WO2020173460A1 - Anticorps de neutralisation 4f1 à large spectre entièrement humain dirigé contre le virus respiratoire syncytial et utilisation associée - Google Patents

Anticorps de neutralisation 4f1 à large spectre entièrement humain dirigé contre le virus respiratoire syncytial et utilisation associée Download PDF

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WO2020173460A1
WO2020173460A1 PCT/CN2020/076767 CN2020076767W WO2020173460A1 WO 2020173460 A1 WO2020173460 A1 WO 2020173460A1 CN 2020076767 W CN2020076767 W CN 2020076767W WO 2020173460 A1 WO2020173460 A1 WO 2020173460A1
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antibody
variable region
protein
present
light chain
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PCT/CN2020/076767
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Chinese (zh)
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孙兵
王宾
凌志洋
赵干
伊春艳
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中国科学院上海生命科学研究院
复旦大学
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Publication of WO2020173460A1 publication Critical patent/WO2020173460A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the field of medicine, in particular to a fully human broad-spectrum neutralizing antibody 4F1 against respiratory syncytial virus and its application. Background technique
  • RSV causes acute upper and lower respiratory tract infections and is one of the most important pathogens of respiratory tract infections in infants and young children worldwide. At the same time, RSV is also recognized as a high-risk adult, such as elderly individuals and adults with chronic lung diseases. An important pathogen in individuals and adults with weakened immune functions (such as bone marrow transplant patients). There are 30 million new infections worldwide each year, and about 200,000 people die from RSV infection. Among Chinese children with pneumonia syndrome under 2 years of age, RSV is the most common viral pathogen (17.0%). The world urgently needs safe, effective, and low-cost preventive treatments for RSV infection.
  • Palivizumab targets the conserved area of F protein and has a broad spectrum of action, but the current dose of palivizumab required for each administration is 15mg/kg (body weight), passive immunization is 5 times a year, and there is a dose It is high, requires multiple administrations, and is expensive.
  • Palivizumab is a humanized antibody, which still retains some mouse-derived sequences. It may have a certain degree of immunogenicity, and has a certain degree of safety. concern. With the development of immunology and molecular biology, genetically engineered antibodies have developed rapidly, and the production technology of chimeric antibodies, humanized antibodies and fully human antibodies has been continuously developed to minimize or even eliminate HAMA reactions, especially fully human antibodies become antibodies The future direction of the drug.
  • the purpose of the present invention is to provide a fully human monoclonal antibody capable of preventing and controlling RSV infection.
  • the first aspect of the present invention provides a heavy chain variable region of an antibody.
  • the heavy chain variable region includes the following three complementarity determining region CDRs:
  • any one of the above amino acid sequences further includes optionally added or missing Loss, modification and/or substitution of at least one (such as 1-3, preferably 1-2, more preferably 1) amino acid and can retain the binding of the respiratory syncytial virus fusion protein (preferably the F protein before fusion) Affinity derived sequence.
  • the heavy chain variable region further includes a human FR region or a murine FR region.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.: 1.
  • the second aspect of the present invention provides a heavy chain of an antibody, which has the heavy chain variable region as described in the first aspect of the present invention.
  • the heavy chain of the antibody also includes a heavy chain constant region.
  • the heavy chain constant region is of human, murine or rabbit origin.
  • the third aspect of the present invention provides a light chain variable region of an antibody.
  • the light chain variable region includes the following three complementarity determining region CDRs:
  • amino acid sequence is CDR2’ of LGS, and
  • any one of the above amino acid sequences further includes at least one (such as 1-3, preferably 1-2, more preferably added, deleted, modified and/or substituted). 1) amino acid and can retain the binding affinity of the respiratory syncytial virus fusion protein (preferably the F protein before fusion).
  • the light chain variable region further includes a human FR region or a murine FR region.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO.: 2.
  • the light chain of the antibody also includes a light chain constant region.
  • the light chain constant region is of human, murine or rabbit origin.
  • the fifth aspect of the present invention provides an antibody, the antibody having:
  • the antibody has: a heavy chain as described in the second aspect of the present invention; and/or a light chain as described in the fourth aspect of the present invention.
  • the antibody is a specific anti-respiratory syncytial virus antibody, preferably a specific anti-respiratory syncytial virus fusion protein (preferably pre-fusion F protein).
  • the antibody is selected from: animal-derived antibodies, chimeric antibodies, humanized antibodies, or a combination thereof.
  • the antibody is a double-chain antibody or a single-chain antibody.
  • the antibody is a monoclonal antibody or a polyclonal antibody.
  • the antibody is a partially or fully humanized monoclonal antibody.
  • the antibody is in the form of a drug conjugate.
  • the heavy chain variable region sequence of the antibody is shown in SEQ ID NO.: 1; and the light chain variable region sequence of the antibody is shown in SEQ ID NO.: 2.
  • the sixth aspect of the present invention provides a recombinant protein, the recombinant protein having:
  • the tag sequence includes 6His tag, GGGS sequence, and FLAG tag.
  • the recombinant protein includes a fusion protein.
  • the recombinant protein is a monomer, dimer, or multimer.
  • the immune cells are selected from the group consisting of NK cells and T cells.
  • the immune cells are derived from human or non-human mammals (such as mice).
  • the ninth aspect of the present invention provides an antibody-drug conjugate, the antibody-drug conjugate containing:
  • a coupling part coupled to the antibody part being selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.
  • the conjugate is selected from the group consisting of fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computed tomography technology) contrast agents, or can produce detectable Product enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, kinner Rice particles/nanorods, viral particles, liposomes, magnetic nanoparticles, prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutics (for example , Cisplatin) or any form of nanoparticles.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the antibody portion and the coupling portion are coupled through a chemical bond or linker.
  • the tenth aspect of the present invention provides a use of an active ingredient selected from the group consisting of the heavy chain variable region according to the first aspect of the present invention, and the heavy chain variable region according to the second aspect of the present invention.
  • Chain, the light chain variable region of the third aspect of the present invention, the light chain of the fourth aspect of the present invention, or the antibody of the fifth aspect of the present invention, the recombinant of the sixth aspect of the present invention Protein, or a combination thereof, the active ingredient is used to prepare a medicament, reagent, detection plate or kit.
  • the reagent, detection plate or kit is used to detect respiratory syncytial virus.
  • the medicament is used to treat or prevent respiratory syncytial virus infection.
  • the reagents include chips and immune particles coated with antibodies.
  • the eleventh aspect of the present invention provides a pharmaceutical composition, the pharmaceutical composition containing:
  • Active ingredient which is selected from the group consisting of: the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, and the heavy chain as described in the third aspect of the present invention
  • the light chain variable region of the fourth aspect of the present invention, the light chain of the fourth aspect of the present invention, or the antibody of the fifth aspect of the present invention, the recombinant protein of the sixth aspect of the present invention, the eighth aspect of the present invention Immune cells, the antibody-drug conjugate according to the ninth aspect of the present invention, or a combination thereof; and
  • the pharmaceutical composition is a liquid preparation.
  • the pharmaceutical composition is an injection.
  • the pharmaceutical composition is used to prevent and/or treat respiratory syncytial virus infection.
  • the twelfth aspect of the present invention provides a polynucleotide, which encodes a polypeptide selected from the following group:
  • the polynucleotide has the sequence shown in SEQ ID NO.: 8 and/or SEQ ID NO.: 9.
  • the thirteenth aspect of the present invention provides a vector containing the polynucleotide according to the twelfth aspect of the present invention.
  • the vector includes: bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.
  • the fourteenth aspect of the present invention provides a genetically engineered host cell, the host cell contains the vector as described in the thirteenth aspect of the present invention or the genome integrates the vector as described in the twelfth aspect of the present invention Polynucleotide.
  • the fifteenth aspect of the present invention provides a method for detecting respiratory syncytial virus in a sample, the method comprising the steps:
  • the detection is for non-therapeutic and non-diagnostic purposes.
  • the present invention also provides a method for detecting respiratory syncytial virus fusion protein in a sample, the method comprising the steps:
  • the respiratory syncytial virus fusion protein is a pre-fusion F protein of respiratory syncytial virus.
  • the detection is for non-therapeutic and non-diagnostic purposes.
  • the sixteenth aspect of the present invention provides a test board, the test board comprising: a substrate (support plate) and a test strip, and the test strip contains the antibody according to the fifth aspect of the present invention or The immunoconjugate according to the ninth aspect of the present invention.
  • the seventeenth aspect of the present invention provides a kit, which includes:
  • a first container which contains the antibody according to the fifth aspect of the present invention.
  • a second container which contains a secondary antibody against the antibody according to the fifth aspect of the present invention; or, the kit contains the detection plate according to the sixteenth aspect of the present invention.
  • the eighteenth aspect of the present invention provides a method for preparing a recombinant polypeptide, the method comprising:
  • Figure 1 shows flow cytometric sorting of memory B cells specifically bound by RSV F protein.
  • Figure 2 shows the agarose gel electrophoresis pattern of matched antibody light and heavy chain genes.
  • Figure 3 shows the binding activity of 4F1 antibody to RSV A2 pre-fusion F protein (A), B9320 pre-fusion F protein (B) and A2 post-fusion F protein (C).
  • 4F1 antibody can bind RSV type A and B pre-fusion
  • Figure 4 shows the dose fitting curve of the antibody neutralization activity of 4F1 antibody to RSV A2 (A) and B9320 (B); and Palivizumab (Palivizumab) to RSV A2 (; C) and B9320 CD) antibody Fit the curve with the active dose.
  • Figure 5 shows the comparison of the ability of 4F1 and Palivizumab to reduce the viral load in the lungs in the mouse prevention experiment.
  • Figure 6 shows the comparison of lung pathological damage after passive immunization with 4F1 and Palivizumab in the mouse prevention experiment.
  • the inventors unexpectedly obtained a fully human monoclonal antibody 4F1 against the fusion protein (F protein) of the respiratory syncytial virus and the pre-fusion F protein (preF protein).
  • the antibody can bind to the pre-fusion F protein of RSV A and B viruses in a broad spectrum, and has high binding and neutralizing activity to respiratory syncytial virus, and has broad-spectrum recognition and broad-spectrum neutralization. Can inhibit or prevent respiratory syncytial virus from infecting susceptible cells.
  • 4F1 antibody can effectively prevent and control RSV infection. On this basis, the present invention has been completed.
  • the term “optional” or “optionally” means that the event or situation described later can occur but does not have to occur.
  • “optionally comprising 1-3 antibody heavy chain variable regions” means that the antibody heavy chain variable regions of a specific sequence may but not necessarily have, and can be 1, 2, or 3.
  • RSV belongs to the family of Paramyxoviridae and the genus of pneumoviruses. It is a negative-strand RNA virus composed of 15222 nucleotides. RSV is an envelope virus. The genome encodes 11 proteins, including fusion protein F, adsorption protein G, small hydrophobin SH, matrix protein M, nucleoprotein N, phosphoprotein P, large polymerase protein L, small matrix protein M2 and The non-structural proteins NS1 and NS2, in which the fusion protein F, the adsorption protein G and the small hydrophobin SH protein are located on the surface of the virus. RSV has two main surface glycoproteins, G and F.
  • the G protein mediates the binding of the virus to the cell surface, and at the same time, it can mimic the effect of the chemokine CX 3 C chemokine and interact with its receptor to enhance the inflammatory response after RSV infection.
  • the F protein mediates the fusion of the virus and the cell membrane. It also promotes the fusion of the membrane of the infected cell with surrounding cells to form syncytia.
  • a and B antigen groups or subtypes of RSV
  • Most RSV proteins are highly conserved between the two subgroups.
  • the fusion F protein shows no 91% amino acid similarity, and the G protein only has 53% homology between A and B.
  • the F protein is highly conserved, and the neutralizing antibodies induced by it can simultaneously inhibit viral infections of the two subtypes A and B, which is of great significance in the development of anti-RSV monoclonal antibody drugs and vaccines.
  • the F protein is a type I transmembrane protein, and the F protein first synthesizes the precursor protein F0.
  • F0 is trimerized in the endoplasmic reticulum and processed by cell furin-like proteases at two conserved sites to produce F1, F2 and Pep27 polypeptides, Finally, two fragments of F1 and F2 connected by disulfide bonds are formed, and the F2-F1 heterodimer forms a trimer to assemble the mature F protein.
  • the F1 subunit consists of heptapeptide repeat region A (HRA), functional domain 1/11, heptapeptide repeat region B (HRB), transmembrane protein (TM), cytoplasmic region (CP), and F2 subunit consists of heptapeptide repeats District C (HRC) composition.
  • HRA heptapeptide repeat region A
  • HRB heptapeptide repeat region B
  • TM transmembrane protein
  • CP cytoplasmic region
  • F2 subunit consists of heptapeptide repeats District C (HRC) composition.
  • HRA heptapeptide repeat region A
  • HRB heptapeptide repeat region B
  • TM transmembrane protein
  • CP cytoplasmic region
  • F2 subunit consists of heptapeptide repeats District C (HRC) composition.
  • the hydrophobic N-terminal (137-155) of F1 plays an important role in mediating fusion.
  • pre-fusion F protein When the G protein binds to the receptor on the target cell membrane, the pre-fusion F protein (pre-fusion F protein) starts to trigger the allosteric to a stable post-fusion form (post-fusion). Due to its key role in RSV invasion and its high degree of conservation, RSV F protein is the target of neutralizing antibodies and the main antigen for vaccine development. A large number of studies have confirmed that the neutralizing antibody recognition site for the F protein is mainly on the pre-fusion F protein. The immunogenicity and protective effect of the recombinant protein based on the pre-fusion F design is significantly better than that of the post-fusion F protein (post-fusion F protein). F protein) designed vaccine.
  • pre-fusion F protein As used herein, the terms "pre-fusion F protein”, “pre-fusion F protein”, and “preF protein” are used interchangeably, and all refer to the pre-fusion form of the fusion protein F of respiratory syncytial virus.
  • the present invention uses single cell RT-PCR technology to isolate a broad-spectrum neutralizing antibody 4F1 from human peripheral blood PBMC.
  • the discovery of new antibodies provides new options for broad-spectrum neutralizing antibody therapeutic applications; on the other hand, the discovery of new epitopes provides new ideas for the development of broad-spectrum vaccines.
  • antibody or "immunoglobulin” is a heterotetrameric glycoprotein of about 150,000 daltons with the same structural characteristics, which consists of two identical light chains (L) and two identical heavy chains. (H) Composition. Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain .
  • Special amino acid residues form the interface between the variable regions of the light and heavy chains.
  • variable means that certain parts of the variable region of the antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three fragments called complementarity determining regions (CDR) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved part of the variable region is called the framework region (FR).
  • CDR complementarity determining regions
  • FR framework region
  • the variable regions of the natural heavy chain and light chain each contain four FR regions, which are roughly in a P-folded configuration and are connected by three CDRs forming a connecting loop, which can form a partially folded structure in some cases.
  • the CDRs in each chain are closely joined together by the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pp. 647-669 (1991)). Constant regions do not directly participate in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.
  • immunoglobulins can be classified as significantly different based on the amino acid sequence of their constant regions One of two categories (called K and people). According to the amino acid sequence of the constant region of their heavy chains, immunoglobulins can be divided into different types. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes), such as IgGl, IgG2, IgG3, IgG4, IgA and IgA2.
  • the heavy chain constant regions corresponding to different classes of immunoglobulins are called a, 5, S , ⁇ , and 1 ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
  • the term "monoclonal antibody (monoclonal antibody)” refers to an antibody obtained from a substantially homogeneous population, that is, the single antibodies contained in the population are the same, except for a few naturally occurring mutations that may exist. Monoclonal antibodies are highly specific to a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which usually have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized through hybridoma culture and will not be contaminated by other immunoglobulins.
  • the modifier "monoclonal” refers to the characteristics of the antibody, which is obtained from a substantially uniform antibody population. This should not be interpreted as requiring any special method to produce antibodies.
  • the present invention also includes a monoclonal antibody having the corresponding amino acid sequence of the anti-respiratory syncytial virus fusion protein (preferably pre-fusion F protein) monoclonal antibody, and a monoclonal antibody having the anti-respiratory syncytial virus fusion protein (more The best pre-fusion F protein) monoclonal antibody of the variable region chain of the monoclonal antibody, and other proteins or protein conjugates and fusion expression products with these chains.
  • a monoclonal antibody having the corresponding amino acid sequence of the anti-respiratory syncytial virus fusion protein preferably pre-fusion F protein
  • a monoclonal antibody having the anti-respiratory syncytial virus fusion protein more The best pre-fusion F protein
  • the present invention includes any protein or protein conjugate and fusion expression product (ie, immunoconjugate and fusion expression product) having a light chain and a heavy chain containing hypervariable regions (complementarity determining regions, CDR), as long as the The hypervariable region is the same or at least 90% homologous to the hypervariable regions of the light chain and heavy chain of the present invention, preferably at least 95% homology.
  • immunoconjugate and fusion expression product having a light chain and a heavy chain containing hypervariable regions (complementarity determining regions, CDR), as long as the The hypervariable region is the same or at least 90% homologous to the hypervariable regions of the light chain and heavy chain of the present invention, preferably at least 95% homology.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules and the anti-RSV fusion protein Conjugates formed by combining monoclonal antibodies or fragments thereof.
  • the present invention also includes cell surface markers or antigens that bind to the anti-respiratory syncytial virus fusion protein monoclonal antibody or fragments thereof.
  • antibody fragment refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of antibodies.
  • binding fragments contained in the term "antigen-binding fragments of antibodies” include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab') 2 fragments, including A bivalent fragment of two Fab fragments connected by a disulfide bridge on the chain region; (iii) Fd fragment composed of VH and CH1 domains; (iv) Fv composed of VH and VL domains of one arm of an antibody Fragment.
  • the Fv antibody contains the variable region of the heavy chain and the variable region of the light chain, but does not have the constant region, and has the smallest antibody fragment with all antigen binding sites.
  • an Fv antibody also contains a polypeptide linker between the VH and VL domains, and can form the structure required for antigen binding.
  • the present invention includes not only complete monoclonal antibodies, but also antibody fragments with immunological activity, such as Fab or (Fab') 2 fragments; antibody heavy chains; antibody light chains.
  • epitopes or “antigenic determinant” refers to the site on an antigen where an immunoglobulin or antibody specifically binds. Epitopes usually include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 continuous or discontinuous ammonia in a unique spatial conformation Base acid.
  • antibodies bind with an affinity (KD) of less than about 10_ 7 M, for example, about less than 10_ 8 M, 109 M, or 10′10 M or less.
  • antigenic determinant refers to a discrete three-dimensional site on the antigen that is recognized by the antibody or antigen-binding fragment of the present invention.
  • the present invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.
  • antibodies include murine, chimeric, humanized, or fully human antibodies prepared by techniques well known to those skilled in the art.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human parts, can be prepared using DNA recombination techniques well known in the art.
  • the term "murine antibody” in the present invention refers to a monoclonal antibody against the fusion protein of respiratory syncytial virus prepared according to the knowledge and skills in the art.
  • chimeric antibody is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • humanized antibody also known as CDR-grafted antibody, refers to the transplantation of mouse CDR sequences into the human antibody variable region framework, that is, different types of human germline antibodies The antibody produced in the framework sequence. Humanized antibody can overcome the heterogeneous reaction induced by chimeric antibody because it carries a large amount of murine protein.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the human antibody variable region framework sequence may be subjected to minimal reverse mutations or back mutations to maintain activity.
  • the antibody may be monospecific, bispecific, trispecific, or more multispecific.
  • the terms "heavy chain variable region” and “VH” are used interchangeably.
  • variable region and “complementarity determining region (CDR)” are used interchangeably.
  • CDR refers to one of the six hypervariable regions in the variable domain of an antibody that mainly contribute to antigen binding.
  • the six CDR as defined in one of the most commonly used by Kabat EA et al., (1991) Sequences of proteins of immunological interest. NIH Publication 9 1- 3242) provided.
  • the heavy chain variable region of the antibody includes the following three complementarity determining region CDRs:
  • CDR2 WYDGNHQ (SEQ ID NO.: 4), and
  • CDR3 TARSLVITLAGAGRDDY (SEQ ID NO.: 5).
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.: 1, wherein the underlined amino acid sequences are the amino acid sequences of the heavy chain variable region CDR1, CDR2, and CDR3.
  • nucleic acid coding sequence of the heavy chain variable region is shown in SEQ ID NO.: 8, wherein the underlined nucleic acid coding sequences of the heavy chain variable region CDR1, CDR2, and CDR3 are in sequence.
  • the heavy chain of the antibody includes the aforementioned heavy chain variable region and heavy chain constant region, and the heavy chain constant region may be of murine or human origin.
  • VL light chain variable region
  • the light chain variable region of the antibody according to the present invention has a complementarity determining region CDR selected from the following group:
  • VQDLQTSLT VQDLQTSLT (SEQ ID NO: 7).
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO.: 2, wherein the double underlined sequence is the amino acid sequence of the light chain variable region CDR1', CDR2', CDR3' .
  • nucleic acid coding sequence of the light chain variable region is shown in SEQ ID NO.: 9, wherein the double underlined nucleic acids are the light chain variable regions CDR1', CDR2', CDR3' in sequence Coding sequence.
  • the terms "antibody of the present invention”, “protein of the present invention”, or “polypeptide of the present invention” are used interchangeably, and all refer to specific binding to an anti-RSV fusion protein (preferably pre-fusi on F Protein), for example, having a heavy chain variable region (as shown in SEQ ID NO.: 8 amino acid sequence encoded by the nucleotide sequence) and/or light chain variable region (as shown in SEQ ID NO.: 9
  • the nucleotide sequence encoding the amino acid sequence) of the protein or polypeptide may or may not contain the starting methionine.
  • the antibody is a mouse or human mouse chimeric monoclonal antibody against respiratory syncytial virus fusion protein (preferably pre-fusion F protein), and its heavy chain constant region and/or light
  • the chain constant region can be a humanized heavy chain constant region or a light chain constant region. More preferably, the humanized heavy chain constant region or light chain constant region is a heavy chain constant region or light chain constant region of human IgG1, IgG2, etc.
  • variable regions which are divided into 4 framework regions (FR)
  • FR framework regions
  • amino acid sequence of FR is relatively conservative and does not directly participate in the binding reaction. These CDRs form a circular structure, and the P folds formed by the FR between them are close to each other in space structure.
  • the CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen binding site of the antibody.
  • the amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.
  • variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding antigen. Therefore, the present invention includes those molecules with CDR-bearing monoclonal antibody light chain and heavy chain variable regions, as long as their CDRs have more than 90% of the CDRs identified here (: preferably more than 95%, most preferably 98%). % Above).
  • the present invention includes not only complete monoclonal antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies. For example, in the modification of the Fc fragment on the basis of the antibody of the present invention, in order to extend the half-life of the antibody, three mutation points M252Y /S254T /T256E are introduced in the CH2 region.
  • fragment refers to polypeptides that substantially retain the same biological function or activity as the antibody of the present invention.
  • the polypeptide fragment, derivative or analogue of the present invention may be (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide with substitution groups in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that prolongs the half-life of the polypeptide, such as Polyethylene glycol) fused to a polypeptide, or (iv) additional amino acid sequence fused to the polypeptide sequence to form a polypeptide (such as a leader sequence or secretory sequence, or a sequence or proprotein sequence used to purify the polypeptide, or with Fusion protein formed by 6His tag
  • the antibody of the present invention refers to a polypeptide that has the binding activity of an anti-respiratory syncytial virus fusion protein (preferably pre-fusion F protein) and includes the above-mentioned CDR regions.
  • the term also includes variant forms of polypeptides containing the above-mentioned CDR regions that have the same function as the antibody of the present invention. These variants include (but are not limited to): One or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid deletion , Insertion and/or substitution, and the addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminal and/or N-terminal.
  • substitution of amino acids with similar or similar properties usually does not change the function of the protein.
  • adding one or several amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • the variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and DNA that can hybridize with the coding DNA of the antibody of the present invention under high or low stringency conditions.
  • the encoded protein, and the polypeptide or protein obtained by using an antiserum against the antibody of the present invention.
  • the present invention also provides other polypeptides, such as fusion proteins containing human antibodies or fragments thereof.
  • the present invention also includes fragments of the antibodies of the present invention.
  • the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 60 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.
  • “conservative variants of the antibody of the present invention” refer to at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 compared to the amino acid sequence of the antibody of the present invention. Two amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are best produced according to Table A by amino acid substitution.
  • the present invention also provides polynucleotide molecules encoding the aforementioned antibodies or fragments or fusion proteins thereof.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the sequence of the coding region encoding the mature polypeptide may be the same as the sequence of the coding region shown in SEQ ID NO.: 8 or 9 or a degenerate variant.
  • degenerate variant in the present invention refers to a nucleic acid sequence encoding a nucleic acid sequence having the same amino acid sequence as the polypeptide of the present invention but different from the coding region sequence shown in SEQ ID NO.: 8 or 9 .
  • the polynucleotide encoding the mature polypeptide of the present invention includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence (and optional additional coding sequence) and non-coding sequences of the mature polypeptide .
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the present invention also relates to polynucleotides that hybridize with the aforementioned sequence and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize with the polynucleotide of the present invention under stringent conditions.
  • stringent conditions refer to: G) Hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (7) Add denaturant during hybridization , Such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 ° C, etc.; or (3) only the identity between the two sequences is at least 90% or more, preferably Hybridization only occurs when more than 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO.: 1 and/or SEQ ID NO.: 2.
  • the full-length nucleotide sequence or fragments of the antibody of the present invention can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • a feasible method is to use artificial synthesis to synthesize relevant sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then connecting them to obtain a very long fragment.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.
  • the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • the biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules that exist in an isolated form.
  • DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
  • the present invention also relates to a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, and 293 cells.
  • Transformation of host cells with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as Escherichia coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCh method.
  • the steps used are well known in the art.
  • Another method is to use MgCh.
  • transformation can also be performed by electroporation.
  • the host is a eukaryote, the following DNA transfection methods can be used: phosphoric acid co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the culture is carried out under conditions suitable for the growth of the host cell.
  • use an appropriate method such as temperature conversion or chemical induction to induce the selected promoter, and then culture the cell for a period of time.
  • the recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic breakage, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the antibody of the present invention can be used alone, or can be combined or coupled with a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modified part, or any combination of these substances.
  • Detectable markers used for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agents, or capable of producing detectable products Of enzymes.
  • Coupling therapeutic agents include, but are not limited to: insulin, IL-2, interferon, calcitonin, GHRH peptide, intestinal peptide analogs, albumin, antibody fragments, cytokines, and hormones.
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or active fragment or fusion protein thereof, and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition which contains the above-mentioned antibody or active fragment or fusion protein thereof, and a pharmaceutically acceptable carrier.
  • these substances are formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, and preferably the pH is about 6-8, although the pH may vary depending on the nature of the substance being formulated And the disease to be treated varies.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): oral, respiratory, intratumor, intraperitoneal, intravenous, or local administration.
  • the pharmaceutical composition of the present invention can be directly used to bind to the fusion protein (preferably pre-fusion F protein) molecule of the respiratory syncytial virus, and thus can be used to prolong the half-life of the drug.
  • fusion protein preferably pre-fusion F protein
  • other therapeutic agents can also be used at the same time.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned monoclonal antibody (or conjugate thereof) of the present invention and a pharmaceutical Acceptable carrier or excipient.
  • a pharmaceutical Acceptable carrier or excipient include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injections, for example, with physiological saline or an aqueous solution containing glucose and other adjuvants for preparation by conventional methods. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions.
  • the dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram/kg body weight to about 10 mg/kg body weight per day.
  • the polypeptide of the present invention
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases, does not exceed about 8 mg/kg body weight, Preferably the dosage is about 10 micrograms/kg body weight to about 1 mg/kg body weight.
  • the specific dosage should also consider factors such as the route of administration, the patient's health status, etc., which are within the skill range of a skilled physician. Test purpose and kit
  • the antibodies of the present invention can be used in detection applications, for example, to detect samples, thereby providing diagnostic information.
  • the samples (samples) used include cells, tissue samples and biopsy specimens.
  • the term "biopsy” used in the present invention shall include all kinds of biopsy known to those skilled in the art. Therefore, the biopsy used in the present invention may include, for example, tissue samples prepared by endoscopic methods or organ puncture or needle biopsy.
  • the samples used in the present invention include fixed or preserved cell or tissue samples.
  • the present invention also provides a kit containing the antibody (or fragment thereof) of the present invention.
  • the kit further includes a container, instructions for use, buffer, and the like.
  • the antibody of the present invention can be immobilized on a detection plate. Main advantages of the invention
  • the fully human monoclonal antibody of the present invention can specifically recognize and bind to the pre-fusion F protein of respiratory syncytial virus, has high neutralizing activity against respiratory syncytial virus, and can bind to and neutralize RSV type A and B Type virus, effectively inhibiting or preventing respiratory syncytial virus from infecting susceptible cells.
  • the fully human monoclonal antibody of the present invention has broad-spectrum binding activity and broad-spectrum neutralization activity against RSV A and B viruses. It can effectively neutralize a variety of respiratory syncytial viruses, and it is significantly better than the current commercially available antibodies (such as Palivizumab antibody) in either the cell-level microneutralization test or the mouse prevention test.
  • the present invention is a fully human monoclonal antibody 4F1, which does not contain mouse-derived parts. It has lower immunogenicity and higher safety for humans, and can avoid the mediation of human anti-mouse and other species-derived antibodies. Immune rejection.
  • the fully human monoclonal antibody 4F1 of the present invention binds to the pre-fusion form of RSV F protein.
  • a large number of studies have confirmed that the neutralizing antibody recognition site for F protein is mainly on the pre-fusion F protein, and the discovery of 4F1 antibody epitopes is also RSV.
  • the design of the vaccine provides some new ideas and references.
  • the present invention will be further explained below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.
  • the experimental methods without specific conditions in the following examples usually follow conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturing The conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and parts by weight.
  • Example 1 Obtaining antibody gene and antibody expression by single cell RT-PCR method
  • PBMC peripheral blood mononuclear cells
  • Peripheral blood was drawn from healthy volunteers and used conventional Ficoll-Paque (manufactured by Lympholyte®-H (CEDARLANE)) density gradient centrifugation to obtain more than 10 7 peripheral blood mononuclear cells (PBMC).
  • Ficoll-Paque manufactured by Lympholyte®-H (CEDARLANE)
  • BD Horizonä Fixable Viability Stain 780 removes dead cells, and obtains specific B cells by flow cytometry to a 96-well RT-PCR plate with one cell per well to obtain F protein-specific memory B cells.
  • RSV A2 pre-fusion F protein is expressed by mammalian cell CHO expression system; refer to Invitrogen ExpiCHO-Sä Expression System manual; A 2 F protein sequence refer to UniProtKB/Swiss-Prot: P03420.1, RSV pre-fusion F protein
  • the design was designed according to the strategy adopted by Jason S. McLellan. Science 2013, and the whole gene was synthesized in Shanghai Jierui Company and constructed on the expression vector of invitrogen pcDNA3.1.
  • PBMC cells are grouped into experimental group + control group. Markers are added according to the number of cells, stained in the dark, labeled, resuspended in PBS, and filtered with 40
  • Sorting of specific B cells Use BD FACS Influx to screen, select lymphocytes from PBMC according to forward and lateral angles, and then adjust and compensate by different control groups to obtain specific memory of RSV F protein B cells are sorted into 96-well plates for RT-PCR (reverse transcription PCR), one cell per well, and the plate is placed on dry ice.
  • RT-PCR reverse transcription PCR
  • the obtained single memory B cell was obtained by RT-PCR to obtain cDNA, and then the antibody gene variable region was obtained by nested-PCR, and the gel was run on an agarose nucleic acid gel, and the gel block with the heavy and light chain was recovered and sequenced.
  • IgBLAST website https://www.ncbi.nlm.nih.gov/projects/igblast/) search to obtain the antibody gene sequence.
  • the antibody gene was linked to the corresponding IgH, Ig K and IgX expression vectors through the Agel and Sail restriction sites, Agel and BsiwI restriction sites, and Agel and Xhol restriction sites.
  • the fully human antibody expression vectors IgH, IgK and Ig express antibody heavy chain, kappa chain, and lambda chain respectively) as a gift from Patrick Wilson laboratory.
  • NCBI GenBank FJ475055, FJ475056 and FJ517647.
  • the antibody was purified using Protein G Agarose 4FF packing (purchased from GE). First, centrifuge the collected CHO cell suspension at 4000 rpm at 4°C for 30 minutes, and filter the collected supernatant with a 0.45um filter for purification. Take weight Force spin column, add Protein G Agarose 4FF packing, use 3 times column volume of 20% ethanol to stabilize packing, then equilibrate the column with 5 times column volume of binding buffer, then load the sample, and then use 10 times column volume of binding buffer Liquid equilibrate the column, and finally elution the column with 3 times the column volume of the elution buffer, and add a neutralization buffer to the eluted antibody solution to make the eluted sample pH 7.5. The antibody solution was dialyzed 3 times in 5L 1*PBS, the antibody can be concentrated and stored to -80°C. Experimental results:
  • the full human antibody 4F1 heavy chain variable region gene sequence is as follows, where underlined are the hypervariable region sequence in the heavy chain gene variable region, followed by the heavy chain gene CDR1, CDR2 and CDR3 sequences.
  • the amino acid sequence of the heavy chain variable region of the fully human antibody 4F1 is as follows, in which underlined are the heavy chain amino acid CDR1, CDR2, and CDR3 sequences.
  • the full human antibody 4F1 light chain variable region gene sequence is as follows, where underlined are the hypervariable region sequence in the light chain gene variable region, followed by CDR1', CDR2' and CDR3' sequences.
  • amino acid sequence of the light chain variable region of the fully human antibody 4F1 is as follows, in which the light chain amino acid CDR1', CDR2' and CDR3' sequences are underlined.
  • ELISA detects the activity of antibody binding antigen
  • ELISA was used to detect whether the expressed antibody recognizes RSV A2 and B9320 pre-fusion F protein and A2 post-fusion F protein.
  • A2 F protein sequence refers to UniProtKB/Swiss-Prot: P03420.1
  • B9320 sequence refers to UniProtKB/Swiss-Prot: Q6V2E7
  • RSV pre-fusion F protein is designed according to the strategy adopted by Jason S. McLellan. Science 2013, RSV post The design of -fusion F protein was designed according to the strategy adopted by Davide Corti. Nature 2013.
  • the whole gene was synthesized in Shanghai Jierui Company and constructed on the expression vector of invitrogen pcDNA3.1. Expressed by mammalian cell CHO expression system; refer to Invitrogen ExpiCHO-STM Expression System manual.
  • Palivizumab (SYNAGIS®) was a positive control antibody, purchased from Abbott.
  • Coated F protein ELISA plate 0.5/mL, 100 pL per well, 4. (: Overnight. The next day, wash the plate 3 times with PBST. 2% BSA block, 200 pL per well, 37 ° C, 2 h o, and wash the plate 3 times with PBST.
  • the results are shown in Figure 3.
  • the 4F1 antibody can bind to the pre-fusion F protein of RSV A and B viruses in a broad spectrum, which is equivalent to the binding ability of the positive control antibody.
  • 4F1 does not bind to type A post-fusion F protein.
  • Palivizumab has been reported to bind to two forms of F protein.
  • the results showed that 4F1 antibody can broadly bind RSV type A and B pre-fusion F forms, and 4F1 antibody and palivizumab bind two different epitopes on F protein.
  • TCID 5 o/fL Number of Antilog 10 U positive wells/3)-0.5 ⁇ x 0.3 ]/2
  • the titer of the diluted virus solution should be 50-2000 TCIDW wells.
  • test virus strains A2 and B9320 purchased from ATCC
  • the diluted antibody and 200 TCIDW wells of the virus were added to a 96-well cell culture plate at equal volume ratios, mixed and incubated in a 37 °C, 5% CO 2 incubator for 2 hours.
  • HEp2 cells were seeded into the test plate at a density of 25,000 cells per well and cultured in a 37°C, 5% CO 2 incubator for 5 days.
  • Antibodies are tested at 9 concentrations, 3-fold serial dilutions, 3 replicate wells, the initial test concentration is 4000 ng/ml o
  • Activity percentage (%) (test hole reading value-virus control average value) / (cell control average value-virus control average value) ⁇ 100
  • the EC5 Q value is calculated by Prism software, and the neutralization activity curve fitting method is sigmoidal dose-response (variable slope).
  • Fig. 4 shows the antibody neutralizing activity dose fitting curve of the 4F 1 antibody and the control antibody Palivizumab against the RSV strain.
  • Palivizumab showed neutralizing activity against RSV A2 and B9320, with IC 5Q values of 384.3 ng/ml and 367.2 ng/ml, respectively.
  • the ICso values of 4F 1 for RSV A2 and B9320 were 4.368 ng/ml and 23.51 ng/ml, respectively.
  • the neutralizing activity of 4F 1 antibody to RSV A2 and B9320 is better than palivizumab, and the IC 5Q is nearly 80-100 times lower.
  • the negative antibody NC showed no neutralizing activity against the tested virus strain within the test concentration, and its ICso value was greater than the highest detection concentration of 4000 ng/ml (see Table 1).
  • mice 6-8 weeks old BalB/c female mice were placed in the biosafety secondary laboratory animal laboratory in advance. On day 0, mice were intraperitoneally injected with 15 mg/kg, 3 mg/kg, 0.6 mg/kg, 0.12 mg/kg 4F1 antibody, Palivizumab and PBS. After 24h, mice were anesthetized with ether, the mice nasally attack RSV A2 virus (10 7 PFU / 50ul / mouse). After five days, the animals were sacrificed and their lungs were collected.
  • the left lung was removed from each mouse and fixed with 4% paraformaldehyde, embedded in paraffin and stained with hematoxylin-eosin (HE), and observed the pathological damage and inflammatory cell infiltration of the lung under light microscope.
  • HE hematoxylin-eosin
  • conservative fragments were selected for primer design, and mouse P actin was used as the internal reference gene (see Table 2). Refer to the Toyobo KOD SYBR ® qPCR Mix manual.

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Abstract

L'invention concerne un anticorps de neutralisation à large spectre entièrement humain dirigé contre le virus respiratoire syncytial et une utilisation associée. Plus particulièrement, la présente invention concerne un anticorps monoclonal 4F1 entièrement humain dirigé contre une protéine de fusion du virus respiratoire syncytial (protéine F) et une protéine de pré-fusion F (protéine preF), une séquence d'acide nucléique codant pour ledit anticorps et un fragment d'anticorps, ainsi que son procédé de préparation. Des expériences in vivo et in vitro confirment que ledit anticorps 4F1 peut prévenir et lutter de manière efficace contre une infection par le VRS, présente une faible immunogénicité dans le corps humain, peut éviter des réactions de rejet immunitaire à médiation par des anticorps hétérogénétiques comme des anti-souris humains, et peut être utilisé sur le plan clinique pour prévenir et traiter une infection par le virus respiratoire syncytial.
PCT/CN2020/076767 2019-02-26 2020-02-26 Anticorps de neutralisation 4f1 à large spectre entièrement humain dirigé contre le virus respiratoire syncytial et utilisation associée WO2020173460A1 (fr)

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WO2024120517A1 (fr) * 2022-12-08 2024-06-13 南京诺唯赞生物科技股份有限公司 Anticorps se liant spécifiquement au vrs
CN117720650B (zh) * 2024-02-04 2024-07-02 北京百普赛斯生物科技股份有限公司 抗人呼吸道合胞病毒抗体及其应用
CN118005783B (zh) * 2024-04-08 2024-06-11 南京医科大学 一种抗呼吸道合胞病毒抗体及其应用

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