CN117924473A - Antibodies neutralizing respiratory syncytial virus - Google Patents

Antibodies neutralizing respiratory syncytial virus Download PDF

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CN117924473A
CN117924473A CN202410174943.XA CN202410174943A CN117924473A CN 117924473 A CN117924473 A CN 117924473A CN 202410174943 A CN202410174943 A CN 202410174943A CN 117924473 A CN117924473 A CN 117924473A
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amino acid
antibody
variable region
chain variable
seq
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李新新
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Beijing Weixing Biotechnology Co ltd
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Beijing Weixing Biotechnology Co ltd
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Abstract

The invention discloses an antibody for neutralizing respiratory syncytial virus, and the antibody ZV28 for neutralizing respiratory syncytial virus can specifically bind respiratory syncytial virus, has strong affinity activity, provides a new scheme for detecting respiratory syncytial virus and treating related diseases, and has wide application prospect.

Description

Antibodies neutralizing respiratory syncytial virus
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to an antibody for neutralizing respiratory syncytial virus.
Background
Respiratory syncytial virus (Respiratory Syncytial Virus, RSV) is the most important viral pathogen for acute lower respiratory tract infections in infants and young children worldwide, and has a broad spectrum and high incidence of diseases, and the respiratory syncytial virus can cause upper respiratory diseases, symptoms including fever, nasal obstruction, pharyngitis and otitis media, asthma, bronchitis, bronchiolitis and pneumonia of respiratory symptoms. Recent studies have found that adults with immune deficiency and the elderly are also susceptible to RSV.
Laboratory detection of pathogenic microorganism infection mainly comprises two methods of detecting pathogenic bacteria at the infection site and detecting antibodies in serum and body fluid of patients. The current culture method is a "gold standard" for respiratory syncytial virus diagnosis, but this method is generally time-consuming, requires strict transportation procedures and complex techniques, and is not suitable for rapid diagnosis. The nucleic acid detection techniques such as Polymerase Chain Reaction (PCR) method have high specificity and sensitivity, but the accuracy is not high, and pollution is very easy to cause. Antibody detection is one of the more desirable methods for detecting respiratory syncytial virus infection. Thus, screening for antibodies against respiratory syncytial virus is necessary for detection of respiratory syncytial virus and for anti-RSV infection.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention provides an antibody for neutralizing respiratory syncytial virus.
In order to achieve the above purpose, the invention adopts the following technical scheme:
In a first aspect the invention provides an antibody for neutralising respiratory syncytial virus, said antibody comprising a heavy chain variable region complementarity determining region of three CDRs and a light chain variable region complementarity determining region of three CDRs, wherein the amino acid sequences of the heavy chain variable region complementarity determining regions CDR1, CDR2, CDR3 have 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 1,2,3, respectively, and the amino acid sequences of the light chain variable region complementarity determining regions CDR1, CDR2, CDR3 have 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 9, 10, 11, respectively.
Furthermore, the amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain are shown as SEQ ID NO. 1, 2 and 3, respectively, and the amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 of the light chain are shown as SEQ ID NO. 9, 10 and 11, respectively.
Further, the heavy chain variable region further comprises four FR heavy chain variable region framework regions, and the light chain variable region further comprises four FR light chain variable region framework regions, wherein the amino acid sequences of the heavy chain variable region framework regions FR1, FR2, FR3 and FR4 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, respectively, and the amino acid sequences of the light chain variable region framework regions FR1, FR2, FR3 and FR4 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, respectively.
Further, the amino acid sequences of the heavy chain variable region framework regions FR1, FR2, FR3 and FR4 are shown as SEQ ID NO. 4, 5, 6 and 7, respectively, and the amino acid sequences of the light chain variable region framework regions FR1, FR2, FR3 and FR4 are shown as SEQ ID NO. 12, 13, 14 and 15, respectively.
Further, the amino acid sequence of the heavy chain variable region has at least 70%, at least 75%, at least 80%, at least 85%, 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 sequence shown in SEQ ID NO. 8,
The amino acid sequence of the light chain variable region has at least 70%, at least 75%, at least 80%, at least 85%, 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 sequence set forth in SEQ ID NO. 16.
Further, the amino acid sequence of the heavy chain variable region 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 antibody specifically binds to the pre-fusion conformation of the F protein of respiratory syncytial virus.
Further, the antibody is afucosylated.
Further, the antibodies have any variant with one or more conservative amino acid substitutions.
In a second aspect the invention provides a nucleic acid molecule encoding an antibody according to the first aspect of the invention.
Further, the nucleotide sequences of the nucleic acid molecules encoding the complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain have 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,
The nucleotide sequences of the nucleic acid molecules encoding the light chain variable region complementarity determining regions CDR1, CDR2, CDR3 have 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 acid molecules encoding the complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region are shown in SEQ ID NOS 17, 18, 19, respectively,
The nucleotide sequences of the nucleic acid molecules for coding the light chain variable region complementarity determining regions CDR1, CDR2 and CDR3 are shown in SEQ ID NOS 20, 21 and 22, respectively.
In a third aspect the invention provides a recombinant expression vector comprising a nucleic acid molecule according to the second aspect of the invention.
Further, the recombinant expression vector also includes a promoter.
Further, the recombinant expression vector includes a plasmid vector, a viral vector, or a phage vector.
In a fourth aspect, the invention provides a host cell comprising a nucleic acid molecule according to the second aspect of the invention or a recombinant expression vector according to the third aspect of the invention.
Further, the host cells include prokaryotic cells and eukaryotic cells.
Further, the eukaryotic cells include lower eukaryotic cells and higher eukaryotic cells.
Further, the higher eukaryotic cells include mammalian cells.
Further, the mammalian cells include myeloma cell lines, huT78 cells, 293T cells, 293F cells, CHO cells, W138, BHK cells.
In a fifth aspect the invention provides a pharmaceutical composition comprising an antibody according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a recombinant expression vector according to the third aspect of the invention or a host cell according to the fourth aspect of the invention.
Further, the pharmaceutical composition further comprises a pharmaceutically compatible carrier.
Further, the pharmaceutical composition further comprises a buffer.
Further, the pharmaceutical composition may be used in combination with one or more antiviral agents.
In a sixth aspect the invention provides a kit for the detection of respiratory syncytial virus, the kit comprising an antibody according to the first aspect of the invention.
Further, the kit further comprises a detectable label conjugated to the antibody.
Further, the detectable label includes a fluorescent label, a radioisotope, a chemiluminescent molecule, a paramagnetic ion, or a spin-trapping reagent.
In a seventh aspect, the invention provides a method of detecting respiratory syncytial virus in a sample, the method comprising contacting an antibody according to 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 is a method for non-diagnostic purposes.
In an eighth aspect, the invention provides the use of an antibody according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a recombinant expression vector according to the third aspect of the invention or a host cell according to the fourth aspect of the invention for the detection of respiratory syncytial virus or for the preparation of a product for the diagnosis of respiratory syncytial virus infection.
The ninth aspect of the invention provides the use of an antibody according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a recombinant expression vector according to the third aspect of the invention or a host cell according to the fourth 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 infection disease.
The invention has the advantages and beneficial effects that:
the antibody ZV28 for neutralizing the respiratory syncytial virus can specifically bind the respiratory syncytial virus, has strong affinity activity, provides a new scheme for detecting the respiratory syncytial virus and treating related diseases, and has 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 present invention provides antibodies that neutralize respiratory syncytial virus, comprising a heavy chain variable region complementarity determining region of three CDRs and a light chain variable region complementarity determining region of three CDRs.
In the present invention, antibodies include, but are not limited to, any particular binding member, immunoglobulin class and/or isotype (e.g., igG1, igG2, igG3, igG4, igM, igA, igD, igE, and IgM); and biologically relevant fragments or specific binding members thereof, including but not limited to Fab, F (ab') 2, fv, and scFv (single chain or related entities). The term "antibody" is understood to include functional antibody fragments thereof, unless otherwise indicated.
Antibodies also include functional variants of the antibodies that bind to respiratory syncytial virus and have neutralizing activity against the subtype or fragment.
In particular, functional variants include, but are not limited to: modified derivatives that are substantially similar in primary structural sequence, but which comprise chemical and/or biochemical modifications, e.g. in vitro or in vivo, not found in the parent antibodies of the invention. Such modifications include, for example, acetylation, acylation, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, cross-linking, disulfide bond formation, glycosylation, hydroxylation, methylation, oxidation, pegylation, proteolytic processing, phosphorylation.
The antibodies specifically bind to the pre-fusion conformation of the F protein of the 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 antibodies are afucosylated.
In the present invention, afucosylation refers to antibodies (preferably of the IgG1 isotype) having an altered glycosylation pattern at Asn297 in the Fc region and having a reduced level of fucose residues of the IgG1 or IgG3 isotype. Glycosylation of human IgG1 or IgG3 occurs at Asn297 as core fucosylated double antennary complex oligosaccharides terminating in up to two Gal residues. Depending on the amount of terminal Gal residues, these structures are referred to as G0, G1 (α1,6 or α1, 3) or G2 glycan residues (Raju, T.S., bioProcess Int.1 (2003) 44-53). CHO-type glycosylation of the Fc portion of antibodies is described, for example, by Routier, f.h., glycoconjugate j.14 (1997) 201-207. Antibodies recombinantly expressed in non-sugar modified CHO host cells are typically fucosylated at Asn297 in an amount of at least 85%. It is to be understood that the term afucosylated antibodies as used in the present invention includes antibodies without fucose in their glycosylation pattern. It is generally known that a typical glycosylation residue position in an antibody is asparagine (Asn 297) at position 297 according to the EU numbering system.
The antibodies have any variant with one or more conservative amino acid substitutions.
In the present invention, a variant refers to a molecule that differs in amino acid sequence from a parent antibody amino acid sequence due to the addition, deletion and/or substitution of one or more amino acid residues in the parent antibody sequence and retains at least one desired activity of the parent antibody. The desired activity may include the ability to specifically bind to an antigen, the ability to reduce, inhibit or neutralize respiratory syncytial virus activity in an animal, and the ability to inhibit respiratory syncytial virus-mediated signaling in a cell-based assay. In one embodiment, the variant comprises one or more amino acid substitutions in one or more hypervariable and/or framework regions of the parent antibody. For example, a variant may comprise at least one, or about one to about ten, or about two to about five substitutions in one or more hypervariable and/or framework regions of a parent antibody. Variants that retain the ability to bind respiratory syncytial virus variants may have greater binding affinity, enhanced ability to reduce, inhibit or neutralize respiratory syncytial virus activity in animals, and/or enhanced ability to inhibit respiratory syncytial virus-mediated signaling in cell-based assays.
The invention provides a recombinant expression vector comprising the nucleic acid molecule.
In the present invention, when a prokaryotic cell is used as a host, the recombinant expression vector generally contains a strong promoter (for example, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLlambda promoter, pRlambda promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter), a ribosome binding site for initiating translation, and a transcription/translation termination sequence. In the case of using E.coli strains (e.coli) (e.g., HB101, BL21, DH 5. Alpha., top10, JM109, etc.) as host cells, promoters and operator sites of E.coli tryptophan biosynthesis pathway (Yanofsky, C., J.bacteriol., (1984) 158:1018-1024) and leftward promoters of phage lambda (pLlambda promoter, herskowitz, I.and Hagen, D., ann.Rev.Genet., (1980) 14:399-445) can be used as regulatory sites. In the case of using Bacillus as a host cell, any promoter capable of being expressed in the toxin protein gene of Bacillus (appl. Environ. Microbiol. (1998) 64:3932-3938; mol. Gen. Genet. (1996) 250:734-741) or Bacillus may be used as a regulatory site.
When eukaryotic cells are used as hosts, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter, β -actin promoter, human hemoglobin promoter, and human creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse Mammary Tumor Virus (MMTV) promoter, HIV LTR promoter, moloney virus promoter, EBV promoter, and Rous Sarcoma Virus (RSV) promoter) may be used, and polyadenylation sequences are usually used as transcription termination sequences.
The recombinant expression vector of the present invention also includes plasmids (for example, ,pCL、pSC101、pGV1106、pACYC177、ColE1、pKT230、pME290、pBR322、pUC8/9、pUC6、pBD9、pHC79、pIJ61、pLAFR1、pHV14、pGEX series, pET series, pUC19, etc.), phages (for example, λgt4, λb, λ -Charon, λΔz1, M13, etc.), and viruses (for example, SV40, etc.).
The present invention provides a host cell comprising the above nucleic acid molecule or the above recombinant expression vector.
In the present invention, the host cell may be a prokaryotic cell, such as e.coli, bacillus subtilis (Bacillus subtilis), streptomyces sp, pseudomonas sp, proteus mirabilis Proteus mirabilis, or Staphylococcus sp. Also, the host cell may be a fungal cell, such as Aspergillus sp, a yeast cell such as saccharomyces cerevisiae, saccharomyces cerevisiae Saccharomyces cerevisiae, schizosaccharomyces pombe, and neurospora crassa Neurospora crassa, a lower eukaryotic cell, and a higher eukaryotic cell such as an insect cell. Also, the host cell may be from a plant and/or mammal. Preferred examples of host cells include, but are not limited to, PER.C6 cells, monkey kidney cells 7 (COS 7, particularly simian COS cells), NSO cells, SP2/0, chinese Hamster Ovary (CHO) cells, W138, baby Hamster Kidney (BHK) cells, madin-Darby canine kidney (MDCK) cells, myeloma cell lines, huT78 cells, 293T cells, 293F cells, and other mammalian host cells that produce antibody proteins according to the invention.
In the present invention, the method of transformation into a host cell includes any method for introducing nucleic acid into an organism, cell, tissue or organ, and may be performed as known in the art using standard techniques selected according to the type of host cell. Such methods include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO 4) precipitation, calcium chloride (CaCl 2) precipitation, oscillation with silicon carbide fibers (agitation), agrobacterium-mediated transformation, and PEG, dextran sulfate, liposomes (lipofectamine) or desiccation/inhibition-mediated transformation.
The present invention provides a pharmaceutical composition comprising the above antibody, the above nucleic acid molecule, the above recombinant expression vector or the above host cell.
The pharmaceutical composition further comprises a pharmaceutically compatible carrier.
In the present invention, pharmaceutically compatible refers to non-toxic materials that do not interact with the action of the active components of the pharmaceutical composition. By pharmaceutically compatible carrier is meant a natural or synthetic, organic or inorganic component, which is used in combination with the active component to facilitate application. According to the present invention, a pharmaceutically compatible carrier comprises one or more compatible solid or liquid fillers, diluents or encapsulating substances, said carrier being suitable for administration to a patient. The components of the pharmaceutical compositions of the present invention generally do not interact with each other to significantly affect the desired therapeutic effect of the drug.
In the present invention, the pharmaceutical composition further comprises buffering agents including, but not limited to, acetate, citrate, borate and phosphate.
In the present invention, the pharmaceutical composition further comprises a salt, which, when used in medicine, should be a pharmaceutically compatible salt. However, pharmaceutically incompatible salts may also be used to prepare pharmaceutically compatible salts and are included in the present invention. Such pharmacologically and pharmaceutically compatible salts include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid. Pharmaceutically compatible salts may also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium, potassium or calcium salts.
In the present invention, the pharmaceutical composition also includes suitable preservatives including, but not limited to, benzalkonium chloride, chlorobutanol, nipagin (paraben), and thimerosal, as appropriate.
In the present invention, the pharmaceutical composition further comprises a supplemental immune enhancing substance, such as an adjuvant, including but not limited to CpG oligonucleotides, cytokines, chemokines, saponins, GM-CSF and/or RNA.
Various delivery systems are known and can be used to administer the pharmaceutical compositions of the invention, for example in liposome encapsulation, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis. Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions may be administered by any convenient route, for example by infusion or bolus injection, absorbed through the epithelium or skin mucosa lining (e.g., oral mucosa, nasal mucosa, rectal and intestinal mucosa, etc.), and may be co-administered with other bioactive agents. Administration may be systemic or local. It may be delivered as an aerosolized formulation.
The pharmaceutical composition may also be delivered in vesicles, particularly liposomes.
In certain instances, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, a polymeric material may be used. In yet another embodiment, the controlled release system may be placed in close proximity to the target of the composition, thus requiring only a fraction of the systemic dose.
Injectable formulations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injection, instillation, and the like. These injectable formulations can be prepared by well known methods. For example, injectable preparations may be prepared, for example, by dissolving, suspending or emulsifying the above-described antibodies or salts thereof in a sterile aqueous or oily medium conventionally used for injection. Aqueous media for injection such as physiological saline, isotonic solution containing glucose, other adjuvants, etc., which may be used in combination with: suitable solubilisers, such as alcohols (e.g. ethanol); polyols (e.g., propylene glycol, polyethylene glycol); nonionic surfactants [ e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adducts of hydrogenated castor oil) ] and the like. Useful oily media are, for example, sesame oil, soybean oil, etc., which can be used in combination with solubilizing agents such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in suitable ampules.
The pharmaceutical compositions of the present invention may be delivered subcutaneously or intravenously by standard needles or syringes.
Pharmaceutical compositions for oral or parenteral use are prepared in dosage forms suitable for unit dosage compounding with dosages of the active ingredient. Unit doses of these dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories and the like.
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 pharmaceutical compositions of the present invention may also be used in combination with one or more antiviral agents for the treatment of respiratory syncytial virus infection. Antiviral agents include, but are not limited to, acyclovir, famciclovir, ganciclovir, penciclovir, valganciclovir, idoside, trifluoracetin, brivudine, cidofovir, behenyl alcohol, fomivir, foscarnet acid, qu Jingang amine, imiquimod, podophyllotoxin, entecavir, lamivudine, tebipdine, clevudine, adefovir, tenofovir, boceprevir, telaprevir, praecoratil, arbidol, amantadine, rimantadine, oseltamivir, zanamivir, peramivir, inosine, interferon (e.g., interferon alpha-2 b, PEG interferon alpha-2 a), ribavirin/taribavirin, abacavir, emtricitabine, lamivudine, didanosine, zidovudine, aliscitabine (apricitabine), stampidine, ifetrobin, racivir, amadoravir, stavudine, zalcitabine, tenofovir, efavirenz, nevirapine, itravirlin, rilpivirine, lopinamine, pivirine, atazanavir, furshanavir, lopinavir, darunavir, nelfinavir, ritonavir, saquinavir, telanavir, aminoprevir, indinavir, enfuvirtide, valiro, PRO 140, ibalizumab, raltegravir, elvitegravir, bevirimat, vivecon, including tautomeric forms, analogues, isomers, polymorphs, solvates, derivatives or salts thereof.
The invention provides a kit for detecting respiratory syncytial virus, which comprises the antibody.
The kit further comprises a detectable label conjugated to the antibody.
In the present invention, detectable labels include, but are not limited to, fluorescent labels, radioisotopes, chemiluminescent molecules, paramagnetic ions, or spin-trapping reagents.
Among them, fluorescent labels include, but are not limited to Alexa 350、Alexa 430、AMCA、BODIPY 630/650、BODIPY 650/665、BODIPY-FL、BODIPY-R6G、BODIPY-TMR、BODIPY-TRX、Cascade Blue、Cy3、Cy5,6-FAM、 fluorescein isothiocyanate, HEX, 6-JOE, oregon Green488, oregon Green 500, oregon Green 514, pacific Blue, REG, rhodamine Green, rhodamine Red, renographin, ROX, TAMRA, TET, tetramethyl rhodamine, and/or Texas Red.
Radioisotopes include, but are not limited to, astatine 211、14 carbon, 51 chromium, 36 chlorine, 57 cobalt, 58 cobalt, copper 67、152 Eu, gallium 67、3 hydrogen, iodine 123, iodine 125, iodine 131, indium 111、59 iron, 32 phosphorus, rhenium 186, rhenium 188、75 selenium, 35 sulfur, technetium 99m (technetium), and/or yttrium 90.
Paramagnetic ions include, but are not limited to, ions of chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and/or erbium (III).
The invention provides the use of the above antibody, the above nucleic acid molecule, the above recombinant expression vector or the above host cell for inhibiting respiratory syncytial virus infection or for preparing a pharmaceutical composition for treating respiratory syncytial virus infection diseases.
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. Examples of diseases falling within the former category include pneumonia and bronchiolitis. Diseases and conditions in the latter category (i.e., those in which the patient is at risk of severe RSV infection) include cystic fibrosis, congenital heart disease, cancer, age-related immunosuppression, transplant recipients, and any condition that generally causes a reduction in immunosuppressive status or immune system function, such as post-operative organ transplant protocols or premature labor.
Treatment means internal or external administration of a therapeutic agent, e.g., a composition comprising any antibody or antigen binding fragment of the invention, to an individual or patient suffering from one or more symptoms of a disease, or suspected of suffering from a disease for which the agent has therapeutic activity. Typically, the agent is administered in an amount effective to reduce one or more symptoms of the disease in the treated individual or population, whether by inducing regression of such symptoms or inhibiting progression of such symptoms via any clinically measurable degree. The amount of therapeutic agent effective to alleviate symptoms of any particular disease may vary depending on factors such as the disease state, age and weight of the patient, and the ability of the drug to elicit a desired response in the individual. Whether a symptom of a disease has been alleviated can be assessed by any clinical measure that is commonly used by a physician or other skilled healthcare provider to assess the severity or state of progression of the symptom. Treatment with anti-RSV antibodies can also be combined with other interventions (antibodies, nucleic acids, vaccines and small molecule compounds) to treat other respiratory pathogens.
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 ZV28 to be tested is diluted 11 times in a 3-fold gradient, the concentration of the first hole is 100 mug/ml, a compound hole is arranged at the same time, and the incubation is carried out for 1 hour at 37 degrees. 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 ZV28 antibody sequences
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TABLE 2 heavy chain novel analysis of antibodies
TABLE 3 analysis of antibody light chain novelty
Single cell sorting of mouse spleen memory B cells immunized with RSV-F pre-fusion conformational immunogen DS2-strep II-His6 was performed using flow cell sorting techniques to obtain a novel neutralizing antibody ZV28 (FIG. 1) of RSV-F protein, the sequences of which are shown in Table 1, and the antibodies were novel (Table 2, table 3) and had the ability to neutralize live virus of RSV-A2 strain.
Half-maximal binding concentration EC50 of ZV28 antibody to RSV-F pre-fusion conformational immunogen DS2-strepII-His6 was 0.009495 μg/ml (FIG. 2), demonstrating that the antibody has higher binding activity to pre-fusion conformational immunogen DS2-strepII-His 6. In addition, the IC50 value of ZV28 for neutralizing RSV-A2 strain virus is 0.6634 μg/ml (FIG. 3), indicating that the antibody has 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 (10)

1. An antibody for neutralizing respiratory syncytial virus, characterized in that the antibody comprises a heavy chain variable region complementarity determining region of three CDRs and a light chain variable region complementarity determining region of three CDRs, wherein the amino acid sequences of the heavy chain variable region complementarity determining regions CDR1, CDR2, CDR3 have 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 1,2,3, respectively, and the amino acid sequences of the light chain variable region complementarity determining regions CDR1, CDR2, CDR3 have 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 9,10, 11, respectively;
Preferably, the amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain are shown in SEQ ID NO. 1, 2 and 3, respectively, and the amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 of the light chain are shown in SEQ ID NO. 9, 10 and 11, respectively.
2. The antibody of claim 1, wherein the heavy chain variable region further comprises four FR heavy chain variable region framework regions, the light chain variable region further comprises four FR light chain variable region framework regions, wherein the amino acid sequences of heavy chain variable region framework regions FR1, FR2, FR3 and FR4 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 set forth in SEQ ID NOs 4, 5, 6, 7, respectively, and the amino acid sequences of light chain variable region framework regions FR1, FR2, FR3 and FR4 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 set forth in SEQ ID NOs 12, 13, 14, 15, respectively;
preferably, the amino acid sequences of the heavy chain variable region framework regions FR1, FR2, FR3 and FR4 are shown as SEQ ID NO. 4, 5, 6 and 7 respectively, and the amino acid sequences of the light chain variable region framework regions FR1, FR2, FR3 and FR4 are shown as SEQ ID NO. 12, 13, 14 and 15 respectively;
Preferably, the amino acid sequence of the heavy chain variable region has at least 70%, at least 75%, at least 80%, at least 85%, 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 sequence shown in SEQ ID NO. 8,
The amino acid sequence of the light chain variable region has at least 70%, at least 75%, at least 80%, at least 85%, 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 sequence set forth in SEQ ID NO. 16;
preferably, the amino acid sequence of the heavy chain variable region 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;
Preferably, the antibody specifically binds to the pre-fusion conformation of the F protein of respiratory syncytial virus;
Preferably, the antibody is afucosylated;
Preferably, the antibody has any variant with one or more conservative amino acid substitutions.
3. A nucleic acid molecule encoding the antibody of claim 1 or 2;
Preferably, the nucleotide sequences of the nucleic acid molecules encoding the complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain have 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,
The nucleotide sequences of the nucleic acid molecules encoding the complementarity determining regions CDR1, CDR2, CDR3 of the light chain have 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;
Preferably, the nucleotide sequences of the nucleic acid molecules encoding the complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region are shown in SEQ ID NOS 17, 18, 19, respectively,
The nucleotide sequences of the nucleic acid molecules for coding the light chain variable region complementarity determining regions CDR1, CDR2 and CDR3 are shown in SEQ ID NOS 20, 21 and 22, respectively.
4. A recombinant expression vector comprising the nucleic acid molecule of claim 3;
Preferably, the recombinant expression vector further comprises a promoter;
Preferably, the recombinant expression vector comprises a plasmid vector, a viral vector or a phage vector.
5. A host cell comprising the nucleic acid molecule of claim 3 or the recombinant expression vector of claim 4;
Preferably, the host cell comprises a prokaryotic cell, a eukaryotic cell;
preferably, the eukaryotic cells include lower eukaryotic cells and higher eukaryotic cells;
preferably, the higher eukaryotic cell comprises a mammalian cell;
Preferably, the mammalian cells include myeloma cell lines, huT78 cells, 293T cells, 293F cells, CHO cells, W138, BHK cells.
6. A pharmaceutical composition comprising the antibody of claim 1 or 2, the nucleic acid molecule of claim 3, the recombinant expression vector of claim 4, or the host cell of claim 5;
preferably, the pharmaceutical composition further comprises a pharmaceutically compatible carrier;
preferably, the pharmaceutical composition further comprises a buffer;
preferably, the pharmaceutical composition may be used in combination with one or more antiviral agents.
7. A kit for detecting respiratory syncytial virus, comprising the antibody of claim 1 or 2;
Preferably, the kit further comprises a detectable label conjugated to an antibody;
preferably, the detectable label comprises a fluorescent label, a radioisotope, a chemiluminescent molecule, a paramagnetic ion or a spin-trapping reagent.
8. A method of detecting respiratory syncytial virus in a sample, comprising contacting the antibody of claim 1 or 2 with a sample to be detected, and detecting the level of respiratory syncytial virus in the sample to be detected.
9. Use of the antibody of claim 1 or 2, the nucleic acid molecule of claim 3, the recombinant expression vector of claim 4 or the host cell of claim 5 for detecting respiratory syncytial virus or for the preparation of a product for diagnosing respiratory syncytial virus infection.
10. Use of an antibody according to claim 1 or 2, a nucleic acid molecule according to claim 3, a recombinant expression vector according to claim 4 or a host cell according to claim 5 for inhibiting respiratory syncytial virus infection or for the preparation of a pharmaceutical composition for the treatment of a respiratory syncytial virus-infected disease.
CN202410174943.XA 2024-02-04 2024-02-07 Antibodies neutralizing respiratory syncytial virus Pending CN117924473A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103097412A (en) * 2010-07-09 2013-05-08 克鲁塞尔荷兰公司 Anti-human respiratory syncytial virus (RSV) antibodies and methods of use
CN116836269A (en) * 2023-03-15 2023-10-03 山西高等创新研究院 Monoclonal antibody for resisting respiratory syncytial virus and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103097412A (en) * 2010-07-09 2013-05-08 克鲁塞尔荷兰公司 Anti-human respiratory syncytial virus (RSV) antibodies and methods of use
CN116836269A (en) * 2023-03-15 2023-10-03 山西高等创新研究院 Monoclonal antibody for resisting respiratory syncytial virus and application thereof

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