CN117964750B

CN117964750B - Monoclonal antibody for resisting respiratory syncytial virus and application thereof

Info

Publication number: CN117964750B
Application number: CN202410174934.0A
Authority: CN
Inventors: 李新新
Original assignee: Beijing Weixing Biotechnology Co ltd
Current assignee: Beijing Weixing Biotechnology Co ltd
Priority date: 2024-02-04
Filing date: 2024-02-07
Publication date: 2024-10-25
Anticipated expiration: 2044-02-07
Also published as: CN117964750A

Abstract

The monoclonal antibody LV26 provided by the invention has strong affinity activity to the respiratory syncytial virus, can be specifically combined with the respiratory syncytial virus, provides a new thought for the detection of the respiratory syncytial virus and the treatment of diseases related to respiratory syncytial virus infection, and has wide application prospect.

Description

Monoclonal antibody for resisting respiratory syncytial virus and application thereof

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to an anti-respiratory syncytial virus monoclonal antibody and application thereof.

Background

Respiratory syncytial virus (Respiratory Syncytial Virus, RSV) is an important cause of severe respiratory disease in infants and young children, and RSV infection can also cause severe disease in the elderly, individuals with chronic lung disease, and immunocompromised adults (e.g., bone marrow transplant recipients).

Monoclonal antibodies are homogeneous antibodies characterized by the property of specifically recognizing a single antigen, specifically binding to the antigen and having a high affinity for the antigen have prompted the development of monoclonal antibodies as a very useful tool for detecting molecules. The development of efficient, rapid and specific monoclonal antibodies to detect RSV is therefore a highly desirable problem in the art.

Disclosure of Invention

In order to make up for the defects of the prior art, the invention provides an anti-respiratory syncytial virus monoclonal antibody and application thereof.

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

In a first aspect the invention provides a monoclonal antibody against respiratory syncytial virus comprising heavy chain variable region complementarity determining regions CDR1, CDR2, CDR3 having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequences shown in SEQ ID NOS 1,2, 3,

And light chain variable region complementarity determining regions CDR1, CDR2, CDR3 having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% 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 monoclonal antibody also comprises heavy chain variable region framework regions FR1, FR2, FR3, FR4 having at least 90%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% 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 90%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to 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 monoclonal antibody has an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO. 8,

The light chain variable region has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 16.

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

Further, the monoclonal antibodies also include all or part of the antibody heavy chain constant region and/or antibody light chain constant region.

Further, the monoclonal antibody specifically binds to the pre-fusion conformation of the F protein of respiratory syncytial virus.

Further, the monoclonal antibody is nonfucosylated.

In a second aspect the invention provides a nucleic acid encoding a monoclonal 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 monoclonal antibody have nucleotide sequences having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% 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 variable region of the monoclonal antibody have nucleotide sequences at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleotide sequences shown in SEQ ID NOS 20, 21, 22, respectively.

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 monoclonal antibody are shown in SEQ ID NO. 17, 18 and 19 respectively,

The nucleotide sequences of nucleic acids for coding the complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region of the monoclonal antibody are shown in SEQ ID NOs 20, 21 and 22 respectively.

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 also include a transcription promoter and/or 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 prokaryotic cell includes escherichia coli.

Further, the eukaryotic cells include protozoan cells, animal cells, plant 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, 293T cells, 293F cells.

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

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 the detection of respiratory syncytial virus, the product comprising a monoclonal antibody according to the first aspect of the invention.

Further, the product comprises a kit and test paper.

Further, the kit further comprises a solid support.

Further, the kit also comprises a buffer solution, a blocking reagent, a washing reagent and an enzyme substrate.

Further, the kit also includes instructions.

In a seventh aspect, the invention provides a pharmaceutical composition comprising a monoclonal 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 also includes a pharmaceutically acceptable carrier and/or excipient.

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

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

(2) A method for detecting respiratory syncytial virus in a sample, the method comprising contacting a monoclonal antibody of the first aspect of the invention with a sample to be detected, and detecting the level of respiratory syncytial virus in the sample to be detected.

Further, the method described in (2) is a method for non-diagnostic purposes.

According to a ninth aspect of the present invention there is provided the use of a monoclonal 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 in the detection of respiratory syncytial virus or in the preparation of a product for the diagnosis of respiratory syncytial virus infection.

In a tenth aspect, the invention provides the use of a monoclonal 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 for inhibiting respiratory syncytial virus infection or for the preparation of a pharmaceutical composition for the treatment of a respiratory syncytial virus-infected disease.

The invention has the advantages and beneficial effects that:

The monoclonal antibody LV26 provided by the invention has strong affinity activity to respiratory syncytial virus, can specifically bind to respiratory syncytial virus, provides a new thought for detection of respiratory syncytial virus and treatment of diseases related to respiratory syncytial virus infection, and has a wide application prospect.

Drawings

FIG. 1 is a SDS-PAGE of protein A magnetic beads after purification of antibodies;

FIG. 2 is a graph showing the detection of binding activity of RSV-F antibody to the antigen DS2-strepII-His 6;

FIG. 3 is a graph showing the detection of the neutralizing activity of RSV-F antibody against the live RSV-A2 virus.

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 a monoclonal antibody for resisting respiratory syncytial virus, which comprises heavy chain variable region complementarity determining regions CDR1, CDR2 and CDR3, light chain variable region complementarity determining regions CDR1, CDR2 and CDR3, and heavy chain variable region framework regions FR1, FR2, FR3 and FR4, and light chain variable region framework regions FR1, FR2, FR3 and FR4.

In the present invention, monoclonal antibody (mAB) or antibody refers to an antibody molecule having a single molecular composition obtained from a population of substantially identical antibodies. Antibodies comprise two heavy (H) chains and two light (L) chains. The mammalian heavy chain consists of a variable region (VH) and first, second, third and optionally fourth constant regions (CH 1, CH2, CH3, CH4, respectively); mammalian light chains consist of a variable region (VL) and a constant region. The antibody is Y-shaped, wherein the stem of Y consists of the second and third constant regions of two heavy chains that are joined together by disulfide bonds. Each arm of Y comprises a variable region and a first constant region of a single heavy chain in combination with a variable region and a constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable region in both chains typically contains three highly variable loops, known as complementarity determining regions (complementaritydetermining region; CDRs), the light chain CDRs comprise LCDR1, LCDR2 and LCDR3, and the heavy chain CDRs comprise HCDR1, HCDR2, HCDR3. The variable regions of the light and heavy chains also include Framework Regions (FRs), the light chain FRs including LFR1, LFR2, LFR3 and LFR4, and the heavy chain FRs including HFR1, HFR2, HFR3 and HFR4. 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 the heavy chain constant region of the antibody.

The monoclonal antibody specifically binds to the pre-fusion conformation of the F protein of respiratory syncytial virus.

Respiratory syncytial virus fusion proteins (RSV-F proteins) are responsible for fusion of viral and host cell membranes and syncytial formation between viral particles. Its sequence is highly conserved among strains. The RSV-F protein undergoes a large conformational change during the mediated membrane Fusion process, transitioning from a metastable pre-Fusion conformation (pre-Fusion) to a stable post-Fusion conformation (post-Fusion). Antibodies to the RSV-F protein include antibodies that bind to the Pre-fusion conformation, neutralizing antibodies that bind to both the Pre-fusion conformation and the Post-fusion conformation. The antibodies of the invention bind to the Pre-fusion conformation.

The monoclonal antibody or fragment thereof is nonfucosylated.

In the present invention, fucosylation refers to the presence of fucose residues within oligosaccharides 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 the present invention, the vector also includes a transcription promoter and optionally an enhancer, a translation signal, 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, e.g., a GST fusion; and sequences for directing protein secretion and/or membrane association.

The vector of the present invention is not particularly limited, and may be a vector capable of replicating and/or expressing a polynucleotide in eukaryotic or prokaryotic cells including mammalian cells (e.g., human, monkey, rabbit, rat, hamster, or mouse cells), plant cells, yeast cells, insect cells, and bacterial cells (e.g., E.coli)). Preferably, the vector comprises at least one selectable marker operably linked to a suitable promoter such that the polynucleotide can be expressed in a host cell. For example, the vector may comprise a polynucleotide into which a phage, plasmid, cosmid, minichromosome, virus, or retroviral vector is introduced, as other vectors conventionally used, for example, in genetic engineering.

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、pSV2-dhfr、pIZD35、Okayama-Berg cDNA expression vectors pcDV1(Pharmacia)、pRc/CMV、pcDNA1、pcDNA3(Invitrogen)、pcDNA3.1、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 the present invention, a host cell is a cell that is used to receive, hold, replicate, and amplify a 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, plant cells, or fungal cells, including mammalian cells, avian cells, insect cells; wherein the 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, 293T cells, 293F cells.

The invention provides a derivative, which comprises the monoclonal antibody or the nucleic acid connected with a detectable reagent and a therapeutic reagent.

In the present 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, streptavidin, 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 Amphotericin (AMC)), antimitotics (e.g., vincristine and vinblastine), and antiviral agents such as, but not limited to, nucleoside analogs such as zidovudine, acyclovir, ganciclovir (gangcyclovir), vidin, and iodine and doxorubicin; foscarnet (foscamet), amantadine, rimantadine, saquinavir, indinavir, ritonavir, and interferon-alpha.

The invention provides a product for detecting respiratory syncytial virus, which comprises the monoclonal antibody.

The product comprises a kit and test paper

In the present invention, the kit comprises one or more reagents for various detection assays, including, for example, immunoassays such as ELISA (sandwich-type or competitive format). The components of the kit may be pre-attached to the solid support or, when the kit is used, may be applied to the surface of the solid support. In some embodiments of the invention, the signal generating means may be pre-conjugated to an antibody or fragment of the invention, or may need to be combined with one or more components such as buffers, antibody-enzyme conjugates, enzyme substrates, etc., prior to use. The kit may also include additional reagents, such as blocking reagents for reducing non-specific binding to the solid phase surface, washing reagents, enzyme substrates, and the like. The solid phase surface may be in the form of a tube, bead, microtiter plate, microsphere or other material suitable for immobilization of proteins, peptides or polypeptides. In particular aspects, enzymes that catalyze the formation of chemiluminescent or chromogenic products or the reduction of chemiluminescent or chromogenic substrates are components of signal generating means. Such enzymes are well known in the art. The kit may include any of the capture reagents and detection reagents described herein. Optionally, the kit may further comprise instructions for performing the method of the invention.

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

The pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.

In the present invention, the pharmaceutical compositions may be administered orally and may be readily formulated by combination with pharmaceutically acceptable carriers well known in the art. The carrier enables the compound to be formulated as lozenges, pills, dragees, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by mixing the compound with solid excipients, optionally grinding the resulting mixture, and treating the mixture of granules after adding suitable adjuvants, if desired, to obtain lozenges or dragee cores. Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, for example corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, for example crosslinked polyvinylpyrrolidone, agar or alginic acid or a salt thereof (e.g., sodium alginate).

The pharmaceutical compositions may be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion. For injection, one or more compounds may be formulated by dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent (e.g., vegetable or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids or propylene glycol) and, if desired, with usual additives (e.g., solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives). In some embodiments, the compounds may be formulated in aqueous solutions, preferably physiologically compatible buffers (e.g., hanks's solution, ringer's solution, or physiological saline buffer). Formulations for injection may be presented in unit dosage form, for example in ampules or in multi-dose containers, with the addition of a preservative. The compositions may be in the form of suspensions, solutions or emulsions, such as in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

The pharmaceutical composition may be administered systemically via transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. For topical administration, the agent is formulated as an ointment, cream, salve, powder, or gel. In one embodiment, the transdermal delivery agent may be DMSO. The transdermal delivery system may include, for example, a patch. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

The pharmaceutical composition is used for treating diseases of respiratory syncytial virus infection.

In the present invention, respiratory syncytial virus-infected disease means any disease directly or indirectly caused by Respiratory Syncytial Virus (RSV) infection, as well as a disease or condition that predisposes a patient to RSV infection. Including but not limited to lower respiratory tract infections, pneumonia, tracheobronchitis, bronchiolitis, bronchitis, and any related infection or inflammatory condition.

In some embodiments, the pharmaceutical composition may be administered with one or more additional anti-viral antibodies selected from anti-RSV antibodies or antigen-binding fragments thereof, such as palivizumab, motavizumab 、AFFF、P12f2、P12f4、P11d4、A1e9、A12a6、A13c4、A17d4、A4B4、A8c7、1X-493L1、FR H3-3F4、M3H9、Y10H6、DG、AFFF(1)、6H8、L1-7E5、L2-15B10、A13a11、A1h5、A4B4(1)、A4B4L1FR-S28R、A4B4-F52S、rsv6、rsv11、rsv13、rsv19、rsv21、rsv22、rsv23、RF-1、RF-2, or antigen-binding fragments thereof.

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.

Examples

1 Experimental materials

Nupraise mouse single cell BCR IgG H/K amplification kit: mouse SINGLE CELL BCR IgG H/K Amplification Kit, vazyme, DD5101;

293F cells and 293T cells are commercially available human embryonic kidney epithelial cells;

pcDNA3.1 (+) vector: invitrogen, cat# V790-20;

HRP anti-Respiratory Syncytial virus antibody：Abcam,Cat:ab20686；

KPL Trueblue color development liquid: sera care, cat:5510-0030.

2 Experimental methods

2.1 Flow sorting of mouse RSV-F antigen specific binding to B cells

Mice were immunized with the pre-fusion conformation of the RSV-F antigen DS2-strep II-His6, and after 3 weeks, secondary immunization was performed, and the spleen of the mice 15 weeks after secondary immunization was taken, and lymphocytes from the mice were obtained using lymphocyte isolates. Lymphocytes were resuspended in 80 μl of 1% BSA in PBS, added to 20 μl l Fc blocking reagent, and blocked for 20min. Adjusting the density of lymphocytes to 1x10e7/ml, adding 200nM of antigen DS2-strepII-His6 and fluorescent antibody of memory B cells, and incubating for 30min in ice and in the absence of light; 1ml of PBS containing 1% BSA was added for resuspension and the cells were washed 3 times. Adding APC-streptavidine, and incubating for 30min in ice in dark place; 1ml of PBS containing 1% BSA was added for resuspension and the cells were washed 3 times. 400 μl of PBS containing 1% BSA was added to resuspend the cells, filtered to flow tubes, and antigen-specific binding memory B cells were collected by sorting using FACS ARIA III flow cytometer.

2.2 Single B cell antibody sequence acquisition, sequence analysis

The sequences of the heavy and light chains of BCR were obtained using a nupraise Mouse single cell BCR IgG H/K amplification kit (Mouse SINGLE CELL BCR IgG H/K Amplification Kit, vazyme, DD 5101).

The method mainly comprises the following steps: (1) reverse transcription, first strand cDNA synthesis was performed. (2) And (3) amplifying by using the first chain cDNA synthesis product as a template to obtain the full-length cDNA of the IgG heavy chain and the light chain. (3) And (3) using the full-length cDNA amplification products of the IgG heavy chain and the light chain as templates to amplify the variable region genes of the heavy chain and the light chain of the antibody. (4) And (3) using the amplified products of the variable regions of the IgG heavy chain and the light chain as templates to amplify the spliced products of the expression frames of the IgG heavy chain and the light chain.

2.3 Detection of antigen-specific binding Activity of antibodies

The heavy and light chain expression cassette splice products were co-transfected into 293T cells. After 48 hours of transfection, the transfection supernatants were collected and assayed for antigen-specific binding activity.

ELISA plates were coated with 1. Mu.g/ml of the RSV-F antigen DS2-strepII-His6, incubated overnight at 4℃and blocked with PBS containing 3% BSA for 1 hour. The 293T supernatant transfected with the linear expression cassette was diluted 1:3 in PBS, added to a 96-well ELISA plate, incubated for 1 hour, and washed 3 times with 200. Mu.l/well by adding PBST (0.05% Tween-80). HRP-Goat-anti-mouse IgG was added, and after 45min incubation, PBST (0.05% Tween-80) was added for 3 washes, 200. Mu.l/well. And constructing heavy chain and light chain sequences of the antibodies into pcDNA3.1 (+) vectors for the antigen specific binding samples obtained by screening, sequencing to obtain variable region sequences of the IgG heavy chain and the light chain, and analyzing sequencing results by utilizing Igblast websites.

2.4 Expression and purification of antibodies

Expression of antibodies: transfection was performed at a density of 2.5X10e6/ml for 293F cells, and 1. Mu.g of antibody heavy chain plasmid and 1. Mu.g of antibody light chain plasmid were added to 20. Mu.l of medium per 2.5X10e6/ml of cells. Mu.g PEI was added to 20. Mu.l medium, after 5min, the dilutions of plasmid and PEI were mixed, left at room temperature for 15min and added to 293F cells. After transfection, the cell supernatants were collected by centrifugation after incubation in a shaker incubator at 37℃and 220rpm at 5% carbon dioxide concentration for 72 hours.

Purification of the antibodies: antibody 293F cell transfection supernatant was incubated with Protein A magnetic beads equilibrated with PBS for 2 hours, the magnetic beads were adsorbed by a magnetic rack, and the supernatant was discarded. And adding 20CV PBS to clean the magnetic beads, repeatedly reversing and uniformly mixing, adsorbing the magnetic beads by using a magnetic frame, discarding the supernatant, and cleaning for 3 times. The antibody was eluted by adding 10CV 0.1M Glycine (ph=3.0), the beads were adsorbed using a magnetic rack, the supernatant was aspirated, and 1M Tris (ph=8.5) was added to neutralize to ph=7. PBS was added to replace buffer.

2.5 Binding Activity of antibodies to Pre-fusion conformational RSV-F protein DS2-strepII-His6

The binding activity of RSV-F antibody to pre-fusion conformation RSV-F protein DS2-strepII-His6 was detected by means of an enzyme-linked adsorption reaction.

The pre-fusion RSV-F protein DS2-strepII-His6 was used as a coating at a concentration of 1. Mu.g/ml, 100. Mu.l/well, coated overnight and the supernatant was discarded. Blocking was performed for 1 hour with 3% BSA in PBS and the blocking solution was discarded. The antibody LV26 to be tested was diluted 11 times with a 3-fold gradient, the initial well concentration was 100. Mu.g/ml, and a multiplex well was set at 37℃and incubated for 1 hour. Mu.l of PBST (0.05% Tween) was added and the plate was washed 3 times. HRP-coat-anti-mouse IgG diluted 1:5K was added, incubated for 45min, 200. Mu.l PBST (0.05% Tween) was added, and the plate was washed 3 times. Adding TMB color development liquid, and after developing for 10min, stopping developing, and reading by an enzyme-labeling instrument.

2.6 Detection of neutralizing Activity of antibodies

1. Vero cells were plated in 96-well plates, 3 x 10e4 per well, and incubated overnight at 37 ℃ in a 5% co ₂ incubator using 200 μl of complete medium containing 10% serum.

2. The antibodies were diluted in 96-well plates using a three-fold gradient of complete medium with 2% serum at a initial well concentration of 100 ng/. Mu.l, ensuring a final volume of 100. Mu.l per well.

3. The RSV A2 live virus was thawed at 4℃and the TCID50 of the live virus was 1.45x10e5 Pfu/ml. Mu.l of the mixture was added to 9.7ml of a complete medium containing 2% serum, and the mixture was mixed and added to 100. Mu.l of the antibody gradient dilution.

4. VC (no antibody, no virus) wells and CC (no antibody, no virus) wells were set in 96-well plates as controls.

5. The overnight medium was discarded, the solution obtained in the 2-4 steps was mixed well, and the mixture was added to Vero cells and cultured in a 5% CO ₂ incubator at 37℃for 3 hours.

6. The solution added in step 5 was discarded, and 200. Mu.l of methylcellulose was used for each well, and the mixture was cultured in a 5% CO ₂ incubator at 37℃for 72 hours.

7. Mu.l of 4% paraformaldehyde was added to each well, and the mixture was fixed at room temperature for 10 minutes, tapped to discard all the supernatant, and 60. Mu.l of 4% paraformaldehyde was added to each well again, and the mixture was fixed at room temperature for 10 minutes.

8. And 200 mu l of PBS solution is used for each hole, the holes are washed for 5 to 8 times, all the supernatant is removed by tapping, all the residual methylcellulose on the cell surface is washed completely, no viscous liquid or viscous bubbles are ensured in the holes, and the effects of the steps such as subsequent antibody staining and the like are prevented from being influenced.

9. 200 Μl of 5% BSA in PBS was added to each well and blocked for 1 hour at room temperature with a shaker.

10. 1 Diluted with 100. Mu.l PBS per well: 500 RSV polyclonal antibody (Abcam, cat: ab 20686), incubated for 2 hours at room temperature in the absence of light.

11. The wells were washed 5-8 times with 200. Mu.l of PBS solution each.

12. And adding 100 mu l Trueblue KPL of color development liquid (Seracare, cat: 5510-0030) into each hole, incubating for 10-20 minutes at room temperature in a dark place, discarding the color development liquid when obvious blue precipitates are formed in cells observed under a microscope, and directly or after the liquid in the holes is dried, performing photographing counting by using an ELISPot plate reader.

Neutralization activity (%) = [1- (number of spots of test group-number of spots of cell control)/(number of spots of virus control-number of spots of cell control) ] ×100%.

The concentration of antibody at 50% neutralization activity, i.e., the IC50 value of the antibody, was calculated using GRAPHPAD PRISM software.

3 Results of experiments

TABLE 1 LV26 antibody sequences

TABLE 2 heavy chain novel analysis of antibodies

TABLE 3 analysis of antibody light chain novelty

The monoclonal cell sorting technology is used for carrying out single cell sorting on mouse spleen memory B cells immunized with RSV-F pre-fusion conformational immunogen DS2-strep II-His6, thus obtaining a novel neutralizing antibody LV26 (figure 1) of RSV-F protein, the sequences of the antibodies are shown in table 1, and the antibodies have novelty (tables 2 and 3) and have the capability of neutralizing live viruses of RSV-A2 strain.

The half-binding concentration EC50 of LV26 antibody to RSV-F pre-fusion conformational immunogen DS2-strepII-His6 was 0.004091 μg/ml (FIG. 2), indicating that the antibody has higher binding activity to pre-fusion conformational immunogen DS2-strepII-His 6. In addition, the IC50 value of LV26 for neutralizing RSV-A2 strain virus was 0.01727. Mu.g/ml (FIG. 3), indicating that the antibody has a strong neutralizing activity.

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. A monoclonal antibody for resisting respiratory syncytial virus is characterized in that the amino acid sequences of CDR1, CDR2 and CDR3 of heavy chain variable region of the monoclonal antibody are respectively shown as SEQ ID NO.1, 2 and 3,

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

2. The monoclonal antibody according to claim 1, further comprising heavy chain variable region framework regions FR1, FR2, FR3, FR4 that have at least 90%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequences shown in SEQ ID NOS.4, 5,6,7,

3. The monoclonal antibody according to 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 monoclonal antibody of claim 3, wherein the heavy chain variable region of the monoclonal antibody has an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 8,

5. The monoclonal antibody of claim 4, wherein the heavy chain variable region of the monoclonal antibody has an amino acid sequence as set forth in SEQ ID NO. 8 and the light chain variable region has an amino acid sequence as set forth in SEQ ID NO. 16.

6. The monoclonal antibody of claim 5, further comprising all or part of an antibody heavy chain constant region and/or an antibody light chain constant region.

7. The monoclonal antibody of claim 6, wherein the monoclonal antibody specifically binds to the pre-fusion conformation of the F protein of respiratory syncytial virus.

8. The monoclonal antibody of claim 7, wherein the monoclonal antibody is nonfucosylated.

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

10. The nucleic acid of claim 9, wherein the nucleic acid encoding the heavy chain variable region complementarity determining regions CDR1, CDR2, CDR3 of the monoclonal antibody has a nucleotide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO 17, 18, 19, respectively,

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

12. A vector comprising the nucleic acid of any one of claims 9-11.

13. The vector of claim 12, further comprising a transcription promoter and/or enhancer.

14. The vector of claim 12, further comprising an operably linked nucleic acid molecule.

15. The vector of claim 12, wherein the operably linked nucleic acid molecule comprises a tag.

16. The vector of claim 15, wherein the tag comprises a localized epitope tag, a tag for purification.

17. A host cell comprising the nucleic acid of any one of claims 9-11 or the vector of any one of claims 12-16.

18. The host cell of claim 17, wherein the host cell comprises a prokaryotic cell, a eukaryotic cell.

19. The host cell of claim 18, wherein the eukaryotic cell comprises a protist cell, an animal cell, a plant cell, or a fungal cell.

20. The host cell of claim 19, wherein the animal cell comprises a mammalian cell, an avian cell, an insect cell.

21. The host cell of claim 20, wherein the mammalian cell comprises a CHO cell, a HeLa cell, a 911 cell, an AT1080 cell, an a549 cell, a 293T cell, a 293F cell.

22. A derivative comprising a detectable agent attached to a monoclonal antibody according to any one of claims 1-8 or to a nucleic acid according to any one of claims 9-11.

23. The derivative of claim 22, wherein the detectable agent comprises a fluorescent dye, a radiolabel, a metal ion, an enzyme, a magnetic bead, a colorimetric label.

24. A product for detecting respiratory syncytial virus, comprising the monoclonal antibody of any one of claims 1-8.

25. The product according to claim 24, wherein the product comprises a kit, a test paper.

26. The product of claim 25, wherein the kit further comprises a solid support.

27. The product of claim 25, wherein the kit further comprises buffers, blocking reagents, washing reagents, enzyme substrates.

28. The product of claim 25, wherein the kit further comprises instructions.

29. A pharmaceutical composition comprising the monoclonal antibody of any one of claims 1-8, the nucleic acid of any one of claims 9-11, the vector of any one of claims 12-16, the host cell of any one of claims 17-21, or the derivative of any one of claims 22-23.

30. The pharmaceutical composition of claim 29, further comprising a pharmaceutically acceptable carrier and/or excipient.

31. The method comprises the following steps:

(1) A method of producing the monoclonal antibody of any one of claims 1-8, comprising culturing the host cell of any one of claims 17-21, and recovering the monoclonal antibody;

(2) A method for detecting respiratory syncytial virus in a sample for non-diagnostic purposes, comprising contacting the monoclonal antibody of any one of claims 1-8 with the sample to be detected, and detecting the level of respiratory syncytial virus in the sample to be detected.

32. Use of the monoclonal antibody of any one of claims 1-8, the nucleic acid of any one of claims 9-11, the vector of any one of claims 12-16, the host cell of any one of claims 17-21, or the derivative of any one of claims 22-23 for the detection of respiratory syncytial virus for non-diagnostic purposes or for the preparation of a product for the diagnosis of respiratory syncytial virus infection.

33. Use of the monoclonal antibody of any one of claims 1-8, the nucleic acid of any one of claims 9-11, the vector of any one of claims 12-16, the host cell of any one of claims 17-21, or the derivative of any one of claims 22-23 in the preparation of a pharmaceutical composition for inhibiting or treating a disease of respiratory syncytial virus infection.