example, the aforementioned antibody or antigen-binding fragment can be directly or indirectly linked to a functional molecule via a spacer of suitable length, either through chemical cross-linking or genetic engineering fusion expression, thereby obtaining the immunoconjugate.

[0054] In some embodiments, the drug compound conjugated to the antibody or antigen-binding fragment may include, but is not limited to, proton pump inhibitors (e.g., omeprazole, pantoprazole, rabeprazole, vonoprazan, etc.), antibiotics (e.g., tetracycline, amoxicillin, clarithromycin, quinolones, etc.) and other small molecule compounds (e.g., rhamnolipids, allicin, etc.).

[0055] In some embodiments, the peptide conjugated to the antibody or antigen-binding fragment may include, but is not limited to, pexiganan, Epinecdin-1 (Epi-1), and Tilapia Piscidin 4 (TP4).

[0056] In some embodiments, the pharmaceutical composition may further include a pharmaceutically acceptable carrier, such as a buffer, antioxidant, surfactant, osmotic pressure regulator, pH adjuster, stabilizer, excipient, flavoring agent, preservative, etc. The dosage form of the pharmaceutical composition may be selected according to application needs, including but not limited to injections, tablets, capsules, oral liquid dosage forms, granules, etc.

[0057] Some embodiments of this specification also provide the use of the aforementioned antibody or antigen-binding fragment, nucleic acid molecule, recombinant vector, host cell, or pharmaceutical composition in the preparation of a medicament for treating Helicobacter pylori infection.

[0058] Some embodiments of this specification also provide a kit for detecting Helicobacter pylori. The kit may contain the aforementioned antibody or antigen-binding fragment, which is capable of binding to the Helicobacter pylori antigen protein urease.

[0059] In some embodiments, the kit is an immunochromatographic kit, such as a colloidal gold immunochromatographic kit and / or a latex microsphere immunochromatographic kit. The antibody or antigen-binding fragment can be used as a labeled pad for coating the chromatographic test strip with a detectable label. In other embodiments, the kit can be other kits for implementing an immunoassay method for Helicobacter pylori, such as immunofluorescence kits, immunohistochemical kits, etc., and is not limited thereto.

[0060] Some embodiments in this specification also provide the use of the aforementioned antibody or antigen-binding fragments, nucleic acid molecules, recombinant vectors, or host cells in the preparation of kits for detecting Helicobacter pylori.

[0061] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent companies. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0062] Example Method

[0063] Methods of animal immunization

[0064] Animal immunization experiments were conducted using Helicobacter pylori whole-cell protein antigen (Holmes (Beijing) Biotechnology Co., Ltd., HA025DC). The specific steps are as follows.

[0065] 1. Balb / c mice with the same weight and age were randomly divided into two groups: an aluminum adjuvant group (aluminum hydroxide adjuvant) and a non-aluminum adjuvant group.

[0066] 2. Before the experiment, pre-immune serum was collected from each mouse (pre-immune serum was collected on the fifth day by blood collection through the eyeball, and an appropriate amount of blood was collected to ensure the normal condition of the mice). The collected serum was stored at -80℃.

[0067] 3. Preparation of aluminum adjuvant (aluminum hydroxide adjuvant) group: Before immunization, each antigen was diluted in 75 μL PBS to the corresponding dose (75 μg / mouse) and mixed with alum adjuvant (1 mg / mouse) at a volume ratio of antigen:adjuvant = 3:1 (i.e., 25 μL adjuvant was added to 75 μL of immunogen dilution). The adjuvant was shaken well before use, and the 25 μL of adjuvant was slowly added dropwise to the immunogen solution. After thorough mixing of the adjuvant and immunogen dilution, the two solutions were allowed to mix thoroughly for 30 minutes to allow for effective adsorption of the antigen. Subsequent procedures were performed according to the animal immunization experiment procedures.

[0068] 4. Preparation method for the group without aluminum adjuvant: The antigen was diluted in 100 μL PBS to the corresponding dose in Table 1 (175 μg / mouse), and 100 μL of immunogen was added. Subsequent procedures were carried out according to the procedures for immunizing animal experiments.

[0069] 5. Subcutaneous injection at 2-week intervals: The experiment was designed as a 3-immunization method, but blood was collected from the eyeballs 7 days after each immunization injection. Part of the mouse supernatant was obtained by centrifugation and the serum titer was first tested. 7 days after the last immunization, the maximum blood volume was collected from the heart, and the supernatant was obtained by centrifugation and stored at -80℃.

[0070] Methods for detecting the antigen-binding ability (antibody titer) of antibodies

[0071] The antigen-binding activity of antibodies was detected using enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows.

[0072] 1. Base plate coating: Dilute Helicobacter pylori urease antigen HP urease Antigen (Feipeng Biotechnology Co., Ltd., HP-Ag1) to 3μg / mL with coating diluent, add 100μL of the prepared coating solution to each well, and place in a 4℃ refrigerator for 24h.

[0073] 2. After 24 hours, remove it from the refrigerator and place it at 37°C for 30 minutes to equilibrate. Then discard the liquid in the well. Wash the well with washing solution three times, for 3 minutes each time.

[0074] 3. Blocking the enzyme-labeled reaction wells: Add 200 μL of 5% fetal bovine serum to each well and incubate at 37°C for 90 min. After blocking, wash the wells three times with washing buffer for 3 min each time.

[0075] 4. Add the sample to be tested: Dilute the sample to be tested according to the required ratio, add 100 μL of the diluted sample to each enzyme-labeled reaction well, and incubate at 37℃ for 90 min; wash the wells three times with washing buffer for 3 min each time.

[0076] 5. Add enzyme-labeled antibody: Add the appropriate concentration of secondary antibody according to the instructions; incubate at 37℃ for 90 min, and wash with 100 μL per well as before.

[0077] 6. Add substrate solution: Add 100 μL of substrate per well and incubate at 37°C in the dark for 15–30 min.

[0078] 7. Termination of reaction: Add 50 μL of stop solution to each well to terminate the reaction, and measure the absorbance OD450 value within 20 min.

[0079] Methods for cell fusion and screening and cloning of fused cells

[0080] Immunosplenic cells from successfully immunized mice were extracted and fused with the mouse myeloma cell line SP2 / 0. The fused cells were screened using HAT selective medium (containing hypoxanthine, aminopterin, and thymine), and the fused cells were subjected to two rounds of ELISA positive screening and subcloning to obtain a hybridoma cell line that stably secretes monoclonal antibodies against Helicobacter pylori urease.

[0081] The specific methods for cell fusion and the screening and cloning of fused cells should be known to those skilled in the art, and can be found, for example, in: Wang Shihua et al., Antibody Technology, 2nd Edition [M]. Beijing: Science Press, 2018.11.

[0082] Establishment of hybridoma cell lines with monoclonal antibodies against Helicobacter pylori urease

[0083] Preparation of cell fusion spare mice

[0084] Healthy female Balb / c mice aged 6-8 weeks were selected and immunized according to the aforementioned animal immunization method. The serum antibody titer data of mice numbered A0, A1, and A2 (A0 and A1 belong to the aluminum adjuvant group, and A2 belongs to the non-aluminum adjuvant group) after 3 immunizations are shown in Table 1.

[0085] Table 1. Serum antibody titer test data

[0086]

[0087] As shown in Table 1, the orbital blood titers of the three mice after the third immunization were all >62500, and all reached more than 50% at a ratio of 1:12500, which can be used for further cell fusion.

[0088] Cell fusion and screening of hybridoma cell lines

[0089] Immunosplenic cells from three mice (A0, A1, and A2) were fused with the myeloma cell line SP2 / 0 for cell fusion, followed by screening and cloning of the fused cells. Specifically:

[0090] Fifteen positive wells from the A0 mouse fusion screening were selected for subcloning. Following fusion screening, 61 positive wells with OD450 values ​​>2.2 were selected. These positive wells were serially diluted to determine antibody titers, and then subjected to a second subcloning screening. Three hybridoma cell lines were obtained, numbered A0-1, A0-2, and A0-3, respectively.

[0091] Eleven positive wells from the A1 mouse fusion screening were selected for subcloning. After further fusion screening, three positive wells with OD450 values ​​>2.1 were selected. These positive wells were serially diluted to determine antibody titers, followed by a second and third subcloning screening. Four hybridoma cell lines were obtained, numbered A1-1, A1-2, A1-3, and A1-4.

[0092] Eighteen positive wells from the A2 mouse fusion screening were selected for subcloning, followed by a second and third subcloning screening. Three hybridoma cell lines were obtained, numbered A2-1, A2-2, and A2-3, respectively.

[0093] Identification of the immunological characteristics of monoclonal antibodies

[0094] (1) Ascites titer detection of monoclonal antibody: Each hybridoma cell line was injected into the peritoneal cavity of F1 generation mice, and a total of 10 ascites samples were collected. The titer data of all ascites samples are shown in Table 2 below.

[0095] Table 2. Ascites antibody titer detection data

[0096]

[0097] (2) Antibody titer test after purification: The ascites fluid was purified by 3.3% octanoic acid-thiamine precipitation method to obtain 10 candidate monoclonal antibodies corresponding to hybridoma cell lines. The titer test data of all candidate monoclonal antibodies are shown in Table 3.

[0098] Table 3. Potency data of candidate monoclonal antibodies

[0099]

[0100]

[0101] The data above show that the 10 candidate monoclonal antibodies have good specific binding ability to urease antigen.

[0102] The ascites fluid was purified using a Protein A / G antibody purification column. The purified antibody had an ELISA titer >1:128,000 and a purity >90%.

[0103] Validation of candidate monoclonal antibodies in Helicobacter pylori antibody detection kit

[0104] Candidate anti-Helicobacter pylori urease monoclonal antibodies were validated using an immunogold platform.

[0105] Experimental group: Candidate monoclonal antibodies numbered A2-1 and A2-3.

[0106] Negative control group: lysis buffer of whole blood.

[0107] The experimental group and negative control group were tested using a Helicobacter pylori antibody detection kit (Hangzhou Anxu Biotechnology Co., Ltd.). After reagent addition, the results were detected using a POCT instrument ACG1000 (Hangzhou Anxu Biotechnology Co., Ltd., ID-A003). The results are shown in Table 4.

[0108] Table 4. Test Results

[0109]

[0110] Note: G1-G10 indicate the level of color intensity of the test strip. The higher the number, the darker the color; + / - indicates a slightly darker or lighter color than that level.

[0111] As shown in Table 4, candidate monoclonal antibodies A2-1 and A2-3 can be detected using the Helicobacter pylori antibody detection kit, indicating that they can specifically recognize Helicobacter pylori. These candidate monoclonal antibodies can be used in kits for detecting Helicobacter pylori. For example, they can be used as detection reagents in Helicobacter pylori antigen detection kits or as quality control materials in Helicobacter pylori antibody detection kits.

[0112] Sequencing analysis of the monoclonal antibody anti-HP-urease-mab1

[0113] The monoclonal antibody A2-3 was named anti-HP-urease-mab1, and its sequencing analysis was performed. The specific steps are as follows.

[0114] (1) Design primers for amplifying the heavy chain variable region (VH) and light chain variable region (VL) genes. The primers for amplifying the VH and VL genes are as follows:

[0115] Forward primer for the variable region of the heavy chain (VH-FOR): GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG (SEQ ID NO:15);

[0116] Heavy chain variable region reverse primer (VH-BACK): GGAAGGTGTGCACACCGCTGGAC (SEQ ID NO:16);

[0117] Light chain variable region forward primer (VL-FOR): GATGGTGGGAAGATGGATACAGTT (SEQ ID NO:17);

[0118] Light chain variable region reverse primer (VL-BACK): TCTTGTTGCTCTGGTTYCCAG (SEQ ID NO:18).

[0119] (2) Take approximately 10 hybridoma cell lines in the logarithmic growth phase that secrete anti-HP-urease-mab1. 7 Total RNA was extracted from cells according to the instructions of the Trizol RNA Extraction Kit (TAKARA, 9767). The total RNA was used as a template to reverse transcribe and synthesize the first strand of cDNA. The VH / VL gene of the antibody was amplified by PCR using the amplification product as a template.

[0120] (3) The VH and VL fragments of anti-HP-urease-mab1 were recovered and sequenced.

[0121] The resulting sequence is as follows:

[0122] Heavy chain variable region coding sequence / anti-HP-urease-mab1-VH:312bp

[0123] CCTGGGGCCTCAGTGAAGATGTCCTGCAGGGCTTCTGACTACACCTTTACTAACTC

[0124] CTGGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGGTTGGATACATT

[0125] AATCCTAGCACTGGTTCTACTGACTACAATCAGAAGTTCAGGGACAAGGCCACTTTGA

[0126] CTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGA

[0127] CTCTGCAGTCTATTACTGTGCAAGCGAGGAGTACGACGGCTTTGACTACTGGGGCCAA

[0128] GGCACCACTCTCACAGTCTCCTCA(SEQ ID NO:8);

[0129] Heavy chain variable region amino acid sequence / anti-HP-urease-mab1-VH-protein: 104aa

[0130] PGASVKMSCRASDYTFTNSWMHWVKQRPGQGLEWVGYINPSTGSTDYNQKFRDKA TLTADKSSSTAYMQLSSLTSEDSAVYYCASEEYDGFDYWGQGTTLTVSS(SEQ ID NO:1);

[0131] Light chain variable region coding sequence / anti-HP-urease-mab1 LVκ: 318bp

[0132] GATATTGTGATAACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAACTCAAGTATAATTTACATGCATTGGTACCAGCAGAAGTCAGGCACGTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGC TTCTGGAGTCCCTGCTCGTTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAGCCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAAATAAAA(SEQ ID NO:12);

[0133] Light chain variable region amino acid sequence / anti-HP-urease-mab1 LVκ protein: 106aa

[0134] DIVITQSPAIMSASPGEKVTMTCSANSSIIYMHWYQQKSGTSPKRWIYDTSKLASGVPA RFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSSPYTFGGGTKLEIK (SEQ ID NO: 5).

[0135] The beneficial effects of the antibody or antigen-binding fragment that binds to Helicobacter pylori antigen protein urease and its related applications disclosed in the embodiments of this specification include, but are not limited to: (1) The antibody or antigen-binding fragment that binds to Helicobacter pylori antigen protein urease provided in the embodiments of this specification has the characteristics of high purity, high specificity and sensitivity, and can improve the reliability of detection results as a raw material in immunological detection methods or products; (2) The antibody or antigen-binding fragment that binds to Helicobacter pylori antigen protein urease provided in the embodiments of this specification can be used for immunological detection such as immunoblotting and immunofluorescence, for example, it can be used in colloidal gold detection platforms as a detection reagent to detect whether Helicobacter pylori infection exists in samples; (3) The good specific binding ability of the antibody or antigen-binding fragment that binds to Helicobacter pylori antigen protein urease provided in the embodiments of this specification to the antigen protein can provide strong support for the development of drugs for the treatment of Helicobacter pylori infection. It should be noted that different embodiments may produce different beneficial effects. In different embodiments, the beneficial effects that may be produced may be any one or a combination of the above, or any other possible beneficial effects.

[0136] Those skilled in the art should understand that the above embodiments are merely illustrative of the present invention and do not constitute a limitation thereof. Any modifications, equivalent substitutions, and variations made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0137] Furthermore, this specification uses specific terms to describe embodiments thereof. For example, "an embodiment," "one embodiment," and / or "some embodiments" refer to a particular feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "an embodiment," "one embodiment," or "an alternative embodiment" in different locations throughout this specification do not necessarily refer to the same embodiment. Moreover, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0138] In some embodiments, numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of embodiments are modified in some examples with the terms "approximately," "approximately," or "generally." Unless otherwise stated, "approximately," "approximately," or "generally" indicates that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may be changed depending on the characteristics required by individual embodiments. In some embodiments, numerical parameters should take into account specified significant digits and employ a general method of digit reservation. Although the numerical ranges and parameters used to confirm their breadth of range in some embodiments of this specification are approximate values, in specific embodiments, such values ​​are set as precisely as feasible.

[0139] For each patent, patent application, patent application publication, and other material, such as articles, books, specifications, publications, and documents, referenced in this specification, the entire contents of which are incorporated herein by reference. This excludes historical application documents that are inconsistent with or conflict with the content of this specification, as well as documents that limit the broadest scope of the claims in this specification (currently or subsequently appended to this specification). It should be noted that in the event of any inconsistency or conflict between the descriptions, definitions, and / or terminology used in the supplementary materials to this specification and the content of this specification, the descriptions, definitions, and / or terminology used in this specification shall prevail.

[0140] Finally, it should be understood that the embodiments described in this specification are merely illustrative of the principles of the embodiments described herein. Other variations may also fall within the scope of this specification. Therefore, alternative configurations of the embodiments described herein are intended to be illustrative rather than limiting, and should be considered consistent with the teachings of this specification. Accordingly, the embodiments described herein are not limited to those explicitly introduced and described herein.

Claims

1. An antibody or antigen-binding fragment that binds to Helicobacter pylori antigen protein urease, characterized in that, The antibody or antigen-binding fragment includes: The heavy chain variable region includes CDRH1 with the amino acid sequence shown in SEQ ID NO: 2, CDRH2 with the amino acid sequence shown in SEQ ID NO: 3, and CDRH3 with the amino acid sequence shown in SEQ ID NO: 4; The light chain variable region includes CDRL1 with the amino acid sequence shown in SEQ ID NO: 6, CDRL2 with the amino acid sequence DTS, and CDRL3 with the amino acid sequence shown in SEQ ID NO:

7.

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

5.

3. The antibody or antigen-binding fragment as described in claim 1, characterized in that, The antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

4. The antibody or antigen-binding fragment as described in claim 1, characterized in that, The antigen-binding fragments are selected from the following group: Fab, Fab', F(ab')2, Fv, scFv, dsFv.

5. A nucleic acid molecule having a nucleotide sequence encoding an antibody or antigen-binding fragment as described in any one of claims 1-4.

6. The nucleic acid molecule as described in claim 5, characterized in that, The nucleic acid molecules include: The nucleotide sequences encoding the variable regions of the heavy chain include the coding sequences for CDRH1 as shown in SEQ ID NO: 9, CDRH2 as shown in SEQ ID NO: 10, and CDRH3 as shown in SEQ ID NO: 11; The nucleotide sequences encoding the variable regions of the light chains include the coding sequence of CDRL1 as shown in SEQ ID NO: 13, the coding sequence of CDRL2 with the sequence GACACATCC, and the coding sequence of CDRL3 as shown in SEQ ID NO:

14.

7. The nucleic acid molecule as described in claim 5, characterized in that, The nucleotide sequence encoding VH is shown in SEQ ID NO:8; the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO:

12.

8. A recombinant vector comprising a nucleic acid molecule as described in any one of claims 5-7.

9. A host cell comprising a nucleic acid molecule as described in any one of claims 5-7 or a recombinant vector as described in claim 8.

10. A pharmaceutical composition comprising an antibody or antigen-binding fragment as described in any one of claims 1-4.

11. A kit for detecting Helicobacter pylori, comprising an antibody or antigen-binding fragment as described in any one of claims 1-4.

12. The use of the antibody or antigen-binding fragment of any one of claims 1-4, the nucleic acid molecule of any one of claims 5-7, the recombinant vector of claim 8, or the host cell of claim 9 in the preparation of a kit for detecting Helicobacter pylori.

Images