WO2020173460A1 - Full human broad-spectrum neutralizing antibody 4f1 against respiratory syncytial virus and use thereof - Google Patents
Full human broad-spectrum neutralizing antibody 4f1 against respiratory syncytial virus and use thereof Download PDFInfo
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Classifications
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- 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
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- C—CHEMISTRY; METALLURGY
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- 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]
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- 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
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- 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
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- 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
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- 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
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- 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
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- 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
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- 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
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- 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]
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- 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
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- 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
Disclosed are a full human broad-spectrum neutralizing antibody against respiratory syncytial virus and use thereof. Specifically, disclosed in the present invention are a full human monoclonal antibody 4F1 against a respiratory syncytial virus fusion protein (F protein) and pre-fusion F protein (preF protein), a nucleic acid sequence encoding said antibody and an antibody fragment, and a preparation method therefor. In vivo and in vitro experiments confirm that said 4F1 antibody can effectively prevent and control RSV infection, has low immunogenicity in the human body, can avoid immune rejection reactions mediated by heterogenetic antibodies such as human anti-mouse, and can be used clinically for preventing and treating respiratory syncytial virus infection.
Description
抗呼吸道合胞病毒的全人广谱中和抗体 4F1及其应用 技术领域 A fully human broad-spectrum neutralizing antibody 4F1 against respiratory syncytial virus and its application Technical field
本发明涉及医药领域， 具体地涉及一株抗呼吸道合胞病毒的全人广谱中和抗体 4F1 及其应用。 背景技术 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 导致急性上呼吸道和下呼吸道感染， 是世界范围内婴幼儿呼吸道感染重要的病 原之一， 同时 RSV还被公认为是某些高风险成年人， 诸如老年个体、 患有慢性肺部疾病 的成年个体以及免疫功能低下的成人（例如骨髓移植病人）的重要病原体。全球每年有 3000 万的新增感染病例， 因 RSV感染而死亡的人数约为 20万人。在中国 2岁以下儿童肺炎症 候群中， RSV是最常见的病毒性病原（17.0%）。全世界都迫切需要对于 RSV病毒感染的安 全、 有效、 价格低廉的预防治疗方法。 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.
在过去几十年里， 已研究预防和治疗 RSV感染的方法， 包括疫苗、抗病毒化合物（利 巴韦林）、反义药物、 RNA干扰技术以及抗体产品例如免疫球蛋白或静脉注射单克隆抗体。 帕利珠单抗是唯一被批准用于高风险儿童中的 RSV预防的抗体药物。 然而， 在中国尚没 有 RSV 的疫苗或可商购的治疗药物， 仅利巴韦林被批准用于治疗 RSV感染，但存在严 重的副作用。 帕利珠单抗靶向 F 蛋白保守的区域， 其作用广谱， 但目前每次给药所需帕 利珠单抗的剂量为 15mg/kg（体重）， 一年被动免疫 5次， 存在剂量高， 需多次给药， 价格 昂贵的缺点， 而且帕利珠单抗属于人源化的抗体， 仍然保留着部分鼠源序列， 可能存在着 一定的免疫原性， 在安全性上有一定的顾虑。 随着免疫学和分子生物学的发展， 基因工程 抗体迅速发展， 嵌合抗体、 人源化抗体和全人抗体生产技术不断发展， 将 HAMA反应降 至最低乃至消除， 尤其以全人抗体成为抗体药物未来的发展方向。 In the past few decades, methods to prevent and treat RSV infection have been studied, including vaccines, antiviral compounds (ribavirin), antisense drugs, RNA interference technology, and antibody products such as immunoglobulin or intravenous monoclonal antibodies . Palivizumab is the only antibody drug approved for RSV prevention in high-risk children. However, there is no RSV vaccine or commercially available therapeutic drug in China. Only ribavirin is approved for the treatment of RSV infection, but there are serious side effects. 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.
因此， 本领域仍然需要开发能够预防及控制 RSV感染的更有效的全人单克隆抗体。 发明内容 Therefore, there is still a need in the art to develop more effective fully human monoclonal antibodies that can prevent and control RSV infection. Summary of the invention
本发明目的是提供了一种能够预防及控制 RSV感染的全人单克隆抗体。 本发明的第一方面，提供了一种抗体的重链可变区，所述的重链可变区包括以下三个 互补决定区 CDR：
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:
在另一优选例中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺
失、 修饰和 /或取代至少一个（如 1-3个， 较佳地 1-2个， 更佳地 1个）氨基酸并能够保留呼 吸道合胞病毒融合蛋白（较佳地融合前 F蛋白）结合亲和力的衍生序列。 In another preferred embodiment, 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.
在另一优选例中， 所述重链可变区还包括人源的 FR区或鼠源的 FR区。 In another preferred embodiment, the heavy chain variable region further includes a human FR region or a murine FR region.
在另一优选例中， 所述重链可变区具有 SEQ ID NO.: 1所示的氨基酸序列。 本发明的第二方面，提供了一种抗体的重链，所述的重链具有如本发明第一方面所述 的重链可变区。 In another preferred example, 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.
在另一优选例中， 所述的抗体的重链还包括重链恒定区。 In another preferred embodiment, the heavy chain of the antibody also includes a heavy chain constant region.
在另一优选例中， 所述的重链恒定区为人源、 鼠源或兔源的。 本发明的第三方面，提供了一种抗体的轻链可变区，所述的轻链可变区包括以下三个 互补决定区 CDR： In another preferred embodiment, 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:
SEQ ID NO.: 6所示的 CDR1’， CDR1' shown in SEQ ID NO.: 6,
氨基酸序列为 LGS的 CDR2’， 和 The amino acid sequence is CDR2’ of LGS, and
SEQ ID NO.: 7所示的 CDR3’。 CDR3' shown in SEQ ID NO.: 7.
在另一优选例中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺 失、 修饰和 /或取代至少一个（如 1-3个， 较佳地 1-2个， 更佳地 1个）氨基酸并能够保留呼 吸道合胞病毒融合蛋白（较佳地融合前 F蛋白）结合亲和力的衍生序列。 In another preferred embodiment, 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).
在另一优选例中， 所述轻链可变区还包括人源的 FR区或鼠源的 FR区。 In another preferred example, the light chain variable region further includes a human FR region or a murine FR region.
在另一优选例中， 所述轻链可变区具有 SEQ ID NO.: 2所示的氨基酸序列。 本发明的第四方面，提供了一种抗体的轻链，所述的轻链具有如本发明第三方面所述 的轻链可变区。 In another preferred example, the light chain variable region has the amino acid sequence shown in SEQ ID NO.: 2. The fourth aspect of the present invention provides a light chain of an antibody, the light chain having the light chain variable region as described in the third aspect of the present invention.
在另一优选例中， 所述的抗体的轻链还包括轻链恒定区。 In another preferred embodiment, the light chain of the antibody also includes a light chain constant region.
在另一优选例中， 所述的轻链恒定区为人源、 鼠源或兔源的。 本发明的第五方面， 提供了一种抗体， 所述抗体具有： In another preferred embodiment, 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:
（1） 如本发明第一方面所述的重链可变区； 和 /或 (1) The heavy chain variable region as described in the first aspect of the present invention; and/or
（2） 如本发明第三方面所述的轻链可变区； (2) The light chain variable region as described in the third aspect of the present invention;
或者， 所述抗体具有： 如本发明第二方面所述的重链； 和 /或如本发明第四方面所述 的轻链。 Alternatively, 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.
在另一优选例中，所述的抗体为特异性抗呼吸道合胞病毒的抗体，较佳地为特异性抗 呼吸道合胞病毒融合蛋白（较佳地融合前 F蛋白）的抗体。 In another preferred embodiment, the antibody is a specific anti-respiratory syncytial virus antibody, preferably a specific anti-respiratory syncytial virus fusion protein (preferably pre-fusion F protein).
在另一优选例中， 所述抗体选自： 动物源抗体、 嵌合抗体、 人源化抗体、 或其组合。 在另一优选例中， 所述的抗体为双链抗体、 或单链抗体。
在另一优选例中， 所述的抗体为单克隆抗体、 或多克隆抗体。 In another preferred embodiment, the antibody is selected from: animal-derived antibodies, chimeric antibodies, humanized antibodies, or a combination thereof. In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody. In another preferred embodiment, the antibody is a monoclonal antibody or a polyclonal antibody.
在另一优选例中， 所述的抗体是部分或全人源化的单克隆抗体。 In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.
在另一优选例中， 所述的抗体为药物偶联物形式。 In another preferred embodiment, the antibody is in the form of a drug conjugate.
在另一优选例中， 所述抗体的重链可变区序列如 SEQ ID NO. : 1所示； 并且所述的抗 体的轻链可变区序列如 SEQ ID NO. : 2所示。 本发明的第六方面， 提供了一种重组蛋白， 所述的重组蛋白具有： In another preferred example, 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:
（i） 如本发明第一方面所述的重链可变区、 如本发明第二方面所述的重链、 如本发明 第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面所述的抗 体； 以及 (I) The heavy chain variable region according to the first aspect of the present invention, the heavy chain according to the second aspect of the present invention, the light chain variable region according to the third aspect of the present invention, and the fourth aspect of the present invention The light chain of the aspect, or the antibody of the fifth aspect of the present invention; and
（ii） 任选的协助表达和 /或纯化的标签序列。 (Ii) Optional tag sequence to assist in expression and/or purification.
在另一优选例中， 所述的标签序列包括 6His标签、 GGGS序列、 FLAG标签。 In another preferred example, the tag sequence includes 6His tag, GGGS sequence, and FLAG tag.
在另一优选例中， 所述的重组蛋白（或多肽）包括融合蛋白。 In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.
在另一优选例中， 所述的重组蛋白为单体、 二聚体、 或多聚体。 In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.
在另一优选例中，所述的重组蛋白特异性结合呼吸道合胞病毒融合蛋白，较佳地结合 融合前 F蛋白。 本发明的第七方面， 提供了一种 CAR构建物， 所述的 CAR构建物的抗原结合区域 的 scFv段为特异性结合于 RSV融合蛋白（较佳地融合前 F蛋白）的结合区， 并且所述 scFv 具有如本发明第一方面所述的重链可变区和如本发明第三方面所述的轻链可变区。 本发明的第八方面，提供了一种重组的免疫细胞，所述的免疫细胞表达外源的如本发 明第七方面所述的 CAR构建物。 In another preferred embodiment, the recombinant protein specifically binds to the respiratory syncytial virus fusion protein, preferably to the pre-fusion F protein. The seventh aspect of the present invention provides a CAR construct, the scFv segment of the antigen binding region of the CAR construct is the binding region that specifically binds to the RSV fusion protein (preferably the pre-fusion F protein), and The scFv has a heavy chain variable region as described in the first aspect of the present invention and a light chain variable region as described in the third aspect of the present invention. The eighth aspect of the present invention provides a recombinant immune cell that expresses an exogenous CAR construct as described in the seventh aspect of the present invention.
在另一优选例中， 所述的免疫细胞选自下组： NK细胞、 T细胞。 In another preferred embodiment, the immune cells are selected from the group consisting of NK cells and T cells.
在另一优选例中， 所述的免疫细胞来自人或非人哺乳动物（如鼠）。 本发明的第九方面， 提供了一种抗体药物偶联物， 所述的抗体药物偶联物含有： In another preferred example, 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）抗体部分， 所述抗体部分选自下组： 如本发明第一方面所述的重链可变区、 如本 发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述 的轻链、 或如本发明第五方面所述的抗体、 或其组合； 和 (A) An antibody portion, wherein the antibody portion 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 variable region of the light chain, the light chain according to the fourth aspect of the present invention, or the antibody according to the fifth aspect of the present invention, or a combination thereof; and
（b） 与所述抗体部分偶联的偶联部分，所述偶联部分选自下组： 可检测标记物、药物、 毒素、 细胞因子、 放射性核素、 酶、 或其组合。 (B) A coupling part coupled to the antibody part, the coupling part being selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.
在另一优选例中， 所述偶联物选自： 荧光或发光标记物、 放射性标记物、 MRI（磁共 振成像）或 CT（电子计算机 X射线断层扫描技术）造影剂、 或能够产生可检测产物的酶、 放 射性核素、 生物毒素、 细胞因子（如 IL-2等）、 抗体、 抗体 Fc片段、 抗体 scFv片段、 金纳
米颗粒 /纳米棒、 病毒颗粒、 脂质体、 纳米磁粒、 前药激活酶 (例如， DT-心肌黄酶 (DTD) 或联苯基水解酶-样蛋白质 (BPHL))、 化疗剂 (例如， 顺铂 )或任何形式的纳米颗粒等。 In another preferred embodiment, 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.
在另一优选例中， 所述的抗体部分与所述的偶联部分通过化学键或接头进行偶联。 本发明的第十方面， 提供了一种活性成分的用途， 所述活性成分选自下组： 如本发明 第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻 链可变区、 如本发明第四方面所述的轻链、 或本发明第五方面所述的抗体、 如本发明第六 方面所述的重组蛋白、 或其组合， 所述活性成分用于制备药剂、 试剂、 检测板或试剂盒。 In another preferred embodiment, 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.
在另一优选例中， 所述试剂、 检测板或试剂盒用于检测呼吸道合胞病毒。 In another preferred embodiment, the reagent, detection plate or kit is used to detect respiratory syncytial virus.
在另一优选例中， 所述药剂用于治疗或预防呼吸道合胞病毒感染。 In another preferred embodiment, the medicament is used to treat or prevent respiratory syncytial virus infection.
在另一优选例中， 所述的试剂包括芯片、 包被抗体的免疫微粒。 本发明的第十一方面， 提供了一种药物组合物， 所述的药物组合物含有： In another preferred embodiment, 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:
(i) 活性成分， 所述活性成分选自下组： 如本发明第一方面所述的重链可变区、 如本 发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述 的轻链、 或本发明第五方面所述的抗体、 如本发明第六方面所述的重组蛋白、 如本发明第 八方面所述的免疫细胞、 如本发明第九方面所述的抗体药物偶联物、 或其组合； 以及 (i) 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
(ii) 药学上可接受的载体。 (ii) A pharmaceutically acceptable carrier.
在另一优选例中， 所述的药物组合物为液态制剂。 In another preferred embodiment, the pharmaceutical composition is a liquid preparation.
在另一优选例中， 所述的药物组合物为注射剂。 In another preferred embodiment, the pharmaceutical composition is an injection.
在另一优选例中， 所述的药物组合物用于预防和 /或治疗呼吸道合胞病毒感染。 本发明的第十二方面， 提供了一种多核苷酸， 所述的多核苷酸编码选自下组的多肽: In another preferred embodiment, 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:
(1) 如本发明第一方面所述的重链可变区、 如本发明第二方面所述的重链、 如本发明 第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面所述的抗 体； 或 (1) The heavy chain variable region described in the first aspect of the present invention, the heavy chain described in the second aspect of the present invention, the light chain variable region described in the third aspect of the present invention, and the fourth aspect of the present invention The light chain of the aspect, or the antibody of the fifth aspect of the present invention; or
(2) 如本发明第六方面所述的重组蛋白； (2) The recombinant protein according to the sixth aspect of the present invention;
(3) 如本发明第七方面所述的 CAR构建物。 (3) The CAR construct according to the seventh aspect of the present invention.
在另一优选例中， 所述的多核苷酸具有 SEQ ID NO. : 8和 /或 SEQ ID NO. : 9所示的序 列。 本发明的第十三方面， 提供了一种载体， 所述的载体含有如本发明第十二方面所述的 多核苷酸。 In another preferred embodiment, 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.
在另一优选例中， 所述的载体包括： 细菌质粒、 噬菌体、 酵母质粒、 植物细胞病毒、 哺乳动物细胞病毒如腺病毒、 逆转录病毒、 或其他载体。
本发明的第十四方面，提供了一种遗传工程化的宿主细胞，所述的宿主细胞含有如本 发明第十三方面所述的载体或基因组中整合有如本发明第十二方面所述的多核苷酸。 本发明的第十五方面，提供了一种检测样品中呼吸道合胞病毒的方法，所述方法包括 步骤： In another preferred embodiment, 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:
(1) 将样品与本发明的第五方面所述的抗体接触； (1) Contacting the sample with the antibody according to the fifth aspect of the present invention;
P) 检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在呼吸道合胞病 毒。 P) Detect whether an antigen-antibody complex is formed. The formation of a complex indicates the presence of respiratory syncytial virus in the sample.
在另一优选例中， 所述检测为非治疗非诊断目的。 In another preferred embodiment, the detection is for non-therapeutic and non-diagnostic purposes.
本发明还提供了一种检测样品中呼吸道合胞病毒融合蛋白的方法， 所述方法包括步 骤： The present invention also provides a method for detecting respiratory syncytial virus fusion protein in a sample, the method comprising the steps:
(1) 将样品与本发明的第五方面所述的抗体接触； (1) Contacting the sample with the antibody according to the fifth aspect of the present invention;
P) 检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在呼吸道合胞病 毒融合蛋白。 P) Detect whether an antigen-antibody complex is formed. The formation of a complex indicates the presence of respiratory syncytial virus fusion protein in the sample.
在另一优选例中， 所述呼吸道合胞病毒融合蛋白为呼吸道合胞病毒融合前 F蛋白。 在另一优选例中， 所述检测为非治疗非诊断目的。 本发明的第十六方面， 提供了一种检测板， 所述的检测板包括： 基片 (支撑板)和测试 条，所述的测试条含有如本发明第五方面所述的抗体或如本发明第九方面所述的免疫偶联 物。 本发明的第十七方面， 提供了一种试剂盒， 所述试剂盒中包括： In another preferred embodiment, the respiratory syncytial virus fusion protein is a pre-fusion F protein of respiratory syncytial virus. In another preferred embodiment, 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:
(1) 第一容器， 所述第一容器中含有如本发明第五方面所述的抗体； 和 /或 (1) A first container, which contains the antibody according to the fifth aspect of the present invention; and/or
(2) 第二容器， 所述第二容器中含有抗如本发明第五方面所述的抗体的二抗； 或者， 所述试剂盒含有如本发明第十六方面所述的检测板。 本发明的第十八方面， 提供了一种重组多肽的制备方法， 所述方法包括： (2) 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:
(a)在适合表达的条件下， 培养如本发明第十四方面所述的宿主细胞； (a) Culturing the host cell according to the fourteenth aspect of the present invention under conditions suitable for expression;
(b) 从培养物中分离出重组多肽， 所述的重组多肽是如本发明第五方面所述的抗体或 如本发明第六方面所述的重组蛋白。 本发明的第十九方面， 提供了一种治疗呼吸道合胞病毒感染的方法， 所述方法包括: 给需要的对象施用如本发明第五方面所述的抗体、 所述抗体的抗体-药物偶联物、 或表达 所述抗体的 CAR-T细胞、 或其组合。
应理解， 在本发明范围内中， 本发明的上述各技术特征和在下文 (如实施例)中具体描 述的各技术特征之间都可以互相组合， 从而构成新的或优选的技术方案。 限于篇幅， 在此 不再—累述。 附图说明 (b) Isolating a recombinant polypeptide from the culture, where the recombinant polypeptide is the antibody according to the fifth aspect of the present invention or the recombinant protein according to the sixth aspect of the present invention. The nineteenth aspect of the present invention provides a method for the treatment of respiratory syncytial virus infection, the method comprising: administering the antibody according to the fifth aspect of the present invention, the antibody-drug pair of the antibody to a subject in need Conjugate, or CAR-T cell expressing the antibody, or a combination thereof. It should be understood that, within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat it here. Description of the drawings
图 1显示了流式分选 RSV F蛋白特异性结合的记忆 B细胞。 Figure 1 shows flow cytometric sorting of memory B cells specifically bound by RSV F protein.
图 2显示了匹配抗体轻重链基因琼脂糖凝胶电泳图谱。 Figure 2 shows the agarose gel electrophoresis pattern of matched antibody light and heavy chain genes.
图 3显示了 4F1抗体对 RSV A2 pre-fusion F蛋白 (A)、 B9320 pre-fusion F蛋白 (B)及 A2 post-fusion F蛋白 (C)的结合活性。其中， 4F1抗体可以结合 RSV A型和 B型的 pre-fusion 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). Among them, 4F1 antibody can bind RSV type A and B pre-fusion
F 蛋白。 F protein.
图 4显示了 4F1抗体对 RSV A2 (A)和 B9320 (B)的抗体中和活性剂量拟合曲线；和帕 利珠单抗 (Palivizumab)对 RSV A2 (；C)和 B9320 CD)的抗体中和活性剂量拟合曲线。 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.
图 5显示了小鼠预防实验中 4F1和帕利珠单抗对降低肺部病毒载量能力的比较。 图 6显示了小鼠预防实验中 4F1和帕利珠单抗被动免疫后肺部病理损伤比较。 具体实施方式 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. detailed description
本发明人通过广泛而深入的研究， 意外地获得一株针对呼吸道合胞病毒融合蛋白 (F 蛋白)及融合前 F蛋白 (preF蛋白)的全人单克隆抗体 4F1。 该抗体能够可以广谱性的结合 RSV A型和 B型病毒的 pre-fusion F蛋白，并对呼吸道合胞病毒具有很高的结合中和活性， 具有识别广谱性和广谱中和性，能够抑制或阻止呼吸道合胞病毒侵染易感细胞。体内外实 验证实 4F1抗体能有效地预防及控制 RSV的感染。 在此基础上， 完成了本发明。 Through extensive and in-depth research, 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. In vivo and in vitro experiments have confirmed that 4F1 antibody can effectively prevent and control RSV infection. On this basis, the present invention has been completed.
本发明从一个健康的志愿者 PBMC中， 通过单细胞 RT-PCR技术筛选到一株抗 RSV 病毒全人广谱性中和抗体 4F1。 ELISA结合实验和细胞水平微中和实验证实 4F1抗体对 RSV A型和 B型蛋白具有广谱结合活性和广谱微中和活性。 比较了 4F1和现在市售的帕 利珠单抗 (Palivizumab)抗体， 发现 4F1无论在细胞水平微中和实验还是小鼠预防实验， 均 显著优于帕利珠单抗抗体， 且 4F1 抗体属于全人源抗体， 不含有鼠源成分， 意味着其有 更低的免疫原性和更高的安全性， 这预示该抗体潜在的抗 RSV感染的临床应用价值， 为 临床上提供新型抗 RSV病毒感染的候选药物。本发明的抗体结合 RSV F蛋白融合前形式， 大量研究证实针对 F蛋白的中和抗体识别位点主要在 pre-fusion F 蛋白上， 4F1抗体表位 的发掘也为 RSV疫苗的设计提供一些新的思路和参考。 术语 In the present invention, a fully human broad-spectrum neutralizing antibody 4F1 against RSV virus was screened from a healthy volunteer PBMC through single-cell RT-PCR technology. ELISA binding experiments and cell-level micro-neutralization experiments confirmed that 4F1 antibody has broad-spectrum binding activity and broad-spectrum micro-neutralization activity to RSV type A and B proteins. Comparing 4F1 with the currently commercially available Palivizumab antibody, it is found that 4F1 is significantly better than the palivizumab antibody in both the cell-level microneutralization experiment and the mouse prevention experiment, and the 4F1 antibody belongs to the whole The human antibody does not contain murine components, which means that it has lower immunogenicity and higher safety, which indicates the potential clinical application value of the antibody against RSV infection and provides a new type of anti-RSV virus infection in the clinic. Drug candidates. The antibody of the present invention binds to the pre-fusion form of the RSV F protein. 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 discovery of the 4F1 antibody epitope also provides some new ideas for the design of RSV vaccines. Ideas and references. the term
为了更容易理解本发明， 以下具体定义了某些技术和科学术语。除非在本文中另有明 确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常
理解的含义。在描述本发明之前， 应当理解本发明不限于所述的具体方法和实验条件， 因 为这类方法和条件可以变动。 还应当理解本文所用的术语其目的仅在于描述具体实施方 案， 并且不意图是限制性的， 本发明的范围将仅由所附的权利要求书限制。 To make it easier to understand the present invention, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise in this document, all other technical and scientific terms used herein have the usual meaning of those of ordinary skill in the art to which the present invention belongs. Understand the meaning. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can vary. It should also be understood that the terms used herein are only intended to describe specific embodiments and are not intended to be limiting, and the scope of the present invention will only be limited by the appended claims.
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普 通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约” 意指该值可以从列举的值变动不多于 1%。例如，如本文所用，表述“约 100”包括 99和 101 和之间的全部值（例如， 99.1、 99.2、 99.3、 99.4等）。 Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary from the recited value by no more than 1%. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (eg, 99.1, 99.2, 99.3, 99.4, etc.).
本发明所用氨基酸三字母代码和单字母代码如 J.biol.chem,243,p3558（1968）中所述。 如本文所用， 术语“治疗”指给予患者内用或外用治疗剂， 包含本发明的针对呼吸道合 胞病毒融合蛋白（较佳地融合前 F蛋白）的单克隆抗体及其组合物，所述患者具有一种或多 种疾病症状， 而已知所述治疗剂对这些症状具有治疗作用。通常， 以有效缓解一种或多种 疾病症状的治疗剂的量（治疗有效量）给予患者。 The three-letter code and one-letter code of amino acids used in the present invention are as described in J. biool. chem, 243, p3558 (1968). As used herein, the term "treatment" refers to the administration of a therapeutic agent for internal or external use to a patient, comprising the monoclonal antibody against the respiratory syncytial virus fusion protein (preferably pre-fusion F protein) of the present invention and the composition thereof. It has one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Usually, the amount of a therapeutic agent (therapeutically effective amount) that is effective to alleviate one or more disease symptoms is administered to the patient.
如本文所用，术语“任选”或“任选地”意味着随后所描述的事件或情况可以发生但不是 必须发生。例如， “任选包含 1-3个抗体重链可变区”是指特定序列的抗体重链可变区可以 有但不是必须有， 可以是 1个、 2个或 3个。 As used herein, the term "optional" or "optionally" means that the event or situation described later can occur but does not have to occur. For example, "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.
本发明所述的“序列同一性”表示当具有适当的替换、插入或缺失等突变的情况下最佳 比对和比较时，两个核酸或两个氨基酸序列之间的同一性程度。本发明中所述的序列和其 具有同一性的序列之间的序列同一性可以至少为 85%、 90%或 95%， 优选至少为 95%。 非限制性实施例包括 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%。 呼吸道合胞病毒（RSV） The "sequence identity" in the present invention refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions or deletions. The sequence identity between the sequence described in the present invention and its identical sequence may be at least 85%, 90% or 95%, and preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , 100%. Respiratory syncytial virus (RSV)
RSV属于副粘病毒科、肺炎病毒属，是由 15222个核苷酸组成的负链 RNA病毒。 RSV 为囊膜病毒， 基因组共编码 11种蛋白， 包括融合蛋白 F、 吸附蛋白 G、 小疏水蛋白 SH、 基质蛋白 M、 核蛋白 N、 憐蛋白 P、 大聚合酶蛋白 L、 小基质蛋白 M2 以及非结构蛋白 NS1和 NS2， 其中融合蛋白 F、 吸附蛋白 G和小疏水蛋白 SH蛋白位于病毒表面。 RSV 具有两种主要表面糖蛋白， G和 F。 G蛋白介导病毒结合到该细胞表面， 同时可模拟趋化 因子 CX3C趋化因子的作用， 与其受体相互作用， 增强 RSV感染后的炎症反应； F蛋白 介导病毒和细胞膜的融合并且还促进感染的细胞的膜与周围细胞融合以形成合胞体。根据 血清学， RSV存在两种不同抗原群或亚型， 即 A和 B， 主要通过 G蛋白的差异进行区 别。 大多数 RSV蛋白在两种亚群间是 度保守的， 其中 fusion F蛋白显不了 91%的氨基 酸相似性， G蛋白在 A和 B之间仅具有 53%的同源性。 F蛋白高度保守， 其诱导的中和 抗体可以同时抑制 A和 B两个亚型的病毒感染，在抗 RSV单克隆抗体药物及疫苗研制方 面均具有重要意义。 F蛋白属于 I型跨膜蛋白， F蛋白首先合成前体蛋白 F0。 F0在内质 网中三聚化并且在两个保守位点被细胞弗林样蛋白酶处理， 产生 Fl、 F2和 Pep27多肽，
最后形成以二硫键相连接的 F1和 F2两个片段， F2-F1异二聚体形成三聚体组装成熟 F蛋 白。 F1亚单位包含七肽重复区 A(HRA)、功能域 1/11、七肽重复区 B (HRB)、跨膜蛋白 (TM)、 胞质区 (CP)， F2亚单位则由七肽重复区 C (HRC)组成。 F1 疏水性 N端 (137-155)在介导融 合中起重要的作用。 F蛋白介导的膜融合过程亦是其结构由融合前状态到融合状态的变化 过程。 其采用亚稳态融合前构象。 当 G蛋白和靶细胞膜上的受体结合后， 融合前 F蛋白 (pre-fusion F蛋白)开始触发变构为稳定的融合后形式 (post-fusion)。 由于其在 RSV入侵中 的关键作用且高度保守， RSV F蛋白是中和抗体的靶标和疫苗开发的主要抗原。大量研究 证实针对 F蛋白的中和抗体识别位点主要在 pre-fusion F 蛋白上， 基于 pre-fusion F设计 的重组蛋白免疫原性和保护效果要显著优于基融合后 F蛋白 (post-fusion F蛋白)设计的疫 苗。 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. According to serology, there are two different antigen groups or subtypes of RSV, namely A and B, which are mainly distinguished by the difference of G protein. 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. The hydrophobic N-terminal (137-155) of F1 plays an important role in mediating fusion. The membrane fusion process mediated by F protein is also the change process of its structure from the pre-fusion state to the fusion state. It adopts a metastable pre-fusion conformation. 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.
如本文所用， 术语“融合前 F蛋白” 、 "pre-fusion F蛋白” 、 “preF蛋白”可互换 使用， 均指呼吸道合胞病毒的融合蛋白 F的融合前形式。 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.
为进一步获得疗效更好的、 全新的 RSV抗体药物及寻找新的抗体识别表位， 本发明 利用单细胞 RT-PCR技术从人的外周血 PBMC中分离到一株广谱性中和抗体 4F1。 新的 抗体的发现一方面为广谱中和抗体治疗应用提供了新的选择，另一方面新的表位的发现为 广谱疫苗的开发提供了新的思路。 抗体 In order to further obtain a new RSV antibody drug with better curative effect and find a new antibody recognition epitope, 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, on the one hand, 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
如本文所用， 术语“抗体”或“免疫球蛋白”是有相同结构特征的约 150000道尔顿的异 四聚糖蛋白， 其由两个相同的轻链 (L)和两个相同的重链 (H)组成。 每条轻链通过一个共价 二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻 链也有规则间隔的链内二硫键。 每条重链的一端有可变区 (VH)， 其后是多个恒定区。 每 条轻链的一端有可变区 (VL)，另一端有恒定区;轻链的恒定区与重链的第一个恒定区相对， 轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界 面。 As used herein, the term "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. 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.
如本文所用， 术语“可变”表示抗体中可变区的某些部分在序列上有所不同， 它形成了 各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可 变区中。 它集中于轻链和重链可变区中称为互补决定区 (CDR)或超变区中的三个片段中。 可变区中较保守的部分称为构架区 (FR)。天然重链和轻链的可变区中各自包含四个 FR区， 它们大致上呈 P-折叠构型， 由形成连接环的三个 CDR相连， 在某些情况下可形成部分 折叠结构。每条链中的 CDR通过 FR区紧密地靠在一起并与另一链的 CDR一起形成了抗 体的抗原结合部位 (参见 Kabat等， NIH Publ. No. 91-3242, 卷 I， 647-669页 (1991))。 恒定 区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖 于抗体的细胞毒性。 As used herein, the term "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). 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.
脊椎动物抗体 (免疫球蛋白 )的“轻链”可根据其恒定区的氨基酸序列归为明显不同的
两类（称为 K和人）中的一类。根据其重链恒定区的氨基酸序列， 免疫球蛋白可以分为不同的 种类。 主要有 5类免疫球蛋白： IgA, IgD, IgE, IgG和 IgM， 其中一些还可进一步分成亚类 （同种型）， 如 IgGl, IgG2, IgG3, IgG4, IgA和 IgA2。 对应于不同类免疫球蛋白的重链恒定 区分别称为 a、 5、 S、 丫、 和 1^。 不同类免疫球蛋白的亚单位结构和三维构型是本领域人员 所熟知的。 The "light chains" of vertebrate antibodies (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 , γ, and 1^, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
如本文所用， 术语“单克隆抗体（单抗）”指从一类基本均一的群体获得的抗体， 即该群 体中包含的单个抗体是相同的， 除少数可能存在的天然发生的突变外。单克隆抗体高特异 性地针对单个抗原位点。 而且， 与常规多克隆抗体制剂（通常是具有针对不同决定簇的不 同抗体）不同， 各单克隆抗体是针对抗原上的单个决定簇。 除了它们的特异性外， 单克隆 抗体的好处还在于它们是通过杂交瘤培养来合成的，不会被其它免疫球蛋白污染。修饰语 “单克隆”表示了抗体的特性， 是从基本均一的抗体群中获得的， 这不应被解释成需要用任 何特殊方法来生产抗体。 As used herein, 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.
本发明还包括具有所述的抗呼吸道合胞病毒融合蛋白（较佳地 pre-fusion F蛋白）单克 隆抗体的相应氨基酸序列的单克隆抗体、 具有所述的抗呼吸道合胞病毒融合蛋白（较佳地 pre-fusion F蛋白）单克隆抗体可变区链的单克隆抗体，以及具有这些链的其他蛋白质或蛋 白质偶联物及融合表达产物。 具体地， 本发明包括具有含超变区（互补决定区， CDR）的轻 链和重链的任何蛋白质或蛋白质偶联物及融合表达产物（即免疫偶联物及融合表达产物）， 只要该超变区与本发明的轻链和重链的超变区相同或至少 90%同源性， 较佳地至少 95% 同源性。 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. Specifically, 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.
如本领域技术人员所知， 免疫偶联物及融合表达产物包括： 药物、 毒素、 细胞因子 （cytokine）, 放射性核素、 酶和其他诊断或治疗分子与所述的抗呼吸道合胞病毒融合蛋白 单克隆抗体或其片段结合的而形成的偶联物。本发明还包括与所述的抗呼吸道合胞病毒融 合蛋白单克隆抗体或其片段结合的细胞表面标记物或抗原。 As those skilled in the art know, 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.
术语“抗体的抗原结合片段”（或简称“抗体片段”）是指抗体的保持特异性结合抗原的能 力的一个或多个片段。己显示可利用全长抗体的片段来进行抗体的抗原结合功能。术语“抗 体的抗原结合片段”中包含的结合片段的实例包括（i）Fab片段， 由 VL、 VH、 CL和 CH1 结构域组成的单价片段； （ii）F（ab’）2片段， 包含通过较链区上的二硫桥连接的两个 Fab片 段的二价片段； （iii）由 VH和 CH1结构域组成的 Fd片段； （iv）由抗体的单臂的 VH和 VL 结构域组成的 Fv片段。 Fv抗体含有抗体重链可变区、 轻链可变区， 但没有恒定区， 并具 有全部抗原结合位点的最小抗体片段。 一般的， Fv抗体还包含 VH和 VL结构域之间的 多肽接头， 且能够形成抗原结合所需的结构。 The term "antigen-binding fragment of an antibody" (or "antibody fragment" for short) 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. Examples of 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. Generally, an Fv antibody also contains a polypeptide linker between the VH and VL domains, and can form the structure required for antigen binding.
本发明不仅包括完整的单克隆抗体， 还包括具有免疫活性的抗体片段， 如 Fab 或 （Fab’）2片段； 抗体重链； 抗体轻链。 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.
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位。表位通 常以独特的空间构象包括至少 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14或 15个连续或非连续的氨
基酸。 The term "epitope" 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.
术语“特异性结合”、 “选择性结合”、 “选择性地结合”和“特异性地结合”是指抗体对预 先确定的抗原上的表位的结合。通常，抗体以大约小于 IO_7M，例如大约小于 10_8M、 10 9M 或 10'10M或更小的亲和力 (KD)结合。 The terms "specifically binds", "selectively binds", "selectively binds" and "specifically binds" refer to the binding of an antibody to an epitope on a predetermined antigen. Generally, 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.
如本文所用， 术语“抗原决定簇”指抗原上不连续的， 由本发明抗体或抗原结合片段识 别的三维空间位点。 As used herein, the term "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.
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形 成的融合蛋白。 因此， 本发明还包括所述抗体的片段、 衍生物和类似物。 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.
在本发明中， 抗体包括用本领域技术人员熟知技术所制备的鼠的、嵌合的、人源化的 或者全人的抗体。重组抗体，例如嵌合的和人源化的单克隆抗体，包括人的和非人的部分， 可以采用本领域熟知的 DNA重组技术制备。 术语“鼠源抗体”在本发明中为根据本领域知 识和技能制备的针对呼吸道合胞病毒融合蛋白的单克隆抗体。 术语“嵌合抗体 (chimeric antibody)”是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体， 可以减轻鼠源性 抗体诱发的免疫应答反应。 术语“人源化抗体 (humanized antibody)”， 也称为 CDR移植抗 体 (CDR-grafted antibody)， 是指将鼠的 CDR序列移植到人的抗体可变区框架， 即不同类 型的人种系抗体构架序列中产生的抗体。人源化抗体可以克服嵌合抗体由于携带大量鼠蛋 白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共 DNA 数据库或公开的参考文献获得。为避免免疫原性下降的同时， 引起的活性下降， 可对所述 的人抗体可变区框架序列进行最少反向突变或回复突变， 以保持活性。 In the present invention, 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. The term "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. The term "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. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. In order to avoid the decrease in immunogenicity and the resulting decrease in activity, the human antibody variable region framework sequence may be subjected to minimal reverse mutations or back mutations to maintain activity.
在本发明中， 抗体可以是单特异性、 双特异性、 三特异性、 或者更多的多重特异性。 如本文所用， 术语“重链可变区”与“VH”可互换使用。 In the present invention, the antibody may be monospecific, bispecific, trispecific, or more multispecific. As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably.
如本文所用， 术语“可变区”与“互补决定区 (complementarity determining region, CDR)” 可互换使用。 As used herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.
术语“CDR”是指抗体的可变结构域内主要促成抗原结合的 6 个高变区之一。 所述 6 个 CDR 的最常用的定义之一由 Kabat E.A 等人， (1991) Sequences of proteins of immunological interest. NIH Publication91-3242)提供。 The term "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.
在本发明的一个优选的实施方式中， 所述抗体的重链可变区包括以下三个互补 决定区 CDR： In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes the following three complementarity determining region CDRs:
CDR1 : GFSFYSYS (SEQ ID NO : 3)， CDR1: GFSFYSYS (SEQ ID NO: 3),
CDR2： WYDGNHQ (SEQ ID NO. : 4)， 和 CDR2: WYDGNHQ (SEQ ID NO.: 4), and
CDR3： TARSLVITLAGAGRDDY (SEQ ID NO. : 5)。 CDR3: TARSLVITLAGAGRDDY (SEQ ID NO.: 5).
在另一优选例中， 所述重链可变区的氨基酸序列如 SEQ ID NO. : 1所示， 其中下 划线标注的依次为重链可变区 CDR1， CDR2， CDR3的氨基酸序列。 In another preferred example, the 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.
EVOLVOSGGGVVRPGRSLRLSCAASGFSFYSYSVHWVROAPGKGLEWVADV
AGRDDYWGOGTRVTVS S (SEQ ID NO : 1) EVOLVOSGGGVVRPGRSLRLSCAASGFSFYSYSVHWVROAPGKGLEWVADV AGRDDYWGOGTRVTVS S (SEQ ID NO: 1)
在另一优选例中， 所述重链可变区的核酸编码序列如 SEQ ID NO. : 8所示， 其中 下划线标注的依次为重链可变区 CDR1， CDR2， CDR3的核酸编码序列。 In another preferred example, the 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.
GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCGGCCTGGGAGGTC CCTGAGACTCTCCTGTGCAGCCTCTGGATTCAGCTTCTATTCCTATTCTGTGCAC TGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGATGTTGTATAT GATGGAAATCATCAACATTACACGGAGTCCGTGAGGGGCCGATTCTCCATCTCC AGAGACACCTCCACCAATACGGTGTATCTGCAAATGGGCAGCCTGAGGCCTGA AGACACGGCTCTTTATTACTGTACGGCCCGCAGTTTGGTCATAACGCTCGCGGG GGCGGGTCGAGATGACTATTGGGGCCAGGGAACTCGGGTCACCGTCTCCTCA GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCGGCCTGGGAGGTC CCTGAGACTCTCCTGTGCAGCCTCTGGATTCAGCTTCTATTCCTATTCTGTGCAC TGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGATGTTGTATAT GATGGAAATCATCAACATTACACGGAGTCCGTGAGGGGCCGATTCTCCATCTCC AGAGACACCTCCACCAATACGGTGTATCTGCAAATGGGCAGCCTGAGGCCTGA AGACACGGCTCTTTATTACTGTACGGCCCGCAGTTTGGTCATAACGCTCGCGGG GGCGGGTCGAGATGACTATTGGGGCCAGGGAACTCGGGTCACCGTCTCCTCA
(SEQ ID NO : 8) (SEQ ID NO: 8)
在本发明的一个优选的实施方式中， 所述抗体的重链包括上述重链可变区和重 链恒定区， 所述重链恒定区可以为鼠源或人源。 In a preferred embodiment of the present invention, 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.
如本文所用， 术语“轻链可变区”与“VL”可互换使用。 As used herein, the terms "light chain variable region" and "VL" are used interchangeably.
在本发明的一个优选的实施方式中， 根据本发明的抗体的轻链可变区， 具有选 自下组的互补决定区 CDR： In a preferred embodiment of the present invention, the light chain variable region of the antibody according to the present invention has a complementarity determining region CDR selected from the following group:
CDR1，： QSLLHSNGYTY (SEQ ID NO : 6)， CDR1,: QSLLHSNGYTY (SEQ ID NO: 6),
CDR2，： LGS， 和 CDR2,: LGS, and
CDR3’： VQDLQTSLT (SEQ ID NO : 7)。 CDR3': VQDLQTSLT (SEQ ID NO: 7).
在另一优选例中， 所述轻链可变区的氨基酸序列如 SEQ ID NO.: 2所示， 其中双 下划线标注的依次为轻链可变区 CDR1’， CDR2’， CDR3’的氨基酸序列。 In another preferred example, the 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' .
DIVMTOSPLSLSVTPGEPASISCKSSOSLLHSNGYTYLDWYLOKPGKSPOLLIFL GSSRASGVPARFSGSGSGTDFTLEISRVEAEDVGVYYCVODLOTSLTFGGGTKVDIK DIVMTOSPLSLSVTPGEPASISCKSSOSLLHSNGYTYLDWYLOKPGKSPOLLIFL GSSRASGVPARFSGSGSGTDFTLEISRVEAEDVGVYYCVODLOTSLTFGGGTKVDIK
(SEQ ID NO : 2) (SEQ ID NO: 2)
在另一优选例中， 所述轻链可变区的核酸编码序列如 SEQ ID NO. : 9所示， 其中 双下划线标注的依次为轻链可变区 CDR1’， CDR2’， CDR3’的核酸编码序列。 In another preferred embodiment, the 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.
GATATTGTGATGACTCAGTCTCCACTCTCCCTGTCCGTCACCCCTGGAGAGC CGGCCTCCATCTCCTGCAAGTCTAGTCAGAGCCTCCTCCATAGTAATGGATACA CTTATTTGGATTGGTACCTGCAGAAGCCAGGGAAGTCTCCACAACTCCTGATCT TTTTGGGTTCTAGTCGGGCCTCCGGGGTCCCTGCCAGGTTCAGTGGCAGTGGAT CAGGCACAGATTTTACACTGGAAATCAGCAGAGTGGAGGCTGAGGATGTTGGG GTTTACTACTGCGTGCAAGATCTACAAACTTCCCTCACTTTCGGCGGAGGGACC AAAGTGGATATCAAA (SEQ ID NO : 9) GATATTGTGATGACTCAGTCTCCACTCTCCCTGTCCGTCACCCCTGGAGAGC CGGCCTCCATCTCCTGCAAGTCTAGTCAGAGCCTCCTCCATAGTAATGGATACA CTTATTTGGATTGGTACCTGCAGAAGCCAGGGAAGTCTCCACAACTCCTGATCT TTTTGGGTTCTAGTCGGGCCTCCGGGGTCCCTGCCAGGTTCAGTGGCAGTGGAT CAGGCACAGATTTTACACTGGAAATCAGCAGAGTGGAGGCTGAGGATGTTGGG GTTTACTACTGCGTGCAAGATCTACAAACTTCCCTCACTTTCGGCGGAGGGACC AAAGTGGATATCAAA (SEQ ID NO: 9)
在本发明的一个优选的实施方式中， 所述抗体的轻链包括上述轻链可变区和轻 链恒定区， 所述轻链恒定区可以为鼠源或人源。
本发明抗体的功能是由此抗体轻链和重链可变区基因特异性基因序列决定，可以广谱 性的结合 RSV A型和 B型病毒的 pre-fusion F蛋白，能够阻止呼吸道合胞病毒侵染易感细 胞。利用此抗体可变区基因或互补决定区（CDR）基因， 可在利用原核和真核细胞的任何表 达系统中改造和生产不同形式的基因工程抗体。 In a preferred embodiment of the present invention, the light chain of the antibody includes the above-mentioned light chain variable region and light chain constant region, and the light chain constant region may be of murine or human origin. The function of the antibody of the present invention is determined by the specific gene sequence of the light chain and heavy chain variable region genes of the antibody, can bind to the pre-fusion F protein of RSV A and B viruses in a broad spectrum, and can prevent respiratory syncytial virus Infect susceptible cells. Using this antibody variable region gene or complementarity determining region (CDR) gene, different forms of genetically engineered antibodies can be modified and produced in any expression system using prokaryotic and eukaryotic cells.
在本发明中， 术语“本发明抗体”、 “本发明蛋白”、 或“本发明多肽”可互换使用， 都指特异性结合抗呼吸道合胞病毒融合蛋白（较佳地 pre-fusi on F蛋白）的抗体，例如具 有重链可变区（如 SEQ ID NO. : 8所示的核苷酸序列编码的氨基酸序列）和 /或轻链可变 区（如 SEQ ID NO. : 9所示的核苷酸序列编码的氨基酸序列）的蛋白或多肽。 它们可含 有或不含起始甲硫氨酸。 In the present invention, 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. They may or may not contain the starting methionine.
在另一优选例中， 所述的抗体为抗呼吸道合胞病毒融合蛋白（较佳地 pre-fusion F 蛋白）的鼠或人鼠嵌合单克隆抗体， 它的重链恒定区和 /或轻链恒定区可以是人源化的 重链恒定区或轻链恒定区。 更优选地， 所述的人源化的重链恒定区或轻链恒定区为人 IgGl、 IgG2等的重链恒定区或轻链恒定区。 In another preferred embodiment, 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.
一般， 抗体的抗原结合特性可由位于重链和轻链可变区的 3 个特定的区域来描 述， 称为可变区域（CDR）， 将该段间隔成 4个框架区域（FR）， 4个 FR的氨基酸序列相 对比较保守， 不直接参与结合反应。 这些 CDR形成环状结构， 通过其间的 FR形成 的 P折叠在空间结构上相互靠近，重链上的 CDR和相应轻链上的 CDR构成了抗体的 抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了 FR或 CDR区域。 Generally, the antigen-binding properties of antibodies can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDR), which are divided into 4 framework regions (FR), and 4 The 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.
本发明抗体的重链和 /或轻链的可变区特别令人感兴趣， 因为它们中至少部分涉 及结合抗原。 因此， 本发明包括那些具有带 CDR的单克隆抗体轻链和重链可变区的 分子， 只要其 CDR与此处鉴定的 CDR具有 90%以上（:较佳地 95%以上， 最佳地 98% 以上）的同源性。 The 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).
本发明不仅包括完整的单克隆抗体， 还包括具有免疫活性的抗体的片段或抗体 与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。 例如在本发明抗体的基础上进行的 Fc片段的改造， 为了延长抗体半衰期， 在 CH2区 域引入三个突变点 M252Y /S254T /T256E。 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.
如本文所用， 术语“片段”、 “衍生物”和“类似物”是指基本上保持本发明抗体相同 的生物学功能或活性的多肽。 本发明的多肽片段、 衍生物或类似物可以是（i）有一个或 多个保守或非保守性氨基酸残基（优选保守性氨基酸残基）被取代的多肽， 而这样的取 代的氨基酸残基可以是也可以不是由遗传密码编码的， 或（ii）在一个或多个氨基酸残 基中具有取代基团的多肽， 或（iii）成熟多肽与另一个化合物（比如延长多肽半衰期的化 合物， 例如聚乙二醇）融合所形成的多肽， 或（iv）附加的氨基酸序列融合到此多肽序列 而形成的多肽（如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列， 或与 6His标签形成的融合蛋白）。 根据本文的教导， 这些片段、 衍生物和类似物属于本领
域熟练技术人员公知的范围。 As used herein, the terms "fragment", "derivative" and "analog" refer 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). According to the teachings of this article, these fragments, derivatives and analogs belong to the art A range known to those skilled in the art.
本发明抗体指具有抗呼吸道合胞病毒融合蛋白（较佳地 pre-fusion F蛋白）结合活 性的、 包括上述 CDR区的多肽。 该术语还包括具有与本发明抗体相同功能的、 包含 上述 CDR区的多肽的变异形式。 这些变异形式包括（但并不限于）： 一个或多个（通常 为 1-50个， 较佳地 1-30个， 更佳地 1-20个， 最佳地 1-10个）氨基酸的缺失、 插入和 /或取代， 以及在 C末端和 /或 N末端添加一个或数个（通常为 20个以内， 较佳地为 10 个以内， 更佳地为 5 个以内）氨基酸。 例如， 在本领域中， 用性能相近或相似的氨基 酸进行取代时， 通常不会改变蛋白质的功能。 又比如， 在 C末端和 /或 N末端添加一 个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段 和活性衍生物。 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. For example, in this field, the substitution of amino acids with similar or similar properties usually does not change the function of the protein. For another example, 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.
该多肽的变异形式包括： 同源序列、 保守性变异体、 等位变异体、 天然突变体、 诱导突变体、 在高或低的严紧度条件下能与本发明抗体的编码 DNA杂交的 DNA所 编码的蛋白、 以及利用抗本发明抗体的抗血清获得的多肽或蛋白。 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.
本发明还提供了其他多肽， 如包含人抗体或其片段的融合蛋白。 除了几乎全长 的多肽外， 本发明还包括了本发明抗体的片段。 通常， 该片段具有本发明抗体的至少 约 50个连续氨基酸， 较佳地至少约 60个连续氨基酸， 更佳地至少约 80个连续氨基 酸， 最佳地至少约 100个连续氨基酸。 The present invention also provides other polypeptides, such as fusion proteins containing human antibodies or fragments thereof. In addition to almost full-length polypeptides, the present invention also includes fragments of the antibodies of the present invention. Generally, 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.
在本发明中， “本发明抗体的保守性变异体”指与本发明抗体的氨基酸序列相比， 有至多 10个， 较佳地至多 8个， 更佳地至多 5个， 最佳地至多 3个氨基酸被性质相 似或相近的氨基酸所替换而形成多肽。 这些保守性变异多肽最好根据表 A进行氨基 酸替换而产生。 In the present invention, "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.
表 A Table A
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。 本发明 的多核苷酸可以是 DNA形式或 RNA形式。 DNA形式包括 cDNA、基因组 DNA或人 工合成的 DNA。 DNA可以是单链的或是双链的。 DNA可以是编码链或非编码链。编 码成熟多肽的编码区序列可以与 SEQ ID NO. : 8或 9所示的编码区序列相同或者是简 并的变异体。 如本文所用， “简并的变异体”在本发明中是指编码具有与本发明的多肽 相同的氨基酸序列， 但与 SEQ ID NO. : 8或 9所示的编码区序列有差别的核酸序列。 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. As used herein, "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 .
编码本发明的成熟多肽的多核苷酸包括： 只编码成熟多肽的编码序列； 成熟多 肽的编码序列和各种附加编码序列； 成熟多肽的编码序列（和任选的附加编码序列）以 及非编码序列。 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 .
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括 附加编码和 /或非编码序列的多核苷酸。 The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or a polynucleotide that also includes additional coding and/or non-coding sequences.
本发明还涉及与上述的序列杂交且两个序列之间具有至少 50%， 较佳地至少 70%， 更佳地至少 80%相同性的多核苷酸。 本发明特别涉及在严格条件下与本发明所 述多核苷酸可杂交的多核苷酸。 在本发明中， “严格条件”是指： G）在较低离子强度 和较高温度下的杂交和洗脱，如 0.2xSSC, 0.1%SDS, 60 °C ;或⑺杂交时加有变性剂， 如 50%（v/v）甲酰胺， 0.1%小牛血清 /0.1% Ficoll， 42°C等； 或（3）仅在两条序列之间的相同 性至少在 90%以上,更好是 95%以上时才发生杂交。 并且， 可杂交的多核苷酸编码的 多肽与 SEQ ID NO. : 1和 /或 SEQ ID NO. : 2所示的成熟多肽有相同的生物学功能和活 性。 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. In the present invention, "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 ⑺ 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%. In addition, 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.
本发明的抗体的核苷酸全长序列或其片段通常可以用 PCR扩增法、 重组法或人 工合成的方法获得。 一种可行的方法是用人工合成的方法来合成有关序列， 尤其是片 段长度较短时。 通常， 通过先合成多个小片段， 然后再进行连接可获得序列很长的片 段。 此外， 还可将重链的编码序列和表达标签（如 6His）融合在一起， 形成融合蛋白。 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. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.
一旦获得了有关的序列， 就可以用重组法来大批量地获得有关序列。 这通常是 将其克隆入载体， 再转入细胞， 然后通过常规方法从增殖后的宿主细胞中分离得到有 关序列。本发明所涉及的生物分子（核酸、蛋白等）包括以分离的形式存在的生物分子。 Once the relevant sequence is obtained, 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.
目前， 已经可以完全通过化学合成来得到编码本发明蛋白（或其片段， 或其衍生
物）的 DNA序列。然后可将该 DNA序列引入本领域中已知的各种现有的 DNA分子（或 如载体）和细胞中。 此外， 还可通过化学合成将突变引入本发明蛋白序列中。 At present, it is possible to obtain the protein (or fragments thereof, or derivatives thereof) of the present invention completely through chemical synthesis. 物) DNA sequence. The DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
本发明还涉及包含上述的适当 DNA序列以及适当启动子或者控制序列的载体。 这些载体可以用于转化适当的宿主细胞， 以使其能够表达蛋白质。 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.
宿主细胞可以是原核细胞， 如细菌细胞； 或是低等真核细胞， 如酵母细胞； 或 是高等真核细胞， 如哺乳动物细胞。 代表性例子有： 大肠杆菌， 链霉菌属； 鼠伤寒沙 门氏菌的细菌细胞； 真菌细胞如酵母； 果蝇 S2或 Sf9的昆虫细胞； CHO、 COS7、 293 细胞的动物细胞等。 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. 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.
用重组 DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。 当宿主为 原核生物如大肠杆菌时，能吸收 DNA的感受态细胞可在指数生长期后收获，用 CaCh 法处理， 所用的步骤在本领域众所周知。 另一种方法是使用 MgCh。 如果需要， 转化 也可用电穿孔的方法进行。 当宿主是真核生物， 可选用如下的 DNA转染方法： 磷酸 共沉淀法， 常规机械方法如显微注射、 电穿孔，脂质体包装等。 Transformation of host cells with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When 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. If necessary, transformation can also be performed by electroporation. When 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.
获得的转化子可以用常规方法培养， 表达本发明的基因所编码的多肽。 根据所 用的宿主细胞， 培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的 条件下进行培养。 当宿主细胞生长到适当的细胞密度后， 用合适的方法（如温度转换 或化学诱导）诱导选择的启动子， 将细胞再培养一段时间。 The obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, 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. When the host cell has grown to an appropriate cell density, 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.
在上面的方法中的重组多肽可在细胞内、 或在细胞膜上表达、 或分泌到细胞外。 如果需要， 可利用其物理的、 化学的和其它特性通过各种分离方法分离和纯化重组的 蛋白。 这些方法是本领域技术人员所熟知的。 这些方法的例子包括但并不限于： 常规 的复性处理、 用蛋白沉淀剂处理（盐析方法）、 离心、 渗透破菌、 超处理、 超离心、 分 子筛层析（凝胶过滤）、 吸附层析、 离子交换层析、 高效液相层析（HPLC）和其它各种液 相层析技术及这些方法的结合。 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.
本发明的抗体可以单独使用，也可与可检测标记物（为诊断目的）、治疗剂、 PK（蛋 白激酶）修饰部分或任何以上这些物质的组合结合或偶联。 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.
用于诊断目的的可检测标记物包括但不限于： 荧光或发光标记物、 放射性标记 物、 MRI（磁共振成像）或 CT（电子计算机 X射线断层扫描技术）造影剂、 或能够产生可 检测产物的酶。 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.
可偶联的治疗剂包括但不限于： 胰岛素、 IL-2、 干扰素、 降钙素、 GHRH肽、 肠 肽类似物、 白蛋白、 抗体片段、 细胞因子、 和激素。 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.
此外还可与本发明抗体结合或偶联的治疗剂包括但不限于： 1. 放射性核素； 2 生物毒； 3. 细胞因子如 IL-2等； 4. 金纳米颗粒 /纳米棒； 5. 病毒颗粒； 6. 脂质体; 7. 纳米磁粒； 8.前药激活酶； 10.化疗剂（例如， 顺铂）或任何形式的纳米颗粒等。 In addition, therapeutic agents that can be combined or coupled with the antibody of the present invention include but are not limited to: 1. Radionuclide; 2 Biological toxicity; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. Virus particles; 6. Liposomes; 7. Nano magnetic particles; 8. Prodrug activating enzymes; 10. Chemotherapy agents (for example, cisplatin) or any form of nanoparticles, etc.
本发明还提供了一种组合物。 在优选例中， 所述的组合物是药物组合物， 它含 有上述的抗体或其活性片段或其融合蛋白， 以及药学上可接受的载体。 通常， 可将这
些物质配制于无毒的、 惰性的和药学上可接受的水性载体介质中， 其中 pH通常约为 5-8， 较佳地 pH约为 6-8， 尽管 pH值可随被配制物质的性质以及待治疗的病症而有 所变化。 配制好的药物组合物可以通过常规途径进行给药， 其中包括（但并不限于）： 口服、 呼吸道、 瘤内、 腹膜内、 静脉内、 或局部给药。 The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or active fragment or fusion protein thereof, and a pharmaceutically acceptable carrier. Usually, this 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.
本发明的药物组合物可直接用于结合呼吸道合胞病毒的融合蛋白（较佳地 pre-fusion F蛋白）分子， 因而可用于延长药物的半衰期， 此外， 还可同时使用其他治 疗剂。 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. In addition, other therapeutic agents can also be used at the same time.
本发明的药物组合物含有安全有效量（如 0.001-99wt%,较佳地 0.01-90wt%， 更佳 地 0.1 -80wt%）的本发明上述的单克隆抗体（或其偶联物）以及药学上可接受的载体或赋 形剂。 这类载体包括（但并不限于）： 盐水、 缓冲液、 葡萄糖、 水、 甘油、 乙醇、 及其 组合。 药物制剂应与给药方式相匹配。 本发明的药物组合物可以被制成针剂形式， 例 如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物 如针剂、 溶液宜在无菌条件下制造。 活性成分的给药量是治疗有效量， 例如每天约 1 微克 /千克体重-约 10毫克 /千克体重。 此外， 本发明的多肽还可与其他治疗剂一起使 用。 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. Such carriers 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. In addition, the polypeptide of the present invention can also be used with other therapeutic agents.
使用药物组合物时， 是将安全有效量的免疫偶联物施用于哺乳动物， 其中该安 全有效量通常至少约 10微克 /千克体重，而且在大多数情况下不超过约 8毫克 /千克体 重， 较佳地该剂量是约 10微克 /千克体重-约 1毫克 /千克体重。 当然， 具体剂量还应 考虑给药途径、 病人健康状况等因素， 这些都是熟练医师技能范围之内的。 检测用途和试剂盒 When using the pharmaceutical composition, 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. Of course, 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
本发明的抗体可用于检测应用， 例如用于检测样本， 从而提供诊断信息。 The antibodies of the present invention can be used in detection applications, for example, to detect samples, thereby providing diagnostic information.
本发明中， 所采用的样本（样品）包括细胞、 组织样本和活检标本。 本发明使用的术语 “活检”应包括本领域技术人员已知的所有种类的活检。因此本发明中使用的活检可以包括 例如通过内窥镜方法或器官的穿刺或针刺活检制备的组织样本。 In the present invention, 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.
本发明中使用的样本包括固定的或保存的细胞或组织样本。 The samples used in the present invention include fixed or preserved cell or tissue samples.
本发明还提供了一种指含有本发明的抗体（或其片段）的试剂盒， 在本发明的一个优选 例中， 所述的试剂盒还包括容器、 使用说明书、 缓冲剂等。 在优选例中， 本发明的抗体可 以固定于检测板。 本发明的主要优点 The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, buffer, and the like. In a preferred embodiment, the antibody of the present invention can be immobilized on a detection plate. Main advantages of the invention
（1）本发明全人单克隆抗体能够特异性识别并结合呼吸道合胞病毒的 pre-fusion F 蛋 白， 对呼吸道合胞病毒具有很高的中和活性， 并且可以结合中和 RSV A型和 B型病毒， 有效抑制或阻止呼吸道合胞病毒侵染易感细胞。 (1) 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.
（2）本发明全人单克隆抗体对 RSV A型和 B型病毒具有广谱结合活性和广谱中和活
性，能有效中和多种呼吸道合胞病毒，并且无论在细胞水平微中和实验还是小鼠预防实验， 均显著优于现在市售的抗体 (如 Palivizumab抗体)。 (2) 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.
(3)本发明是全人单克隆抗体 4F1，不含鼠源部分，对人体来说具有更低的免疫原性和 更高的安全性， 可以避免人抗鼠等其它物种来源的抗体介导免疫排斥反应。 (3) 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.
(4) 本发明全人单克隆抗体 4F1结合 RSV F蛋白融合前形式， 大量研究证实针对 F 蛋白的中和抗体识别位点主要在 pre-fusion F 蛋白上， 4F1抗体表位的发掘也为 RSV疫苗 的设计提供一些新的思路和参考。 下面结合具体实施例， 进一步阐述本发明。应理解， 这些实施例仅用于说明本发明而 不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件， 例如 Sambrook等人， 分子克隆： 实验室手册 (New York: Cold Spring Harbor Laboratory Press, 1989)中所述的条件， 或按照制造厂商所建议的条件。 除非另外说明， 否则百分比和 份数是重量百分比和重量份数。 (4) 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.
本发明实施例或测试例中未注明具体条件的实验，通常按常规条件进行，或按照原料 /商品制造商建议的条件； 未注明具体来源的试剂， 为市场购买的常规试剂。 Experiments without specifying specific conditions in the embodiments or test examples of the present invention are usually carried out under conventional conditions or according to the conditions recommended by the raw material/commodity manufacturer; reagents without specific sources are conventional reagents purchased on the market.
以下说明本发明能够中和呼吸道合胞病毒融合蛋白的中和性全人单克隆抗体制备以 及抗体特性分析过程。 实施例 1 单细胞 RT-PCR法获得抗体基因以及抗体表达 The preparation of the neutralizing fully human monoclonal antibody capable of neutralizing the respiratory syncytial virus fusion protein of the present invention and the antibody characteristic analysis process are described below. Example 1 Obtaining antibody gene and antibody expression by single cell RT-PCR method
1、 外周血单核细胞 (PBMC)的获得 1. Acquisition of peripheral blood mononuclear cells (PBMC)
从健康的志愿者体内抽取外周血， 采用常规 Ficoll-Paque (厂家为 Lympholyte®-H (CEDARLANE)公司)密度梯度离心， 得到 107以上个外周血单核细胞 (PBMC)。 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分离方法: Ficoll separation method:
(1)收集血液， 于 50ml 离心管 (预含 4%梓檬酸钠 1ml)， 收集全血 20ml， 颠倒混勾 8-10次。 (即使柠檬酸钠终浓度为 0.4%)； (1) Collect blood, in a 50ml centrifuge tube (pre-contained 4% sodium citrate 1ml), collect 20ml of whole blood, invert and mix 8-10 times. (Even if the final concentration of sodium citrate is 0.4%);
(2)加等体积 RPMI1640(含柠檬酸钠)， 混匀； (2) Add an equal volume of RPMI1640 (containing sodium citrate) and mix well;
(3)用 15ml透明离心管， 铺 3ml淋巴细胞分离液， 在其上小心加 6ml血样。 形成分 离界面 (或 4ml分离液加 8ml血样)； (3) Use a 15ml transparent centrifuge tube to spread 3ml lymphocyte separation solution, and carefully add 6ml blood sample on it. Form a separation interface (or 4ml separation solution plus 8ml blood sample);
(4)室温离心 800g， 20min(2000rpm， 20min)； (4) Centrifuge at 800g at room temperature, 20min (2000rpm, 20min);
(5)小心吸取界面层细胞， 转移至新管； (5) Carefully aspirate the cells in the interface layer and transfer to a new tube;
(6)加 RPMI1640(含柠檬酸钠)， 稀释减小液体密度。 离心， 800g/2000rpm， 10min。 去上清； (6) Add RPMI1640 (containing sodium citrate) to dilute to reduce liquid density. Centrifuge, 800g/2000rpm, 10min. Go to supernatant
(7)RPMI 1640洗细胞 2-3次， 备用 (7) Wash cells with RPMI 1640 2-3 times, spare
2、 RSV 融合蛋白 F特异性记忆 B细胞的获得 2. The acquisition of RSV fusion protein F specific memory B cells
使用 FITC-CD19/APC-IgG/BV421 和 PE -RSV F 蛋白为标志物， BD Horizonä
Fixable Viability Stain 780 去掉死细胞， 经流式细胞仪获得特异性 B 细胞至 96 孔 RT-PCR板， 每孔一个细胞， 获得 F蛋白特异性记忆 B细胞。 Using FITC-CD19/APC-IgG/BV421 and PE-RSV F protein as markers, 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.
（1） RSV A2 pre-fusion F 蛋白由哺乳动物细胞 CHO表达系统表达； 参考 Invitrogen ExpiCHO-Sä Expression System 手册； A2 F 蛋白序列参考 UniProtKB/Swiss-Prot: P03420.1， RSV pre-fusion F蛋白的设计根据 Jason S. McLellan. Science 2013采用的策略进 行设计， 在上海捷瑞公司进行全基因合成， 构建到 invitrogen pcDNA3.1的表达载体上。 (1) 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.
（2） RSV A2 pre-fusion F蛋白进行生物素（Biotin）标记： No-Weigh Sulfo-NHS-LC-Biotin （购自 PIERCE, 参考 PIERCE公司 EZ-Link Sulfo-NHS-LC-Biotin生物素标记 Protocol） 10mM试剂； 另两个标志物 FITC-CD19 和 APC-IgG均购自 BD Bioscience 公司； SA Streptavidin PE和 streptavidin BV 421（贝勾自 BD公司） 用来检测生物素标记的 F蛋白； (2) Biotin labeling of RSV A2 pre-fusion F protein: No-Weigh Sulfo-NHS-LC-Biotin (purchased from PIERCE, refer to PIERCE company EZ-Link Sulfo-NHS-LC-Biotin Biotin labeling Protocol ) 10mM reagent; the other two markers, FITC-CD19 and APC-IgG, were purchased from BD Bioscience; SA Streptavidin PE and streptavidin BV 421 (from BD) were used to detect biotin-labeled F protein;
（3）分选细胞的标记： PBMC细胞分组， 实验组 +对照组， 按细胞数加入标志物， 避光 染色， 进行标记， 用 PBS重悬后， 使用 40 |im BD falcon滤膜过滤； (3) Marking of sorted cells: 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 |im BD falcon filter;
（4）特异性 B细胞的分选 :使用 BD FACS Influx筛选，根据前向角和侧向角从 PBMC 中筛选到淋巴细胞， 然后通过不同对照组的调节补偿， 获得 RSV F蛋白的特异性记忆 B 细胞，将其分选到 96孔板中进行 RT-PCR（反转录 PCR）,每孔一个细胞，板置于干冰上。 (4) 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.
3、 抗体基因获得及载体构建 3. Antibody gene acquisition and vector construction
将获得的单个记忆 B细胞通过 RT-PCR获得 cDNA,然后通过 nested-PCR获得抗 体基因可变区， 跑琼脂糖核酸凝胶， 将重轻链可以配对的胶块回收并测序。 通过 IgBLAST网站（https://www.ncbi. nlm.nih.gov/projects/igblast/）检索获得抗体基因序列。 接下来通过 Agel及 Sail酶切位点， Agel及 BsiwI酶切位点， Agel及 Xhol酶切位点 将抗体基因分别连接到相应的 IgH， IgK和 IgX表达载体上。 全人抗体表达载体 IgH， IgK和 Ig 分别表达抗体 heavy链、 kappa链、 lambda链）由 Patrick Wilson实验室馈 赠， 载体序列见 NCBI GenBank:FJ475055、 FJ475056和 FJ517647。 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. Through the IgBLAST website (https://www.ncbi.nlm.nih.gov/projects/igblast/) search to obtain the antibody gene sequence. Next, 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. For the vector sequence, see NCBI GenBank: FJ475055, FJ475056 and FJ517647.
4、 抗体表达和纯化 4. Antibody expression and purification
瞬时转染 CHO 细胞， 进行全人抗体表达。 转染前一天 （第 -1 天）， 分种 ExpiCHO-Sä 细胞， 最终密度为 3 x io6-4 x 106 个活细胞 /mL， 使细胞过夜生长。 次 日 （第 0 天）， 测定活细胞密度和存活率百分比。 细胞密度应达到约 7 x io6-10 x io6 个活细胞 /mL。 存活率应为 95-99%， 方可继续转染。 将细胞稀释至最终密度为 6 x lO6 个活细胞 /mL。 使用预冷的试剂 （4°C） 配制 Expi Fectamineä CHO/质粒 DNA 复合物。 室温孵育 Expi Fectamineä CHO/质粒 DNA 复合物 1-5 分钟， 然后将溶液 慢慢转移至 CHO细胞培养瓶中， 在添加过程中轻轻晃动培养瓶。 将细胞置于轨道摇 床上 （37°C 培养箱含 8% C02 的湿化空气条件下） 培养。 培养 7-1 1天， 待细胞死亡 一半时即可收上清， 开始纯化抗体。 Transfect CHO cells transiently to express fully human antibodies. One day before transfection (day -1), separate ExpiCHO-Sä cells to a final density of 3 x io 6 -4 x 10 6 viable cells/mL, and allow the cells to grow overnight. On the next day (day 0), the viable cell density and survival rate percentage were determined. The cell density should reach about 7 x io 6 -10 x io 6 viable cells/mL. The survival rate should be 95-99% before transfection can continue. Dilute the cells to a final density of 6 x 10 6 viable cells/mL. Prepare the Expi Fectamineä CHO/plasmid DNA complex using pre-chilled reagents (4°C). Incubate the Expi Fectamineä CHO/plasmid DNA complex at room temperature for 1-5 minutes, and then slowly transfer the solution to the CHO cell culture flask, shaking the flask gently during the addition. Place the cells on an orbital shaker (37°C incubator with 8% CO 2 in humidified air) for cultivation. After culturing for 7-1 for 1 day, the supernatant can be collected when the cells are half dead, and the antibody can be purified.
使用 Protein G Agarose 4FF填料 （购自 GE）纯化抗体。 首先将收集的 CHO细胞 悬液 4000rpm,4°C离心 30min， 将收集的上清再用 0.45um filter过滤， 待纯化。 取重
力型离心柱，加入 Protein G Agarose 4FF填料，使用 3倍柱体积的 20%乙醇稳定填料， 之后用 5倍柱体积的结合缓冲液平衡柱子， 然后上样品， 再用 10倍柱体积的结合缓 冲液平衡柱子， 最后用 3倍柱体积的洗脱缓冲液洗脱柱子， 向洗脱下来的抗体溶液中 加入中和 buffer使洗脱下来的样品 pH达 7.5左右。 将抗体溶液在 5L 1 *PBS中透析 3 次， 即可将抗体浓缩保存至 -80°C。 实验结果： 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:
结果如图 1所示， 使用 FITC-CD19/APC-IgG/BV421和 PE双阳性标记为特异性 标志物，获得了若干个 RSV-F蛋白的特异性 B细胞，约占总记忆 B细胞数量的 0.3% o 单细胞 RT-PCR和 Nested-PCR法获得匹配抗体重轻链可变区基因， 分子量为 400bp 左右， 电泳图谱见图 2。 琼脂糖割胶回收并进行测序。 将测序结果在 http://www.ncbi.nlm.nih.gov/igblast 及 http://www.imgt.org/ 进行比对,获得抗体胚系 基因信息和抗体重轻链基因高可变区信息， 并进行表达载体构建及后续表达纯化。 最 后， 通过该技术， 成功获得一株广谱性中和 RSV A型和 B型病毒的全人单克隆抗体， 命名为 4F1。 The results are shown in Figure 1. Using FITC-CD19/APC-IgG/BV421 and PE double positive markers as specific markers, several RSV-F protein-specific B cells were obtained, which accounted for about the total number of memory B cells 0.3% o Single-cell RT-PCR and Nested-PCR methods to obtain matching antibody heavy and light chain variable region genes, the molecular weight is about 400bp, the electrophoresis pattern is shown in Figure 2. Agarose gum tapping is recovered and sequenced. Compare the sequencing results at http://www.ncbi.nlm.nih.gov/igblast and http://www.imgt.org/ to obtain antibody germline gene information and antibody heavy and light chain gene high variable regions Information, and carry out expression vector construction and subsequent expression purification. Finally, through this technology, a broad-spectrum fully human monoclonal antibody that neutralizes RSV A and B viruses was successfully obtained, named 4F1.
全人抗体 4F1重链可变区基因序列如下， 其中使用下划线标注的为重链基因可变区 中的高变区序列， 依次为重链基因 CDR1， CDR2和 CDR3序列。 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.
GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCGGCCTGGGAGGTCCC TGAGACTCTCCTGTGCAGCCTCTGGATTCAGCTTCTATTCCTATTCTGTGCACTGGG TCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGATGTTGTATATGATGG AAATCATCAACATTACACGGAGTCCGTGAGGGGCCGATTCTCCATCTCCAGAGACA CCTCCACCAATACGGTGTATCTGCAAATGGGCAGCCTGAGGCCTGAAGACACGGCT CTTTATTACTGTACGGCCCGCAGTTTGGTCATAACGCTCGCGGGGGCGGGTCGAGA TGACTATTGGGGCCAGGGAACTCGGGTCACCGTCTCCTCA (SEQ ID NO : 8) GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCGGCCTGGGAGGTCCC TGAGACTCTCCTGTGCAGCCTCTGGATTCAGCTTCTATTCCTATTCTGTGCACTGGG TCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGATGTTGTATATGATGG AAATCATCAACATTACACGGAGTCCGTGAGGGGCCGATTCTCCATCTCCAGAGACA CCTCCACCAATACGGTGTATCTGCAAATGGGCAGCCTGAGGCCTGAAGACACGGCT CTTTATTACTGTACGGCCCGCAGTTTGGTCATAACGCTCGCGGGGGCGGGTCGAGA TGACTATTGGGGCCAGGGAACTCGGGTCACCGTCTCCTCA (SEQ ID NO: 8)
全人抗体 4F1重链可变区氨基酸序列如下， 其中使用下划线标注的依次为重链氨基 酸 CDR1， CDR2， CDR3序列。 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.
EVOLVOSGGGVVRPGRSLRLSCAASGFSFYSYSVHWVROAPGKGLEWVADVVY DGNHOHYTESVRGRFSISRDTSTNTVYLOMGSLRPEDTALYYCTARSLVITLAGAGRD DYWGOGTRVTVSS (SEQ ID NO : 1) EVOLVOSGGGVVRPGRSLRLSCAASGFSFYSYSVHWVROAPGKGLEWVADVVY DGNHOHYTESVRGRFSISRDTSTNTVYLOMGSLRPEDTALYYCTARSLVITLAGAGRD DYWGOGTRVTVSS (SEQ ID NO: 1)
全人抗体 4F1轻链可变区基因序列如下， 其中下划线标注的为轻链基因可变区中的 高变区序列， 依次为 CDR1'， CDR2'和 CDR3'序列。 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.
GATATTGTGATGACTCAGTCTCCACTCTCCCTGTCCGTCACCCCTGGAGAGCC GATATTGTGATGACTCAGTCTCCACTCTCCCTGTCCGTCACCCCTGGAGAGCC
GGCCTCCATCTCCTGCAAGTCTAGTCAGAGCCTCCTCCATAGTAATGGATACACTTAGGCCTCCATCTCCTGCAAGTCTAGTCAGAGCCTCCTCCATAGTAATGGATACACTTA
TTTGGATTGGTACCTGCAGAAGCCAGGGAAGTCTCCACAACTCCTGATCTTTTTGGTTTGGATTGGTACCTGCAGAAGCCAGGGAAGTCTCCACAACTCCTGATCTTTTTGG
GTTCTAGTCGGGCCTCCGGGGTCCCTGCCAGGTTCAGTGGCAGTGGATCAGGCACA
GATTTTACACTGGAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTACTACTG CGTGCAAGATCTACAAACTTCCCTCACTTTCGGCGGAGGGACCAAAGTGGATATCA AA (SEQ ID NO : 9) GTTCTAGTCGGGCCTCCGGGGTCCCTGCCAGGTTCAGTGGCAGTGGATCAGGCACA GATTTTACACTGGAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTACTACTG CGTGCAAGATCTACAAACTTCCCTCACTTTCGGCGGAGGGACCAAAGTGGATATCA AA (SEQ ID NO: 9)
全人抗体 4F1轻链可变区氨基酸序列如下， 其中使用下划线标注的依次为轻链氨基 酸 CDR1'， CDR2'和 CDR3'序列。 The 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.
DIVMTOSPLSLSVTPGEPASISCKSSOSLLHSNGYTYLDWYLOKPGKSPOLLIFLGS SRASGVPARFSGSGSGTDFTLEISRVEAEDVGVYYCVODLOTSLTFGGGTKVDIK (SEQ ID NO.: 2) 实施例 2抗体特性分析 DIVMTOSPLSLSVTPGEPASISCKSSOSLLHSNGYTYLDWYLOKPGKSPOLLIFLGS SRASGVPARFSGSGSGTDFTLEISRVEAEDVGVYYCVODLOTSLTFGGGTKVDIK (SEQ ID NO.: 2) Example 2 Analysis of antibody characteristics
1、 ELISA检测抗体结合抗原的活性 1. ELISA detects the activity of antibody binding antigen
为了研究 4F1 抗体结合 RSV A型和 B型病毒 F蛋白的能力， 使用 ELISA检测表达 的抗体是否识别 RSV A2和 B9320 pre-fusion F蛋白及 A2 post-fusion F蛋白。 A2 F蛋白序 列参考 UniProtKB/Swiss-Prot: P03420.1,B9320序列参考 UniProtKB/Swiss-Prot: Q6V2E7, RSV pre-fusion F蛋白的设计根据 Jason S. McLellan. Science 2013采用的策略进行设计， RSV post-fusion F蛋白的设计根据 Davide Corti. Nature 2013 采用的策略进行设计， 在上 海捷瑞公司进行全基因合成， 构建到 invitrogen pcDNA3.1的表达载体上。 由哺乳动物细 胞 CHO表达系统表达； 参考 Invitrogen ExpiCHO-S™ Expression System手册。 帕利珠单 抗 (Palivizumab， SYNAGIS®)为阳性对照抗体， 购自雅培公司。 包被 F蛋白 ELISA板， 0. 5 /mL，每孔 100 pL， 4。(:过夜。次日 PBST洗板 3 次。 2% BSA封闭，每孔 200 pL， 37 °C, 2 ho 再次 PBST洗板 3 次。 4F1及对照抗体测试 12个浓度， 3倍梯度稀释， 2复 孔， 起始测试浓度为 30 /ml。 上样， 每孔 100 ， 37 °C， 2 h。 PBST洗板 3 次。 羊 抗人 IgG (Goat Anti-Human IgG) (Fc specific)- Peroxidase antibody (sigma)， 1 :5000 稀释， 每孔 100 ， 37 °C， 1 ho PBST 洗板 3 次。 加底物 TMB 100 ^L /孔显色， 若颜色较浅 可 37°C避光反应 15 min。 加入 2M H2S04 终止反应， 每孔 50 。 测定 OD45o, 并进行 数据处理。 帕利珠单抗 (Palivizumab)简称 PVZ 用来作为阳性对照抗体， 抗无关病毒的全 人抗体作为阴性同型对照抗体 (NC)。 In order to study the ability of 4F1 antibody to bind RSV A and B virus F protein, 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-S™ 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. 4F1 and control antibody test 12 concentrations, 3-fold gradient dilution, 2 replicate wells, the initial test concentration is 30/ml. Load the sample, 100 per well, 37 ° C, 2 h. Wash the plate 3 times with PBST. Goat Anti-Human IgG (Fc specific)- Peroxidase Antibody (sigma), diluted 1:5000, 100 per well, 37 ° C, 1 h o PBST wash 3 times. Add substrate TMB 100 ^L/well to develop color, if the color is light, 37 ° C to avoid light 15 min. Add 2M H 2 S0 4 to stop the reaction, 50 per well. Measure OD 45 o, and perform data processing. Palivizumab (Palivizumab) referred to as PVZ is used as a positive control antibody, a fully human antibody against unrelated viruses As a negative isotype control antibody (NC).
结果如图 3所示， 4F1抗体可以广谱性的结合 RSV A型和 B型病毒的 pre-fusion F蛋 白， 与阳性对照抗体结合能力相当。 4F1不结合 A型 post-fusion F 蛋白， 帕利珠单抗被 报道能结合两种形式的 F蛋白。结果表明， 4F1抗体能广谱结合 RSV A型和 B型 pre-fusion F 形式， 并且 4F1抗体和帕利珠单抗结合 F蛋白上两种不同的表位。 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.
2、 病毒 TCID5Q测定 2. Virus TCID 5Q determination
使用 TCID5o实验核实 RSV微中和实验中稀释病毒液的滴度。将 200 |il稀释病毒 液加入 96孔细胞板 B2-D2孔内， 其它孔内加入 100 |il细胞培养液。从 B2-D2孔内吸 取 100 |il病毒液加入 B3-D3孔内， 混匀后并依次做 2倍梯度稀释， 病毒共稀释 18个
浓度点， 3复孔。 随后测试板于 37 °C、 5% C02培养箱中孵育 2个小时。 HEp2细胞 以每孔 25,000个细胞的密度接种到测试板中并于 37 °C、 5% C02培养箱中培养 5天。 Use the TCID 5 o experiment to verify the titer of the diluted virus solution in the RSV microneutralization experiment. Add 200 |i l of the diluted virus solution to wells B2-D2 of the 96-well cell plate, and add 100 |i l of cell culture solution to other wells. Pipette 100 |i l of virus liquid from well B2-D2 into well B3-D3, mix well and perform 2-fold serial dilutions in sequence, and dilute 18 viruses in total Concentration point, 3 replicate holes. The test plate was then 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.
培养 5天后， 弃去上清， 用 80%丙酮固定测试板中细胞。 使用 ELISA检测细胞 内的病毒含量。 原始数据用于病毒 TCID5Q计算（Karber法）。 计算公式如下： After culturing for 5 days, discard the supernatant and fix the cells in the test plate with 80% acetone. ELISA was used to detect the virus content in the cells. The original data is used for virus TCID 5Q calculation (Karber method). Calculated as follows:
TCID5o/fL= Antilog 10 U阳性孔数量 / 3） - 0.5} x 0.3 ]/2 TCID 5 o/fL= Number of Antilog 10 U positive wells/3)-0.5} x 0.3 ]/2
阳性孔判定标准: 测试孔读值 ＞（培养液对照平均值 + 3 培养液对照 SD值） Criteria for positive wells: Reading value of test wells> (average value of culture medium control + 3 medium control SD value)
QC标准： 稀释病毒液的滴度应为 50 - 2000 TCIDW孔。 QC standard: The titer of the diluted virus solution should be 50-2000 TCIDW wells.
3、 病毒微中和实验 3. Virus micro-neutralization experiment
为了研究 4F1抗体中和 RSV 病毒的能力及广谱性， 将梯度稀释的 4F1抗体与不 同的病毒孵育， 通过微中和实验检测 4F1 抗体中和病毒的能力。 测试病毒株 A2 和 B9320（购自 ATCC）分别用细胞培养液稀释到 4000 TCIDWml。倍比稀释的抗体和 200 TCIDW孔的病毒以等体积比加入 96孔细胞培养板内， 混匀后于 37 °C、 5% C02培养 箱中孵育 2个小时。随后 HEp2细胞以每孔 25,000个细胞的密度接种到测试板中并于 37 °C、 5% C02培养箱中培养 5天。 抗体均测试 9个浓度， 3倍梯度稀释， 3复孔， 起始测试浓度为 4000 ng/ml o In order to study the ability of 4F1 antibody to neutralize RSV virus and its broad spectrum, the 4F1 antibody was incubated with different viruses, and the ability of 4F1 antibody to neutralize the virus was tested by a micro-neutralization experiment. Test virus strains A2 and B9320 (purchased from ATCC) were diluted with cell culture medium to 4000 TCIDWml. 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. Subsequently, 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
培养 5天后， 弃去上清， 用 80%丙酮固定测试板中细胞。 使用 ELISA检测细胞 内的病毒含量。 原始数据用于抗体在不同浓度下的中和活性计算。 计算公式如下： 活性百分比（％） =（测试孔读值 - 病毒对照平均值）/（细胞对照平均值 - 病毒对照 平均值）乂 100 After culturing for 5 days, discard the supernatant and fix the cells in the test plate with 80% acetone. Use ELISA to detect the virus content in the cells. The raw data is used to calculate the neutralizing activity of the antibody at different concentrations. The calculation formula is as follows: Activity percentage (%) = (test hole reading value-virus control average value) / (cell control average value-virus control average value) 乂 100
EC5Q值通过 Prism软件计算， 中和活性曲线拟合方法为 sigmoidal dose-response （variable slope）。 The EC5 Q value is calculated by Prism software, and the neutralization activity curve fitting method is sigmoidal dose-response (variable slope).
结果如图 4和表 1所示。 4F 1抗体和对照抗体帕利珠单抗 Palivizumab对 RSV病 毒株的抗体中和活性剂量拟合曲线见图 4。 帕利珠单抗对 RSV A2和 B9320均显示出 中和活性， 其 IC5Q值分别为 384.3 ng/ml和 367.2 ng/ml。 4F 1对 RSV A2和 B9320的 ICso值分别为 4.368 ng/ml和 23.51 ng/ml。 4F 1抗体对 RSV A2和 B9320的中和活性 均优于帕利珠单抗， IC5Q低近 80-100倍。 阴性抗体 NC在测试浓度内没有显示出对测 试病毒株的中和活性， 其 ICso值大于最高检测浓度 4000 ng/ml（见表 1）。 The results are shown in Figure 4 and Table 1. 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).
_ 表 1 抗体 ICso值 _ _ Table 1 Antibody ICso value _
抗体 ECso值（ng/ml） RSV A2 RSV B9320 Antibody ECso value (ng/ml) RSV A2 RSV B9320
帕利珠单抗 Palivizumab 384.3 367.2 Palivizumab Palivizumab 384.3 367.2
4F1抗体 4.368 23.51 NC抗体 >4000 >4000
4、 4F1抗体在动物预防效果检测 4F1 antibody 4.368 23.51 NC antibody>4000 >4000 4. Detection of 4F1 antibody preventive effect in animals
本实施例检测了本发明的抗体 4F1和 Palivizumab预防小鼠感染 RSV的能力。 6-8 周龄的 BalB/c雌鼠提前放置到生物安全二级实验室动物实验房。 dayO天小鼠分别腹 腔注射 15mg/kg， 3 mg/kg, 0.6mg/kg， 0.12mg/kg的 4F1抗体、 Palivizumab及 PBS。 24h后， 用乙醚麻醉小鼠， 对小鼠经鼻腔攻击 RSV A2 病毒（107 PFU/50ul/小鼠）。 五 天后处死动物采集肺。 从每只小鼠去除左肺并且用 4%的多聚甲醛固定， 用石蜡包埋 并用常规苏木素 -伊红（HE）染色， 光镜观察肺部病理损伤和炎性细胞浸润情况。 取每 只小鼠右边肺组织进行组织匀浆，用 EZ-press RNA Purification Kit 进行总 RNA抽提， 然后反转成 cDNA, 用实时定量 RT-PCR的方法对 RSV的基因组进行相对定量，比对 编码 RSV的 NP蛋白的基因序列， 选取保守片段进行引物设计， 小鼠的 P actin作为 内参基因 （见表 2）。 参考 Toyobo KOD SYBR®qPCR Mix 使用手册。 This example tested the ability of the antibodies 4F1 and Palivizumab of the present invention to prevent RSV infection in 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. Take the right lung tissue of each mouse for tissue homogenization, use EZ-press RNA Purification Kit for total RNA extraction, and then invert it into cDNA, use real-time quantitative RT-PCR method for relative quantification and comparison of RSV genome For the gene sequence encoding the NP protein of RSV, 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.
表 2 实时定量 RT-PCR 引物 Table 2 Real-time quantitative RT-PCR primers
结果如图 5所示， 与 PBS组相比， 不同剂量的 4F1和帕利珠单抗均能有效地降 低肺部病毒的 RNA水平。 相比帕利珠单抗， 4F1抗体对肺部病毒载量的降低更为明 显。 0.12mg/kg 给药剂量的 4F1 比 15mg/kg给药剂量的帕利珠单抗对 RSV病毒的抑 制效果明显。 The results are shown in Figure 5. Compared with the PBS group, different doses of 4F1 and Palivizumab can effectively reduce the level of lung virus RNA. Compared with palivizumab, 4F1 antibody reduces the viral load of the lungs more significantly. The inhibitory effect of 4F1 at a dose of 0.12 mg/kg on the RSV virus was greater than that of palivizumab at a dose of 15 mg/kg.
结果如图 6所示。 相对于 PBS对照组， 肺组织病理学的结果显示 4F1抗体在高 剂量和低剂量组能显著地降低小鼠肺部的病理损伤情况，包括减少肺间质和支气管周 围炎性细胞浸润情况， 维持肺泡的正常形态结构， 减少出血点等指标。 而帕利珠单抗 只有在高剂量组 15mg/kg起到较明显的保护作用。 在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献被单独引 用作为参考那样。此外应理解， 在阅读了本发明的上述讲授内容之后， 本领域技术人员可 以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范 围。
The result is shown in Figure 6. Compared with the PBS control group, the results of lung histopathology show that the 4F1 antibody in the high-dose and low-dose groups can significantly reduce the pathological damage of the lungs of mice, including reducing the lung interstitium and peribronchial inflammatory cell infiltration, and maintaining The normal shape and structure of the alveoli reduce bleeding points and other indicators. However, palivizumab only had a significant protective effect in the high-dose group at 15 mg/kg. All documents mentioned in the present invention are cited as references in this application, just as each document is individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.
Claims
1. 一种抗体的重链可变区， 其特征在于， 所述的重链可变区包括以下三个互 补决定区 CDR： 1. A heavy chain variable region of an antibody, characterized in that the heavy chain variable region includes the following three complementary determining region CDRs:
SEQ ID NO.: 3所示的 CDR1， CDR1 shown in SEQ ID NO.: 3,
SEQ ID NO.: 4所示的CDR2， 和 CDR2 shown in SEQ ID NO.: 4, and
SEQ ID NO.: 5所示的 CDR3。 SEQ ID NO.: CDR3 shown in 5.
2. —种抗体的重链， 其特征在于， 所述的重链具有如权利要求 1所述的重链 可变区。 2. A heavy chain of an antibody, wherein the heavy chain has the heavy chain variable region of claim 1.
3. 一种抗体的轻链可变区， 其特征在于， 所述的轻链可变区包括以下三个互 补决定区 CDR： 3. A light chain variable region of an antibody, characterized in that the light chain variable region includes the following three complementary determining region CDRs:
SEQ ID NO.: 6所示的 CDR1，， CDR1 shown in SEQ ID NO.: 6,
氨基酸序列为 LGS的 CDR2’， 和 The amino acid sequence is CDR2’ of LGS, and
SEQ ID NO.: 7所示的 CDR3，。 SEQ ID NO.: CDR3 shown in 7.
4. 一种抗体的轻链， 其特征在于， 所述的轻链具有如权利要求 3所述的轻链 可变区。 4. A light chain of an antibody, characterized in that the light chain has the light chain variable region of claim 3.
5. 一种抗体， 其特征在于， 所述抗体具有： 5. An antibody, characterized in that the antibody has:
(1) 如权利要求 1所述的重链可变区； 和 /或 (1) The heavy chain variable region of claim 1; and/or
(2) 如权利要求 3所述的轻链可变区； (2) The light chain variable region of claim 3;
或者， 所述抗体具有： 如权利要求 2所述的重链； 和 /或如权利要求 4所述的 轻链。 Alternatively, the antibody has: the heavy chain according to claim 2; and/or the light chain according to claim 4.
6. 如权利要求 5所述的抗体，其特征在于，所述抗体的重链可变区序列如 SEQ ID NO.: 1所示； 并且所述的抗体的轻链可变区序列如 SEQ ID NO.: 2所示。 6. The antibody of claim 5, wherein 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.: As shown in 2.
7. —种重组蛋白， 其特征在于， 所述的重组蛋白具有： 7. A recombinant protein, characterized in that the recombinant protein has:
(i) 如权利要求 1所述的重链可变区、 如权利要求 2所述的重链、 如权利要求 3所述的轻链可变区、 如权利要求 4所述的轻链、 或如权利要求 5所述的抗体； 以 及 (i) The heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, or The antibody of claim 5; and
(ii) 任选的协助表达和 /或纯化的标签序列。 (ii) Optional tag sequence to assist expression and/or purification.
8. 一种 CAR构建物，其特征在于，所述的 CAR构建物的抗原结合区域的 scFv 段为特异性结合于 RSV融合蛋白的结合区，并且所述 scFv具有如权利要求 1所述 的重链可变区和如权利要求 3所述的轻链可变区。
8. A CAR construct, characterized in that, the scFv segment of the antigen binding region of the CAR construct is a binding region that specifically binds to the RSV fusion protein, and the scFv has the weight as claimed in claim 1. Chain variable region and the light chain variable region of claim 3.
9.一种抗体药物偶联物， 其特征在于， 所述的抗体药物偶联物含有： 9. An antibody drug conjugate, characterized in that the antibody drug conjugate contains:
(a) 抗体部分， 所述抗体部分选自下组： 如权利要求 1所述的重链可变区、 如 权利要求 2所述的重链、如权利要求 3所述的轻链可变区、如权利要求 4所述的轻 链、 或如权利要求 5所述的抗体、 或其组合； 和 (a) An antibody part, which is selected from the group consisting of the heavy chain variable region according to claim 1, the heavy chain according to claim 2, and the light chain variable region according to claim 3 , The light chain of claim 4, or the antibody of claim 5, or a combination thereof; and
(b) 与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、 药物、 毒素、 细胞因子、 放射性核素、 酶、 或其组合。 (b) A coupling part coupled to the antibody part, the coupling part being selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.
10. —种活性成分的用途， 所述活性成分选自下组： 如权利要求 1所述的重链 可变区、如权利要求 2所述的重链、如权利要求 3所述的轻链可变区、如权利要求 4所述的轻链、 或如权利要求 5所述的抗体、 如权利要求 7所述的重组蛋白、 或其 组合， 其特征在于， 所述活性成分用于制备药剂、 试剂、 检测板或试剂盒。
10. Use of an active ingredient, the active ingredient selected from the group consisting of: the variable region of the heavy chain as claimed in claim 1, the heavy chain as claimed in claim 2, and the light chain as claimed in claim 3 The variable region, the light chain as claimed in claim 4, or the antibody as claimed in claim 5, the recombinant protein as claimed in claim 7, or a combination thereof, characterized in that the active ingredient is used for preparing a medicament , Reagents, test panels or kits.
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| CN118725053A (en) * | 2024-09-03 | 2024-10-01 | 北京民海生物科技有限公司 | F protein mutant before fusion of respiratory syncytial virus and application thereof |
| EP4353744A4 (en) * | 2021-06-11 | 2024-10-30 | Inst Microbiology Cas | Antibody against respiratory syncytial virus and use thereof |
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| CN114644708B (en) * | 2020-12-18 | 2023-06-27 | 珠海泰诺麦博生物技术有限公司 | Respiratory syncytial virus specific binding molecules |
| CN115960214A (en) * | 2021-10-12 | 2023-04-14 | 中国科学院分子细胞科学卓越创新中心 | Design and application of fully human antibody for neutralizing respiratory syncytial virus |
| CN116514960A (en) * | 2022-01-30 | 2023-08-01 | 中国科学院微生物研究所 | Fully human monoclonal antibody of respiratory syncytial virus and application thereof |
| CN115960216B (en) * | 2022-09-27 | 2023-07-07 | 深圳重链生物科技有限公司 | Anti-respiratory syncytial virus antibodies and related uses thereof |
| WO2024120516A1 (en) * | 2022-12-08 | 2024-06-13 | 南京诺唯赞生物科技股份有限公司 | Antibodies specifically binding to rsv |
| CN117720650B (en) * | 2024-02-04 | 2024-07-02 | 北京百普赛斯生物科技股份有限公司 | Anti-human respiratory syncytial virus antibody and application thereof |
| CN118005783B (en) * | 2024-04-08 | 2024-06-11 | 南京医科大学 | Anti-respiratory syncytial virus antibody and application thereof |
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