CN118440191B

CN118440191B - Preparation and application of anti-pseudomonas aeruginosa pcrV antibody

Info

Publication number: CN118440191B
Application number: CN202410581293.0A
Authority: CN
Inventors: 杨峰; 肖斐斐; 杨茜; 张家瑞; 尚德丽; 刘唯; 蔡昌芝; 陈鑫
Original assignee: CHONGQING YUANLUN BIO-TECHNOLOGY CO LTD
Current assignee: CHONGQING YUANLUN BIO-TECHNOLOGY CO LTD
Priority date: 2024-05-11
Filing date: 2024-05-11
Publication date: 2025-06-10
Anticipated expiration: 2044-05-11
Also published as: CN118440191A

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 PcrV lysis of A549 cells, and has the ability to neutralize PcrV-mediated rabbit red blood cell lysis, and has a preventive/therapeutic effect on systemic infection and acute pneumonia.

Description

Preparation and application of anti-pseudomonas aeruginosa pcrV antibody

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to preparation and application of an anti-pseudomonas aeruginosa pcrV antibody.

Background

Pseudomonas aeruginosa (Pseudomonas aeruginosa, PA) is opportunistic pathogen, is one of the most common gram-negative bacteria in hospital acquired infection, has strong inherent drug resistance to various antibiotics, and compared with pulmonary and blood flow infection caused by other pathogens, the PA infection has narrow selection range of antibiotics for clinical treatment and higher patient death rate. PA chronic infection is one of the main causes of morbidity and mortality in cystic fibrosis patients, and by forming a biofilm on the upper respiratory tract of the cystic fibrosis patient, the lower respiratory tract repeatedly colonizes, ultimately leading to chronic pulmonary infection. In addition, it is a common pathogen, and is associated with burn wound infections, pneumonia, urinary tract infections, bacteremia and aids patients. The clinical treatment of pseudomonas aeruginosa infection mainly uses antibiotics as a treatment means, but due to the characteristics of natural drug resistance and acquired drug resistance, effective treatment and control strategies are still lacking at present. According to the statistics result of the drug resistance monitoring data of the Chinese bacteria in the Chinese in 2020, the detection rate of the pseudomonas aeruginosa in clinical specimens is 8.42%, and the fourth rank is achieved. Therefore, it is extremely important to develop new anti-infective strategies based on PA infection. The antibody medicine can eliminate pathogens through toxin neutralization, phagocytosis opsonization and the like, and the antibody has the advantages of high specificity, small side effect and the like.

Pseudomonas aeruginosa primarily utilizes a type III secretion (type III secretion) system, which was first discovered in gram-negative bacteria, to inject toxins into host cells to initiate infection. Clinical studies have shown that PA expressing T3SS has a higher mortality rate. The results in vitro cell culture experiments show that PA expressing T3SS exhibits greater toxicity. Thus, blocking toxins associated with T3SS is critical in controlling infection. The PcrV protein is a transporter protein of the type III secretion system, which interferes with the normal function of the host cell by delivering the virulence protein molecules produced by the bacteria directly into the host cell, triggering the death of the host cell. Gene mutation studies have demonstrated that bacterial type III secretion systems cannot bind to the host cell membrane after deletion of PcrV and thus cannot damage the host cell. The PcrV can be used as a protective antigen of an acute lung infection model, and the active and passive immunity of the PcrV has a protective effect on lung injury caused by PA.

Disclosure of Invention

In order to make up the defects of the prior art, the invention provides a preparation method and application of an anti-pseudomonas aeruginosa pcrV antibody.

In order to achieve the above purpose, the invention adopts the following technical scheme:

In a first aspect the invention provides an antibody against PcrV of Pseudomonas aeruginosa comprising heavy chain variable region complementarity determining regions CDR1, CDR2, CDR3 having at least 95% sequence identity to the amino acid sequences shown in SEQ ID NO. 1,2,3,

And light chain variable region complementarity determining regions CDR1, CDR2, CDR3 having at least 95% sequence identity to the amino acid sequences shown in SEQ ID NOS 9, 10, 11.

Furthermore, the amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown as SEQ ID NO. 1, 2 and 3 respectively,

The amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO. 9, 10 and 11 respectively.

Further, the antibody further comprises heavy chain variable region framework regions FR1, FR2, FR3, FR4 that have at least 70% sequence identity with the amino acid sequences shown in SEQ ID NOS.4, 5, 6, 7,

And light chain variable region framework regions FR1, FR2, FR3, FR4 that have at least 70% sequence identity with the amino acid sequences shown in SEQ ID NOS 12, 13, 14, 15.

Further, the amino acid sequences of the heavy chain variable region framework regions FR1, FR2, FR3 and FR4 are respectively shown as SEQ ID NO. 4, 5, 6 and 7,

The amino acid sequences of the framework regions FR1, FR2, FR3 and FR4 of the light chain variable region are respectively shown as SEQ ID NO. 12, 13, 14 and 15.

Further, the heavy chain variable region of the antibody has an amino acid sequence having at least 70% sequence identity to 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 set forth in SEQ ID NO. 16.

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

Further, the epitope of the antibody is a linear epitope.

Further, the antibody is nonfucosylated.

In a second aspect the invention provides a nucleic acid encoding an antibody according to the first aspect of the invention.

Further, the nucleic acids encoding the complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region of the antibody have nucleotide sequences having at least 95% sequence identity to the nucleotide sequences shown in SEQ ID NOS 17, 18, 19, respectively,

Nucleic acids encoding the complementarity determining regions CDR1, CDR2, CDR3 of the light chain of the antibody have nucleotide sequences that are at least 95% sequence identical to the nucleotide sequences shown in SEQ ID NOS 25, 26, 27, respectively.

Further, the nucleotide sequences of the nucleic acids encoding the complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region of the antibody are shown in SEQ ID NOS 17, 18, 19, respectively,

The nucleotide sequences of the nucleic acids encoding the complementarity determining regions CDR1, CDR2 and CDR3 of the light chain of the antibody are shown in SEQ ID NOS 25, 26 and 27, respectively.

Further, nucleic acids encoding heavy chain variable region framework regions FR1, FR2, FR3, FR4 have nucleotide sequences that have at least 70% sequence identity to the nucleotide sequences shown in SEQ ID NOs 20, 21, 22, 23, respectively,

Nucleic acids encoding the framework regions FR1, FR2, FR3, FR4 of the light chain variable region have a nucleotide sequence that has at least 70% sequence identity to the nucleotide sequences shown in SEQ ID NOS 28, 29, 30, 31, respectively.

Further, 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 nucleotide sequences of the nucleic acids encoding the framework regions FR1, FR2, FR3 and FR4 of the light chain variable region are shown as SEQ ID NOs 28, 29, 30 and 31 respectively.

Further, the nucleic acid encoding the heavy chain variable region has a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence set forth 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 set forth in SEQ ID NO. 32.

Further, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown as SEQ ID NO. 24,

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

In a third aspect the invention provides a vector comprising a nucleic acid according to the second aspect of the invention.

Further, the vector may further comprise a promoter and/or an enhancer.

Further, the vector also includes an operably linked nucleic acid molecule.

Further, the operably linked nucleic acid molecule comprises a tag.

Further, the tag includes a localized epitope tag, a tag for purification.

In a fourth aspect the invention provides a host cell comprising a nucleic acid according to the second aspect of the invention or a vector according to the third aspect of the invention.

Further, the host cells include prokaryotic cells and eukaryotic cells.

Further, the eukaryotic cells include protozoan cells, animal cells, or fungal cells.

Further, the animal cells include mammalian cells, avian cells, insect cells.

Further, the mammalian cells include CHO cells, heLa cells, 911 cells, AT1080 cells, a549 cells, 293 cells.

In a fifth aspect the invention provides a derivative comprising a detectable agent, a therapeutic agent, attached to an antibody according to the first aspect of the invention or to a nucleic acid according to the second aspect of the invention.

Further, the detectable agent includes fluorescent dyes, radiolabels, metal ions, enzymes, magnetic beads, colorimetric labels.

Further, the therapeutic agent includes cytotoxins, therapeutic agents.

In a sixth aspect the invention provides a product for detecting pseudomonas aeruginosa comprising an antibody according to the first aspect of the invention.

Further, the product comprises a kit and test paper.

Further, the kit further comprises a buffer solution.

Further, the kit also includes instructions.

The seventh aspect of the invention provides a pharmaceutical composition comprising an antibody according to the first aspect of the invention, a nucleic acid according to the second aspect of the invention, a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention or a derivative according to the fifth aspect of the invention.

Further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

Further, the pharmaceutical composition also includes other therapeutic agents.

Further, the additional therapeutic agents include one or more of antibiotics, anti-inflammatory drugs, different antibodies against pseudomonas aeruginosa, and therapeutic agents useful in treating co-infections.

An eighth aspect of the invention provides a method of any one of:

(1) A method of producing an antibody according to the first aspect of the invention, the method comprising culturing a host cell according to the fourth aspect of the invention, recovering the antibody;

(2) A method of detecting pseudomonas aeruginosa in a sample, the method comprising contacting an antibody according to the first aspect of the invention with a sample to be detected, detecting the level of pseudomonas aeruginosa in the sample;

(3) A method of neutralizing pseudomonas aeruginosa comprising contacting a cell infected with pseudomonas aeruginosa with an antibody according to the first aspect of the invention.

Further, the method described in (1) further comprises purifying the antibody.

Further, the cells described in (3) include a549 cells and erythrocytes.

Further, the erythrocytes are rabbit erythrocytes.

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

The ninth aspect of the invention provides the use of an antibody according to the first aspect of the invention, a nucleic acid according to the second aspect of the invention, a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention or a derivative according to the fifth aspect of the invention in the detection of pseudomonas aeruginosa or in the preparation of a product for detecting pseudomonas aeruginosa.

According to a tenth aspect of the present invention there is provided the use of an antibody according to the first aspect of the present invention, a nucleic acid according to the second aspect of the present invention, a vector according to the third aspect of the present invention, a host cell according to the fourth aspect of the present invention or a derivative according to the fifth aspect of the present invention for inhibiting pseudomonas aeruginosa or for the preparation of a pharmaceutical composition for the prophylaxis and/or treatment of a disease associated with pseudomonas aeruginosa infection.

Further, the pseudomonas aeruginosa infection related diseases include one or more of fever, chills, fatigue, muscle and joint pain, joint swelling, headache, diarrhea, rash, wound pus, bacteremia, acute pneumonia, systemic infection, burn wound infection, intraperitoneal infection, respiratory tract infection, septic shock, suppurative arthritis, enteritis, skin and soft tissue infection, urinary tract infection, intestinal tract infection, CNS infection (central nervous system infection), ulcerative keratitis, chronic suppurative otitis media, mastoiditis, sinusitis, endocarditis.

Further, the pseudomonas aeruginosa infection related disease is selected from one or more of acute pneumonia, systemic infection and bacteremia.

The invention has the advantages and beneficial effects that:

experiments prove that the PCRV antibody can be specifically combined with the PCRV antigen, has a neutralization effect on the PCRV-cracked A549 cells, has the capability of neutralizing the PCRV-mediated rabbit erythrocyte cracking, and has a prevention/treatment effect on systemic infection and acute pneumonia.

Drawings

FIG. 1 is a diagram showing the result of detecting the purity of the PCRV protein by SDS-PAGE;

FIG. 2 is a diagram of the identification result of agarose gel electrophoresis of PCR products established by a human specific anti-Pseudomonas aeruginosa antibody ScFv library, wherein 2A is a diagram of nucleic acid gel electrophoresis of k-ScFv PCR products, 2B is a diagram of nucleic acid gel electrophoresis of lambda-ScFv PCR products, and 2C is a diagram of nucleic acid gel electrophoresis of PCR products;

FIG. 3 is a graph showing the results of expression and purification of the monoclonal antibody PcrV-A039;

FIG. 4 is a graph showing the results of detection of the binding activity of the monoclonal antibody PcrV-A039;

FIG. 5 is a graph showing the results of epitope type identification of the monoclonal antibody PcrV-A039;

FIG. 6 is a graph showing the results of detection of the killing of A549 cells by the monoclonal antibody of PcrV-A039;

FIG. 7 is a graph showing the results of detection of the lysis of rabbit erythrocytes by the monoclonal antibody PcrV-A039;

FIG. 8 is a graph of the establishment and in vivo protection in a model of systemic infection by Pseudomonas aeruginosa, wherein 8A is a graph of experimental results of infection dose fumbling in the model of systemic infection, and 8B is a graph of analysis of survival rate results in the model of systemic infection;

FIG. 9 is a graph showing the establishment and in vivo protection of a Pseudomonas aeruginosa model for pneumonia infection, wherein 9A is a graph showing experimental results of infection dose fumbling of the model for pneumonia infection, and 9B is a graph showing analysis of survival rate of the model for pneumonia infection.

Detailed Description

The following provides definitions of some of the terms used in this specification. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The invention provides an antibody for resisting pseudomonas aeruginosa PcrV.

In one embodiment of the invention, the antibodies include full length antibodies and antigen binding fragments thereof. Full length antibodies include two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable region in both chains typically comprises 3 hypervariable loops, known as Complementarity Determining Regions (CDRs) (light chain (LC) CDRs comprise LC-CDR1, LC-CDR2 and LC-CDR3, and Heavy Chain (HC) CDRs comprise HC-CDR1, HC-CDR2 and HC-CDR 3). The 3 CDR regions of the heavy or light chain are inserted between flanking segments called Framework Regions (FRs) and form a scaffold supporting the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit multiple 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 alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (gamma 1 heavy chain), igG2 (gamma 2 heavy chain), igG3 (gamma 3 heavy chain), igG4 (gamma 4 heavy chain), igA1 (alpha 1 heavy chain n) or IgA2 (alpha 2 heavy chain).

In one embodiment of the invention, an antigen binding fragment refers to an antibody fragment, including, for example, diabody (diabody), 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), a multispecific antibody consisting of an antibody fragment comprising one or more CDRs, a single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment capable of binding an antigen but not comprising an intact antibody structure. The antigen binding fragment is capable of binding the same antigen as the parent antibody or parent antibody fragment (e.g., parent scFv). In some embodiments, an antigen binding fragment may include one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.

The antibody is a non-rock and (3) fucosylation.

In one embodiment of the invention, fucosylation refers to the presence of fucose residues within an oligosaccharide attached to the peptide backbone of an antibody. Specifically, the fucosylated antibody comprises an alpha (l, 6) linked fucose at the innermost N-acetylglucosamine (GlcNAc) residue in one or both of the N-linked oligosaccharides attached to the Fc region of the antibody, e.g., position Asn297 of the human IgG1 Fc domain (EU numbering of the Fc region residues). Asn297 may also be located about +3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in immunoglobulins.

Non-fucosylated or fucose deficient antibodies include glycosylated antibody variants of an Fc region in which 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. Non-fucosylated or fucose-deficient antibodies have reduced fucose relative to the amount of fucose on the same antibodies produced in the cell line.

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

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

As an alternative to the invention, the vector is a virus. Viral vectors are used to introduce non-endogenous nucleic acid sequences encoding target-specific polypeptides. The viral vector may be a retroviral vector or a lentiviral vector. Viral vectors may also include nucleic acid sequences encoding transduction markers. Suitable viral vectors include RNA virus-based vectors, such as retroviral-derived vectors, such as moloney Murine Leukemia Virus (MLV) -derived vectors, and more complex retroviral-derived vectors, such as lentiviral-derived vectors. HIV-1 derived vectors belong to this class.

Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative strand RNA viruses (e.g., orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and sendai viruses), positive strand RNA viruses (e.g., picornaviruses and A-type viruses) and double stranded DNA viruses, including adenoviruses, herpesviruses (e.g., type 1 and type 2 herpes simplex viruses and Epstein-Barr viruses and cytomegalovirus) and poxviruses (e.g., vaccinia, chicken pox and canary pox). Other viruses include, but are not limited to, norwalk viruses, togaviruses, flaviviruses, reoviruses, papillomaviruses, hepatitis viruses and hepatitis viruses, examples of retroviruses include avian leukemias, mammalian type C, type B viruses, type D viruses, HTLV group, lentiviruses or foamy viruses.

As an alternative to the invention, the vector is an expression vector. Expression vectors according to the invention are capable of directing replication and expression of the nucleic acid molecules of the invention in a host.

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 (L45 a) vector system, pREP (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5

(Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV-dhfr, pIDD 35, okayama-Berg cDNA expression vectors 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 Invitrogen). Another vector suitable for expression of proteins in Xenopus (Xenopus) embryos, zebra fish embryos, and a wide variety of mammalian and avian cells is the multipurpose expression vector pCS2+.

The present invention provides a host cell comprising the nucleic acid described above or the vector described above.

In one embodiment of the invention, the host cell is a cell for receiving, holding, replicating and amplifying the vector. Including prokaryotic cells and eukaryotic cells. Among these, prokaryotic cells include gram-negative or gram-positive organisms, such as E.coli (DH 5. Alpha., BL21DE3pLysS, JM109, TOP 10) or Bacillus. Eukaryotic cells include, but are not limited to, protozoan cells, animal cells including mammalian cells including but not limited to CHO cells, F2N cells, CSO cells, BHK cells, bowes melanoma cells, heLa cells, 911 cells, AT1080 cells, a549 cells, 293T cells, HEK 293F cells, or fungal cells.

The invention provides a derivative, which comprises the steps of connecting a detectable reagent and a therapeutic reagent to the antibody or the nucleic acid.

In one embodiment of the invention, the detectable agent may be any substance having a detectable physical or chemical property. Such detectable reagents have been well developed in the field of immunoassays, and in general, a large portion of any label useful in such methods can be applied to the provided methods. Thus, the label may 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.), radiolabels (e.g., ³H、¹²⁵I、³⁵S、¹⁴ C or ³² P), particularly radiolabels (e.g., ¹⁵⁷Gd、⁵⁵Mn、¹⁶²Dy、⁵² Cr and ⁵⁶ Fe), metal ions (e.g., ¹¹¹In、⁹⁷Ru、⁶⁷Ga、⁶⁸Ga、⁷²As、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-dihydrophthalazine (2, 3-dihydrophtahlazinediones), e.g., luminol), magnetic beads (e.g., DYNABEADS ^TM),

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

Therapeutic agents include, but are not limited to, cytotoxins, therapeutic agents, or radiometal ions, wherein cytotoxins include, but are not limited to, paclitaxel, cytochalasin B, poncirin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide (tenoposide), vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroanthracenedione (dihydroxy anthracin dione), mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine (decarbazine)), alkylating agents (e.g., nitrogen mustard, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU)), cyclophosphamide (cyclothosphamide), busulfan, dibromomannitol, streptozocin, mitomycin C and cisplatin (cis-dichlorodiamine platinum) (II) (DDP) cisplatin (cispratin)), anthracyclines (e.g., daunorubicin (formerly dactinomycin) and doxorubicin), antibiotics (e.g., actinomycin D (dactinomycin) (formerly actinomycin), bleomycin, optical mycin and angustaine)), antimitotics (e.g., vincristine and vinblastine), and antiviral agents such as, but not limited to, nucleoside analogs such as zidovudine, acyclovir, ganciclovir (24), valicarb, dulcin, ribavirin, and amantadine (4, fludromycin, and amantadine).

The invention provides a product for detecting pseudomonas aeruginosa, which comprises the antibody.

The product comprises a kit and test paper.

In one embodiment of the application, the kit is packaged in a suitable form. Suitable packages include, but are not limited to, vials, bottles, jars, flexible packages (e.g., sealed mylar or plastic bags), and the like. The kit may optionally provide other components, such as buffers. And in some embodiments, further comprises another agent (e.g., an agent described herein) and/or instructions for use. The instructions for use attached to the kits of the application are typically written instructions on labels or packaging instructions (e.g., paper sheets contained within the kit), and machine-readable instructions (e.g., instructions on a magnetic or optical storage disc) are also acceptable.

The present invention provides a pharmaceutical composition comprising the above antibody, the above nucleic acid, the above vector, the above host cell or the above derivative.

In one embodiment of the invention, the pharmaceutically acceptable carrier is non-toxic to the recipient at the dosage and concentration used. Pharmaceutically acceptable carriers include buffers such as phosphates, citric acid and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethyl ammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl p-hydroxybenzoates such as methyl 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 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).

The pharmaceutical composition also includes other therapeutic agents.

Such other therapeutic agents include one or more of antibiotics, anti-inflammatory drugs, different antibodies against pseudomonas aeruginosa, and therapeutic agents useful in the treatment of co-infections.

The antibiotics include any one or more of penicillins (piperacillin, piperacillin/tazobactam, mezlocillin, ticarcillin/clavulanic acid), cephalosporins (ceftazidime, cefpirome, cefepime), carbapenems (imipenem/cilastatin; meropenem), monoamide rings (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, an Qu south or carbapeneplus aminoglycoside or ciprofloxacin, for CNS infections, ceftazidime, optionally plus an amino sugar alcohol, cefepime, ciprofloxacin, an Quna, meropenem, for bone or joint infections, penicillin plus aminoglycoside or ciprofloxacin, cephalosporin, an Quna, fluoroquinolone, carbapenem, otitis externa, cephalosporin, carbapenem, ciprofloxacin, cephalosporin plus aminoglycoside, keratitis/corneal ulceration (eye), tobramycin (topical), optionally piperacillin or ticarcillin (topical), ciprofloxacin or ofloxacin (topical), and urinary tract infections, ciprofloxacin, aminoglycoside, penicillin, cephalosporin, carbapenem.

Such anti-inflammatory drugs include, but are not limited to, corticosteroids and non-steroidal anti-inflammatory drugs.

In one embodiment of the present invention, the pharmaceutical composition may be administered by a variety of routes including, for example, intravenous, intra-arterial, intraperitoneal, intrapulmonary, oral, inhalation, intravascular, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, mucosal or transdermal. In some embodiments, a slow 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 through an artery. In some embodiments, the composition is administered intraperitoneally. In some embodiments, the composition is administered intrahepatially. In some embodiments, the composition is administered by hepatic arterial infusion. In some embodiments, the composition is applied to a site remote from the first lesion.

The invention is further illustrated below in connection with specific embodiments. It should be understood that the particular embodiments described herein are presented by way of example and not limitation. The principal features of the invention may be used in various embodiments without departing from the scope of the invention.

EXAMPLE 1 expression and purification of recombinant Pseudomonas aeruginosa pcrV protein

The full length sequence of the PcrV gene (Shanghai Jierui) was synthesized and cloned into the expression vector pGEX-6P-2 using restriction enzyme recognition sites BamHI and EcoRI, and the recombinant plasmid was transformed into XL1-Blue E.coli for expression. Inoculating the pcrV engineering bacteria into LB culture medium containing ampicillin, culturing overnight (37 ℃ C., 100 rpm), performing expansion culture to OD ₆₀₀ at a ratio of 1:100 (V/V) the next day until OD ₆₀₀ is about 0.8, adding IPTG to induce expression overnight (4 ℃ C., 200 rpm), centrifuging, collecting bacteria, and performing ultrasonic lysis on the bacteria. The cleavage supernatant was added with GST-4FF affinity filler and mixed vertically at 4℃overnight, glutathione eluted to obtain pcrV-GST fusion protein, and the GST tag was cleaved by presision protease, pcrV protein was obtained by P HP chromatography, and the buffer was replaced with PBS. The purity of the PcrV protein was checked by SDS-PAGE gel electrophoresis.

The molecular weight and purity of PcrV are shown in fig. 1, which shows that PcrV protein has a purity of 100.0%.

EXAMPLE 2 isolation of PBMC cells

Healthy volunteers and those recovering after severe infection with pseudomonas aeruginosa were recruited and peripheral blood samples were collected for plasma cell isolation.

Collecting venous blood sample of the volunteer, separating plasma and PBMC cells by density centrifugation, wherein the method comprises collecting venous blood at 400g, 22 ℃ for 15min, sucking the supernatant transparent plasma layer after centrifugation, sub-packaging to 80 ℃ for freezing, sucking the supernatant, fully mixing with equal amount of RPMI1640 (Gibco), slowly adding into a sterile centrifuge tube containing lymphocyte separation liquid, keeping the liquid level layering intact, centrifuging at 2000rpm for 20min, sucking mononuclear cells by a capillary tube at cloud fog layer, placing into another sterile centrifuge tube, adding more than 5 times of RPMI1640, centrifuging at 1500rpm for 10min, washing the cells twice, and freezing for standby at 1X 10 ⁷ per branch after cell counting.

EXAMPLE 3 construction of human-specific anti-Pseudomonas aeruginosa antibody ScFv library

1. CDNA preparation

The peripheral blood lymphocytes isolated in example 2 were taken for total RNA cell extraction, and then reverse transcription was performed to synthesize cDNA using total RNA as a template.

2. Gene amplification

And (3) performing PCR amplification on the VK and the VH by taking cDNA as a template, adding a forward primer and a reverse primer, performing PCR amplification on the VK and the VH, preparing 1.5% nucleic acid gel, and collecting and purifying target bands near 400 bp.

And (3) performing PCR amplification by using the VK and VH PCR products as templates, adding forward primers and reverse primers for PCR amplification, preparing 1.0% of nucleic acid gel, and collecting and purifying target bands of about 750 bp. The results of nucleic acid gel electrophoresis of the k-ScFv PCR products are shown in FIG. 2A.

Lambda PCR amplification, namely, taking cDNA as a template, adding a forward primer and a reverse primer, respectively amplifying VK and VH by PCR, preparing 1.0% nucleic acid gel, and collecting and purifying target bands near 400 bp.

Lambda-ScFv PCR amplification, namely, adding forward primer and reverse primer by taking lambda and VH PCR products as templates, carrying out PCR amplification on lambda-ScFv, preparing 1.0% nucleic acid gel, and collecting and purifying target bands near 750 bp. The results of nucleic acid gel electrophoresis of lambda-ScFv PCR products are shown in FIG. 2B.

3. Construction of phage display libraries

Connecting ScFv to pcomb XSS through double enzyme digestion, adding TG1 competent cells into the connection product, uniformly mixing, adding a precooled electric shock cup, performing electric shock conversion by using a conversion program preset by an electric shock converter, adding a preheated SOB culture medium into the electric shock cup to resuspend the cells, converting the products to be at 37 ℃ for 120r/min, performing shake culture for 40min, uniformly coating the culture on LB/AMPGLU plates, culturing for 6h at 37 ℃ at 1.5mL each plate, adding 2mL of LB/AMP-GLU culture medium into the plates, collecting lawn by using a cell scraper, adding 1/3 volume of 50% glycerol, uniformly mixing, subpackaging and storing at-80 ℃ to obtain the ScFv antibody phage library.

4. Positive rate identification

The ScFv antibody library bacterial solution is used as a template, RSH-F and RSH-B primers are added, PCR amplification is carried out, 1.0% nucleic acid gel is prepared, and the positive rate of the PCR product is identified. The result of the PCR product nucleic acid electrophoresis shows (figure 2C) that the positive rate of the PCR identification of the ScFv antibody library bacterial liquid is 100%. EXAMPLE 4 screening of Natural library of human-specific anti-Pseudomonas aeruginosa antibodies

1. Screening human library by solid-liquid phase method

1) Solid phase screening

Antigen concentrations were coated three times at 4 ℃ overnight, antibody libraries were diluted at a ratio of phage: 5% pbsm=1:3, and blocked for 1h after addition of 5% PBSM. Then the corresponding blocked phage library was added to the corresponding immune tube, incubated at room temperature for 1h, 800. Mu.L pancreatin was added to the immune tube, and eluted at room temperature for 20min.

2) Liquid phase screening

Adding 1mL of PBST into a centrifuge tube with 1.5mL of needed Beads, cleaning, uniformly mixing for 5min by using a four-dimensional rotating mixer, adsorbing the supernatant for about 2min, adding 1mL of 5% BSA for heavy suspension, placing the supernatant on the four-dimensional rotating mixer for 1h, diluting the Phage according to the ratio of Phage to PBS to 5% BSA=1:1:2, adding the blocked Beads, and uniformly mixing on a vertical rotating device for about 1 h. The magnetic beads are adsorbed by a magnetic rack, and the supernatant is collected.

3) Phage infection SS320

Mixing 5mL SS320 bacteria solution with OD ₆₀₀ of about 0.5 with the eluate, standing in a 37 deg.C incubator for 30min. 90 mu L of SS320 bacterial liquid with the OD ₆₀₀ of about 0.5 is added into a 96-hole round-bottomed dilution plate, 10 mu L of prepared phage is taken, the dilution is sequentially and multiply diluted from a first row of holes to a last row, and the dilution plate is placed into a 37 ℃ constant temperature incubator for standing for 30min.

After the infection is completed, the centrifuge tube is taken out from the constant temperature incubator, and centrifuged for 5min at 5000 g. A portion of the supernatant from the centrifuge tube was discarded, leaving about 300. Mu.L of resuspended cells. And uniformly dripping the resuspended thalli onto a plate, horizontally shaking the plate back and forth in different directions to ensure that the bacterial liquid is uniformly coated on the plate, and culturing overnight in a 37 ℃ inverted constant temperature incubator when the bacterial liquid is not obvious on the plate. Taking out the warehouse plate the next day, checking whether the warehouse plate has plaque, if not, sucking 3mL 2YT culture medium by using a Pasteur pipette, slightly shaking left and right to uniformly distribute the culture medium, and scraping the surface thalli of the culture medium by using a disposable coating rod. The dilution factor corresponding to 5×10 ³ for row a was sequentially increased 10 times down according to the previous dilution factor, so the number of clones (n) titer=5×10× 10 ³×10^X-1 ×n was counted for row (X) where the number of clones was clear was chosen. The wavelength of the visible spectrophotometer is adjusted to 600nm, 2mL of 2YT culture medium is added for zeroing (OA/100%T mode), 1 tube of the split-packed kumquat is taken to be 50 mu L into a 15mL centrifuge tube which is pre-filled with 1950mL of 2YT culture medium, and after uniform mixing, detection is carried out, and OD value is recorded. The obtained value was multiplied by 40 to obtain the concentration of the corresponding cell.

2. Screening test of the level of Phage of the full-human antibody library by ELISA method

The antigen was diluted to 2. Mu.g/mL with 1 XPBS, incubated at 4℃overnight, PBST plates were washed 3 times, the corresponding blocking solution was selected, 180. Mu.L/well, 1h at RT, PBST plates were washed 3 times, primary antibodies after centrifugation in advance were added, 30. Mu.L/well, 1h at RT, PBST plates were washed 3 times, different secondary antibodies were selected according to the experimental requirements, 30. Mu.L/well, 1h at RT, PBST plates were washed 6 times, chromogenic solution TMB was added, 30. Mu.L/well, the reaction was performed at room temperature for 5-10min, 2M stop solution was added, 30. Mu.L/well, and data were read using an ELISA reader at OD ₄₅₀ nm. Positive clones were defined according to a certain background value and submitted for analysis.

EXAMPLE 5 cloning, expression and purification of the PCRV-A039 fully human antibody

1. Experimental method

The screened positive clone display vector is used as a template, human Ig VH and VK/L are respectively amplified by utilizing PCR through synthetic vector primers, a PCR product is identified by 1.2% agarose gel electrophoresis, the antibody genes which are identified as positive and can be matched with a light chain and a heavy chain in pairs are purified by using a QIAGEN PCR product purification kit, the purified products are subjected to bidirectional sequence determination, and the structures of an antibody gene family, mutation rate, CDR region and the like are predicted by using an IMGT online server (http:// IMGT. Cis. Fr /).

The PCR purified product was ligated to pcDNA3.4 vector (light chain type Kappa, heavy chain type hIgG 1) by TA cloning, the constructed recombinant humanized anti-PcrV antibody expression vector was transformed into DH 5. Alpha. Competent cells, cultured on ampicillin-containing LB plate, 10 single colonies were picked and PCR was performed with specific primers under conditions of 94℃pre-denaturation for 3min, 94℃denaturation for 30s,55℃annealing for 30s,72℃extension for 100s,28 cycles, 72℃extension for 5min. The PCR products were electrophoretically detected on a 1% agarose gel.

Transforming the vector plasmid in the obtained positive transformant into DH5 alpha for mass amplification, extracting the plasmid, transfecting the recombinant plasmid into HEK 293F cells by using a transfection reagent PEI, carrying out shake culture for 5 days in a 37 ℃ and 5% CO ₂ incubator, centrifuging the culture to collect cell supernatant, and carrying out protein A affinity chromatography on the supernatant to obtain the PcrV antibody. The expression and purification of the antibodies were detected by SDS-PAGE gel electrophoresis.

2. Experimental results

The purified recombinant monoclonal antibody was designated as the fully human PcrV-a039 monoclonal antibody (fig. 3), and the relative molecular weight was about 140-160 kDa, the heavy chain was about 48kDa, the light chain was about 23kDa, and the sequences of the antibodies are shown in table 1.

TABLE 1PcrV-A039 monoclonal antibody sequences

EXAMPLE 6 detection of binding Activity of monoclonal antibody PcrV-A039

1. Experimental method

The PcrV protein stock was diluted to 3 μg/mL with coating solution, 96 well elisa plates were added at 100 μl/well and coated overnight at 4 ℃.

Plates were washed 3 times with PBST buffer, 200. Mu.L of 3% BSA blocking solution was added to each well and incubated at 37℃for 2 hours. PBST buffer washing plate 3 times, PCRV-A039 monoclonal antibody diluted to 100 mug/mL with antibody diluent, adding into ELISA plate for 3 times serial dilution, each concentration is 3 times repeated, vaccine serum diluted 2000 times is used as positive control, irrelevant antibody IgG1 (100 mug/mL) is used as negative control, PBST diluent is used as blank control, each 100 mug of each well is 3 multiple wells, and incubation is carried out for 1 hour at 37 ℃. Plates were washed 3 times with PBST, anti-human HRP-IgG was diluted 5000-fold with PBST, 96-well ELISA plates were added at 100. Mu.L/well, and incubated at 37℃for 1 hour. PBST plates were washed 3 times, 100. Mu.L/well of TMB color development solution was added, left at 37℃in the dark for 10min, and 50. Mu.L of sulfuric acid (2M) was added to each well to terminate the reaction. The OD ₄₅₀ values were read by placing the microplate into a microplate reader and the absorbance values were analyzed by four parameter logistic equation (GRAPHPAD PRISM) and EC50 was calculated.

2. Experimental results

As a result, the monoclonal antibody PcrV-A039 was able to bind specifically to the PcrV antigen, and the EC50 was calculated to be 46.35nM (0.00695. Mu.g/mL), as shown in FIG. 4.

Example 7 epitope type determination of the PcrV-a039 monoclonal antibody

1. Experimental method

Mixing protein PcrV with protein loading buffer solution (reduced form) according to a ratio of 1:5, performing SDS-PAGE gel electrophoresis, transferring membrane after electrophoresis, soaking filter paper in electrotransfer buffer solution, soaking PVDF membrane in anhydrous methanol, transferring with semi-dry transfer printing instrument for 23V,20 min, taking out PVDF membrane after electrotransfer, washing membrane 3 times with TBST buffer solution, sealing overnight in 1% bovine serum albumin at 4 ℃, washing membrane 3 times with TBST buffer solution after sealing, adding anti-human PcrV-A039 diluted with TBST according to 1:5000, incubating at 37 ℃ for 1h, washing membrane 3 times with TBST buffer solution, placing in HRP-marked goat anti-human IgG secondary antibody, incubating at 37 ℃ for 40 min, developing color with DAB color development liquid droplet on PVDF membrane, and washing with water to terminate reaction when strip color development is obvious.

2. Experimental results

The results are shown in fig. 5, where the PcrV-a039 antibody was able to bind to the PcrV antigen, indicating that the PcrV antibody is a linear epitope.

EXAMPLE 8 neutralization of PcrV-A039 monoclonal antibodies on PcrV-lysed A549 cells

1. Experimental method

The ability of anti-PcrV-a 039 monoclonal antibodies to prevent PcrV-mediated lysis of human lung epithelial cell line a549 cells was assessed using the CCK-8 kit. A549 cell concentration was adjusted to 2×10 ⁵/mL with DMEM (glutamine +10% fetal bovine serum) medium, added to 96-well U-plates at 100 μl/well and incubated overnight in a 37 ℃ 5% CO ₂ incubator. The supernatant was removed by centrifugation, pcrV-a039 monoclonal antibody and isotype control (IgG 1) were added to the cells and incubated with 5% CO ₂ for 30min at 37 ℃ in an incubator.

At the same time, a log phase culture of Pseudomonas aeruginosa strain PAO1 was prepared. The PAO1 strain was inoculated into 10mL of LB medium, cultured overnight at 37℃and 150rpm, diluted 1:50, and then inoculated into LB medium for secondary activation, and cultured with shaking at 37℃until OD ₆₀₀ =1. After centrifugation, the cultures were washed once with PBS, and then OD ₆₀₀ was diluted to 0.03 with PBS and added to wells containing cells and antibody at 100. Mu.L/well, and incubated at 37℃for 2 hours with 5% CO ₂.

The amount of Lactate Dehydrogenase (LDH) released was measured according to the CCK-8 kit instructions and cell lysis was quantified by% inhibition of lactate dehydrogenase release.

2. Experimental results

As shown in FIG. 6, the PCRV-A039 antibody shows the effect of preventing the death of A549 cells, the monoclonal antibody has a protective effect on the pseudomonas aeruginosa strain PAO1, the IC50 is 8.20X10 ^-8 M, and the isotype control IgG1 has no neutralizing toxic effect.

EXAMPLE 9 ability of the monoclonal antibody to PcrV-A039 to neutralize PcrV-mediated Rabbit erythrocyte lysis

1. Experimental method

The ability of the PcrV-a039 monoclonal antibody to prevent PcrV-mediated lysis of rabbit erythrocytes was assessed. Pseudomonas aeruginosa strain PA14 was inoculated into LB medium, cultured overnight at 37℃and 150rpm, diluted 1:50 and inoculated into LB medium, and shake-cultured at 37℃to OD ₆₀₀ =1. After centrifugation of the culture, the culture was washed once with PBS and the OD ₆₀₀ was diluted to 0.15 with PBS. After centrifugation of 50% rabbit red blood cell (rRBC) suspension for 10min at 4℃and 2000 Xg, the supernatant was discarded and rRBC was diluted to 5% with PBS.

The PcrV-a039 mab, isotype control (IgG 1), positive lysis control (Triton X100) were mixed with PA14 broth separately, 100 μl/well was added to a 96-well plate round bottom plate, and then 100 μl/well of 5% rrbc was added, and the ELISA plate was incubated for 2 hours at 37 ℃ with shaking. After incubation, 200g was centrifuged at 25 ℃ for one minute, 100 μl of supernatant was transferred to a new 96-well plate round bottom plate and OD ₅₄₀ was detected and absorbance was analyzed by four parameter logistic equation (GRAPHPAD PRISM).

2. Experimental results

The results show that the PcrV-a039 antibody showed efficacy in preventing rRBC hemolysis (see fig. 7), with an IC50 of 9.30 x 10 ^- ⁸ M, isotype control IgG1 with no neutralizing toxic effects.

EXAMPLE 10 effect of the monoclonal antibody PCRV-A039 on systemic infection model

1. Experimental method

1) Pseudomonas aeruginosa systemic infection model (bacteremia model) infection dose fumbling the experiment was divided into 6 groups of 10 BALB/c mice each. PAO1 bacteria solution concentrations were adjusted to 6.3×10⁷CFUs/mL、1.3×10⁸CFUs/mL、2.5×10⁸CFUs/mL、5.0×10⁸CFUs/mL、1.0×10⁹CFUs/mL, physiological saline as a control, and 100. Mu.L of each mouse was intravenously injected. Mice survived every 12 hours after challenge and survival was calculated and observed for a total of 7 days.

2) The survival rate analysis of the Pseudomonas aeruginosa systemic infection model comprises the steps of taking 40 BALB/c mice with the weight of 20 g+/-1 g, carrying out intravenous injection on 100 mu L of PAO1 bacterial liquid (7.0X10 ⁸ CFUs/mouse) for each mouse, carrying out antibody injection on the mice according to the following groups after 2h, carrying out antibody injection on 10mg/kg of the BALB/c mice, carrying out tail intravenous injection on 200 mu g of PCRV-A039 antibody, carrying out 100 mu L of the volume, carrying out hIgG1 (Hla-20) control group (Hla-20 is a specific fully human IgG1 type monoclonal antibody of anti-staphylococcus aureus antigen Hla), carrying out tail intravenous injection on 10 BALB/c mice, carrying out tail intravenous injection on 200 mu g of hIgG1 (Hla-20), carrying out 100 mu L of physiological saline control group, carrying out tail intravenous injection on 10 BALB/c mice, and carrying out 100 mu L physiological saline. Mice survival time was observed every 12 hours, for a total of 168 hours.

2. Experimental results

The results of infection dose of the whole body infection model (bacteremia model) of pseudomonas aeruginosa are shown in fig. 8A, and the mortality of mice is 0%, 10%, 30%, 60% and 100% when the PAO1 infection amount is 6.3×10⁶CFUs/mouse、1.3×10⁷CFUs/mouse、2.5×10⁷CFUs/mouse、5.0×10⁷CFUs/mouse、1.0×10⁸CFUs/mouse. The probability weighted regression method (Bliss method) was performed using SPSS13.0 software to calculate that the LD50 of PAO1 tracheal instillation challenge in BALB/c mice was 3.5X10 ⁷ CFUs/mouse, and the 95% confidence interval was [ 2.5X10 ⁷,5.1×10⁷ ] CFUs/mouse. Therefore, the dose LD50 (3.5X10 ⁷ CFus/mouse) of PAO1 in the systemic infection model is suitable for the index evaluation of bacterial colonization, pathological change, inflammation level and the like of the organs of the mice after bacterial infection, and the dose LD50 (7.0X10 ⁷ CFus/mouse) is suitable for the survival rate evaluation of the mice after bacterial infection.

The results of the survival analysis of the PA systemic infection model show that the survival rate of mice in the PCRV-A039 antibody group of 10mg/kg is 70%, the survival rate is obviously higher than that of mice in the negative control group (0%), the difference has statistical significance (p < 0.05), and the survival rate of mice in the hIgG1 (Hla-20) control group is 10%. The fully human anti-PcrV antibodies were shown to be resistant to systemic attack by PA.

EXAMPLE 11 in vivo prophylactic effect of the monoclonal antibody PCRV-A039 on the model of Pseudomonas aeruginosa PAO1 acute pneumonia

1. Experimental method

1) The infection dosage of the pseudomonas aeruginosa pneumonia infection model is fuelled by taking 60 BALB/c mice and dividing the mice into 6 groups of 10 mice each. PAO1 bacteria solution concentration was adjusted to 5.0×10⁷CFUs/mL、1.3×10⁸CFUs/mL、2.5×10⁸CFUs/mL、5.0×10⁸CFUs/mL、1.0×10⁹CFUs/mL, physiological saline as control with physiological saline, and 20. Mu.L of bacteria solution was injected into each mouse through the trachea. Mice were observed for 7 days, and their mortality was recorded every 12h and survival was calculated.

2) The survival rate analysis of the Pseudomonas aeruginosa pneumonia infection model is that 40 BALB/c mice (weight 20 g+ -1 g) are taken, and PAO1 bacterial liquid (1.0X10 ⁷ CFUs/mouse) is intravenously injected into each mouse, and the volume is 100 mu L. After 2 hours, mice were grouped and antibody was injected at a concentration of 200. Mu.g of PCRV-A039 antibody, 100. Mu.L in the tail vein, 200. Mu.g of IgG1 in the tail vein, 100. Mu.L in the normal saline control group (10), and 100. Mu.L in the tail vein. The test period was 7 days, and the survival time of the mice was observed every 12 hours and the survival rate was calculated.

2. Experimental results

The observation of the pseudomonas aeruginosa pneumonia infection model for seven days shows that the experimental results are shown in fig. 9A, and the death rate of mice is 0%,10%, 50%, 90% and 100% when the PAO1 infection amount is 1.0×10⁶CFUs/mouse、2.5×10⁶CFUs/mouse、5.0×10⁶CFUs/mouse、1.0×10⁷CFUs/mouse、2.0×10⁷CFUs/mouse. The probability weighted regression method (Bliss method) was performed using SPSS13.0 software to calculate that the LD50 of PAO1 tracheal instillation challenge in BALB/c mice was 5.0X10 ⁶ CFUs/mouse, and the 95% confidence interval was [ 3.6X10 ⁶,6.8×10⁶ ] CFUs/mouse.

The results of the survival analysis of the PA pneumonia infection model show that the survival rate of mice in the PCRV-A039 antibody group of 10mg/kg is 75%, the survival rate is obviously higher than that of mice in the negative control group (0%), the difference has statistical significance (p < 0.05), and the survival rate of mice in the hIgG1 (Hla-20) control group is 10%.

The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims

1. An anti-pseudomonas aeruginosa PcrV antibody is characterized in that the amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region of the antibody are respectively shown as SEQ ID NO. 1,2 and 3,

The amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO. 9, 10 and 11, respectively.

2. The antibody of claim 1, further comprising heavy chain variable region framework regions FR1, FR2, FR3, FR4 that have at least 70% sequence identity to the amino acid sequences shown in SEQ ID NOS 4,5, 6, 7,

3. The antibody of claim 2, wherein the amino acid sequences of the heavy chain variable region framework regions FR1, FR2, FR3 and FR4 are shown in SEQ ID NO. 4, 5, 6 and 7, respectively,

4. The antibody of claim 3, wherein the heavy chain variable region of the antibody has an amino acid sequence having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO. 8,

5. The antibody of claim 4, wherein 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 of claim 1, wherein the epitope of the antibody is a linear epitope.

7. The antibody according to claim 1, wherein, the antibody is a non-rock and (3) fucosylation.

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

9. The nucleic acid of claim 8, wherein the nucleic acids encoding CDR1, CDR2, CDR3 of the heavy chain variable region of said antibody have a nucleotide sequence that is at least 95% sequence identical to the nucleotide sequence shown in SEQ ID NOS 17, 18, 19, respectively,

10. The nucleic acid of claim 9, wherein the nucleotide sequences of the nucleic acids encoding CDR1, CDR2, CDR3 of the heavy chain variable region of the antibody are shown in SEQ ID NOS 17, 18, 19, respectively,

11. The nucleic acid according to claim 8, wherein the nucleic acids encoding the heavy chain variable region framework regions FR1, FR2, FR3 and FR4 have a nucleotide sequence that has at least 70% sequence identity to the nucleotide sequence shown in SEQ ID NOS 20, 21, 22 and 23, respectively,

12. The nucleic acid according to claim 11, wherein 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,

13. The nucleic acid of claim 8, wherein the nucleic acid encoding the heavy chain variable region has a nucleotide sequence that is at least 70% sequence identical to the nucleotide sequence set forth in SEQ ID NO. 24,

14. The nucleic acid of claim 13, wherein the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as set forth in SEQ ID NO. 24,

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

16. The vector of claim 15, further comprising a promoter and/or enhancer.

17. The vector of claim 15, further comprising an operably linked nucleic acid molecule.

18. The vector of claim 17, wherein the operably linked nucleic acid molecule comprises a tag.

19. The vector of claim 18, wherein the tag comprises a localized epitope tag, a tag for purification.

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

21. The host cell of claim 20, wherein the host cell comprises a prokaryotic cell, a eukaryotic cell.

22. The host cell of claim 21, wherein the eukaryotic cell comprises a protozoan cell, an animal cell, or a fungal cell.

23. The host cell of claim 22, wherein the animal cell comprises a mammalian cell, an avian cell, an insect cell.

24. The host cell of claim 23, wherein the mammalian cell comprises a CHO cell, a HeLa cell, an a549 cell, a 293 cell.

25. A derivative comprising a detectable agent, a therapeutic agent attached to an antibody according to any one of claims 1 to 7.

26. The derivative of claim 25, wherein the detectable agent comprises a fluorescent dye, a radiolabel, a metal ion, an enzyme, a magnetic bead, a colorimetric label.

27. The derivative of claim 25, wherein the therapeutic agent comprises a therapeutic agent.

28. A product for detecting pseudomonas aeruginosa, comprising the antibody of any one of claims 1-7.

29. The product of claim 28, wherein the product comprises a kit, a test paper.

30. The product of claim 29, wherein the kit further comprises a buffer.

31. The product of claim 29, wherein the kit further comprises instructions.

32. A pharmaceutical composition comprising 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 of claim 32, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

34. The pharmaceutical composition of claim 32, wherein the pharmaceutical composition further comprises an additional therapeutic agent.

35. The pharmaceutical composition of claim 34, wherein the additional therapeutic agent comprises one or more of an antibiotic, an anti-inflammatory agent, a different antibody against pseudomonas aeruginosa.

36. The method comprises the following steps:

(1) A method of producing the antibody of any one of claims 1-7, comprising culturing the host cell of any one of claims 20-24, recovering the antibody;

(2) A method for detecting pseudomonas aeruginosa in a sample for non-diagnostic purposes, comprising contacting the antibody of any one of claims 1-7 with a sample to be detected, detecting the level of pseudomonas aeruginosa in the sample;

(3) A method of neutralizing pseudomonas aeruginosa for non-therapeutic purposes, comprising contacting a cell infected with pseudomonas aeruginosa with the antibody of any one of claims 1-7.

37. The method of claim 36, wherein the method of (1) further comprises purifying the antibody.

38. The method of claim 36, wherein the cells in (3) comprise a549 cells, red blood cells.

39. The method of claim 38, wherein the red blood cells are rabbit red blood cells.

40. Use of 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 for the detection of pseudomonas aeruginosa for non-diagnostic purposes or for the preparation of a product for the detection of pseudomonas aeruginosa.

41. Use of 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 for inhibiting pseudomonas aeruginosa or for preparing a pharmaceutical composition for preventing and/or treating a pseudomonas aeruginosa infection-related disease for non-therapeutic purposes;

the pseudomonas aeruginosa infection related diseases are selected from one or more of acute pneumonia and bacteremia.