Anti-multi-serotype high-virulence klebsiella pneumoniae antibody and application thereof

Abstract

The antibody disclosed by the invention can specifically target high-virulence klebsiella pneumoniae of different serotypes, and specifically targets klebsiella pneumoniae of K1 and K2 serotypes. The antibody disclosed by the invention can be combined with high-toxicity klebsiella pneumoniae extracellular capsular polysaccharide with high affinity, has very strong neutralizing activity on klebsiella pneumoniae, and can mediate complement and mature neutrophil to neutralize and kill bacteria in an in-vitro killing experiment, so that the bacterial density is reduced, and the killing effect is improved. The traditional Chinese medicine composition can effectively prevent and treat high-toxicity klebsiella pneumoniae clinical infection and effectively improve pulmonary infection, provides a way for solving the problems of klebsiella pneumoniae infection and multiple drug resistance, has remarkable significance in preventing, diagnosing and treating klebsiella pneumoniae infection, and has important value in development of a new generation of klebsiella pneumoniae medicine.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and particularly relates to an antibody against hypervirulent Klebsiella pneumoniae of multiple serotypes and its use. Background Art

[0002] Klebsiella pneumoniae (KP) is a Gram-negative bacterium and one of the most common opportunistic pathogens in clinical practice. KP can colonize in the intestines, nasopharynx, axilla and other parts, and the digestive tract is the main colonization site. KP can cause infections in various parts of the body, commonly seen in elderly patients, malnourished, patients with chronic diseases and systemic failure, and can cause systemic or local infections such as pneumonia, urinary tract infection, meningitis, sepsis, etc. (Lee CR, et al. Front Cell Infect Microbiol. 2017). According to different virulence and pathogenic characteristics, KP is currently divided into two categories. One is classical Klebsiella pneumoniae (cKP), which mainly causes hospital-acquired infections such as pneumonia, urinary tract infection, sepsis, etc., with a high drug resistance rate, and is commonly seen in people with underlying diseases or low immunity. The other is hypervirulent Klebsiella pneumoniae (hvKP), which mainly causes infections in healthy people without underlying diseases in the community, and the most common is liver abscess, 66% of which is caused by KP infection (Russo TA, et al. Clin Microbiol Rev. 2019). Among them, Klebsiella pneumoniae of serotypes K1 and K2 are the most important hypervirulent serotypes of Klebsiella pneumoniae, and their pathogenicity is significantly higher than that of other serotype strains of Klebsiella pneumoniae, and is often associated with severe infections and high mortality. K1 serotype strains are widely prevalent in East Asia and are strongly associated with invasive diseases such as liver abscess, meningitis, sepsis, etc., and are the main cause of liver abscess in East Asia. The distribution of K2 serotype strains is more extensive than that of K1 serotype and is closely related to pneumonia, urinary tract infection and hospital infection.

[0003] In recent years, the drug resistance situation of Klebsiella pneumoniae (KP) to various common antibiotics has become increasingly severe. The results of the CHINET China Bacterial Resistance Surveillance in 2021 showed that the isolation rate of KP has jumped to the second among Gram-negative bacilli, second only to Escherichia coli, reaching 19.8% in 2021. Its resistance rate to ampicillin has reached as high as 91.8%, and the resistance rate to piperacillin is close to 50%. Especially with the widespread clinical application of carbapenems, the detection rate of carbapenem-resistant Klebsiella pneumoniae (CRKP) has increased year by year. The resistance rates of KP in China to imipenem and meropenem have increased from about 3% in 2005 to 23.1% and 24.4% respectively in 2021, and the situation is very serious (https: / / www.chinets.com / Document).

[0004] Considering that the drug resistance situation of KP is already very serious, especially the widespread prevalence of CRKP, which makes antibiotic treatment extremely difficult, it has become urgent to develop new treatment strategies. Antibody drugs against Klebsiella pneumoniae, as a new type of treatment method, aim to activate the host's immune response by targeting specific bacterial surface antigens or toxins, thereby achieving the therapeutic effect. Antibody drugs usually have high specificity and low side effects, and can be designed as monoclonal antibodies or polyclonal antibodies against different antigen epitopes. The research on antibody drugs against Klebsiella pneumoniae mainly focuses on developing antibodies that can neutralize bacterial toxins or block bacterial adhesion and invasion of host cells.

[0005] Currently, there is still no specific drug for the treatment of hypervirulent Klebsiella pneumoniae. It is very important to develop alternative drugs or therapies to antibiotics to reduce the usage rate of antibiotics in the medical process. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art. For this reason, the present invention provides an antibody against hypervirulent Klebsiella pneumoniae of multiple serotypes and its use. This antibody against hypervirulent Klebsiella pneumoniae of multiple serotypes can specifically recognize Klebsiella pneumoniae of multiple serotypes and their capsular polysaccharides, and can also improve pulmonary infection caused by Klebsiella pneumoniae.

[0007] In a first aspect of the present invention, there is provided an isolated antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment specifically binds to hypervirulent Klebsiella pneumoniae of a serotype or its capsular polysaccharide, and the antibody or its antigen-binding fragment comprises a heavy-chain variable region containing complementary-determining regions HCDR1, HCDR2, and HCDR3 and a light-chain variable region containing complementary-determining regions LCDR1, LCDR2, and LCDR3, wherein:

[0008] (I) HCDR1 comprises the amino acid sequence GYSFTGYN (SEQ ID NO: 1), HCDR2 comprises the amino acid sequence IDPYYGGT (SEQ ID NO: 2), and HCDR3 comprises the amino acid sequence ARWGGNYYAMDY (SEQ ID NO: 3); LCDR1 comprises the amino acid sequence QSLVHSNGNTY (SEQ ID NO: 4), LCDR2 comprises the amino acid sequence KVS (SEQ ID NO: 5), and LCDR3 comprises the amino acid sequence SQSTHVPFT (SEQ ID NO: 6); or

[0009] (II) HCDR1 comprises the amino acid sequence GFSLSRYS (SEQ ID NO: 9), HCDR2 comprises the amino acid sequence IWGGGST (SEQ ID NO: 10), and HCDR3 comprises the amino acid sequence ARKGGNYGWYFDV (SEQ ID NO: 11); LCDR1 comprises the amino acid sequence ESVDSYGNSF (SEQ ID NO: 12), LCDR2 comprises the amino acid sequence RAS (SEQ ID NO: 13), and LCDR3 comprises the amino acid sequence QQSNE (SEQ ID NO: 14).

[0010] In some embodiments of the present invention, the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are as shown in SEQ ID NOs: 1-3 respectively, and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NOs: 4-6 respectively.

[0011] In some embodiments of the present invention, the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are as shown in SEQ ID NOs: 9-11 respectively, and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NOs: 12-14 respectively.

[0012] In some embodiments of the present invention, the antibody or its antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence SEQ ID NO: 7; in some embodiments of the present invention, the antibody comprises a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 7.

[0013] In some embodiments of the present invention, the antibody or its antigen-binding fragment comprises a light chain variable region comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence SEQ ID NO: 8; in some embodiments of the present invention, the antibody comprises a light chain variable region comprising the amino acid sequence SEQ ID NO: 8.

[0014] In some embodiments of the present invention, the antibody or its antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence SEQ ID NO: 15; in some embodiments of the present invention, the antibody comprises a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 15.

[0015] In some embodiments of the present invention, the antibody or its antigen-binding fragment comprises a light chain variable region comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence SEQ ID NO: 16; in some embodiments of the present invention, the antibody comprises a light chain variable region comprising the amino acid sequence SEQ ID NO: 16.

[0016] In some embodiments of the present invention, the antibody or its antigen-binding fragment comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 7, and / or a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 8.

[0017] In some embodiments of the present invention, the antibody or its antigen-binding fragment comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 15, and / or a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 16.

[0018] In some embodiments of the present invention, the antibody or its antigen-binding fragment is selected from the group consisting of: whole antibody, bispecific antibody, monoclonal antibody, chimeric antibody, humanized antibody, and fully human antibody.

[0019] In some embodiments of the present invention, the antibody or its antigen-binding fragment is selected from the group consisting of: Fab fragment, Fab' fragment, F(ab)2 fragment, Fv fragment, and ScFv.

[0020] In some embodiments of the present invention, the antibody or its antigen-binding fragment is selected from any one of A1 to A4:

[0021] (A1) A single-chain antibody obtained by linking the heavy-chain variable region and the light-chain variable region;

[0022] (A2) A fusion antibody containing the single-chain antibody described in (A1);

[0023] (A3) A Fab containing the heavy-chain variable region and the light-chain variable region;

[0024] (A4) A complete antibody containing the heavy-chain variable region and the light-chain variable region.

[0025] In some embodiments of the present invention, the antibody or its antigen-binding fragment of the present invention may further comprise one or more of a heavy-chain constant region, a light-chain constant region, and an Fc region. In a further preferred embodiment, the light-chain constant region is a lambda-chain or kappa-chain constant region.

[0026] In some embodiments of the present invention, the antibody or its antigen-binding fragment comprises an IgG constant region, and the IgG constant region is selected from IgG1, IgG2, IgG3, or IgG4 constant regions.

[0027] In some embodiments of the present invention, the antibody or its antigen-binding fragment comprises an IgG1 constant region.

[0028] In the present invention, the production of monoclonal antibodies is not limited by any specific method for producing monoclonal antibodies. For example: hybridoma cells capable of secreting the above-mentioned monoclonal antibodies can be cultivated and purified to obtain the monoclonal antibodies; or recombinant DNA technology can be used to obtain them (see, for example, Journal of virological methods, 2009, 158(1-2): 171-179).

[0029] In a second aspect of the present invention, an isolated nucleic acid molecule is provided, comprising a nucleotide sequence encoding the antibody or its antigen-binding fragment described in the first aspect. In some embodiments, the nucleic acid molecule encodes the heavy-chain variable region and / or the light-chain variable region of the antibody or its antigen-binding fragment.

[0030] In some embodiments of the present invention, the nucleic acid molecule encodes a heavy-chain variable region, and its nucleotide sequence is as shown in SEQ ID NO: 17, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the above sequence. In some embodiments of the present invention, the nucleic acid molecule encodes a light-chain variable region, and its nucleotide sequence is as shown in SEQ ID NO: 18, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the above sequence.

[0031] In some embodiments of the present invention, the nucleic acid molecule encodes a heavy chain variable region, the nucleotide sequence of which is shown in SEQ ID NO: 19, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the above sequence. In some embodiments of the present invention, the nucleic acid molecule encodes a light chain variable region, the nucleotide sequence of which is shown in SEQ ID NO: 20, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the above sequence.

[0032] In a third aspect of the present invention, there is provided a biological material, which comprises:

[0033] (B1) A nucleic acid molecule encoding the antibody or its antigen-binding fragment described in the first aspect;

[0034] (B2) An expression cassette containing the nucleic acid molecule described in (B1);

[0035] (B3) A recombinant vector containing the nucleic acid molecule described in (B1) or the expression cassette described in (B2);

[0036] (B4) A recombinant biological cell containing the nucleic acid molecule described in (B1) or the expression cassette described in (B2) or the recombinant vector described in (B3).

[0037] In some embodiments of the present invention, the nucleic acid molecule described in (B1) includes DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can be RNA, such as mRNA and hnRNA.

[0038] In some embodiments of the present invention, the nucleic acid molecule described in (B1) includes the isolated nucleic acid molecule described in the second aspect of the present invention.

[0039] In some embodiments of the present invention, the expression cassette described in (B2) refers to DNA that can express the antibody or its antigen-binding fragment in a host cell. The DNA can not only include a promoter that initiates the transcription of the antibody or its antigen-binding fragment encoding gene, but also include a terminator that terminates the transcription of the antibody or its antigen-binding fragment encoding gene. Further, the expression cassette can also include an origin of replication, a transcription initiation sequence, an enhancer sequence, a selection element or a reporter gene.

[0040] In some embodiments of the present invention, the recombinant vector described in (B3) includes plasmids, cosmids, phages and viral vectors; the recombinant vector includes cloning vectors and expression vectors.

[0041] In some embodiments of the present invention, the recombinant biological cells described in (B4) include bacteria (such as Escherichia coli and Bacillus subtilis), algae, fungi (such as yeast and Aspergillus), insect cells (such as Drosophila S2 cells and Sf9 cells), and animal cells (such as CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, and HEK293 cells), and the biological cells do not include reproductive materials.

[0042] In some embodiments of the present invention, the recombinant biological cell described in (B4) is a hybridoma cell, and the hybridoma cell can be obtained by fusing splenocytes with SP2 / 0 cells.

[0043] In a fourth aspect of the present invention, there is provided a composition comprising the antibody or antigen-binding fragment thereof described in the first aspect, or the isolated nucleic acid molecule described in the second aspect, or the biological material described in the third aspect.

[0044] In some embodiments of the present invention, the composition is a kit.

[0045] In some embodiments of the present invention, the composition is a pharmaceutical composition, further comprising a second medicament.

[0046] In some embodiments of the present invention, the second medicament includes sulbactam, polymyxin E, and tigecycline.

[0047] In some embodiments of the present invention, the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier.

[0048] In a fifth aspect of the present invention, there is provided a method for preparing the antibody or antigen-binding fragment thereof of the present invention, comprising: culturing the above-mentioned recombinant biological cells to express the antibody or antigen-binding fragment thereof, and isolating the antibody or antigen-binding fragment thereof.

[0049] In a sixth aspect of the present invention, there is provided the use of the antibody or antigen-binding fragment thereof according to the first aspect, the nucleic acid molecule according to the second aspect, the biological material according to the third aspect, or the composition according to the fourth aspect in the preparation of any one of (C1)-(C3):

[0050] (C1) A product for detecting hypervirulent Klebsiella pneumoniae;

[0051] (C2) A product for detecting the capsular polysaccharide of hypervirulent Klebsiella pneumoniae;

[0052] (C3) A drug for preventing, treating, or adjuvantly treating hypervirulent Klebsiella pneumoniae infection or related diseases.

[0053] In some embodiments of the present invention, the highly virulent Klebsiella pneumoniae includes at least one of Klebsiella pneumoniae serotype K1 and Klebsiella pneumoniae serotype K2.

[0054] In some embodiments of the present invention, the related diseases include sepsis, bacteremia, pneumonia, and urinary tract infection.

[0055] In some embodiments of the present invention, the product further includes at least one of an enzyme-linked immunosorbent assay (ELISA) detection reagent, an immunohistochemical detection reagent, an immunofluorescence detection reagent, an immunoblotting detection reagent, and an immunoaffinity chromatography reagent. It can be understood that the product of the present invention may also contain reagents not listed above, and other common detection reagents in the art can also be applied to the present invention.

[0056] In some embodiments of the present invention, the detection samples of the product include excreta, sputum, nasal secretions, bronchoalveolar lavage fluid, puncture fluid, wound exudate, and blood of the subject.

[0057] In the seventh aspect of the present invention, there is provided a method for neutralizing / killing highly virulent Klebsiella pneumoniae for non-diagnostic and non-therapeutic purposes, including the following steps: contacting the highly virulent Klebsiella pneumoniae with the antibody or its antigen-binding fragment described in the first aspect, or the composition described in the fourth aspect, or the product or drug described in the sixth aspect.

[0058] The present invention has the following beneficial effects:

[0059] The monoclonal antibodies 2D4 and 2E10 disclosed in the present invention can specifically target highly virulent Klebsiella pneumoniae of different serotypes, including specifically targeting Klebsiella pneumoniae serotype K1 and K2. In cases of invasive Klebsiella pneumoniae infection, the serotypes of the isolated strains are mainly type K1 and type K2. The monoclonal antibodies 2D4 and 2E10 against highly virulent Klebsiella pneumoniae provided by the present invention can effectively prevent and treat Klebsiella pneumoniae serotype K1 and K2 infections in in-vivo and in-vitro tests, providing a way to solve the problems of Klebsiella pneumoniae infection and multi-drug resistance, which is of great significance for the prevention, diagnosis, and treatment of Klebsiella pneumoniae infection and has important value for the development of new-generation drugs against Klebsiella pneumoniae.

[0060] The monoclonal antibodies 2D4 and 2E10 can bind to the extracellular capsular polysaccharide of highly virulent Klebsiella pneumoniae with high affinity and have strong neutralizing activity against Klebsiella pneumoniae. In the in vitro killing experiment, the monoclonal antibodies 2D4 and 2E10 in the examples can mediate complement and mature neutrophils to neutralize and kill bacteria, reducing the bacterial density. The monoclonal antibodies 2D4 and 2E10 can provide effective neutralizing and killing activity for broad protection against various Klebsiella pneumoniae infections, effectively prevent and treat clinical infections of highly virulent Klebsiella pneumoniae, effectively improve lung infections, can mediate phagocytes to enhance phagocytosis, reduce the lung burden in the early stage of Klebsiella pneumoniae infection, shorten the recovery period, and have clinical application value for preventing and treating highly virulent Klebsiella pneumoniae infections. Brief Description of the Drawings

[0061] The present invention will be further described below in conjunction with the drawings and examples, where:

[0062] Figure 1 It is the SDS-PAGE detection result of the monoclonal antibodies 2D4 and 2E10.

[0063] Figure 2 It shows the detection results of the binding activities of the monoclonal antibodies 2D4 and 2E10 to the extracellular capsular polysaccharide of highly virulent Klebsiella pneumoniae of serotypes K1 and K2.

[0064] Figure 3 It shows the detection results of the binding activities of the monoclonal antibodies 2D4 and 2E10 and the isotype control antibody to the intact bacterial particles of highly virulent Klebsiella pneumoniae of serotype K1.

[0065] Figure 4 It shows the detection results of the binding activities of the monoclonal antibodies 2D4 and 2E10 and the isotype control antibody to the intact bacterial particles of highly virulent Klebsiella pneumoniae of serotype K2.

[0066] Figure 5 It shows the neutralizing and killing detection of the monoclonal antibodies 2D4 and 2E10 against highly virulent Klebsiella pneumoniae of serotype K1.

[0067] Figure 6 It shows the neutralizing and killing detection of the monoclonal antibodies 2D4 and 2E10 against highly virulent Klebsiella pneumoniae of serotype K2.

[0068] Figure 7 It shows the results of the body weight changes of mice in the therapeutic test of the monoclonal antibodies 2D4 and 2E10 against highly virulent Klebsiella pneumoniae of serotype K1.

[0069] Figure 8 It shows the survival curves of mice in the therapeutic test of the monoclonal antibodies 2D4 and 2E10 against highly virulent Klebsiella pneumoniae of serotype K1.

[0070] Figure 9 Showing the HE staining results of mouse lung tissue pathological sections in the therapeutic trial of 2D4 and 2E10 monoclonal antibodies against highly virulent Klebsiella pneumoniae of serotype K1.

[0071] Figure 10 Showing the results of changes in body weight of mice in the therapeutic trial of 2D4 and 2E10 monoclonal antibodies against highly virulent Klebsiella pneumoniae of serotype K2.

[0072] Figure 11 Showing the survival curves of mice in the therapeutic trial of 2D4 and 2E10 monoclonal antibodies against highly virulent Klebsiella pneumoniae of serotype K2.

[0073] Figure 12 Showing the HE staining results of mouse lung tissue pathological sections in the therapeutic trial of 2D4 and 2E10 monoclonal antibodies against highly virulent Klebsiella pneumoniae of serotype K2. Detailed implementation manners

[0074] The content of the present invention will be further described in detail below through specific examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The technical solutions in the embodiments of the present invention will be described clearly and completely below. If no specific conditions are indicated in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the reagents or instruments used are not indicated for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0075] Unless otherwise specified, the molecular biology experimental methods and immunoassay methods used in the present invention are basically carried out according to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995.

[0076] Unless otherwise specified, "room temperature" means 25°C ± 5°C.

[0077] The term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). The VH and VL regions can also be further divided into regions of high variability (called complementarity-determining regions (CDRs)), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antibody binding site. The assignment of amino acids to each region or domain follows the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0078] In the present invention, an antibody also includes an antigen-binding portion (used interchangeably with the term "antigen-binding fragment"). An antigen-binding portion refers to a polypeptide containing a fragment of a complete antibody that retains the ability to specifically bind an antigen that specifically binds to the full-length or complete antibody, and / or competes with the full-length antibody for binding to the same antigen. Under some conditions, the antigen-binding portion includes, but is not limited to, Fab, Fab’, F(ab’)2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (such as scFv), chimeric antibodies, diabodies, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability. The antigen-binding portion of an antibody can be obtained from a given antibody by conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and can be screened for specificity in the same manner as the complete antibody.

[0079] The term "fusion antibody" refers to a product obtained by genetically engineering a single-chain antibody to fuse with a functional protein molecule having biological activity. The fusion antibody does not affect the antigen-binding ability of the single-chain antibody nor the biological activity of the functional protein molecule fused thereto.

[0080] The term "complete antibody" is composed of two identical heavy chains and light chains, each chain containing a variable region and one or more constant regions (C regions). The variable region is responsible for binding to the antigen, while the constant region is mainly responsible for binding effector molecules. It can be antibodies of different isotypes, e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0081] The term "neutralizing antibody" refers to an antibody or antibody fragment that can eliminate or significantly reduce the virulence of a target virus (e.g., the ability to infect cells).

[0082] The term "hybridoma cell" refers to a cell formed by fusing a myeloma cell and a B lymphocyte. Hybridoma cells are generally prepared by culturing tumor cells. The technique for preparing hybridoma cells refers to the phenomenon of combining two or more cells to form a single cell; it enables the nuclei of two different origins to express their functions in the same cell. By fusing two types of cells while maintaining their main characteristics, namely, mouse spleen cells immunized with an antigen and mouse myeloma cells. The main characteristic of its B lymphocytes is its antibody-secreting function and the ability to grow in a selective medium; while mouse myeloma cells can divide and proliferate indefinitely under culture conditions, i.e., the so-called immortality. Under the action of the selective medium, only the hybrid cells formed by the fusion of B lymphocytes and myeloma cells have the ability to continuously proliferate, forming cells that possess both the antibody-secreting function and the characteristic of cell immortality.

[0083] The term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) refers to an antibody that binds to the antigen with an affinity of less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less.

[0084] The term "neutralizing activity" refers to the functional activity of an antibody or antibody fragment to bind to the antigen protein on a virus, thereby preventing the virus from infecting cells and / or the maturation of virus progeny and / or the release of virus progeny. An antibody or antibody fragment with neutralizing activity can prevent the amplification of the virus, thereby inhibiting or eliminating virus infection.

[0085] The term "serotype" means that when classifying bacteria, a group of bacteria with the same biological characteristics constitutes a bacterial species. Several bacterial species with similar and closely related characteristics form a genus. Although the individual bacteria of the same bacterial species have basically the same characteristics, there are still certain differences in some aspects. Those with obvious differences are called subspecies (subspecies, subsp.) or varieties (variety, var.); those with small differences are called types. For example, they are divided into different serotypes according to different antigen structures.

[0086] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0087] Example 1: Establishment of Klebsiella pneumoniae hybridoma cell line, preparation and purification of monoclonal antibody ascites

[0088] The strain of Klebsiella pneumoniae of serotype K20 (obtained from Zhuhai Hospital of Guangdong Provincial Hospital of Traditional Chinese Medicine, Kp00198874) was inoculated into LB liquid medium and cultured with shaking at 37°C until the logarithmic growth phase (OD600≈0.6 - 1.0). After inactivating at 60°C in a water bath for 30 min, it was diluted with PBS and directly subcutaneously immunized into Balb / c mice (1×10 7(CFU / mouse), with a two-week interval each time. On the 12th day after three immunizations, blood was collected from the mouse orbits, and the serum titer was detected using the indirect ELISA method. The serum obtained by collecting blood from the mouse orbits before the first immunization was used as a negative control. When the immunization titer reached over 100,000, the mouse with the highest titer was selected 3 days before cell fusion, and the mouse was immunized subcutaneously with an antigen dose of 1×10 7 CFU / mouse to obtain boost-immunized mice. Three days after the boost immunization, spleen cells of the boost-immunized mice were collected, and according to the conventional operation in the art, their spleen cells were fused with SP2 / 0 cells to obtain fused cells (hybridoma cells).

[0089] Among them, the specific steps of the indirect ELISA method are as follows:

[0090] After pre-treating a 96-well polystyrene enzyme-linked immunosorbent assay (ELISA) plate with 100 μL of 5% glutaraldehyde per well at 37 °C for 2 h, 1×10 8 CFU / mL of inactivated bacteria of complete Klebsiella pneumoniae serotypes K1 and K2 were coated on each well at 100 μL / well. The inactivation step of the bacteria was resuspension after water bath at 65 °C for 20 min and ultraviolet irradiation for 10 min. The inactivated bacteria were diluted with 1×PBS solution. After incubation at 37 °C for 2 h, the plate was washed 3 times with PBST, and then 200 μL of 5% skim milk powder solution was added to each well to block the 96-well ELISA plate, and it was incubated in a 37 °C incubator for 2 h or blocked overnight at 4 °C. The mouse serum treated in a 56 °C metal bath for 30 min was used as a sample of the antibody to be detected and was diluted 10 3 times; the diluted sample of the antibody to be detected was added to the wells and incubated in a 37 °C incubator for 30 min; after washing, 100 μL of appropriately diluted horseradish peroxidase (HRP)-labeled secondary antibody (GoatAnti-Mouse IgG+IgM+IgA H&L(HRP), Abcam, ab102448) was added to each well and incubated in a 37 °C incubator for 30 min; after thorough washing, 100 μL of ELISA chromogenic solution (Sangon Biotech, product number: E661007) was added to each well and incubated in a 37 °C incubator in the dark for 10 min to 30 min, and then 50 μL of H2SO4 solution was added to terminate the reaction, and the absorbance OD450 was measured within 15 min. The ratio of the OD450 of the sample well to the OD450 of the negative control well > 2.1 was judged as positive.

[0091] 50 μL of the cell supernatant obtained by culturing hybridoma cells was taken as the sample to be detected, and the hybridoma cells were screened with reference to the established indirect ELISA method above. The identified positive hybridoma cell lines were subcloned 2-3 times using the limiting dilution method, and finally, strongly positive single cell lines were selected for expansion culture.

[0092] After cell fusion, screening, and several subclonings, 2 monoclonal antibody cell lines (2D4 and 2E10) were obtained. The ELISA test results of the cell culture supernatants binding to Klebsiella pneumoniae serotypes K1 and K2 are shown in Table 1 below.

[0093] Table 1. ELISA test results of the cell line culture supernatants binding to Klebsiella pneumoniae serotypes K1 and K2

[0094] Cell line number OD450 value K1-2D4 1.63 K1-2E10 1.35 K2-2D4 1.13 K2-2E10 1.03

[0095] As shown in Table 1, the titers of monoclonal antibodies 2D4 and 2E10 against Klebsiella pneumoniae serotypes were detected by indirect ELISA. It can be seen that the two monoclonal antibodies have good binding ability to Klebsiella pneumoniae serotypes K1 and K2.

[0096] Preparation and purification of monoclonal antibody ascites: One week in advance, mice were intraperitoneally injected with 500 μL / mouse of incomplete Freund's adjuvant (or paraffin oil) for pretreatment. Each mouse was injected with 5×10 5 ~1×10 6 hybridoma cells (the injection volume was not higher than 500 μL), and then waited for ascites to be produced. The ascites produced by in vivo culture in mice was collected and centrifuged initially to remove impurities. Saturated ammonium sulfate solution was added at a ratio of 1:1 to precipitate the antibody, and then centrifuged to remove the supernatant. An appropriate amount of 1×PBS was added for reconstitution and filtered through a 0.22 μm filter membrane, and then purified by affinity chromatography (Protein A agarose purification resin). The monoclonal antibodies secreted by the above hybridoma cells were labeled as 2D4 and 2E10 respectively. 20 μL of each antibody was taken and 5 μL of SDS-PAGE Loading Buffer (5×) (product number: G3422-2) was added. After mixing, it was placed in a metal bath at 100 °C for 10 min. The antibody would unwind at high temperature, and the heavy chain and light chain would dissociate. AmyKD-PAGE gel rapid preparation kit (product number: ATG0046) was used to prepare the gel. The sample loading volume was 20 μL, and electrophoresis was carried out at 90 V for 120 min.

[0097] The SDS-PAGE test results are as Figure 1 shown. Among them, the heavy chain and light chain of the two monoclonal antibodies dissociated, each showing two bands. The heavy chain was about 55 kD and the light chain was about 25 kD. The hybridoma cells prepared by the above method could continuously and efficiently secrete monoclonal antibody 2D4 and monoclonal antibody 2E10, and the method for preparing hybridoma cells was simple and easy to operate. Compared with in vitro culture, culturing hybridoma cells in mice could effectively reduce the adverse effects of external environmental factors on the proliferation of hybridoma cells and the expression of monoclonal antibodies, thus providing a relatively stable survival environment for the survival of hybridoma cells, being not easily contaminated, and having high production efficiency.

[0098] Example 2: Detection of Gene Sequences and Amino Acid Sequences of Klebsiella pneumoniae Monoclonal Antibodies

[0099] This example aims to detect the gene sequences and amino acid sequences of the variable regions of two Klebsiella pneumoniae monoclonal antibodies (monoclonal antibody 2D4 and monoclonal antibody 2E10).

[0100] The hybridoma cells prepared in Example 1 were cultured in a T75 cell culture flask until a density of 70%-90% was reached. The cells were collected by centrifugation, washed twice with 1×PBS, 10 mL each time, and the hybridoma cells were reserved after centrifugation. After extracting the RNA of the hybridoma cells using the AGSteadyPure Universal RNA Extraction Kit II (product number AG21022), Takara PrimeScript TM II 1st Strand cDNA Synthesis Kit (product number 6210A) was used to reverse transcribe the extracted RNA into cDNA, and then Takara Taq TM Version 2.0plus dye premixed enzyme (product number RR901A) was used to perform PCR amplification on the light and heavy chains of monoclonal antibodies 2D4 and 2E10 respectively. The PCR reaction system was prepared according to the product manual. After the amplified PCR products were analyzed by 1%-2% agarose gel electrophoresis, the bands with a size of 300 bp - 500 bp were sequenced. The sequencing results were analyzed for gene sequences on the VBASE2 website to obtain the variable region sequences of the heavy and light chains of monoclonal antibody 2D4 and monoclonal antibody 2E10.

[0101] Among them, the PCR amplification conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 40 s, annealing at 58°C for 40 s, extension at 72°C for 1 min, for a total of 35 cycles; extension at 72°C for another 10 min.

[0102] The primer sequences used for PCR amplification are shown in Table 2. The downstream primers of the heavy chain were combined with 15 upstream primers of the heavy chain for sequencing respectively; the downstream primers of the light chain were combined with 16 upstream primers of the light chain for sequencing respectively.

[0103] Table 2. Primer Sequences Used for PCR Amplification

[0104]

[0105]

[0106] After sequencing, the sequence information of the 2 Klebsiella pneumoniae monoclonal antibodies is as follows:

[0107] The monoclonal antibody 2D4 consists of a light chain and a heavy chain. The heavy chain variable region has three complementarity-determining regions, HCDR1, HCDR2, and HCDR3. Among them, the amino acid sequence of HCDR1 is GYSFTGYN (SEQ ID NO: 1), the amino acid sequence of HCDR2 is IDPYYGGT (SEQ ID NO: 2), and the amino acid sequence of HCDR3 is ARWGGNYYAMDY (SEQ ID NO: 3). The light chain variable region has three complementarity-determining regions, LCDR1, LCDR2, and LCDR3. Among them, the amino acid sequence of LCDR1 is QSLVHSNGNTY (SEQ ID NO: 4), the amino acid sequence of LCDR2 is KVS (SEQ ID NO: 5), and the amino acid sequence of LCDR3 is SQSTHVPFT (SEQ ID NO: 6). The amino acid sequence of the heavy chain variable region of the monoclonal antibody 2D4 is: EVQLQQSGPELEKPGASVKISCKASGYSFTGYNMNWVKQSNGKSLEWIGNIDPYYGGTSYNQKFKGKAT LTVDKSSSTAYMQLKSLTSEDSAVYYCARWGGNYYAMDYWGQGTSVTVSS (SEQ ID NO: 7). The amino acid sequence of the light chain variable region of the monoclonal antibody 2D4 is: DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPFTFGSGTKLEIK (SEQ ID NO: 8).

[0108] The monoclonal antibody 2E10 consists of a light chain and a heavy chain. There are three complementarity-determining regions HCDR1, HCDR2, and HCDR3 in the heavy chain variable region. Among them, the amino acid sequence of HCDR1 is GFSLSRYS (SEQ ID NO: 9), the amino acid sequence of HCDR2 is IWGGGST (SEQ ID NO: 10), and the amino acid sequence of HCDR3 is ARKGGNYGWYFDV (SEQ ID NO: 11); there are three complementarity-determining regions LCDR1, LCDR2, and LCDR3 in the light chain variable region. Among them, the amino acid sequence of LCDR1 is ESVDSYGNSF (SEQ ID NO: 12), the amino acid sequence of LCDR2 is RAS (SEQ ID NO: 13), and the amino acid sequence of LCDR3 is QQSNE (SEQ ID NO: 14). The amino acid sequence of the heavy chain variable region of the monoclonal antibody 2E10 is: EVKLQESGPGLVAPSQSLSITCTVSGFSLSRYSVHWVRQPPGKGLEWLGMIWGGGSTDYNSALKSRLSIS KDNSKSQVFLKMNSLQTDDTAMYYCARKGGNYGWYFDVWGAGTTVTVSS (SEQ ID NO: 15). The amino acid sequence of the light chain variable region of the monoclonal antibody 2E10 is: DIVLTQTPASLAVSLGQRATISCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSR TDFTLTINPVEADDVATYYCQQSNEDPRGRSVEAPSWK (SEQ ID NO: 16).

[0109] The nucleotide sequence of the heavy chain variable region of the monoclonal antibody 2D4 is shown in SEQ ID NO: 17, and the nucleotide sequence of the light chain variable region of the monoclonal antibody 2D4 is shown in SEQ ID NO: 18.

[0110] The nucleotide sequence of the heavy chain variable region of the monoclonal antibody 2E10 is shown in SEQ ID NO: 19, and the nucleotide sequence of the light chain variable region of the monoclonal antibody 2E10 is shown in SEQ ID NO: 20.

[0111] Example 3: Detection of the binding activity of Klebsiella pneumoniae monoclonal antibodies to the capsular polysaccharides of Klebsiella pneumoniae serotypes K1 and K2

[0112] After culturing Klebsiella pneumoniae serotypes K1 and K2 to the logarithmic growth phase respectively, they were placed in an ultrasonic crusher. The ultrasonic power was set to 200 W, and they were crushed for 8 s, with an interval of 12 s, and the cycle was repeated 99 times to completely crush Klebsiella pneumoniae, so that a large amount of capsular polysaccharide was released into the supernatant. After high-speed centrifugation, the precipitate was discarded. A 10% CTAB solution (v / w, prepared by dissolving cetyltrimethylammonium bromide (CTAB) in 0.7 mol / L NaCl aqueous solution) was mixed with the supernatant at a volume ratio of 1:9. After sufficient shaking, it was left to stand at room temperature for 0.5 h - 1 h to form a precipitate, and the precipitate was collected by centrifugation. 5 mol / L CaCl2 solution was added to the precipitate to a final concentration of 1 mol / L, and it was stirred for 1 h - 3 h until the precipitate dissolved. After centrifugation at 8000 rpm and 4 °C for 20 min, the capsular polysaccharide supernatant was collected. Absolute ethanol was added to the capsular polysaccharide supernatant to a final ethanol concentration of 25% (v / v). After standing overnight at 4 °C, it was centrifuged at 8000 rpm and 4 °C for 20 min. The supernatant was taken, and ice-cold absolute ethanol was added to the supernatant to a final ethanol concentration of 80% (v / v). After thorough mixing at room temperature to precipitate the capsular polysaccharide, it was centrifuged at 8000 rpm and 4 °C for 20 min, and the capsular polysaccharide precipitate was collected. After washing the capsular polysaccharide precipitate with absolute ethanol twice, the capsular polysaccharide precipitate was dissolved in 10 mL of ultrapure water, 2 mg / mL Proteinase K was added, and after incubation at 37 °C for 3 h, it was centrifuged at 8000 rpm and 4 °C for 20 min. The supernatant was taken to obtain the capsular polysaccharides of Klebsiella pneumoniae serotypes K1 and K2.

[0113] The 96-well enzyme-linked immunosorbent assay (ELISA) plates were coated with the capsular polysaccharides of Klebsiella pneumoniae serotypes K1 and K2 respectively, and the 96-well ELISA plates were blocked with 5% skim milk solution. The monoclonal antibodies 2D4 and 2E10 to be tested were diluted to 0.1 mg / mL respectively and added to the above 96-well ELISA plates at 100 μL / well. The control group was sterile phosphate solution, and it was incubated at 37 °C for 30 min. After washing the 96-well ELISA plates 5 times with ELISA washing solution PBST, 100 μL of appropriately diluted secondary antibody labeled with horseradish peroxidase (HRP) (Goat Anti-Mouse IgG+IgM+IgA H&L(HRP), Abcam, ab102448) was added to each well, and incubation was continued at 37 °C for 30 min. After washing the 96-well ELISA plates 5 times with PBST, a chromogenic agent was added. After chromogenesis for 15 min, the reaction was terminated, and the absorbance (OD450) at 450 nm was read on an enzyme-linked immunosorbent assay reader.

[0114] The test results are as Figure 2As shown, the results showed that both monoclonal antibodies 2D4 and 2E10 could specifically target and bind to Klebsiella pneumoniae serotypes K1 and K2, while the phosphate solution control did not show specific binding activity.

[0115] Example 4: Detection of the binding activity of Klebsiella pneumoniae monoclonal antibodies to intact bacterial particles of Klebsiella pneumoniae serotypes K1 and K2

[0116] The indirect ELISA method of Example 1 was used to analyze the reaction of Klebsiella pneumoniae monoclonal antibodies with heat-inactivated bacteria of intact Klebsiella pneumoniae serotypes K1 and K2 to evaluate their specificity. After pretreatment of a 96-well polystyrene microtiter plate with 5% glutaraldehyde, each well was coated with 1×10 8 CFU of heat-inactivated bacteria of intact Klebsiella pneumoniae serotypes K1 and K2. Subsequently, the 96-well plate was blocked with 5% non-fat milk powder. After diluting the monoclonal antibodies 2D4 and 2E10 to be tested to 0.1 mg / mL respectively, 11 successive two-fold serial dilutions were continued, and 100 μL / well was added to the 96-well polystyrene microtiter plate and incubated at 37 °C for 30 min; the same treatment was performed with isotype IgG antibody as the isotype IgG antibody group (isotype control group), and phosphate buffer treatment was used as the PBS group. After washing the 96-well microtiter plate 5 times with ELISA washing solution PBST, 100 μL of appropriately diluted horseradish peroxidase (HRP)-labeled secondary antibody (Goat Anti-Mouse IgG+IgM+IgA H&L(HRP), Abcam, ab102448) was added to each well and incubated at 37 °C for another 30 min. After washing the microtiter plate 5 times with PBST, the chromogenic agent was added, and the reaction was terminated after 15 min of color development. The absorbance at 450 nm was read on a microplate reader.

[0117] The test results are as Figures 3 to 4 shown. The results showed that both monoclonal antibodies 2D4 and 2E10 could specifically bind to 2 strains of intact Klebsiella pneumoniae serotypes K1 and K2. Among them, 2D4 and 2E10 broadly targeted and bound to Klebsiella pneumoniae serotype K1 ( Figure 3 ) and Klebsiella pneumoniae serotype K2 ( Figure 4 ); the isotype IgG antibody group did not show binding to Klebsiella pneumoniae serotypes K1 and K2. Even, the monoclonal antibody 2D4 still showed specific reactivity to intact bacterial particles when diluted to a concentration of 0.19 μg / mL (0.019 μg / well). The isotype IgG antibody group and the PBS group did not show specific binding activity.

[0118] Example 5: Neutralization and killing of Klebsiella pneumoniae serotypes K1 and K2 by Klebsiella pneumoniae monoclonal antibodies

[0119] The HL60 cells (human promyelocytic leukemia cell line) were subcultured, and dimethyl sulfoxide (DMSO, MedChemExpress, CATNO: HY-15392) was added for 5 days of induction culture to differentiate the cells into mature neutrophils. The induced mature neutrophils were collected, and the cell number was adjusted to 1.0×10 7 cells / mL of mature neutrophil suspension with 1×PBS solution. Guinea pig blood was centrifuged at 4°C to collect serum, which was filtered and used as a complement source. The serum was diluted 10-fold, 25-fold, and 50-fold with double-distilled water. Klebsiella pneumoniae serotype K1 (from the Third People's Hospital of Shenzhen, KpGZ-8) and Klebsiella pneumoniae serotype K2 (from the Third People's Hospital of Shenzhen, ATCC43816) were cultured in LB liquid medium until the logarithmic growth phase, and the bacterial density was adjusted to 5.0×10 5 CFU / mL.

[0120] To 10 μL of Klebsiella pneumoniae serotype K1 or K2 bacterial solution, 125 μg of monoclonal antibodies 2D4 and 2E10 were added. After mixing, it was incubated at 200 rpm / min at room temperature for 10 min; the same treatment was performed with isotype IgG antibody as the isotype IgG antibody group (isotype control group), and phosphate buffer treatment was used as the PBS group. To each treatment group, 10 μL of guinea pig serum at different dilution degrees and 50 μL of mature neutrophil suspension were added. After incubating and mixing at 200 rpm / min at room temperature for 10 min, it was transferred to a biochemical incubator and incubated at 37°C for 45 min. After the incubation ended, it was placed on ice for 25 min to end phagocytosis. In addition, inactivated guinea pig serum (guinea pig serum was treated at 58°C for 30 min) was used to replace guinea pig serum for the same treatment as the corresponding inactivated group. After phagocytosis ended, 10 μL was taken for plating, and the colony numbers of each control group and the non-specific killing rate (NSK) of the complement were counted the next day. Among them, NSK = [1 - (colony number of antibody group / colony number of inactivated group)] × 100%. Isotype antibody has no specific killing ability, and complement has non-specific killing ability. If the non-specific killing rate is controlled below 25%, the part exceeding 25% in the antibody experimental group is marked as the specific killing ability of the corresponding monoclonal antibody.

[0121] The neutralizing killing test results of Klebsiella pneumoniae serotype K1 are as Figure 5As shown, the results showed that for Klebsiella pneumoniae serotype K1, the non-specific killing rate of the isotype IgG antibody control group was less than 25%. The number of bacterial colonies of monoclonal antibody 2D4 and monoclonal antibody 2E10 was lower compared to the isotype IgG antibody or PBS group; the control groups did not show specific neutralizing bactericidal activity. This indicates that monoclonal antibodies 2D4 and 2E10 can specifically target and mediate the neutralizing killing of Klebsiella pneumoniae serotype K1 by complement and neutrophils.

[0122] The results of the neutralizing killing assay for Klebsiella pneumoniae serotype K2 are as Figure 6 As shown, the results showed that for Klebsiella pneumoniae serotype K2, the non-specific killing rate of the isotype IgG antibody control group was less than 25%. The number of bacterial colonies of monoclonal antibody 2D4 and monoclonal antibody 2E10 was lower compared to the isotype IgG antibody or PBS group; the control groups did not show specific neutralizing bactericidal activity. This indicates that monoclonal antibodies 2D4 and 2E10 can specifically target and mediate the neutralizing killing of Klebsiella pneumoniae serotype K2 by complement and neutrophils.

[0123] Example 6: Therapeutic trial of monoclonal antibodies against Klebsiella pneumoniae serotype K1

[0124] This example shows the protective trial of monoclonal antibodies against Klebsiella pneumoniae. The specific steps are as follows: Balb / c mice were intraperitoneally infected with a Klebsiella pneumoniae serotype K1 strain at a bacterial dose of 5×10 4 CFU. Six hours after infection, two monoclonal antibodies against Klebsiella pneumoniae were administered by intraperitoneal injection at a dose of 0.2 mg per mouse. There were 6 mice in each group. One group of mice was injected with antibody 2D4, and the other group was injected with antibody 2E10. PBS was used as the negative control group. At 72 hours after pulmonary infection, lung tissues of two randomly selected mice from each group were subjected to pathological sectioning and HE staining for observation, and the remaining mice were used to calculate the survival rate over 14 days and draw a survival curve.

[0125] The test results are as Figures 7 to 9 shown. The survival rate of the 2D4 antibody group was 100%, and the survival rate of the 2E10 antibody group was 75%. The results of the body weight change curve of the antibody groups were significantly better than those of the control group. The HE staining results showed that in the tissue pathological sections of the 2D4 antibody group and the 2E10 antibody group, scattered mild inflammation in the mouse lung tissue, bronchiolar inflammatory exudation (black arrow), and inflammatory cell infiltration in the lung interstitium and alveoli (red arrow) could be observed. At the same time, the lung tissue of the control group mice showed extensive moderate inflammation, bronchiolar inflammatory exudation (black arrow), inflammatory cell infiltration in the lung interstitium and alveoli, necrosis and fusion of alveolar tissue (red arrow), and alveolar hemorrhage (yellow arrow). This indicates that both strains of antibodies can play a therapeutic role in the infection of Klebsiella pneumoniae serotype K1.

[0126] Example 7: Therapeutic test of Klebsiella pneumoniae monoclonal antibody against Klebsiella pneumoniae serotype K2

[0127] The experimental steps of this example are substantially the same as those of Example 6, except that the Klebsiella pneumoniae serotype K1 strain is replaced with the Klebsiella pneumoniae serotype K2 strain.

[0128] The test results are as Figures 10 to 12 shown. The survival rate of the antibody group was 25%, and the results of the body weight change curve were significantly better than those of the control group. The HE staining results showed that scattered mild inflammation, bronchiolar inflammatory exudation (black arrow), and interstitial and alveolar inflammatory cell infiltration (red arrow) could be observed in the histopathological sections of the 2D4 antibody group and the 2E10 antibody group. At the same time, extensive moderate inflammation, bronchiolar inflammatory exudation (black arrow), interstitial and alveolar inflammatory cell infiltration, and necrosis and fusion of alveolar tissue (red arrow) were shown in the lung tissue of the control group mice. This indicates that both of the two antibodies can play a therapeutic role in the infection of Klebsiella pneumoniae serotype K2.

[0129] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge scope of those of ordinary skill in the art. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. An isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to serotype hypervirulent Klebsiella pneumoniae or its capsular polysaccharide, and the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising complementarity-determining regions HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising complementarity-determining regions LCDR1, LCDR2 and LCDR3, wherein: (I) HCDR1 comprises the amino acid sequence GYSFTGYN (SEQ ID NO: 1), HCDR2 comprises the amino acid sequence IDPYYGGT (SEQ ID NO: 2), HCDR3 comprises the amino acid sequence ARWGGNYYAMDY (SEQ ID NO: 3); LCDR1 comprises the amino acid sequence QSLVHSNGNTY (SEQ ID NO: 4), LCDR2 comprises the amino acid sequence KVS (SEQ ID NO: 5), LCDR3 comprises the amino acid sequence SQSTHVPFT (SEQ ID NO: 6); or (II) HCDR1 comprises the amino acid sequence GFSLSRYS (SEQ ID NO: 9), HCDR2 comprises the amino acid sequence IWGGGST (SEQ ID NO: 10), HCDR3 comprises the amino acid sequence ARKGGNYGWYFDV (SEQ ID NO: 11); LCDR1 comprises the amino acid sequence ESVDSYGNSF (SEQ ID NO: 12), LCDR2 comprises the amino acid sequence RAS (SEQ ID NO: 13), LCDR3 comprises the amino acid sequence QQSNE (SEQ ID NO: 14).

2. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence SEQ ID NO: 7, and the isolated antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence SEQ ID NO: 8; or the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence SEQ ID NO: 15, and the isolated antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence SEQ ID NO:

16.

3. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: whole antibody, bispecific antibody, monoclonal antibody, chimeric antibody, humanized antibody, and fully human antibody.

4. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: Fab fragment, Fab' fragment, F(ab)2 fragment, Fv fragment, and ScFv.

5. A biological material, comprising: (B1) A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-4; (B2) An expression cassette containing the nucleic acid molecule of (B1); (B3) A recombinant vector containing the nucleic acid molecule of (B1) or the expression cassette of (B2); (B4) A recombinant biological cell containing the nucleic acid molecule of (B1) or the expression cassette of (B2) or the recombinant vector of (B3).

6. The biological material according to claim 5, wherein the nucleic acid molecule comprises a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide sequence SEQ ID NO: 17, a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide sequence SEQ ID NO: 18; or the nucleic acid molecule comprises a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide sequence SEQ ID NO: 19, a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide sequence SEQ ID NO:

20.

7. A composition, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-4, or the biological material according to any one of claims 5-6.

8. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-4, or the biological material according to any one of claims 5-6, or the composition according to claim 7 for the preparation of any one of (C1)-(C3): (C1) A product for detecting Klebsiella pneumoniae with high virulence; (C2) A product for detecting the capsular polysaccharide of Klebsiella pneumoniae with high virulence; (C3) A drug for preventing, treating, or adjuvantly treating Klebsiella pneumoniae with high virulence infection or its related diseases.

9. The use according to claim 8, wherein the Klebsiella pneumoniae with high virulence comprises at least one of Klebsiella pneumoniae serotype K1 and Klebsiella pneumoniae serotype K2.

10. The use according to claim 8, wherein the related diseases include sepsis, bacteremia, pneumonia, and urinary tract infection.

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