Preparation and Application of an Anti-Pseudomonas aeruginosa PcrV Antibody

Abstract

The present invention discloses the preparation and application of an anti-Pseudomonas aeruginosa PcrV antibody. The present invention proves through experiments that the PcrV antibody can specifically bind to the PcrV antigen, has a neutralizing effect on the lysis of A549 cells by PcrV, and also has the ability to neutralize the lysis of rabbit red blood cells mediated by PcrV, and has a preventive / therapeutic effect on systemic infection and acute pneumonia.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the preparation and application of an anti-Pseudomonas aeruginosa PcrV antibody. Background Art

[0002] Pseudomonas aeruginosa (PA) is an opportunistic pathogen and one of the most common Gram-negative bacteria in hospital-acquired infections. Due to its strong inherent resistance to a variety of antibiotics, compared with pulmonary and bloodstream infections caused by other pathogens, the range of antibiotic choices for the clinical treatment of PA infections is narrow, and the mortality rate of patients is relatively high. Chronic PA infection is one of the main causes of morbidity and high mortality in patients with cystic fibrosis. By forming biofilms in the upper respiratory tract of cystic fibrosis patients and repeatedly colonizing the lower respiratory tract, it ultimately leads to chronic pulmonary infections. In addition, it is also a common pathogen associated with burn wound infections, pneumonia, urinary tract infections, bacteremia, and AIDS patients. The clinical treatment of Pseudomonas aeruginosa infection mainly relies on antibiotics. However, due to its natural resistance and acquired resistance characteristics, there is still a lack of effective treatment and control strategies. According to the statistical results of the 2020 CHINET China Bacterial Resistance Surveillance data, the detection rate of Pseudomonas aeruginosa in clinical specimens was 8.42%, ranking fourth. Therefore, it is extremely important to develop new anti-infection strategies against PA infections. Antibody drugs can eliminate pathogens through neutralizing toxins, phagocytic opsonization, etc., and antibodies have the advantages of high specificity and low side effects.

[0003] Pseudomonas aeruginosa mainly uses the type III secretion system to inject toxins into host cells to trigger infections. The type III secretion system was first discovered in Gram-negative bacteria. Clinical studies have shown that PA expressing T3SS has a higher lethality rate. The results of in vitro cell culture experiments have shown that PA expressing T3SS exhibits stronger toxicity. Therefore, blocking the toxins related to T3SS is the key to controlling infections. The PcrV protein is a transporter protein of the type III secretion system. By directly transporting the virulence protein molecules produced by bacteria into host cells, it interferes with the normal functions of host cells and causes the death of host cells. Gene mutation studies have confirmed that after the deletion of PcrV, the bacterial type III secretion system cannot bind to the host cell membrane, and thus cannot damage host cells. PcrV can be used as a protective antigen in an acute lung infection model, and active and passive immunization with PcrV has a protective effect on lung injury caused by PA. Summary of the Invention

[0004] To make up for the deficiencies of the prior art, the present invention provides the preparation and application of an anti-Pseudomonas aeruginosa PcrV antibody.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect of the present invention, there is provided an antibody against Pseudomonas aeruginosa PcrV, wherein the antibody comprises heavy chain variable region complementarity determining regions CDR1, CDR2, and CDR3 having at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3,

[0007] and light chain variable region complementarity determining regions CDR1, CDR2, and CDR3 having at least 95% sequence identity with the amino acid sequences shown in SEQ ID NOs: 9, 10, and 11.

[0008] Further, the amino acid sequences of the heavy chain variable region complementarity determining regions CDR1, CDR2, and CDR3 are respectively as shown in SEQ ID NOs: 1, 2, and 3,

[0009] and the amino acid sequences of the light chain variable region complementarity determining regions CDR1, CDR2, and CDR3 are respectively as shown in SEQ ID NOs: 9, 10, and 11.

[0010] Further, the antibody further comprises heavy chain variable region framework regions FR1, FR2, FR3, and FR4 having at least 70% sequence identity with the amino acid sequences shown in SEQ ID NOs: 4, 5, 6, and 7,

[0011] and light chain variable region framework regions FR1, FR2, FR3, and FR4 having at least 70% sequence identity with the amino acid sequences shown in SEQ ID NOs: 12, 13, 14, and 15.

[0012] Further, the amino acid sequences of the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 are respectively as shown in SEQ ID NOs: 4, 5, 6, and 7,

[0013] and the amino acid sequences of the light chain variable region framework regions FR1, FR2, FR3, and FR4 are respectively as shown in SEQ ID NOs: 12, 13, 14, and 15.

[0014] Further, the heavy chain variable region of the antibody has an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 8,

[0015] and the light chain variable region has an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 16.

[0016] Further, the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO: 8, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 16.

[0017] Further, the epitope of the antibody is a linear epitope.

[0018] Further, the antibody is non-fucosylated.

[0019] The second aspect of the present invention provides a nucleic acid encoding the antibody described in the first aspect of the present invention.

[0020] Further, the nucleic acids encoding the complementarity determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody respectively have nucleotide sequences having at least 95% sequence identity with the nucleotide sequences shown in SEQ ID NO: 17, 18, and 19,

[0021] and the nucleic acids encoding the complementarity determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the antibody respectively have nucleotide sequences having at least 95% sequence identity with the nucleotide sequences shown in SEQ ID NO: 25, 26, and 27.

[0022] Further, the nucleotide sequences of the nucleic acids encoding the complementarity determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody are respectively as shown in SEQ ID NO: 17, 18, and 19,

[0023] and the nucleotide sequences of the nucleic acids encoding the complementarity determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the antibody are respectively as shown in SEQ ID NO: 25, 26, and 27.

[0024] Further, the nucleic acids encoding the framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region respectively have nucleotide sequences having at least 70% sequence identity with the nucleotide sequences shown in SEQ ID NO: 20, 21, 22, and 23,

[0025] and the nucleic acids encoding the framework regions FR1, FR2, FR3, and FR4 of the light chain variable region respectively have nucleotide sequences having at least 70% sequence identity with the nucleotide sequences shown in SEQ ID NO: 28, 29, 30, and 31.

[0026] Further, the nucleotide sequences of the nucleic acids encoding the framework regions FR1, FR2, FR3, and FR4 of the heavy chain variable region are respectively as shown in SEQ ID NO: 20, 21, 22, and 23,

[0027] and the nucleotide sequences of the nucleic acids encoding the framework regions FR1, FR2, FR3, and FR4 of the light chain variable region are respectively as shown in SEQ ID NO: 28, 29, 30, and 31.

[0028] Furthermore, the nucleic acid encoding the heavy chain variable region has a nucleotide sequence with at least 70% sequence identity to the nucleotide sequence shown in SEQ ID NO: 24.

[0029] The nucleic acid encoding the light chain variable region has a nucleotide sequence with at least 70% sequence identity to the nucleotide sequence shown in SEQ ID NO: 32.

[0030] Furthermore, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID NO: 24.

[0031] The nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID NO: 32.

[0032] The third aspect of the present invention provides a vector comprising the nucleic acid described in the second aspect of the present invention.

[0033] Furthermore, the vector further comprises a promoter and / or an enhancer.

[0034] Furthermore, the vector further comprises an operably linked nucleic acid molecule.

[0035] Furthermore, the operably linked nucleic acid molecule comprises a tag.

[0036] Furthermore, the tag comprises a localized epitope tag, a tag for purification.

[0037] The fourth aspect of the present invention provides a host cell comprising the nucleic acid described in the second aspect of the present invention or the vector described in the third aspect of the present invention.

[0038] Furthermore, the host cell comprises a prokaryotic cell, a eukaryotic cell.

[0039] Furthermore, the eukaryotic cell comprises a protist cell, an animal cell or a fungal cell.

[0040] Furthermore, the animal cell comprises a mammalian cell, a bird cell, an insect cell.

[0041] Furthermore, the mammalian cell comprises a CHO cell, a HeLa cell, a 911 cell, an AT1080 cell, an A549 cell, a 293 cell.

[0042] The fifth aspect of the present invention provides a derivative, which comprises a detectable reagent or a therapeutic reagent linked to the antibody described in the first aspect of the present invention or the nucleic acid described in the second aspect of the present invention.

[0043] Furthermore, the detectable reagent comprises a fluorescent dye, a radioactive label, a metal ion, an enzyme, a magnetic bead, a colorimetric label.

[0044] Further, the therapeutic reagent includes cytotoxins and therapeutic agents.

[0045] The sixth aspect of the present invention provides a product for detecting Pseudomonas aeruginosa, and the product includes the antibody described in the first aspect of the present invention.

[0046] Further, the product includes a kit and a test strip.

[0047] Further, the kit further includes a buffer.

[0048] Further, the kit further includes an instruction manual.

[0049] The seventh aspect of the present invention provides a pharmaceutical composition, and the pharmaceutical composition includes the antibody described in the first aspect of the present invention, the nucleic acid described in the second aspect of the present invention, the vector described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, or the derivative described in the fifth aspect of the present invention.

[0050] Further, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0051] Further, the pharmaceutical composition further includes other therapeutic agents.

[0052] Further, the other therapeutic agents include one or more of antibiotics, anti-inflammatory drugs, different antibodies against Pseudomonas aeruginosa, and therapeutic agents for treating co-infections.

[0053] The eighth aspect of the present invention provides any one of the following methods:

[0054] (1) A method for preparing the antibody described in the first aspect of the present invention, the method including culturing the host cell described in the fourth aspect of the present invention and recovering the antibody;

[0055] (2) A method for detecting Pseudomonas aeruginosa in a sample, the method including contacting the antibody described in the first aspect of the present invention with the sample to be detected and detecting the level of Pseudomonas aeruginosa in the sample;

[0056] (3) A method for neutralizing Pseudomonas aeruginosa, the method including contacting the cells infected with Pseudomonas aeruginosa with the antibody described in the first aspect of the present invention.

[0057] Further, the method described in (1) further includes purifying the antibody.

[0058] Further, the cells described in (3) include A549 cells and red blood cells.

[0059] Further, the red blood cells are rabbit red blood cells.

[0060] Further, the method is a method for non-diagnostic or therapeutic purposes.

[0061] The ninth aspect of the present invention provides the use of the antibody described in the first aspect of the present invention, the nucleic acid described in the second aspect of the present invention, the vector described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, or the derivative described in the fifth aspect of the present invention in detecting Pseudomonas aeruginosa or in preparing a product for detecting Pseudomonas aeruginosa.

[0062] The tenth aspect of the present invention provides the use of the antibody described in the first aspect of the present invention, the nucleic acid described in the second aspect of the present invention, the vector described in the third aspect of the present invention, the host cell described in the fourth aspect of the present invention, or the derivative described in the fifth aspect of the present invention in inhibiting Pseudomonas aeruginosa or in preparing a pharmaceutical composition for preventing and / or treating diseases related to Pseudomonas aeruginosa infection.

[0063] Furthermore, the diseases related to Pseudomonas aeruginosa infection include one or more of fever, chills, fatigue, muscle and joint pain, joint swelling, headache, diarrhea, rash, wound suppuration, bacteremia, acute pneumonia, systemic infection, burn wound infection, intra-abdominal infection, respiratory tract infection, septic shock, suppurative arthritis, enteritis, skin and soft tissue infection, urinary tract infection, intestinal infection, CNS infection (central nervous system infection), ulcerative keratitis, chronic suppurative otitis media, mastoiditis, sinusitis, endocarditis.

[0064] Furthermore, the diseases related to Pseudomonas aeruginosa infection are selected from one or more of acute pneumonia, systemic infection, bacteremia.

[0065] Advantages and beneficial effects of the present invention:

[0066] The present invention proves through experiments that the PcrV antibody can specifically bind to the PcrV antigen, has a neutralizing effect on PcrV lysing A549 cells, and at the same time has the ability to neutralize PcrV-mediated rabbit red blood cell lysis, and has a preventive / therapeutic effect on both systemic infection and acute pneumonia. Description of the drawings

[0067] Figure 1 It is a diagram of the SDS-PAGE purity detection result of the PcrV protein;

[0068] Figure 2 It is a diagram of the agarose gel electrophoresis identification result of the PCR product for establishing the human-specific anti-Pseudomonas aeruginosa antibody ScFv library. Among them, 2A is the nucleic acid gel electrophoresis diagram of the k-ScFv PCR product, 2B is the nucleic acid gel electrophoresis diagram of the λ-ScFv PCR product, and 2C is the nucleic acid gel electrophoresis diagram of the PCR product;

[0069] Figure 3 It is a diagram of the expression and purification result of the PcrV-A039 monoclonal antibody.

[0070] Figure 4 It is a graph showing the detection results of the binding activity of the PcrV-A039 monoclonal antibody;

[0071] Figure 5 It is a graph showing the identification results of the epitope type of the PcrV-A039 monoclonal antibody;

[0072] Figure 6 It is a graph showing the detection results of the PcrV-A039 monoclonal antibody killing A549 cells;

[0073] Figure 7 It is a graph showing the detection results of the PcrV-A039 monoclonal antibody lysing rabbit red blood cells;

[0074] Figure 8 It is a graph showing the establishment and in vivo protective effect in a Pseudomonas aeruginosa systemic infection model. Among them, 8A is a graph showing the experimental results of exploring the infection dose of the systemic infection model, and 8B is a graph showing the analysis of the survival rate results of the systemic infection model;

[0075] Figure 9 It is a graph showing the establishment and in vivo protective effect in a Pseudomonas aeruginosa pneumonia infection model. Among them, 9A is a graph showing the experimental results of exploring the infection dose of the pneumonia infection model, and 9B is a graph showing the analysis of the survival rate results of the pneumonia infection model. Detailed implementation manners

[0076] The following provides definitions of some terms used in this specification. Unless otherwise specified, all technical and scientific terms used herein generally have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains.

[0077] The present invention provides an antibody against Pseudomonas aeruginosa PcrV.

[0078] In one embodiment of the present invention, the antibody includes a full-length antibody and its antigen-binding fragments. The full-length antibody includes two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains typically include 3 hypervariable loops, known as complementarity-determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2, and LC-CDR3, and heavy chain (HC) CDRs include HC-CDR1, HC-CDR2, and HC-CDR3). The 3 CDR regions of the heavy or light chain are inserted between flanking segments known as framework regions (FRs) and form a scaffold that supports the hypervariable loops. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant regions. The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by having α, δ, ε, γ, and μ heavy chains, respectively. Several of the main antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0079] In one embodiment of the present invention, an antigen-binding fragment refers to an antibody fragment that includes, for example, a diabody, Fab, Fab’, F(ab’)2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv) 2 , bispecific dsFv (dsFv-dsFv’), disulfide-stabilized diabody (ds diabody), single-chain antibody (scFv), scFv dimer (bivalent diabody), multispecific antibody composed of antibody fragments containing one or more CDRs, single-domain antibody, nanobody, domain antibody, bivalent domain antibody, or any other antibody fragment that can bind to an antigen but does not contain the complete antibody structure. The antigen-binding fragment can bind to the same antigen as the parental antibody or parental antibody fragment (such as the parental scFv). In some embodiments, the antigen-binding fragment may include one or more CDRs from a specific human antibody that are grafted into the framework regions from one or more different human antibodies.

[0080] The antibody is non-fucosylated.

[0081] In one embodiment of the present invention, fucosylation refers to the presence of fucose residues within the oligosaccharides attached to the peptide backbone of the antibody. Specifically, a fucosylated antibody contains an α(1,6)-linked fucose at the innermost N-acetylglucosamine (GlcNAc) residue of one or both of the N-linked oligosaccharides attached to the Fc region of the antibody, such as at position Asn 297 (EU numbering of Fc region residues) of the human IgG1 Fc domain. Due to minor sequence variations in immunoglobulins, Asn297 can also be located approximately +3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300.

[0082] Non-fucosylated or fucose-deficient antibodies include glycosylated antibody variants of the following Fc regions, wherein the carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose. In some embodiments, antibodies with reduced fucose or lacking fucose have improved ADCC function. Relative to the amount of fucose on the same antibody produced in a cell line, non-fucosylated or fucose-deficient antibodies have reduced fucose.

[0083] The present invention provides a vector comprising the above nucleic acid.

[0084] In one embodiment of the present invention, the vector further comprises a transcriptional promoter and optionally an enhancer, translation signals, and transcriptional and translational termination signals. Expression vectors for stable transformation typically have selectable markers that allow the selection and maintenance of transformed cells. In some cases, an origin of replication can be used to amplify the copy number of the vector in the cell. The vector can also include additional nucleotide sequences that are operably linked to the linked nucleic acid molecule, such as epitope tags for localization, such as 6-his tags or myc tags, or tags for purification, such as GST fusions; and sequences for directing protein secretion and / or membrane association.

[0085] As an alternative embodiment of the present invention, the vector is a virus. Viral vectors are used to introduce non-endogenous nucleic acid sequences encoding target-specific polypeptides. Viral vectors can be retroviral vectors or lentiviral vectors. Viral vectors can also include nucleic acid sequences encoding transduction markers. Suitable viral vectors include RNA virus-based vectors, such as vectors derived from retroviruses, such as vectors derived from Moloney murine leukemia virus (MLV), and include more complex retrovirus-derived vectors, such as vectors derived from lentiviruses. Vectors derived from HIV-1 belong to this category.

[0086] Viral vectors include retroviruses, adenoviruses, parvoviruses (such as adeno-associated viruses), coronaviruses, negative-strand RNA viruses (such as orthomyxoviruses (such as influenza viruses), rhabdoviruses (such as rabies and vesicular stomatitis viruses), paramyxoviruses (such as measles and Sendai viruses)), positive-strand RNA viruses (such as picornaviruses and alphaviruses), and double-stranded DNA viruses, including adenoviruses, herpesviruses (such as herpes simplex virus type 1 and 2, Epstein-Barr virus, and cytomegalovirus), and poxviruses (such as vaccinia, fowlpox, and canarypox). Other viruses include, but are not limited to, Norwalk virus, togavirus, flavivirus, reovirus, papillomavirus, hepatitis virus, and hepadnavirus. Examples of retroviruses include avian leukosis-sarcoma, mammalian type C, type B viruses, type D viruses, HTLV-BLV group, lentiviruses, or foamy viruses.

[0087] As an alternative embodiment of the present invention, the vector is an expression vector. The expression vector according to the present invention is capable of directing the replication and expression of the nucleic acid molecule of the present invention in a host.

[0088] Non-limiting examples of vectors include pQE-12, pUC-series, pBluescript (Stratagene), pET-series expression vectors (Novagen), or pCRTOPO (Invitrogen), λgt11, pJOE, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL-n-EK, pESP-1, pOP13CAT, E-027pCAG Kosak-Cherry (L45a) vector system, pREP (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5

[0089] (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, the Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), pcDNA3.1, pcDNA3.4, pSPORT1 (GIBCO BRL), pGEMHE (Promega), pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (EdgeBiosystems), pTriEx-Hygro (Novagen), and pCINeo (Promega). Non-limiting examples of plasmid vectors suitable for Pichia pastoris include, for example, plasmids pAO815, pPIC9K, and pPIC3.5K (all from Invitrogen). Another vector suitable for expressing proteins in Xenopus embryos, zebrafish embryos, and a variety of mammalian and avian cells is the multi-purpose expression vector pCS2+.

[0090] The present invention provides host cells comprising the above nucleic acids or the above vectors.

[0091] In one embodiment of the present invention, the host cell is a cell for receiving, retaining, replicating, and amplifying a vector. It includes prokaryotic cells and eukaryotic cells. Among them, prokaryotic cells include Gram-negative or Gram-positive organisms, such as Escherichia coli (DH5α, BL21DE3, BL21DE3pLysS, JM109, TOP10) or Bacilli. Eukaryotic cells include, but are not limited to, protist cells, animal cells, or fungal cells, and the animal cells include mammalian cells, avian cells, and insect cells; among them, mammalian cells include, but are not limited to, CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, 293 cells, 293T cells, and HEK293F cells.

[0092] The present invention provides a derivative, which comprises a detectable reagent or a therapeutic reagent linked to the above antibody or nucleic acid.

[0093] In one embodiment of the present invention, the detectable reagent can be any substance having detectable physical or chemical properties. Such detectable reagents have been well developed in the field of immunoassays, and in general, most of the labels useful in such methods can be applied to the provided method. Thus, the label can be any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Detectable reagents include, but are not limited to, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, etc.), radioactive labels (e.g., 3 H, 125 I, 35 S, 14 C or 32 P), in particular, radioactive labels (e.g., 157 Gd, 55 Mn, 162 Dy, 52 Cr and 56 Fe), metal ions (e.g., 111 In, 97 Ru, 67 Ga, 68 Ga, 72 As, 89 Zr and 201 Tl), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), electron transfer agents (e.g., including metal-binding proteins and compounds), luminescent and chemiluminescent labels (e.g., luciferin and 2,3-dihydrophtahlazinediones, e.g., luminol), magnetic beads (e.g., DYNABEADS TM ),

[0094] and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex).

[0095] Therapeutic agents include, but are not limited to, cytotoxins, therapeutic agents, or radioactive metal ions, wherein the cytotoxins include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogs or homologs. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agents (e.g., nitrogen mustard, thioepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., actinomycin D (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), antimitotic agents (e.g., vincristine and vinblastine), and antiviral agents, such as, but not limited to, nucleoside analogs, such as zidovudine, acyclovir, ganciclovir, vidarabine, idoxuridine, trifluridine, and ribavirin; foscamet, amantadine, rimantadine, saquinavir, indinavir, ritonavir, and α-interferon.

[0096] The present invention provides a product for detecting Pseudomonas aeruginosa, and the product includes the above-mentioned antibody.

[0097] The product includes a kit, a test strip.

[0098] In one embodiment of the present invention, the kit is packaged in a suitable form. Suitable packages include, but are not limited to, vials, bottles, jars, flexible packages (e.g., sealed polyester films or plastic bags), etc. The kit may optionally provide other components, such as buffers. And in some embodiments, it further includes another medicament (e.g., the medicament described herein) and / or an instruction manual. The instruction manual attached to the kit in the present application is usually a written instruction on a label or package insert (e.g., a sheet included in the kit), and machine-readable instructions (e.g., instructions on a magnetic or optical storage optical disc) are also acceptable.

[0099] The present invention provides a pharmaceutical composition, which comprises the above-mentioned antibody, the above-mentioned nucleic acid, the above-mentioned carrier, the above-mentioned host cell or the above-mentioned derivative.

[0100] In one embodiment of the present invention, the pharmaceutically acceptable carrier is non-toxic to the recipient at the used dosage and concentration. Pharmaceutically acceptable carriers include buffering agents such as phosphates, citric acid and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butanol or benzyl alcohol; alkyl esters of p-hydroxybenzoic acid, such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (such as zinc-protein complexes); and / or nonionic surfactants such as TWEEN TM , PLURONICS TM or polyethylene glycol (PEG).

[0101] The pharmaceutical composition further comprises other therapeutic agents.

[0102] The other therapeutic agents include one or more of antibiotics, anti-inflammatory drugs, different antibodies against Pseudomonas aeruginosa and therapeutic agents useful for treating co-infections.

[0103] The antibiotics include any one or more of the following: penicillins (piperacillin, piperacillin / tazobactam, mezlocillin, ticarcillin, ticarcillin / clavulanic acid), cephalosporins (ceftazidime, cefpirome, cefepime), carbapenems (imipenem / cilastatin; meropenem), monobactams (aztreonam), aminoglycosides (tobramycin, gentamicin, amikacin), quinolones (ciprofloxacin, levofloxacin), and other antibiotics (polymyxin B, colistin). Common treatment regimens include: for bacteremia: penicillin plus aminoglycoside; penicillin plus ciprofloxacin; cephalosporin, aztreonam or carbapenem plus aminoglycoside or ciprofloxacin; for CNS infections: ceftazidime, optionally plus aminoglycoside; cefepime; ciprofloxacin; aztreonam; meropenem; for bone or joint infections: penicillin plus aminoglycoside or ciprofloxacin; cephalosporin; aztreonam; fluoroquinolone; carbapenem; for otitis externa: cephalosporin; carbapenem; ciprofloxacin; cephalosporin plus aminoglycoside; for keratitis / corneal ulcer (eye): tobramycin (topical), optionally piperacillin or ticarcillin (topical); ciprofloxacin or ofloxacin (topical); and for urinary tract infections: ciprofloxacin; aminoglycoside; penicillin; cephalosporin; carbapenem.

[0104] The anti-inflammatory drugs include, but are not limited to, corticosteroids and non-steroidal anti-inflammatory drugs.

[0105] In one embodiment of the present invention, the pharmaceutical composition can be administered by various routes, including, for example, intravenous injection, intra-arterial administration, intraperitoneal injection, intratracheal administration, oral administration, inhalation administration, intravascular administration, intramuscular injection, intratracheal administration, subcutaneous injection, intraocular administration, intrathecal administration, mucosal administration or transdermal administration. In some embodiments, a sustained-release formulation of the composition is used. In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered via the portal vein. In some embodiments, the composition is administered by artery. In some embodiments, the composition is administered intraperitoneally. In some embodiments, the composition is administered intrahepatically. In some embodiments, the composition is administered by hepatic artery infusion. In some embodiments, the composition is administered to a site remote from the first lesion.

[0106] The present invention will be further illustrated with specific examples below. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the present invention. Without departing from the scope of the present invention, the main features of the present invention can be used in various embodiments.

[0107] Example 1 Expression and purification of recombinant Pseudomonas aeruginosa PcrV protein

[0108] The full-length sequence of the PcrV gene was synthesized (by Shanghai Jierui), and it was cloned into the expression vector pGEX-6P-2 using the restriction endonuclease recognition sites BamHI and EcoRI. The recombinant plasmid was transformed into XL1-Blue Escherichia coli for expression. The PcrV engineering bacteria were inoculated into LB medium containing ampicillin and cultured overnight (37 °C, 100 rpm). The next day, the culture was expanded at a ratio of 1:100 (V / V) until the OD 600 reached approximately 0.8, and IPTG was added to induce expression overnight (4 °C, 200 rpm). The bacteria were collected by centrifugation and lysed by sonication. The supernatant of the lysate was added to GST-4FF affinity packing material and mixed vertically at 4 °C overnight. The PcrV-GST fusion protein was obtained by elution with glutathione, and then Prescission protease was added to remove the GST tag. The PcrV protein was obtained by PHP chromatography, and the buffer was replaced with PBS. The purity of the PcrV protein was detected by SDS-PAGE gel electrophoresis.

[0109] The detection results of the molecular weight and purity of PcrV are as Figure 1 shown, and the results show that the purity of the PcrV protein is 100.0%.

[0110] Example 2 Isolation of PBMC cells

[0111] Healthy volunteers and volunteers who had recovered after severe Pseudomonas aeruginosa infection were recruited, and peripheral blood samples were collected for the isolation of plasma cells.

[0112] The venous blood samples of the above volunteers were collected into anticoagulant tubes containing heparin, and plasma and PBMC cells were separated by density centrifugation. The specific operations were as follows: Take venous blood and centrifuge at 400 g, 22 °C for 15 min; Aspirate the clear plasma layer after centrifugation and aliquot it for storage at -80 °C; After aspirating the supernatant, mix it well with an equal volume of RPMI1640 (Gibco), and slowly add it to a sterile centrifuge tube containing lymphocyte separation medium, and keep the liquid layer intact; Centrifuge at 2000 rpm for 20 min, and use a capillary tube to aspirate the mononuclear cells located in the cloudy layer and place them in another sterile centrifuge tube. Add more than 5 times the volume of RPMI1640, centrifuge at 1500 rpm for 10 min, wash the cells twice, and after cell counting, freeze them for standby at 1×10 7 / tube.

[0113] Example 3 Establishment of a human-specific anti-Pseudomonas aeruginosa antibody ScFv library

[0114] 1. cDNA preparation

[0115] Total RNA was extracted from the peripheral blood lymphocytes isolated in Example 2, and then reverse transcription was performed using the total RNA as a template to synthesize cDNA.

[0116] 2. Gene Amplification

[0117] VK and VH PCR Amplification: Using cDNA as a template, add forward and reverse primers, and perform PCR amplification of VK and VH. Prepare a 1.5% nucleic acid gel, and collect and purify the target band around 400 bp.

[0118] k-ScFv PCR Amplification: Using the VK and VH PCR products as templates, add forward and reverse primers for PCR amplification. Prepare a 1.0% nucleic acid gel, and collect and purify the target band around 750 bp. The nucleic acid gel electrophoresis result of the k-ScFv PCR product is as Figure 2 shown in A.

[0119] λ PCR Amplification: Using cDNA as a template, add forward and reverse primers, and perform PCR amplification of VK and VH respectively. Prepare a 1.0% nucleic acid gel, and collect and purify the target band around 400 bp.

[0120] λ-ScFv PCR Amplification: Using the λ and VH PCR products as templates, add forward and reverse primers, and perform PCR amplification of λ-ScFv. Prepare a 1.0% nucleic acid gel, and collect and purify the target band around 750 bp. The nucleic acid gel electrophoresis result of the λ-ScFv PCR product is as Figure 2 shown in B.

[0121] 3. Construction of Phage Display Library

[0122] Link ScFv to pcomb3XSS by double digestion. Take TG1 competent cells, add the ligation product, mix well, add to a pre-chilled electroporation cuvette, and perform electroporation transformation using the preset transformation program of the electroporator. After electroporation, add pre-warmed SOB medium to the electroporation cuvette to resuspend the cells. Incubate the transformation product at 37°C with shaking at 120 r / min for 40 min; evenly spread the culture on LB / AMPGLU plates, 1.5 mL per plate, and culture at 37°C for 6 h. Add 2 mL of LB / AMP-GLU medium to the petri dish, collect the bacterial lawn with a cell scraper, add 1 / 3 volume of 50% glycerol, mix well and aliquot, and store at -80°C, which is the ScFv antibody phage library.

[0123] 4. Identification of Positive Rate

[0124] Using the ScFv antibody library bacterial solution as a template, add RSH-F and RSH-B primers, and perform PCR amplification. Prepare a 1.0% nucleic acid gel to identify the positive rate of the PCR product. The nucleic acid electrophoresis result of the PCR product shows ( Figure 2 C), and the positive rate of the ScFv antibody library bacterial solution PCR identification is 100%. Example 4 Screening of Human-Specific Anti-Pseudomonas aeruginosa Antibody Natural Library

[0125] 1. Screen the human-derived library using solid-phase and liquid-phase methods

[0126] 1) Solid-phase screening

[0127] Coat with the antigen at three different concentrations overnight at 4°C with rotation. Dilute the antibody library according to the ratio of phage: 5% PBSM = 1:3, add 5% PBSM and then block for 1 h. Then add the corresponding blocked phage library to the corresponding immunotube, incubate at room temperature for 1 h, add 800 μL of trypsin to the immunotube, and elute at room temperature for 20 min.

[0128] 2) Liquid-phase screening

[0129] Take the required beads into a 1.5 mL centrifuge tube, wash with 1 mL of PBST, mix well with a four-dimensional rotary mixer for 5 min, adsorb for about 2 min, discard the supernatant, resuspend with 1 mL of 5% BSA, place on the four-dimensional rotary mixer and rotate for 1 h, then discard the supernatant. Dilute the phage according to the ratio of Phage: PBS: 5% BSA = 1:1:2, add to the blocked beads, and mix well on a vertical rotary shaker for about 1 h. Adsorb the magnetic beads with a magnetic stand and collect the supernatant.

[0130] 3) Phage infection of SS320

[0131] Take 5 mL of SS320 bacterial solution with an OD 600 of about 0.5 and invert and mix well with the elution liquid, then let it stand in a 37°C constant temperature incubator for 30 min. Add 90 μL of SS320 bacterial solution with an OD 600 of about 0.5 to a 96-well round-bottom dilution plate. Take 10 μL of the prepared phage, serially dilute it from the first row of wells to the last row in a 10-fold manner, and place the dilution plate in a 37°C constant temperature incubator for 30 min.

[0132] After the infection, take out the centrifuge tube from the constant temperature incubator and centrifuge at 5000 g for 5 min. Discard part of the supernatant in the centrifuge tube, leaving about 300 μL to resuspend the bacteria. Drop the resuspended bacteria evenly onto the plate, horizontally shake the plate back and forth in different directions to ensure that the bacterial solution is evenly spread on the plate. When there is no obvious bacterial solution on the plate, invert the plate and place it in a 37°C constant temperature incubator overnight. Take out the library plate the next day, check if there are plaques on the library plate. If not, use a Pasteur pipette to suck about 3 mL of 2YT medium and gently shake it to make it evenly distributed, then scrape the bacteria on the surface of the medium with a disposable spreader. According to the previous dilution factor, the dilution factor increases by 10 times successively downward corresponding to row A at 5×10 3 Therefore, select a row (X) with clear clone numbers and count the number of clones (n). Titer = 5×10 3 ×10 X-1×n. Adjust the wavelength of the visible spectrophotometer to 600 nm, add 2 mL of 2YT medium for zero calibration (OA / 100% T mode), take 50 μL of the stored bacteria in one tube and transfer it to a 15 mL centrifuge tube pre-filled with 1950 mL of 2YT medium. After mixing, conduct the detection and record the OD value. Multiply the obtained value by 40 to obtain the concentration of the corresponding bacteria.

[0133] 2. Use the ELISA method to perform a preliminary screening test on the trillion-scale fully human antibody library at the phage level

[0134] Dilute the antigen to 2 μg / mL with 1×PBS, add 30 μL per well, and incubate overnight at 4°C; Wash the plate 3 times with PBST, select the corresponding blocking solution, add 180 μL per well, and incubate at room temperature for 1 h; Wash the plate 3 times with PBST, add the primary antibody after centrifugation in advance, add 30 μL per well, and incubate at room temperature for 1 h; Wash the plate 3 times with PBST, select different secondary antibodies according to the experimental requirements, add 30 μL per well, and incubate at room temperature for 1 h; Wash the plate 6 times with PBST, add the chromogenic solution TMB, add 30 μL per well, and react at room temperature for 5 - 10 min; Add 2M termination solution, add 30 μL per well; Use an enzyme-linked immunosorbent assay reader to read the data at OD 450 nm. Define positive clones according to a certain background value and send them for testing and analysis.

[0135] Example 5 Cloning, expression, and purification of the PcrV-A039 fully human antibody

[0136] 1. Experimental method

[0137] Use the screened positive clone display vector as a template, and use PCR to amplify human Ig VH and VK / L respectively by synthesizing vector primers. Identify the PCR products by 1.2% agarose gel electrophoresis. Purify the antibody genes identified as positive and with the light and heavy chains matching in pairs using the Qiagen PCR product purification kit. Perform bidirectional sequence determination on the purified products, and predict the antibody gene family, mutation rate, CDR regions, etc. using the IMGT online server (http: / / imgt.cines.fr / ).

[0138] Connect the PCR purified products to the pcDNA3.4 vector using the TA cloning method (light chain type Kappa, heavy chain type hIgG1). Transform the constructed recombinant humanized anti-PcrV antibody expression vector into DH5α competent cells, culture them on an LB plate containing ampicillin, pick 10 single colonies and perform PCR with specific primers. The reaction conditions are: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 100 s, for 28 cycles; extension at 72°C for 5 min. Detect the PCR products by electrophoresis on a 1% agarose gel.

[0139] The vector plasmid in the obtained positive transformants was transformed into DH5α for large-scale amplification, and then the plasmid was extracted. The recombinant plasmid was transfected into HEK 293F cells using the transfection reagent PEI and cultured with shaking in an incubator at 37°C and 5% CO 2 for 5 days. The culture was centrifuged to collect the cell supernatant, and the PcrV antibody was obtained by protein A affinity chromatography of the supernatant. The expression and purification of the antibody were detected by SDS-PAGE gel electrophoresis.

[0140] 2. Experimental results

[0141] The purified recombinant monoclonal antibody was named the fully human PcrV-A039 monoclonal antibody ( Figure 3 ), with a relative molecular weight of approximately 140 - 160 kDa, a heavy chain of approximately 48 kDa, and a light chain of approximately 23 kDa. The sequence of the antibody is shown in Table 1.

[0142] Table 1 Sequence of PcrV-A039 monoclonal antibody

[0143]

[0144]

[0145]

[0146] Example 6 Detection of the binding activity of PcrV-A039 monoclonal antibody

[0147] 1. Experimental method

[0148] The stock solution of PcrV protein was diluted to 3 μg / mL with the coating solution and added to a 96-well ELISA plate at 100 μL / well, and coated overnight at 4°C.

[0149] The plate was washed 3 times with PBST buffer. 200 μL of 3% BSA blocking solution was added to each well and incubated at 37°C for 2 hours. The plate was washed 3 times with PBST buffer. The PcrV-A039 monoclonal antibody was diluted to 100 μg / mL with the antibody dilution solution and added to the ELISA plate for 3-fold serial dilution. Each concentration was replicated 3 times; the vaccine serum was diluted 2000-fold as a positive control, the irrelevant antibody IgG1 (100 μg / mL) was used as a negative control, and the PBST dilution solution was used as a blank control. 100 μL was added to each well, and all were in 3 replicates. Incubate at 37°C for 1 hour. The plate was washed 3 times with PBST. Anti-human HRP-IgG was diluted 5000-fold with PBST and added to the 96-well ELISA plate at 100 μL / well, and incubated at 37°C for 1 hour. The plate was washed 3 times with PBST. 100 μL of TMB chromogenic solution was added to each well and placed in the dark at 37°C for 10 min. 50 μL of sulfuric acid (2 M) was added to each well to terminate the reaction. The ELISA plate was placed in an ELISA reader to read the OD 450Values, and the absorbance values were analyzed by a four-parameter logistic equation (GraphPad Prism) and the EC50 was calculated.

[0150] 2. Experimental results

[0151] The results are as Figure 4 shown. The PcrV-A039 monoclonal antibody can specifically bind to the PcrV antigen, and the calculated EC50 is 46.35 nM (0.00695 μg / mL).

[0152] Example 7 Determination of the epitope type of the PcrV-A039 monoclonal antibody

[0153] 1. Experimental method

[0154] The PcrV protein was mixed with protein loading buffer (reduced type) at a ratio of 1:5 and then subjected to SDS-PAGE gel electrophoresis. After electrophoresis, transfer the membrane. The filter paper was soaked with electrotransfer buffer, and the PVDF membrane was soaked with anhydrous methanol. Transfer was carried out using a semi-dry transfer instrument at 23 V for 20 minutes. After the electrotransfer was completed, take out the PVDF membrane, wash the membrane 3 times with TBST buffer, place it in 1% bovine serum albumin and block overnight at 4 °C. After blocking, wash the membrane 3 times with TBST buffer, then add anti-human PcrV-A039 diluted 1:5000 with TBST, incubate at 37 °C for 1 hour, wash the membrane 3 times with TBST buffer, place it in HRP-labeled goat anti-human IgG secondary antibody, incubate at 37 °C for 40 minutes, and drop DAB chromogenic solution on the PVDF membrane for color development. When the bands were clearly developed, wash with water to terminate the reaction.

[0155] 2. Experimental results

[0156] The results are as Figure 5 shown. The PcrV-A039 antibody can bind to the PcrV antigen, indicating that the PcrV antibody is a linear epitope.

[0157] Example 8 Neutralizing effect of the PcrV-A039 monoclonal antibody on PcrV-lysed A549 cells

[0158] 1. Experimental method

[0159] The CCK-8 kit was used to evaluate the ability of the anti-PcrV-A039 monoclonal antibody to prevent PcrV-mediated lysis of human lung epithelial cell line A549 cells. The concentration of A549 cells was adjusted to 2×10 5 / mL with DMEM (supplemented with glutamine + 10% fetal bovine serum) medium, and added to a 96-well U-bottom plate at 100 μL / well. Incubate at 37 °C, 5% CO 2Cultivate overnight in an incubator. Centrifuge to remove the supernatant, add the PcrV-A039 monoclonal antibody and isotype control (IgG1) to the cells, and incubate in a 37°C incubator with 5% CO 2 for 30 minutes.

[0160] Meanwhile, prepare a logarithmic-phase culture of Pseudomonas aeruginosa strain PAO1. Inoculate the PAO1 strain into 10 mL of LB medium and culture overnight at 37°C and 150 rpm. Dilute it 1:50 and inoculate it into LB medium for secondary activation, and shake culture at 37°C until OD 600 = 1. After centrifuging the culture, wash it once with PBS, and then dilute the OD 600 to 0.03 with PBS. Add 100 μL / well to the wells containing cells and antibodies, and incubate at 37°C and 5% CO 2 for 2 hours.

[0161] Measure the release of lactate dehydrogenase (LDH) according to the instructions of the CCK-8 kit, and quantify cell lysis by the inhibition rate % of lactate dehydrogenase release.

[0162] 2. Experimental results

[0163] The experimental results are as Figure 6 shown. The PcrV-A039 antibody showed the efficacy of preventing A549 cell death. This monoclonal antibody has a protective effect against Pseudomonas aeruginosa strain PAO1, with an IC50 of 8.20×10 -8 M, and the isotype control IgG1 has no neutralizing toxicity effect.

[0164] Example 9 Ability of the PcrV-A039 monoclonal antibody to neutralize PcrV-mediated rabbit red blood cell lysis

[0165] 1. Experimental method

[0166] Evaluate the ability of the PcrV-A039 monoclonal antibody to prevent PcrV-mediated lysis of rabbit red blood cells. Inoculate Pseudomonas aeruginosa strain PA14 into LB medium and culture overnight at 37°C and 150 rpm. Dilute it 1:50 and inoculate it into LB medium, and shake culture at 37°C until OD 600 = 1. After centrifuging the culture, wash it once with PBS, and then dilute the OD 600 to 0.15 with PBS. After centrifuging the 50% rabbit red blood cell (rRBC) suspension at 4°C and 2000×g for 10 minutes, discard the supernatant and dilute the rRBC to 5% with PBS.

[0167] Mix the PcrV-A039 monoclonal antibody, isotype control (IgG1), and positive lysis control (Triton X 100) with the PA14 bacterial solution respectively, add 100 μL per well to the round bottom of a 96-well plate, then add 100 μL of 5% rRBC to each well, and incubate the ELISA plate at 37 °C with shaking for 2 hours. After incubation, centrifuge at 200 g at 25 °C for one minute, transfer 100 μL of the supernatant from each well to a new round bottom of a 96-well plate and measure the OD 540 , and analyze the absorbance values using a four-parameter logistic equation (GraphPad Prism).

[0168] 2. Experimental results

[0169] The results showed that the PcrV-A039 antibody exhibited the efficacy of preventing rRBC hemolysis (as Figure 7 ), with an IC50 of 9.30×10 - 8 M, and the isotype control IgG1 had no neutralizing toxicity effect.

[0170] Example 10 Role of the PcrV-A039 monoclonal antibody in a systemic infection model

[0171] 1. Experimental method

[0172] 1) Exploration of the infection dose of the Pseudomonas aeruginosa systemic infection model (bacteremia model): The experiment was divided into 6 groups, with 10 BALB / c mice in each group. Adjust the concentration of the PAO1 bacterial solution to 6.3×10 7 CFUs / mL, 1.3×10 8 CFUs / mL, 2.5×10 8 CFUs / mL, 5.0×10 8 CFUs / mL, 1.0×10 9 CFUs / mL with normal saline as the control, and inject 100 μL intravenously into each mouse. Observe and record the survival of the mice every 12 hours after challenge and calculate the survival rate, and observe for 7 days in total.

[0173] 2) Analysis of the survival rate of the Pseudomonas aeruginosa systemic infection model: Take 40 BALB / c mice with a body weight of 20 g ± 1 g, and inject 100 μL of the PAO1 bacterial solution (7.0×10 8CFUs / mouse); After 2 h, the mice were injected with antibodies in the following groups: 10 mg / kg group: 20 BALB / c mice were injected with 200 μg of PcrV-A039 antibody via the tail vein, with a volume of 100 μL; hIgG1(Hla-20) control group (Hla-20 is a specific fully human IgG1 monoclonal antibody against Staphylococcus aureus antigen Hla and serves as an isotype control antibody for the specific fully human IgG1 monoclonal antibody of PcrV): 10 BALB / c mice were injected with 200 μg of hIgG1(Hla-20) via the tail vein, with a volume of 100 μL; normal saline control group: 10 BALB / c mice were injected with 100 μL of normal saline. The survival time of the mice was observed every 12 h for a total of 168 h.

[0174] 2. Experimental results

[0175] The infection dose results of the Pseudomonas aeruginosa systemic infection model (bacteremia model) are as Figure 8 shown in A. When the infection dose of PAO1 was 6.3×10 6 CFUs / mouse, 1.3×10 7 CFUs / mouse, 2.5×10 7 CFUs / mouse, 5.0×10 7 CFUs / mouse, 1.0×10 8 CFUs / mouse, the mortality rates of the mice were 0, 10%, 30%, 60%, and 100% respectively. Using the probit weighted regression method (Bliss method) with SPSS 13.0 software, the LD50 of tracheal instillation challenge with PAO1 in BALB / c mice was calculated to be 3.5×10 7 CFUs / mouse, and the 95% confidence interval was [2.5×10 7 , 5.1×10 7 CFUs / mouse. Therefore, the infection dose LD50 (3.5×10 7 CFUs / mouse) of PAO1 in the systemic infection model is applicable to the evaluation of indicators such as bacterial colonization, pathological changes, and inflammation levels in the organs of mice after bacterial infection; the infection dose 2×LD50 (7.0×10 7 CFUs / mouse) is applicable to the evaluation of the survival rate of mice after bacterial infection.

[0176] The analysis results of the survival rate of the PA systemic infection model showed ( Figure 8B), the survival rate of mice in the PcrV-A039 antibody group at 10 mg / kg was 70%, which was significantly higher than that of the negative control group (0%), and the difference was statistically significant (p < 0.05). The survival rate of mice in the hIgG1 (Hla-20) control group was 10%. It shows that the fully human anti-PcrV antibody can resist the systemic invasion of PA.

[0177] Example 11 In Vivo Prophylactic Effect of PcrV-A039 Monoclonal Antibody on Pseudomonas aeruginosa PAO1 Acute Pneumonia Model

[0178] 1. Experimental Method

[0179] 1) Exploration of the infection dose of the Pseudomonas aeruginosa pneumonia infection model: Take 60 BALB / c mice and divide them into 6 groups with 10 mice in each group. Use normal saline to adjust the PAO1 bacterial solution concentration to 5.0×10 7 CFUs / mL, 1.3×10 8 CFUs / mL, 2.5×10 8 CFUs / mL, 5.0×10 8 CFUs / mL, 1.0×10 9 CFUs / mL respectively, with normal saline as the control. Inject 20 μL of the bacterial solution into each mouse through tracheal injection. Observe for 7 days, and record the death of mice every 12 h and calculate the survival rate.

[0180] 2) Survival rate analysis of the Pseudomonas aeruginosa pneumonia infection model: Take 40 BALB / c mice (body weight 20 g ± 1 g), and inject 100 μL of PAO1 bacterial solution (1.0×10 7 CFUs / mouse) into each mouse via intravenous injection. After 2 h, group the mice and inject antibodies at the following concentrations: 10 mg / kg group (20 mice): Inject 200 μg of PcrV-A039 antibody into the tail vein, with a volume of 100 μL;; hIgG1 (Hla-20) control group (10 mice): Inject 200 μg of IgG1 into the tail vein, with a volume of 100 μL; normal saline control group (10 mice): Inject 100 μL of normal saline into the tail vein. The test period is 7 days, and observe the survival time of mice every 12 h and calculate the survival rate.

[0181] 2. Experimental Results

[0182] After seven days of observation of the Pseudomonas aeruginosa pneumonia infection model, the experimental results are as Figure 9 shown in A. The PAO1 infection dose is 1.0×10 6 CFUs / mouse, 2.5×10 6 CFUs / mouse, 5.0×10 6 CFUs / mouse, 1.0×10 7CFUs / mouse, 2.0×10 7 CFUs / mouse, the mortality rates of the mice were 0, 10%, 50%, 90%, and 100% respectively. The LD50 of intratracheal instillation challenge with PAO1 in BALB / c mice was calculated to be 5.0×10 6 CFUs / mouse by the probit weighted regression method (Bliss method) using SPSS 13.0 software, and the 95% confidence interval was [3.6×10 6 , 6.8×10 6 CFUs / mouse.

[0183] Analysis of the survival rate of the PA pneumonia infection model showed that ( Figure 9 B), the survival rate of the mice in the PcrV-A039 antibody group at 10 mg / kg was 75%, which was significantly higher than that of the negative control group (0%), and the difference was statistically significant (p < 0.05). The survival rate of the mice in the hIgG1 (Hla-20) control group was 10%.

[0184] The description of the above embodiments is only for understanding the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. An antibody against Pseudomonas aeruginosa PcrV, characterized in that: The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the antibody heavy chain variable region are shown in SEQ ID NOs: 1, 2, and 3, respectively. The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID NOs: 9, 10, and 11, respectively.

2. The antibody according to claim 1, characterized in that The antibody further comprises heavy chain variable region framework regions FR1, FR2, FR3, and FR4 having at least 70% sequence identity with the amino acid sequences shown in SEQ ID NOs: 4, 5, 6, and 7, And light chain variable region framework regions FR1, FR2, FR3, and FR4 having at least 70% sequence identity with the amino acid sequences shown in SEQ ID NOs: 12, 13, 14, and 15.

3. The antibody according to claim 2, characterized in that The amino acid sequences of the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 are shown in SEQ ID NOs: 4, 5, 6, and 7, respectively. The amino acid sequences of the light chain variable region framework regions FR1, FR2, FR3, and FR4 are shown in SEQ ID NOs: 12, 13, 14, and 15, respectively.

4. The antibody according to claim 3, characterized in that The heavy chain variable region of the antibody has an amino acid sequence that has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 8, The light chain variable region has an amino acid sequence that has at least 70% sequence identity to the amino acid sequence shown in SEQ ID NO:

16.

5. The antibody according to claim 4, characterized in that The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

16.

6. The antibody according to claim 1, characterized in that The epitope of the antibody is a linear epitope.

7. The antibody according to claim 1, characterized in that The antibody is afucosylated.

8. A nucleic acid encoding the antibody according to any one of claims 1 to 7.

9. The nucleic acid according to claim 8, characterized in that The nucleic acids encoding the antibody heavy chain variable region complementary determining regions CDR1, CDR2, and CDR3 have nucleotide sequences that are at least 95% identical to the nucleotide sequences shown in SEQ ID NOs: 17, 18, and 19, respectively. The nucleic acids encoding the complementary determining regions CDR1, CDR2, and CDR3 of the antibody light chain variable region have nucleotide sequences that have at least 95% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 25, 26, and 27, respectively.

10. The nucleic acid according to claim 9, characterized in that The nucleotide sequences of the nucleic acids encoding the complementary determining regions CDR1, CDR2, and CDR3 of the antibody heavy chain variable region are shown in SEQ ID NOs: 17, 18, and 19, respectively. The nucleotide sequences of the nucleic acids encoding the complementary determining regions CDR1, CDR2 and CDR3 of the antibody light chain variable region are shown in SEQ ID NOs: 25, 26 and 27, respectively.

11. The nucleic acid according to claim 8, characterized in that The nucleic acids encoding the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 have nucleotide sequences that are at least 70% identical to the nucleotide sequences shown in SEQ ID NOs: 20, 21, 22, and 23, respectively. The nucleic acids encoding the light chain variable region framework regions FR1, FR2, FR3, and FR4 have nucleotide sequences that have at least 70% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 28, 29, 30, and 31, respectively.

12. The nucleic acid according to claim 11, characterized in that The nucleotide sequences of the nucleic acids encoding the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 are shown in SEQ ID NOs: 20, 21, 22, and 23, respectively. The nucleic acid nucleotide sequences encoding the light chain variable region framework regions FR1, FR2, FR3, and FR4 are shown in SEQ ID NOs: 28, 29, 30, and 31, respectively.

13. The nucleic acid according to claim 8, characterized in that The nucleic acid encoding the heavy chain variable region has a nucleotide sequence that is at least 70% identical to the nucleotide sequence shown in SEQ ID NO: 24, The nucleic acid encoding the light chain variable region has a nucleotide sequence that has at least 70% sequence identity to the nucleotide sequence shown in SEQ ID NO:

32.

14. The nucleic acid according to claim 13, characterized in that The nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID NO: 24, The nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in SEQ ID NO:

32.

15. A vector comprising the nucleic acid of any one of claims 8 to 14.

16. The carrier according to claim 15, characterized in that The vector also includes a promoter and / or an enhancer.

17. The carrier according to claim 15, characterized in that The vector also includes an operably linked nucleic acid molecule.

18. The carrier according to claim 17, characterized in that The operably linked nucleic acid molecule includes a tag.

19. The carrier according to claim 18, characterized in that The tags include epitope tags for localization and tags for purification.

20. A host cell comprising the nucleic acid of any one of claims 8 to 14 or the vector of any one of claims 15 to 19.

21. The host cell according to claim 20, characterized in that The host cells include prokaryotic cells and eukaryotic cells.

22. The host cell according to claim 21, characterized in that The eukaryotic cell includes a protist cell, an animal cell or a fungal cell.

23. The host cell according to claim 22, characterized in that The animal cells include mammalian cells, avian cells, and insect cells.

24. The host cell according to claim 23, characterized in that The mammalian cells include CHO cells, HeLa cells, A549 cells, and 293 cells.

25. A derivative, characterized in that The derivative comprises a detectable agent or a therapeutic agent linked to the antibody according to any one of claims 1 to 7.

26. The derivative according to claim 25, characterized in that The detectable agents include fluorescent dyes, radioactive labels, metal ions, enzymes, magnetic beads, and colorimetric labels.

27. The derivative according to claim 25, characterized in that The therapeutic agent comprises a therapeutic agent.

28. A product for detecting Pseudomonas aeruginosa, characterized in that: The product comprises the antibody according to any one of claims 1-7.

29. The product according to claim 28, characterized in that The products include test kits and test strips.

30. The product according to claim 29, characterized in that The kit also includes a buffer.

31. The product according to claim 29, characterized in that The kit also includes instructions.

32. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the antibody of any one of claims 1-7, the nucleic acid of any one of claims 8-14, the vector of any one of claims 15-19, the host cell of any one of claims 20-24 or the derivative of any one of claims 25-27.

33. The pharmaceutical composition according to claim 32, characterized in that The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

34. The pharmaceutical composition according to claim 32, characterized in that The pharmaceutical composition may also include other therapeutic agents.

35. The pharmaceutical composition according to claim 34, characterized in that The other therapeutic agents include one or more of antibiotics, anti-inflammatory drugs, and different antibodies against Pseudomonas aeruginosa.

36. Any of the following methods: (1) A method for preparing the antibody according to any one of claims 1 to 7, characterized in that: The method comprises culturing the host cell of any one of claims 20 to 24, and recovering the antibody; (2) A method for detecting Pseudomonas aeruginosa in a sample for non-diagnostic purposes, characterized in that the method comprises contacting the antibody according to any one of claims 1 to 7 with a sample to be detected, and detecting the level of Pseudomonas aeruginosa in the sample; (3) A method for neutralizing Pseudomonas aeruginosa for non-therapeutic purposes, characterized in that the method comprises contacting cells infected with Pseudomonas aeruginosa with the antibody according to any one of claims 1 to 7.

37. The method according to claim 36, characterized in that The method described in (1) further comprises purifying the antibody.

38. The method according to claim 36, characterized in that The cells described in (3) include A549 cells and red blood cells.

39. The method according to claim 38, characterized in that The red blood cells are rabbit red blood cells.

40. Use of the antibody of any one of claims 1 to 7, the nucleic acid of any one of claims 8 to 14, the vector of any one of claims 15 to 19, the host cell of any one of claims 20 to 24, or the derivative of any one of claims 25 to 27 for detecting Pseudomonas aeruginosa for non-diagnostic purposes or in the preparation of a product for detecting Pseudomonas aeruginosa.

41. Use of the antibody of any one of claims 1 to 7, the nucleic acid of any one of claims 8 to 14, the vector of any one of claims 15 to 19, the host cell of any one of claims 20 to 24, or the derivative of any one of claims 25 to 27 for inhibiting Pseudomonas aeruginosa for non-therapeutic purposes or in the preparation of a pharmaceutical composition for preventing and / or treating diseases related to Pseudomonas aeruginosa infection; The Pseudomonas aeruginosa infection-related diseases are selected from one or more of acute pneumonia and bacteremia.

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