Helicobacter pylori bispecific antibody

Abstract

The helicobacter pylori bispecific antibody comprises a first antibody, a connector and a second antibody, the first antibody (VHH) is specifically combined with a helicobacter pylori urease B subunit (UreB) antigen, the second antibody (ScFv) is specifically combined with a helicobacter pylori vacuolar toxin protein (VacA) antigen, the first antibody (VHH) is connected with the second antibody (ScFv) through the connector, and the second antibody (ScFv) is connected with the second antibody (ScFv) through the connector. The whole or part of the connector is a heavy chain constant region derived from humanized lgG1 or lgG2 or lgG3 or lgG4; the bispecific antibody can significantly reduce adhesion of helicobacter pylori in the stomach, and reduce infiltration of inflammatory cells and damage of stomach tissues; the helicobacter pylori bispecific antibody can quickly and obviously reduce the infection ability of helicobacter pylori, and does not generate other harmful effects on stomach tissues and cells.

Description

Technical Field

[0001] The present invention relates to the field of antibodies against Helicobacter pylori, and in particular to a Helicobacter pylori bispecific antibody and uses thereof. Background Art

[0002] Helicobacter pylori (Hp) is a Gram-negative spiral bacterium isolated from the gastric mucosa in 1982. It is mainly transmitted among people through the oral-oral route. Helicobacter pylori is a highly contagious gastrointestinal pathogen that can colonize in the human gastric epithelium, causing gastric mucosal inflammation. In some patients, it can also cause a series of diseases such as peptic ulcers and gastric cancer.

[0003] Antibiotic treatment is currently the main treatment for Helicobacter pylori, but due to the widespread abuse of antibiotics and the easy recurrence of Helicobacter pylori infection, drug resistance has become a major challenge for antibiotic treatment. New methods for the treatment of Helicobacter pylori mainly include: probiotic adjuvant therapy, Helicobacter pylori vaccine and Helicobacter pylori antibody. However, probiotics cannot completely cure Helicobacter pylori and can only be used as an auxiliary means. Vaccines require multiple immunizations and are slow to take effect, so they are more suitable for preventing infection.

[0004] Therapeutic antibodies can specifically bind to target proteins and neutralize the activity of target proteins, thereby reducing the virulence of Helicobacter pylori in a short period of time or even killing Helicobacter pylori. However, the mechanism of antibody elimination of Helicobacter pylori is still unclear, and the effects are also uneven. There are few studies on existing therapeutic antibodies, and they are all concentrated on chicken antibodies IgY, with limited types and limited effects. There is a need for more effective therapeutic antibodies, especially in the field of genetic engineering, where there is still a lack of Helicobacter pylori therapeutic antibodies. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a Helicobacter pylori bispecific antibody in the field of genetic engineering, which can simultaneously neutralize two virulence proteins to achieve a better therapeutic effect.

[0006] A Helicobacter pylori bispecific antibody comprises a first antibody, a connector and a second antibody; the first antibody (VHH) is connected to the second antibody (ScFv) via the connector;

[0007] The first antibody (VHH) specifically binds to the Helicobacter pylori urease B subunit (UreB) antigen, the first antibody (VHH) is a nanobody, and all of them are derived from the heavy chain variable region of IgG1 or IgG2 or IgG3 or IgG4;

[0008] The second antibody (ScFv) specifically binds to the Helicobacter pylori vacuolating toxin protein (VacA) antigen. The second antibody (ScFv) is a single-chain antibody, and the whole or part of it is derived from IgG1, IgG2, IgG3 or IgG4.

[0009] Preferably, the second antibody (ScFv) is composed of a light chain variable region (VL), a linker peptide (Linker) and a heavy chain variable region (VH);

[0010] The light chain variable region (VL) is connected to the heavy chain variable region (VH) through a linker peptide;

[0011] The light chain variable region (VL) is derived in whole or in part from the light chain variable region of IgG1, IgG2, IgG3, or IgG4; the light chain variable region (VL) comprises a sequence as shown in SEA ID NO.1;

[0012] The heavy chain variable region (VH) is derived in whole or in part from the heavy chain variable region of IgG1, IgG2, IgG3 or IgG4; the heavy chain variable region (VH) comprises the sequence shown in SEA ID NO.2.

[0013] Preferably,

[0014] The linker peptide sequence is shown in SEA ID NO.3;

[0015] That is, the sequence of the second antibody (ScFv) is as shown in SEA ID NO.4;

[0016] The sequence of the first antibody (VHH) is shown in SEA ID NO.5.

[0017] Preferably, the second antibody (ScFv) comprises a light chain variable region (VL), a linker peptide (Linker) and a heavy chain variable region (VH) from N-terminus to C-terminus;

[0018] Alternatively, the second antibody (ScFv) comprises a heavy chain variable region (VH), a linker peptide (Linker) and a light chain variable region (VL) from N-terminus to C-terminus.

[0019] Preferably, the linker is derived in whole or in part from the heavy chain constant region of humanized IgG1, IgG2, IgG3 or IgG4;

[0020] The connector includes a humanized IgG hinge peptide, a humanized IgG Fc domain and a linker peptide;

[0021] The C-terminus of the first antibody (VHH) is connected to the humanized IgG Fc domain via a humanized IgG hinge peptide;

[0022] The N-terminus of the second antibody (ScFv) is connected to the humanized IgG Fc domain via a linker peptide.

[0023] Preferably,

[0024] The humanized IgG hinge peptide sequence is shown in SEA ID NO.6;

[0025] The linker peptide sequence is shown in SEA ID NO.3;

[0026] The humanized IgG Fc domain sequence is shown in SEA ID NO.7.

[0027] The Helicobacter pylori bispecific antibody according to claims 3 and 6, characterized in that:

[0028] The nucleotide sequence of the light chain variable region (VL) of the second antibody (ScFv) is shown in SEA ID NO.8;

[0029] The nucleotide sequence of the heavy chain variable region (VH) of the second antibody (ScFv) is shown in SEA ID NO.9;

[0030] The nucleotide sequence of the second antibody (ScFv) is shown in SEA ID NO.19;

[0031] The nucleotide sequence of the linker peptide is shown in SEA ID NO.11;

[0032] The nucleotide sequence of the humanized IgG hinge peptide is shown in SEA ID NO.12;

[0033] The nucleotide sequence of the humanized IgG Fc domain is shown in SEA ID NO.13;

[0034] The overall nucleotide sequence of the Helicobacter pylori bispecific antibody is shown in SEA ID NO.14.

[0035] A homologous dimer of a Helicobacter pylori bispecific antibody. When the Helicobacter pylori bispecific antibody is expressed in a host cell, the humanized IgG hinge peptide undergoes homologous dimerization to form a homologous dimer of the Helicobacter pylori bispecific antibody.

[0036] Use of at least one of the Helicobacter pylori bispecific antibodies described above or homologous dimers of the Helicobacter pylori bispecific antibodies described above in the preparation of a medicament for treating Helicobacter pylori.

[0037] A pharmaceutical composition comprising at least one of the Helicobacter pylori bispecific antibodies described above and a homodimer of the Helicobacter pylori bispecific antibody.

[0038] A detection product, comprising at least one of the Helicobacter pylori bispecific antibodies described above or homologous dimers of the Helicobacter pylori bispecific antibodies, for detecting the presence or amount of Helicobacter pylori in a sample.

[0039] The amino acid sequence in any of the above sequences has a high homology with the original sequence, preferably more than 80% homology, after substitution, deletion or addition of one or several amino acids, and is a derived protein that retains the original properties and functions of the sequence. It is believed that these mutant sequences can achieve the same function and the same effect as the original sequence and are the same protein.

[0040] The nucleotide sequence in any of the above sequences has been replaced, deleted or added with one or several nucleotides, and has a high homology with the original sequence, preferably more than 80% homology, and a derived gene that retains the original properties and functions of the sequence. It is believed that these mutant sequences can achieve the same function and the same effect as the original sequence and are the same gene.

[0041] The infection and pathogenicity of Helicobacter pylori depend on its multiple virulence factors. Based on our laboratory's many years of experience in Helicobacter pylori research, we selected nanoantibodies targeting UreB and single-chain antibodies targeting VacA from among the numerous Helicobacter pylori virulence factors, added humanized linkers, and designed them into bispecific antibodies. The purpose is to effectively inhibit the colonization and virulence of Helicobacter pylori and provide adequate protection for the human body.

[0042] The designed Helicobacter pylori bispecific antibody (BsUV) has a stable structure and can be expressed normally. Both antibodies have antigen binding ability. Through the co-culture experiment of Helicobacter pylori bispecific antibody and Helicobacter pylori infected normal human gastric epithelial cells GES-1, it can be seen that the Helicobacter pylori bispecific antibody can inhibit the infection of Helicobacter pylori (Hp) to human normal gastric epithelial cells GES-1 in a very short time.

[0043] According to the results of mouse experiments, this Helicobacter pylori bispecific antibody can significantly reduce the adhesion of Helicobacter pylori in the stomach, reduce the infiltration of inflammatory cells and damage to gastric tissue; the Helicobacter pylori bispecific antibody can quickly and significantly reduce the infection ability of Helicobacter pylori, and will not have other harmful effects on gastric tissue and cells. In summary, it can be seen that this Helicobacter pylori bispecific antibody is very successfully designed and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is the result of three-dimensional modeling of Helicobacter pylori bispecific antibody (BsUV).

[0045] Figure 2 It is the analysis result of local distance difference test (LDDT) of Helicobacter pylori bispecific antibody (BsUV) three-dimensional modeling.

[0046] Figure 3 It is the result of Ramachandran Plot analysis of three-dimensional modeling of Helicobacter pylori bispecific antibody (BsUV).

[0047] Figure 4 It is the result of docking of Helicobacter pylori urease B subunit (UreB) antigen and Helicobacter pylori bispecific antibody (BsUV).

[0048] Figure 5 It is the result of docking of Helicobacter pylori vacuolating toxin protein (VacA) antigen and Helicobacter pylori bispecific antibody (BsUV).

[0049] Figure 6 This is a diagram of the pPICZαA vector plasmid structure.

[0050] Figure 7 It is the structure diagram of the recombinant plasmid pPICZαA-BsUV.

[0051] Figure 8 This is the result of double digestion of the recombinant plasmid pPICZαA-BsUV with Bgl II / Sal I and electrophoresis detection.

[0052] Fig. 9 This is the sequencing result of the recombinant plasmid pPICZαA-BsUV after double digestion with Bgl II / Sal I.

[0053] Fig.10 This is the result of colony PCR identification of the eukaryotic expression vector X33-pPICZαA-BsUV.

[0054] Fig.11This is the result of Western Blot identification of the expression of Helicobacter pylori bispecific antibody (BsUV).

[0055] Fig.12 It is the purification result of Helicobacter pylori bispecific antibody (BsUV).

[0056] Fig.13 It is the result of Western Blot detection of the binding ability of Helicobacter pylori bispecific antibody (BsUV) with Helicobacter pylori vacuolating toxin protein (VacA) antigen and Helicobacter pylori urease B subunit (UreB) antigen.

[0057] Fig.14 This is the result of indirect ELISA testing the binding ability of BsUV to antigen UreB. (***, p<0.001; **, p<0.01; *, p<0.05)

[0058] Fig.15 This is the result of indirect ELISA to detect the binding ability of BsUV and antigen VacA. (***, p<0.001; **, p<0.01; *, p<0.05)

[0059] Fig.16 The expression levels of Hp-16s-RNA and COX-2 in the GES-1 cell model of Hp infection treated with antibodies. (***, p<0.001; **, p<0.01; *, p<0.05)

[0060] Fig.17 Giemsa staining was used to identify the effect of BsUV on Hp adhesion function.

[0061] Fig.18 This is the identification result of the Hp infection mouse model.

[0062] Fig.19 This is the result of RT-qPCR detection of the expression of Hp-16sRNA and COX-2 in the stomach of each group of mice.

[0063] Fig. 20 This is the result of ELISA detection of the expression of inflammatory factors COX-2 and TNF-α in the stomach of each group of mice.

[0064] Fig.21 HE staining was used to identify the damage of mouse gastric tissue.

[0065] Fig. 22 Immunohistochemistry was used to identify Hp infection in mouse gastric tissue. DETAILED DESCRIPTION

[0066] The technical solutions and technical effects of the embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings of the present invention.

[0067] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field (for example, refer to "Molecular Cloning Experiment Guide" 3rd edition, written by J. Sambrook et al., translated by Huang Peitang et al., Science Press) or the product instructions are used.

[0068] The infection and pathogenicity of Helicobacter pylori depend on its multiple virulence factors. After entering the stomach, Helicobacter pylori first faces a harsh acidic environment with a low pH value, and the first to play a role are urease and flagella. Urease can catalyze urea to produce ammonia, which can form an ammonia cloud around Helicobacter pylori, helping Helicobacter pylori neutralize the acidic environment in the stomach. Urease is mainly composed of two subunits, UreA and UreB, of which UreB is the active center of urease. Urease plays a vital role in Helicobacter pylori's recognition of environmental pH, colonization in the stomach and long-term existence; the presence of monopolar flagella gives Helicobacter pylori a strong motility, which can penetrate the mucus layer covering the surface of the gastric mucosa, and is one of the key colonization factors of Helicobacter pylori. After entering the gastric mucosa, Helicobacter pylori can secrete a variety of outer membrane proteins (OMPs) as adhesins to assist the adhesion and colonization of Helicobacter pylori. After successful colonization, Helicobacter pylori can secrete a variety of soluble components to destroy the gastric barrier and stimulate a variety of cellular pathways, leading to varying degrees of tissue damage and other related diseases.

[0069] VacA is one of the most important virulence factors in Helicobacter pylori-induced host cell pathology, and is named for its ability to induce vacuole formation in eukaryotic cells. Vacuolating toxins are present in all known strains of Helicobacter pylori. Vacuolating toxins have multiple functions. First, vacuolating toxins can cause vacuolization of target cells; second, vacuolating toxins can inhibit cell proliferation, thereby affecting the repair of gastric mucosal cells; third, vacuolating toxins can cause mitochondrial depolarization and induce cell apoptosis; fourth, vacuolating toxins can also inhibit the activation of T lymphocytes in the lamina propria to destroy the immune response; fifth, VacA inhibits the expression of IL-23 in dendritic cells and induces the expression of IL-10 and TGF-β in macrophages. Therefore, Helicobacter pylori can create a tolerant environment through the immunomodulatory activity of VacA. This may cause T cells to deviate from the response of Tregs. This mechanism helps Helicobacter pylori to persist in an environment that affects host cell immunity. Studies have shown that VacA can inhibit autophagy in gastric mucosal epithelial cells, resulting in a weakened ability of cells to clear Helicobacter pylori and its virulence factors. In addition, the toxicity of VacA is affected by urease. The ammonia produced by urease-catalyzed urea can improve the stability of VacA and increase the toxicity of VacA.

[0070] Helicobacter pylori bispecific antibodies (BsUV) targeting Helicobacter pylori urease B subunit (UreB) and Helicobacter pylori vacuolating toxin protein (VacA) are designed to effectively inhibit the colonization and virulence of Helicobacter pylori and provide adequate protection for the human body. On the one hand, targeting the antigen UreB can reduce the adhesion and survival ability of Helicobacter pylori from the source and inhibit the infection of Helicobacter pylori to the gastric mucosa; on the other hand, targeting the antigen VacA can reduce the virulence of Helicobacter pylori and reduce cell damage, apoptosis and immune escape caused by VacA. Reducing the virulence of VacA can protect gastric mucosal cells and reduce gastric tissue inflammation; at the same time, humanized IgG is selected to increase the content of Helicobacter pylori therapeutic antibodies in serum and improve the ability of the immune system to kill Helicobacter pylori. The inventor also believes that inhibiting urease activity can not only reduce the colonization ability of Helicobacter pylori from the source, but also reduce the virulence of Helicobacter pylori.

[0071] From the existing gene library in the laboratory, various first antibody (VHH) genes that specifically bind to the Helicobacter pylori urease B subunit (UreB) antigen and second antibody (ScFv) genes that specifically bind to the Helicobacter pylori vacuolating toxin protein (VacA) antigen were retrieved, and suitable linkers were designed. Through software screening, the Helicobacter pylori bispecific antibody (BsUV) was finally determined.

[0072] Specifically, trRosetta was used to perform three-dimensional modeling on the Helicobacter pylori bispecific antibody (BsUV), and the results were as follows: Figure 1 As shown in Figure 2, the three-dimensional modeling score (Tm-score) of BsUV is 0.534, which meets the topological standard. The results of the local distance difference test (LDDT) of the three-dimensional modeling of Helicobacter pylori bispecific antibody (BsUV) are shown in Figure 2. Figure 2 As shown in Figure 2, the credibility of each residue is described. The higher the score, the higher the confidence. Finally, the Pull-out diagram was used to evaluate the conformation of BsUV. The Pull-out diagram results of the Helicobacter pylori bispecific antibody (BsUV) are shown in Figure 2. Figure 3 As shown, 90.2% of the residues fall in the optimal region, 9.2% of the residues fall in the allowed region, and less than 1% of the residues fall in the unallowed region, meeting the requirements of stereochemical rules. In summary, the predicted three-dimensional structure of the Helicobacter pylori bispecific antibody (BsUV) has sufficient confidence and can be used for subsequent protein docking.

[0073] The spatial structural information of Helicobacter pylori urease B subunit (UreB) antigen (PDBID: 6ZJA) and Helicobacter pylori vacuolating toxin protein (VacA) antigen domain (PDB ID: 2QV3) were obtained from the RCSB protein database (https: / / www.rcsb.org / ) and input into Z-Dock (https: / / zdock.umassmed.edu / ) together with the predicted model of Helicobacter pylori bispecific antibody (BsUV) for protein docking. The binding surface residues of the protein docking model were analyzed using the bioinformatics software PyMOL to predict the binding ability and binding mode of Helicobacter pylori bispecific antibody (BsUV) with Helicobacter pylori urease B subunit (UreB) antigen and Helicobacter pylori vacuolating toxin protein (VacA) antigen, respectively.

[0074] The results of protein-protein docking between Helicobacter pylori bispecific antibody (BsUV) and Helicobacter pylori urease B subunit (UreB) antigen are shown in Figure 2. Figure 4 As shown, the sky blue is UreB, the green is BsUV, the ball-and-stick model is the binding site between UreB and BsUV, and the yellow dotted line is the hydrogen bond. The protein-protein docking results of Helicobacter pylori bispecific antibody (BsUV) and Helicobacter pylori vacuolating toxin protein (VacA) antigen are shown in Figure 5 As shown, the sky blue is the VacA-P55 domain, the green is BsUV, the ball-and-stick model is the binding site of VacA-P55 and BsUV, the yellow dotted line is the hydrogen bond, and the blue dotted line is the π bond interaction. It can be seen that the structure of the Helicobacter pylori bispecific antibody (BsUV) does not affect its function of binding to the antigen.

[0075] Based on the above design and structural simulation verification process, the inventors determined a Helicobacter pylori bispecific antibody, including a first antibody, a linker and a second antibody; the first antibody (VHH) is connected to the second antibody (ScFv) through a linker.

[0076] The first antibody (VHH) specifically binds to the Helicobacter pylori urease B subunit (UreB) antigen. The first antibody (VHH) is a nanobody and all are derived from the heavy chain variable region of IgG1 or IgG2 or IgG3 or IgG4; the sequence of the first antibody (VHH) is shown in SEA ID NO.5.

[0077] The second antibody (ScFv) specifically binds to the Helicobacter pylori vacuolating toxin protein (VacA) antigen. The second antibody (ScFv) is a single-chain antibody, and the whole or part of it is derived from IgG1, IgG2, IgG3 or IgG4.

[0078] Specifically, the second antibody (ScFv) is composed of a light chain variable region (VL), a linker peptide (Linker) and a heavy chain variable region (VH); the light chain variable region (VL) is connected to the heavy chain variable region (VH) through a linker peptide (Linker).

[0079] The second antibody (ScFv) consists of a light chain variable region (VL), a linker peptide (Linker) and a heavy chain variable region (VH) from N-terminus to C-terminus; alternatively, the second antibody (ScFv) consists of a heavy chain variable region (VH), a linker peptide (Linker) and a light chain variable region (VL) from N-terminus to C-terminus.

[0080] The light chain variable region (VL) of the second antibody (ScFv) is derived in whole or in part from the light chain variable region of IgG1, IgG2, IgG3 or IgG4; the light chain variable region (VL) comprises a sequence as shown in SEA ID NO.1; the nucleotide sequence of the light chain variable region (VL) of the second antibody (ScFv) is shown in SEA ID NO.8.

[0081] The second antibody (ScFv) heavy chain variable region (VH) is derived in whole or in part from the heavy chain variable region of IgG1 or IgG2 or IgG3 or IgG4; the heavy chain variable region (VH) comprises the sequence shown in SEA ID NO.2; the nucleotide sequence of the second antibody (ScFv) heavy chain variable region (VH) is shown in SEA ID NO.9.

[0082] The linker peptide sequence is shown in SEA ID NO.3; the nucleotide sequence of the linker peptide is shown in SEA ID NO.11.

[0083] In general, the sequence of the second antibody (ScFv) is shown in SEA ID NO.4; the nucleotide sequence of the second antibody (ScFv) is shown in SEA ID NO.10.

[0084] The linker of the Helicobacter pylori bispecific antibody is derived in whole or in part from the heavy chain constant region of humanized IgG1, IgG2, IgG3 or IgG4.

[0085] Specifically, the connector includes a humanized IgG hinge peptide, a humanized IgG Fc domain and a linker peptide; the C-terminus of the first antibody (VHH) is connected to the humanized IgG Fc domain through the humanized IgG hinge peptide; the N-terminus of the second antibody (ScFv) is connected to the humanized IgG Fc domain through the linker peptide.

[0086] The humanized IgG hinge peptide sequence is shown in SEA ID NO.6; the nucleotide sequence of the humanized IgG hinge peptide is shown in SEA ID NO.12.

[0087] The linker peptide sequence is shown in SEA ID NO.3; the linker peptide nucleotide sequence is shown in SEA ID NO.11.

[0088] The humanized IgG Fc domain sequence is shown in SEA ID NO.7; the nucleotide sequence of the humanized IgG Fc domain is shown in SEA ID NO.13.

[0089] In summary, the overall nucleotide sequence of the Helicobacter pylori bispecific antibody is shown in SEA ID NO.14.

[0090] The designed Helicobacter pylori bispecific antibody (BsUV) will then be specifically constructed, expressed, purified and immunologically analyzed. The specific experimental process is as follows:

[0091] The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. The vector can be transformed, transduced or transfected into a host cell so that the genetic material elements it carries can be expressed in the host cell. The vector can contain a variety of elements that control expression, such as a promoter sequence, a transcription start sequence, an enhancer sequence, a selection element and a reporter gene. In addition, the vector may also contain a replication start site. The vector may also include components that assist it in entering the cell, such as viral particles, liposomes or protein shells, but not only these substances. The vector can be selected from, but not limited to: plasmids, phagemids, cosmids, artificial chromosomes (such as yeast artificial chromosomes YAC, bacterial artificial chromosomes BAC or P1-derived artificial chromosomes PAC), bacteriophages (such as lambda phages or M13 phages) and animal viruses used as vectors, for example, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). Regarding the "host cell", that is, the host cell containing the above-mentioned vector, it can be selected from, but not limited to: prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cell models such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, etc.

[0092] The initially obtained Helicobacter pylori bispecific antibody gene was predicted for signal peptide using Signal P 4.0 and 5.1 predictions, and the signal peptide sequence was removed; the target gene was codon optimized; and the determined full gene sequence of the Helicobacter pylori bispecific antibody (BsUV) was sent to a gene company for synthesis.

[0093] Extract pPICZαA vector plasmid, pPICZαA vector plasmid as Figure 6 As shown, the pPICZαA vector plasmid and the Helicobacter pylori bispecific antibody BsUV were double-digested with EcoR I / Sac I; 37°C, 2h; the BsUV gene and pPICZαA vector double-digested products were recovered using a DNA recovery kit. The recovered ppPICZαA linearized vector and BsUV gene fragment were connected to a closed circular DNA molecule through complementary sticky ends under the action of T4 DNA ligase, namely the yeast plasmid pPICZαA-BsUV. The structure of the recombinant plasmid pPICZαA-BsUV is shown in Figure 7 shown.

[0094] The recombinant plasmid pPICZαA-BsUV was digested with double enzymes and tested by electrophoresis. Figure 8As shown in the figure, lane 1 is the yeast plasmid pPICZαA-BsUV, lane 2 is the result of double enzyme digestion, and lane M is maker; a 3008bp large fragment containing the BsUV gene and a 2279bp small fragment without the BsUV gene are formed. The double enzyme digestion result is consistent with the actual result. The sample was sent for sequencing, and the result is as follows Fig. 9 As shown, the overall nucleotide sequence of the Helicobacter pylori bispecific antibody is shown in SEA ID NO.14; the sequencing results are consistent with the prediction, and there is no frameshift mutation.

[0095] The recombinant plasmid was linearized by Sal I digestion (37°C, 5h), and electroporated into the prepared competent yeast cells X33 at 1.5kv, 200KΩ, 25μF to obtain the expression vector X33-pPICZαA-BsUV. The electroporated bacterial solution was spread on a 15cm YPD plate containing 100mg / L Zeocin and cultured at 30°C until clones grew. Eight clones were randomly selected from the plate and identified by colony PCR. The results are as follows: Fig.10 As shown, all 8 amplified fragments had bands, which were the target gene bands (1812 bp). The PCR products were then sequenced, and the expression vector X33-pPICZαA-BsUV with the correct sequencing was retained.

[0096] The expression vector X33-pPICZαA-BsUV obtained by screening was cultured with 50ml YPG medium at 30℃ and 220rpm for 1-2 days until the bacterial solution was saturated. Centrifuge at 4000rpm for 5min and discard the supernatant; resuspend the bacteria with 50ml BMMY medium, transfer to a new sterile 250ml conical flask, add methanol to a final concentration of 0.5%, and culture at 28℃ and 220rpm for 6 days, adding methanol every 24h (final concentration of 0.75%); collect the bacterial solution on the morning of the 6th day, centrifuge at 4000rpm for 5min, and collect the supernatant. The protein electrophoresis results after induced expression are as follows Fig.11 As shown in the figure, lanes 1-7 are target proteins expressed by different clones, lane 8 is a positive control, and lane M is a maker. The results show that the secreted protein contains the target protein. Clone No. 1 successfully expressed the bispecific antibody BsUV, while the expression of other clones was not obvious. In order to obtain enough target proteins, clone No. 1 was expanded for expression.

[0097] beads; balance the column with 5ml Lysis buffer (50mM NaH2PO4, 300mM NaCl, 10mM imidazole, pH=8.0, filter and sterilize. Repeat 3 times; add 50ml of the collected protein solution and pass it through the column twice; add 10ml Wash buffer (50mM NaH2PO4, 300mM NaCl, 20mM imidazole, pH=8.0, filter and sterilize.) to wash, repeat 5 times; add 5ml (Elution buffer 50mM NaH2PO4, 300mM NaCl, 250mM imidazole, pH=8.0, filter and sterilize.) to elute and collect the eluate.

[0098] After purification, WB detection was performed, and the results were as follows Fig.12 As shown, the size of the protein is 65KD. The concentration was determined by BCA method. The final concentration was about 154μg / mL.

[0099] Purified Helicobacter pylori bispecific antibody protein was obtained. In order to study whether the bispecific antibody BsUV and its components VHH and ScFv can bind to the target antigen in vitro, Western Blot and indirect ELISA methods were used to analyze their in vitro binding ability.

[0100] Western Blot is to transfer UreB antigen and VacA antigen to nitrocellulose membrane (NC) after SDS-PAGE electrophoresis at 100V for 1h, incubate in 5% skim milk powder blocking solution for 1.5 hours; react with 1:1000 purified BsUV protein for 1h, wash with PBST three times, each time for 5min; then react with HRP-labeled his tag antibody at 1:80000 at room temperature for 1h, wash with PBST three times, and develop color with color developer. Fig.13 As shown; Lane 1: coated with antigen UreB 5μg, incubated with BsUV (1:1000) as primary antibody and HRP-Anti-His Tag ((1:80000) as secondary antibody: Lane 2: coated with antigen VacA 5μg, incubated with BsUV (1:1000) as primary antibody and HRP-Anti-His Tag ((1:80000) as secondary antibody: Lane M: Maker. Western Blot showed that BsUV protein can specifically bind to UreB and VacA.

[0101] Indirect ELISA is to add UreB antigen and VacA antigen to a 96-well plate, coat overnight at 4°C; wash five times with PBST, 5 minutes each time; add 5% skim milk powder blocking solution and incubate for 1.5 hours; after washing five times with PBST, add BsUV, positive control, and negative control for 1 hour; then react with HRP-labeled his tag antibody at 1:80000 at room temperature for 1 hour, wash three times with PBST, use TMB colorimetric agent to develop for 5-10 minutes, and add Elisa stop solution to terminate the reaction. Use an enzyme reader to read the OD value at 450nm. Fig.14 and Fig.15 Indirect Elisa showed that BsUV protein could specifically bind to UreB and VacA.

[0102] After confirming that the Helicobacter pylori bispecific antibody (BsUV) can be correctly expressed and has antigen binding ability in the above experiments, the effect of treating Helicobacter pylori was then verified in cell culture and mouse models. The specific experimental process is as follows:

[0103] The effect of treating Helicobacter pylori was verified at the cell culture level. The first step was to culture the normal human gastric epithelial cells GES-1. The normal human gastric epithelial cells GES-1 in liquid nitrogen were resuscitated and cultured. The GES-1 cells frozen in the liquid nitrogen tank were taken out and quickly thawed in a 37°C water bath. After centrifuging the cells in the clean bench, the supernatant was discarded and an appropriate amount of pre-configured complete culture medium (DMEM: fetal bovine serum (FBS): penicillin-streptomycin = 89:10:1) was added and mixed. The mixed cells were transferred to a 25T culture flask and supplemented with an appropriate amount of DMEM complete culture medium (final volume of about 5-6mL). After mixing by blowing and marking, they were placed in a cell culture incubator for culture.

[0104] Then the medium of human gastric normal epithelial cells GES-1 was replaced and subcultured.

[0105] At the same time, Helicobacter pylori (Hp) was cultured to the logarithmic growth phase and the bacterial culture was collected.

[0106] In order to study the inhibitory effect of bispecific antibodies on Hp-infected GES-1 cells, Hp was co-cultured with GES-1 by co-culture technology. Specifically, GES-1 cells were cultured until their cell confluence reached 80%, and the cells were counted using a hemocytometer, and 3×106 cells / well were added to a six-well plate. After the cells adhered to the wall, fresh Hp bacterial solution was added at a ratio of MOI=100:1, and then PBS / BsUV / ScFv / VHH were added respectively, and placed in an incubator with conditions of 37°C and 5% CO2, so that Hp and cells were co-cultured for 12-24h. Among them, BsUV is a Helicobacter pylori bispecific antibody that can specifically bind to the Helicobacter pylori vacuolating toxin protein (VacA) antigen and the Helicobacter pylori urease B subunit (UreB) antigen at the same time. The second antibody ScFv is an antibody that specifically binds to the Helicobacter pylori vacuolating toxin protein (VacA) antigen. The first antibody VHH is an antibody that specifically binds to the Helicobacter pylori urease B subunit (UreB) antigen.

[0107] Hp was co-cultured with GES-1 and uninfected normal cells were used as controls. Samples were collected after 12h and 24h, and the mRNA expression levels of Hp-16sRNA and inflammatory factor COX-2 in different groups were identified. The results are shown in Fig.16 The results show that the expression of Hp-16 sRNA and COX-2 was significantly reduced after 12h and 24h of treatment with the three antibodies, indicating that the bispecific antibody BsUV can inhibit the infection of Hp to GES-1 in a very short time.

[0108] The antibody treatment model of Hp infection GES-1 was established, and Giemsa staining was used to study whether the bispecific antibody BsUV could inhibit the adhesion function of Hp to GES-1. Fig.17 As shown, after Giemsa staining, microscopic observation showed that Hp was able to adhere to the periphery and surface of GES-1, with infected normal cells as negative control, and almost no Hp adhesion was visible in the antibody-treated group. The results suggested that the bispecific antibody BsUV can inhibit the adhesion function of Hp in the in vitro model.

[0109] In summary, in the Hp-infected GES-1 cell model, BsUV can rapidly and significantly reduce the infection ability of Hp and protect GES-1 cells from Hp infection, indicating that BsUV retains the ability to bind to the antigen without losing its ability to neutralize the antigen.

[0110] The efficacy of the treatment against Helicobacter pylori was verified in a mouse model.

[0111] Fresh Hp cultured in advance was taken out and the bacterial solution concentration was adjusted to 1x108 CFU / mL using an ultraviolet spectrophotometer. 3mL of Hp was gavaged into the mice on the 1st and 8th days. On the 15th day, one mouse was randomly selected and killed to obtain the stomach. The quantitative culture ( Fig.18 -A) and Hp identification test strips to identify Hp infection ( Fig.18 -B), the results showed that the Hp infection mouse model was successfully constructed.

[0112] The Hp-infected mice were divided into groups according to their body weight: BsUV treatment group; ScFv treatment group; VHH treatment group; PBS control group. The mice were gavaged with BsUV, ScFv, VHH, and PBS 1 mL (antibody concentration was pre-diluted to 50 μg / mL) on days 15, 22, 29, and 36, respectively. The mice were killed on day 43 and the gastric tissue was removed. The first gastric tissue was weighed after the contents were removed, transferred to a sterile test tube, an appropriate amount of physiological saline was added, and homogenized with an electric homogenizer, and the homogenate was collected for Hp detection; the second gastric tissue was transferred to a sterile test tube after the contents were removed, fixed with 4% paraformaldehyde, and used for mouse gastric tissue pathology detection.

[0113] In order to prove whether the Helicobacter pylori bispecific antibody BsUV can treat C57 mice infected with Hp, the total RNA in the stomach homogenate of each group of mice was extracted by Trizol method, and the expression of Hp-16sRNA and inflammatory factor COX-2 in the stomach of each group of mice was detected by RT-qPCR. Fig.19 The results showed that the expression of Hp-16sRNA in the antibody treatment group was significantly lower than that in the Hp infection group, which was consistent with the expression trend of the inflammatory factor COX-2, and had no significant difference from the negative control group not infected with Hp. This indicates that the bispecific antibody BsUV has a therapeutic effect on C57 mice infected with Hp.

[0114] In order to further verify the level of inflammation in the stomach of each group of mice, total RNA and total protein were extracted from the mouse stomach homogenate, and the expression of inflammatory factors COX-2 and TNF-α in the mouse stomach homogenate was detected by ELISA. Fig. 20 As shown: After BsUV antibody treatment, the level of inflammatory factors in the mouse stomach was significantly reduced, indicating that BsUV antibody inhibited Hp infection, thereby reducing the expression of COX-2 and TNF-α proteins in the mouse stomach and alleviating the gastritis of the mice.

[0115] For pathological examination of mouse stomach tissue, the mouse stomach tissue was divided into two parts along the greater curvature, washed 2-3 times with PBS, fixed with 4% paraformaldehyde, and then stained with HE. The results are as follows Fig.21As shown: compared with the Hp infection group, the infiltration of inflammatory cells in the antibody treatment group was significantly reduced, the gastric mucosal epithelium was more intact, and the damage and shedding were lighter, indicating that the bispecific BsUV can reduce the infection of Hp, thereby reducing the inflammatory response of the gastric mucosa of mice.

[0116] In order to further observe the infection of Hp in the stomach of mice, immunohistochemistry was performed on the wax blocks of stomach tissue prepared above. The results are as follows: Fig.21 As shown: Hp infection was almost invisible in the BsUV treatment group, which further demonstrated that the bispecific antibody BsUV can treat Hp infection in the mouse model and reduce the adhesion of Hp in the mouse stomach.

[0117] In summary, BsUV can significantly reduce the adhesion of Hp in the stomach, reduce the infiltration of inflammatory cells and damage to gastric tissue. Combined with the results in the cell model, the Hp bispecific antibody BsUV can quickly and significantly reduce the infection ability of Hp, and it will not have other harmful effects on gastric tissue and cells. Various experiments have shown that BsUV, as an Hp therapeutic antibody, can have a therapeutic effect on Hp infection.

[0118] A homodimer of a Helicobacter pylori bispecific antibody. When the Helicobacter pylori bispecific antibody is expressed in a host cell, a humanized IgG hinge peptide undergoes homodimerization to form a homodimer of the Helicobacter pylori bispecific antibody.

[0119] Use of at least one of the above-mentioned Helicobacter pylori bispecific antibody and the homologous dimer of the Helicobacter pylori bispecific antibody in the preparation of a drug for treating Helicobacter pylori.

[0120] A pharmaceutical composition comprises at least one of the above-mentioned Helicobacter pylori bispecific antibody and the homologous dimer of the Helicobacter pylori bispecific antibody.

[0121] A detection product, specifically, at least one of the above-mentioned Helicobacter pylori bispecific antibodies and homologous dimers of Helicobacter pylori bispecific antibodies can be labeled by chemical methods or genetic engineering methods, and the labeled antibodies or antigen-binding fragments thereof can be used to detect the presence or amount of Helicobacter pylori in a sample. The detection products include, but are not limited to, detection reagents, detection kits, detection chips or test strips. The specific detection method can adopt the following steps: 1) providing a sample; 2) contacting the sample with the above-mentioned Helicobacter pylori bispecific antibody or antigen-binding fragment thereof of the present invention; 3) detecting the immune reaction between the sample and the antibody or its antigen-binding fragment. 4) obtaining the result.

[0122] What is disclosed above is only a preferred embodiment of the present invention, which certainly cannot be used to limit the scope of rights of the present invention. A person skilled in the art can understand that all or part of the processes of the above embodiments and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. A bispecific antibody against Helicobacter pylori, characterized in that: The method comprises a first antibody, a connector and a second antibody; the first antibody (VHH) is connected to the second antibody (ScFv) via the connector; The first antibody (VHH) specifically binds to the Helicobacter pylori urease B subunit (UreB) antigen. The first antibody (VHH) is a nanobody and all of them are derived from the heavy chain variable region of IgG1, IgG2, IgG3 or IgG4; The second antibody (ScFv) specifically binds to the Helicobacter pylori vacuolating toxin protein (VacA) antigen. The second antibody (ScFv) is a single-chain antibody, and the whole or part of the second antibody is derived from IgG1, IgG2, IgG3 or IgG4.

2. The Helicobacter pylori bispecific antibody according to claim 1, characterized in that: The second antibody (ScFv) is composed of a light chain variable region (VL), a linker peptide (Linker) and a heavy chain variable region (VH); The light chain variable region (VL) is connected to the heavy chain variable region (VH) through a linker peptide; The light chain variable region (VL) is derived in whole or in part from the light chain variable region of IgG1, IgG2, IgG3 or IgG4; the light chain variable region (VL) comprises a sequence as shown in SEA ID NO.1; The heavy chain variable region (VH) is derived in whole or in part from the heavy chain variable region of IgG1, IgG2, IgG3 or IgG4; the heavy chain variable region (VH) comprises a sequence as shown in SEA ID NO.

2.

3. The Helicobacter pylori bispecific antibody according to claim 2, characterized in that: The second antibody (ScFv) comprises a light chain variable region (VL), a linker peptide (Linker) and a heavy chain variable region (VH) from N-terminus to C-terminus; Alternatively, the second antibody (ScFv) comprises a heavy chain variable region (VH), a linker peptide (Linker) and a light chain variable region (VL) from N-terminus to C-terminus.

4. The Helicobacter pylori bispecific antibody according to claim 2, characterized in that: The linker peptide sequence is shown in SEA ID NO.3; That is, the sequence of the second antibody (ScFv) is as shown in SEA ID NO.4; The sequence of the first antibody (VHH) is shown in SEA ID NO.

5.

5. The Helicobacter pylori bispecific antibody according to claim 1, characterized in that: The linker is derived in whole or in part from the heavy chain constant region of humanized IgG1, IgG2, IgG3 or IgG4; The connector includes a humanized IgG hinge peptide, a humanized IgG Fc domain and a linker peptide; The C-terminus of the first antibody (VHH) is connected to the humanized IgG Fc domain via a humanized IgG hinge peptide; The N-terminus of the second antibody (ScFv) is connected to the humanized IgG Fc domain through a linker peptide.

6. The Helicobacter pylori bispecific antibody according to claim 5, characterized in that: The humanized IgG hinge peptide sequence is shown in SEA ID NO.6; The linker peptide sequence is shown in SEA ID NO.3; The humanized IgG Fc domain sequence is shown in SEA ID NO.

7.

7. The Helicobacter pylori bispecific antibody according to claims 3 and 6, characterized in that: The nucleotide sequence of the light chain variable region (VL) of the second antibody (ScFv) is shown in SEA ID NO.8; The nucleotide sequence of the heavy chain variable region (VH) of the second antibody (ScFv) is shown in SEA ID NO.9; The nucleotide sequence of the second antibody (ScFv) is shown in SEA ID NO.10; The nucleotide sequence of the linker peptide is shown in SEA ID NO.11; The nucleotide sequence of the humanized IgG hinge peptide is shown in SEA ID NO.12; The nucleotide sequence of the humanized IgG Fc domain is shown in SEA ID NO.13; The overall nucleotide sequence of the Helicobacter pylori bispecific antibody is shown in SEA ID NO.

14.

8. A homodimer of a Helicobacter pylori bispecific antibody, characterized in that: The Helicobacter pylori bispecific antibody of claim 5, when expressed in a host cell, the humanized IgG hinge peptide undergoes homodimerization to form a homodimer of the Helicobacter pylori bispecific antibody.

9. Use of at least one of the Helicobacter pylori bispecific antibody according to any one of claims 1 to 7 and the homodimer of the Helicobacter pylori bispecific antibody according to claim 8 in the preparation of a medicament for treating Helicobacter pylori.

10. A pharmaceutical composition, characterized in that: The invention comprises at least one of the Helicobacter pylori bispecific antibody according to any one of claims 1 to 7 and the homologous dimer of the Helicobacter pylori bispecific antibody according to claim 8.

11. A detection product, characterized in that: The method comprises at least one of the Helicobacter pylori bispecific antibody according to any one of claims 1 to 7 and the homologous dimer of the Helicobacter pylori bispecific antibody according to claim 8, and is used for detecting the presence or amount of Helicobacter pylori in a sample.

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