Neutralizing antibodies against respiratory syncytial virus and uses thereof

Abstract

The present invention discloses an antibody against respiratory syncytial virus (RSV), a nucleic acid molecule encoding the antibody, an expression vector containing the nucleic acid molecule, a host cell containing the nucleic acid molecule or the vector, and a pharmaceutical composition containing the antibody. The present invention further provides a method for producing and purifying the antibody, and the use of the antibody.

Description

Cross - reference to related applications

[0001] This application claims the priority of Chinese Patent Application No. 202210871810.9 filed on July 22, 2022, and all of its contents are incorporated herein by reference in their entirety.

Technical Field

[0002] The present invention generally relates to the fields of genetic engineering and antibody pharmaceuticals, and specifically relates to antibodies against respiratory syncytial virus (RSV) and the use of said antibodies in the prevention or treatment of respiratory syncytial virus (RSV) - related diseases.

Background Art

[0003] Large - volume and high - quality antibody libraries provide an important molecular source for screening high - affinity antibodies against any antigen and have high commercial and applied value. Currently, many of the high - quality antibody libraries reported at home and abroad in China are constructed by electrotransformation technology. However, the antibody libraries constructed using electrotransformation technology are limited in size by the transformation efficiency, require the production of multiple batches of antibody libraries, take a long time, and require the consumption of a large amount of human and material resources.

[0004] Respiratory syncytial virus (RSV) is the most important viral pathogen that causes acute lower respiratory tract infections (ALRTI) in children under 5 years old worldwide. 1 RS virus infection is the main cause of hospitalization due to viral airway infections in infants and young children, and seriously endangers the health of children, especially premature infants, infants and young children suffering from congenital heart disease or primary immunodeficiency diseases. The five countries with the highest incidence of RSV infection are Pakistan, India, Nigeria, China, and Indonesia, and these countries account for nearly half of the world's RSV - ALRTI diseases. 2 。

[0005] Respiratory syncytial virus (RSV) belongs to the family Pneumoviridae, genus Orthopneumovirus, and is a non-segmented single-stranded negative-sense RNA virus 3 and can be classified into two subtypes, A and B, based on differences in surface antigens 4 The RSV genome contains 10 genes encoding 11 proteins. Among them, the attachment protein G and the fusion protein F are the main protective antigens on the virus surface and can stimulate the body to produce neutralizing antibodies 5 Since the G protein has significant differences between subtypes, antibodies against the G protein are mostly subtype-specific antibodies 6 On the other hand, the F protein is highly conserved between subtypes, and antibodies induced by the F protein can simultaneously inhibit infections of both A / B type RSV viruses

[0006] The F protein is a type I transmembrane protein, and its inactive precursor (F0) is composed of 574 amino acids 7 When three F0s form a trimer and are transported through the Golgi apparatus, host furin cleaves between amino acids 109 and 110, and between amino acids 136 and 137 of F0. After cleavage, a short peptide (P27) of 27 amino acids in the middle is released, and the remaining two segments, F2 and F1, form an active F protein via disulfide bonds (Cys69-Cys212 and Cys37-Cys439) 8 There is a highly hydrophobic fusion peptide (FP: Fusion peptide) at the N-terminus of the F1 protein, which is located in the hydrophobic cavity of the protein and is protected from the influence of the external hydrophilic environment. When the F protein appears on the virion surface or the cell surface, its structure is not stable and is in a high-energy metastable pre-fusion conformation (Pre-F: Prefusion glyprotein) 9Subsequently, a series of dramatic structural changes occur at the N-terminus of the F1 protein, inducing membrane fusion and achieving the goal of viral infection of cells. Also, through this process, the F protein is converted from a high-energy metastable Pre-F structure to a stable post-fusion F protein structure (Post-F: Postfusion glyprotein).

[0007] Currently, there is no vaccine to prevent RSV. The US Food and Drug Administration (FDA) has approved palivizumab (trade name: Synagis) to prevent RSV infection in high-risk infants. This requires up to 5 injections to cover a typical RSV season. Due to the high cost, it cannot be widely used in infants.

[0008] Based on clinical needs, the development of anti-respiratory syncytial virus antibodies is of great medical importance in preventing related diseases caused by RSV infection.

Summary of the Invention

[0009] According to a first aspect, the present invention provides an antibody against respiratory syncytial virus (RSV) comprising a heavy chain variable region containing the amino acid sequences of HCDR1, HCDR2, and HCDR3, and a light chain variable region containing the amino acid sequences of LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of the HCDR1 is represented by SEQ ID NO: 32, the amino acid sequence of the HCDR2 is represented by SEQ ID NO: 33, the amino acid sequence of the HCDR3 is represented by SEQ ID NO: 34, the amino acid sequence of the LCDR1 is represented by SEQ ID NO: 35, the amino acid sequence of the LCDR2 is represented by SEQ ID NO: 36, the amino acid sequence of the LCDR3 is represented by SEQ ID NO: 37, or The amino acid sequence of the HCDR1 is represented by SEQ ID NO:38, the amino acid sequence of the HCDR2 is represented by SEQ ID NO:39, the amino acid sequence of the HCDR3 is represented by SEQ ID NO:40, the amino acid sequence of the LCDR1 is represented by SEQ ID NO:41, the amino acid sequence of the LCDR2 is represented by SEQ ID NO:42, and the amino acid sequence of the LCDR3 is represented by SEQ ID NO:43. Here, the HCDR and LCDR amino acid sequences provide an antibody as defined according to Kabat.

[0010] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody is represented by SEQ ID NO:28 or 30.

[0011] In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the antibody is represented by SEQ ID NO:29 or 31.

[0012] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody is represented by SEQ ID NO:28, and the amino acid sequence of the light chain variable region of the antibody is represented by SEQ ID NO:29, or the amino acid sequence of the heavy chain variable region of the antibody is represented by SEQ ID NO:30, and the amino acid sequence of the light chain variable region of the antibody is represented by SEQ ID NO:31.

[0013] According to a second aspect, the present invention provides an antibody against respiratory syncytial virus (RSV), wherein the amino acid sequence of the heavy chain variable region of the antibody has at least 90% identity with SEQ ID NO:28 or 30, and the amino acid sequence of the light chain variable region of the antibody has at least 90% identity with SEQ ID NO:29 or 31.

[0014] In some embodiments of either the first aspect or the second aspect, the antibody is a neutralizing antibody. In some embodiments of either the first aspect or the second aspect, the antibody can bind to the F protein of human respiratory syncytial virus (RSV).

[0015] In some embodiments of either the first aspect or the second aspect, the antibody is a Fab fragment, a full antibody, an F(ab’)2 fragment, or a single-chain Fv fragment (scFv). In some embodiments of either the first aspect or the second aspect, the antibody is a monoclonal antibody.

[0016] In some embodiments of either the first aspect or the second aspect, the antibody comprises a heavy chain constant region selected from the IgG1 subtype, IgG2 subtype, or IgG4 subtype.

[0017] In some embodiments of either the first aspect or the second aspect, the heavy chain constant region comprises the sequence of the Fc fragment of the IgG1 subtype heavy chain constant region, and the amino acid sequences at positions 252, 254, and 256 of the sequence of the Fc fragment are Y, T, and E, respectively, and the order of the constant region amino acids of the antibody is determined according to EU numbering.

[0018] In some embodiments of either the first aspect or the second aspect, the antibody comprises a light chain constant region selected from the κ subtype or the λ subtype. According to a third aspect, the present invention provides a nucleic acid molecule encoding the antibody described in the first aspect or the second aspect.

[0019] According to a fourth aspect, the present invention provides a pharmaceutical composition comprising the antibody described in the first aspect or the second aspect and a pharmaceutically acceptable excipient, diluent, or carrier.

[0020] According to a fifth aspect, the present invention provides the use of the antibody according to the first aspect or the second aspect, the nucleic acid molecule according to the third aspect, or the pharmaceutical composition according to the fourth aspect in the manufacture of a medicament for preventing or treating RSV-related diseases.

[0021] According to a sixth aspect, the present invention provides a method for preventing or treating RSV-related diseases, comprising administering to an individual in need thereof the antibody according to the first aspect or the second aspect, or the pharmaceutical composition according to the fourth aspect.

[0022] According to a seventh aspect, the present invention provides a combination of a first plasmid and a second plasmid, wherein the first plasmid comprises an antibody heavy chain VH-CH1 expression unit and a first recombination site, the second plasmid comprises an antibody light chain VL-CL expression unit and a second recombination site, and the first recombination site and the second recombination site are different, providing a combination of plasmids for expressing an antibody Fab fragment library.

[0023] In some embodiments of the seventh aspect, the combination of plasmids comprises a plurality of the first plasmids, and the plurality of the first plasmids have different antibody heavy chain VH-CH1 expression units and the same first recombination site.

[0024] In some embodiments of the seventh aspect, the combination of plasmids comprises a plurality of the second plasmids, and the plurality of the second plasmids have different antibody light chain VL-CL expression units and the same second recombination site.

[0025] In some embodiments of the seventh aspect, the first plasmid and / or the second plasmid is a phagemid. In some embodiments of the seventh aspect, the first plasmid and the second plasmid comprise different origins of replication.

[0026] In some embodiments of the seventh aspect, the replication origin is one or more selected from pBR ori, CDF ori, the replication origin of filamentous phage M13 (f1 ori), and p15A ori.

[0027] In some embodiments of the seventh aspect, the first recombination site and the second recombination site are respectively sites for the phage to attach in the respective genomes of the phage and its host bacterium. In some embodiments of the seventh aspect, the first recombination site is attP and the second recombination site is attB.

[0028] In some embodiments of the seventh aspect, the first recombination site and the second recombination site are respectively located at the polyclonal cleavage sites (restriction enzyme cleavage sites) of the first plasmid and the second plasmid. In some embodiments of the seventh aspect, the light chain constant region CL of the antibody is a human κ light chain constant region or a human λ light chain constant region.

[0029] In some embodiments of the seventh aspect, the CH1 fragment of the heavy chain constant region of the antibody is selected from IgG1, IgG2, IgG3, or IgG4 subtypes. In some embodiments of the seventh aspect, the first plasmid and / or the second plasmid contains a resistance gene coding region.

[0030] In some embodiments of the seventh aspect, the 3' end containing the nucleic acid molecule encoding the CH1 fragment of the heavy chain constant region of the antibody is fused to the 5' end of the nucleic acid molecule encoding the gIII protein of filamentous phage M13. In some embodiments of the seventh aspect, the resistance gene is an antibiotic resistance gene.

[0031] In some embodiments of the seventh aspect, the resistance gene is selected from a chloramphenicol resistance gene (CmR), an ampicillin resistance gene (Ampr), a kanamycin resistance gene (KaR), and a tetracycline resistance gene (TetR).

[0032] According to an eighth aspect, the present invention includes a first plasmid, a second plasmid, and a cell that expresses a phage integrase, the first plasmid includes an antibody heavy chain VH-CH1 expression unit and a first recombination site, the second plasmid includes an antibody light chain VL-CL expression unit and a second recombination site, the first recombination site and the second recombination site are different, and provides a recombination system for expressing an antibody Fab fragment library.

[0033] In some embodiments of the eighth aspect, the recombination system includes a plurality of the first plasmids, and the plurality of the first plasmids have different antibody heavy chain VH-CH1 expression units and have the same first recombination site.

[0034] In some embodiments of the eighth aspect, the recombination system includes a plurality of the second plasmids, and the plurality of the second plasmids have different antibody light chain VL-CL expression units and have the same second recombination site.

[0035] In some embodiments of the eighth aspect, the first plasmid and / or the second plasmid is a phagemid. In some embodiments of the eighth aspect, the first plasmid and the second plasmid include different origins of replication.

[0036] In some embodiments of the eighth aspect, the origin of replication is one or more selected from pBR ori, CDF ori, the origin of replication of filamentous phage M13 (f1 ori), and p15A ori. In some embodiments of the eighth aspect, the first recombination site and the second recombination site are sites for phage attachment in the respective genomes of the phage and its host bacterium, respectively.

[0037] In some embodiments of the eighth aspect, the first recombination site is attP and the second recombination site is attB. In some embodiments of the eighth aspect, the first recombination site and the second recombination site are located at the polyclonal cleavage sites of the first plasmid and the second plasmid, respectively.

[0038] In some embodiments of the eighth aspect, the light chain constant region CL of the antibody is a human κ light chain constant region or a human λ light chain constant region. In some embodiments of the eighth aspect, the heavy chain constant region CH1 fragment of the antibody is selected from the IgG1, IgG2, IgG3, or IgG4 subtype.

[0039] In some embodiments of the eighth aspect, the first plasmid and / or the second plasmid contains a resistance gene coding region. In some embodiments of the eighth aspect, the 3' end containing the nucleic acid molecule encoding the heavy chain constant region CH1 fragment of the antibody is fused to the 5' end of the nucleic acid molecule encoding the gIII protein of filamentous phage M13.

[0040] In some embodiments of the eighth aspect, the resistance gene is an antibiotic resistance gene. In some embodiments of the eighth aspect, the resistance gene is selected from the chloramphenicol resistance gene (CmR), ampicillin resistance gene (Ampr), kanamycin resistance gene (KaR), and tetracycline resistance gene (TetR).

[0041] In some embodiments of the eighth aspect, the phage integrase is a tyrosine integrase. In some embodiments of the eighth aspect, the phage integrase is a λ phage integrase.

[0042] In some embodiments of the eighth aspect, the phage integrase is an inducible expression or a constitutive expression.

[0043] In some embodiments of the eighth aspect, the phage integrase is an inducible expression. In some embodiments of the eighth aspect, the cell expressing the phage integrase is a prokaryotic cell.

[0044] In some embodiments of the eighth aspect, the cell expressing the phage integrase is Escherichia coli. In some embodiments of the eighth aspect, the cell expressing the phage integrase is a genetically engineered bacterium.

[0045] In some embodiments of the eighth aspect, the first plasmid, the second plasmid, and the cell expressing the phage integrase exist independently of each other, or at least one of the first plasmid and the second plasmid is introduced into the cell expressing the phage integrase.

[0046] According to the ninth aspect, the present invention provides a recombinant cell that includes a first plasmid and a second plasmid, expresses a phage integrase, the first plasmid includes an antibody heavy chain VH-CH1 expression unit and a first recombination site, the second plasmid includes an antibody light chain VL-CL expression unit and a second recombination site, the first recombination site and the second recombination site are different, and expresses an antibody Fab fragment library.

[0047] In some embodiments of the ninth aspect, the recombinant cell contains a plurality of the first plasmids, and the plurality of the first plasmids have different heavy chain VH-CH1 expression units of the antibody and have the same first recombination site.

[0048] In some embodiments of the ninth aspect, the recombinant cell contains a plurality of the second plasmids, and the plurality of the second plasmids have different light chain VL-CL expression units of the antibody and have the same second recombination site.

[0049] In some embodiments of the ninth aspect, the first plasmid and / or the second plasmid is a phagemid. In some embodiments of the ninth aspect, the first plasmid and the second plasmid contain different origins of replication.

[0050] In some embodiments of the ninth aspect, the origin of replication is one or more selected from pBR ori, CDF ori, the origin of replication of filamentous phage M13 (f1 ori), and p15A ori. In some embodiments of the ninth aspect, the first recombination site and the second recombination site are sites for the phage to attach in the respective genomes of the phage and its host bacterium, respectively.

[0051] In some embodiments of the ninth aspect, the first recombination site is attP and the second recombination site is attB. In some embodiments of the ninth aspect, the first recombination site and the second recombination site are respectively located at the polyclonal cleavage sites of the first plasmid and the second plasmid.

[0052] In some embodiments of the ninth aspect, the light chain constant region CL of the antibody is a human κ light chain constant region or a human λ light chain constant region. In some embodiments of the ninth aspect, the CH1 fragment of the heavy chain constant region of the antibody is selected from IgG1, IgG2, IgG3, or IgG4 subtypes.

[0053] In some embodiments of the ninth aspect, the first plasmid and / or the second plasmid comprises a resistance gene coding region. In some embodiments of the ninth aspect, the 3′ end comprising the nucleic acid molecule encoding the CH1 fragment of the heavy chain constant region of the antibody is fused to the 5′ end of the nucleic acid molecule encoding the gIII protein of filamentous phage M13.

[0054] In some embodiments of the ninth aspect, the resistance gene is an antibiotic resistance gene. In some embodiments of the ninth aspect, the resistance gene is selected from the chloramphenicol resistance gene (CmR), ampicillin resistance gene (Ampr), kanamycin resistance gene (KaR), and tetracycline resistance gene (TetR).

[0055] In some embodiments of the ninth aspect, the phage integrase is a tyrosine integrase. In some embodiments of the ninth aspect, the phage integrase is a λ phage integrase.

[0056] In some embodiments of the ninth aspect, the phage integrase is inducible expression or constitutive expression.

[0057] In some embodiments of the ninth aspect, the phage integrase is inducible expression. In some embodiments of the ninth aspect, the recombinant cell is a prokaryotic cell. In some embodiments of the ninth aspect, the recombinant cell is Escherichia coli. In some embodiments of the ninth aspect, the recombinant cell is a genetically engineered bacterium.

[0058] According to the tenth aspect, the present invention includes transducing a first plasmid and a second plasmid into a cell that expresses a phage integrase, wherein the first plasmid includes an antibody heavy chain VH-CH1 expression unit and a first recombination site, the second plasmid includes an antibody light chain VL-CL expression unit and a second recombination site, the first recombination site and the second recombination site are different, and provides a method for producing an antibody Fab fragment library.

[0059] In some embodiments according to the tenth aspect, a plurality of the first plasmids are transduced into a cell that expresses the phage integrase, and the plurality of the first plasmids have different antibody heavy chain VH-CH1 expression units and have the same first recombination site.

[0060] In some embodiments according to the tenth aspect, a plurality of the second plasmids are transduced into a cell that expresses the phage integrase, and the plurality of the second plasmids have different antibody light chain VL-CL expression units and have the same second recombination site.

[0061] In some embodiments of the tenth aspect, transducing the first plasmid and / or the second plasmid into a cell that expresses the phage integrase comprises: (1) transducing the first plasmid and / or the second plasmid into competent cells; (2) infecting the competent cells obtained in step (1) with a helper phage to construct a phage library; and (3) transducing the phage library obtained in step (2) into a cell that expresses the phage integrase.

[0062] In some embodiments of the tenth aspect, the competent cell is a prokaryotic cell. In some embodiments of the tenth aspect, the competent cell does not express phage integrase.

[0063] In some embodiments of the tenth aspect, the helper phage is an M13 helper phage. In some embodiments of the tenth aspect, the first plasmid and / or the second plasmid is a phagemid.

[0064] In some embodiments of the tenth aspect, the first plasmid and the second plasmid contain different origins of replication. In some embodiments of the tenth aspect, the origin of replication is one or more selected from pBR ori, CDF ori, the origin of replication of filamentous phage M13 (f1 ori), and p15A ori.

[0065] In some embodiments of the tenth aspect, the first recombination site and the second recombination site are, respectively, sites for the phage to attach in the respective genomes of the phage and its host bacterium. In some embodiments of the tenth aspect, the first recombination site is attP and the second recombination site is attB.

[0066] In some embodiments of the tenth aspect, the first recombination site and the second recombination site are respectively located at the polyclonal cleavage sites of the first plasmid and the second plasmid. In some embodiments of the tenth aspect, the light chain constant region CL of the antibody is a human κ light chain constant region or a human λ light chain constant region.

[0067] In some embodiments of the tenth aspect, the heavy chain constant region CH1 fragment of the antibody is selected from IgG1, IgG2, IgG3, or IgG4 subtypes. In some embodiments of the tenth aspect, the first plasmid and / or the second plasmid comprises a resistance gene coding region.

[0068] In some embodiments of the tenth aspect, the resistance gene is selected from a chloramphenicol resistance gene (CmR), an ampicillin resistance gene (Ampr), a kanamycin resistance gene (KaR), and a tetracycline resistance gene (TetR).

[0069] In some embodiments of the tenth aspect, the phage integrase is a tyrosine integrase. In some embodiments of the tenth aspect, the phage integrase is a λ phage integrase. In some embodiments of the tenth aspect, the phage integrase is inducible expression or constitutive expression.

[0070] In some embodiments of the tenth aspect, the phage integrase is inducible expression. In some embodiments of the tenth aspect, the cell expressing the phage integrase is a prokaryotic cell. In some embodiments of the tenth aspect, the cell expressing the phage integrase is Escherichia coli.

[0071] In some embodiments of the tenth aspect, the cell expressing the phage integrase is a genetically engineered bacterium. According to the eleventh aspect, the present invention provides the use of the combination of plasmids according to the seventh aspect, the recombinant system according to the eighth aspect, or the recombinant cell according to the ninth aspect in the production of an antibody Fab fragment library.

Brief Description of the Drawings

[0072]

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Figure 7

[0073] 〔Description of the sequence〕 SEQ ID NO:1 shows the nucleotide sequence derived from Escherichia coli encoding the chloramphenicol resistance gene promoter (Cat promoter). SEQ ID NO:2 shows the nucleotide sequence derived from Escherichia phage Lambda encoding the attP attachment site. SEQ ID NO:3 shows the nucleotide sequence encoding CDF ori derived from Escherichia coli.

[0074] SEQ ID NO:4 shows the nucleotide sequence encoding the attB attachment site derived from Escherichia coli. SEQ ID NO:5 shows the nucleotide sequence encoding the chloramphenicol resistance gene derived from Escherichia coli. SEQ ID NO:6 shows the nucleotide sequence encoding the complete ampicillin resistance gene expression element derived from Escherichia coli.

[0075] SEQ ID NO:7 shows the nucleotide sequence encoding the λ phage integrase expression element induced by the λ phage lactose operon derived from the genus Escherichia. SEQ ID NO:8 shows the nucleotide sequence encoding the attL recombination site. SEQ ID NO:9 shows the nucleotide sequence encoding the attR recombination site.

[0076] SEQ ID NO:10 shows the amino acid sequence of the F protein of RSV A subtype strain A2. SEQ ID NO:11 shows the amino acid sequence of the F protein of RSV B subtype strain 18537. SEQ ID NO:12 shows the amino acid sequence of the prefusion F protein mutant RSV-DS-Cav1-A of the DS-Cav1 structure.

[0077] SEQ ID NO:13 shows the amino acid sequence of the prefusion F protein mutant RSV-DS-Cav1-B of the DS-Cav1 structure.

[0078] SEQ ID NO:14 shows the amino acid sequence of the His tag.

[0079] SEQ ID NO:15 shows the amino acid sequence of the constant region of the human (homo sapiens) IgG1 subtype heavy chain.

[0080] SEQ ID NO:16 shows the amino acid sequence of the variant IgG1 - YTE of the human (homo sapiens) IgG1 subtype heavy chain constant region.

[0081] SEQ ID NO:17 shows the amino acid sequence of the mouse (mus musculus) IgG2a subtype heavy chain constant region. SEQ ID NO:18 shows the amino acid sequence of the human (homo sapiens) κ subtype light chain constant region. SEQ ID NO:19 shows the amino acid sequence of the human (homo sapiens) λ subtype light chain constant region.

[0082] SEQ ID NO:20 shows the amino acid sequence of the mouse (mus musculus) κ subtype light chain constant region. SEQ ID NO:21 shows the amino acid sequence of the mouse (mus musculus) λ subtype light chain constant region. SEQ ID NO:22 shows the amino acid sequence of the heavy chain variable region of the humanized anti - RSV monoclonal antibody palivizumab (Hu1129).

[0083] SEQ ID NO:23 shows the amino acid sequence of the light chain variable region of the humanized anti - RSV monoclonal antibody palivizumab (Hu1129). SEQ ID NO:24 shows the amino acid sequence of the heavy chain variable region of the fully humanized anti - RSV monoclonal antibody nirsevimab (MEDI8897). SEQ ID NO:25 shows the amino acid sequence of the light chain variable region of the fully humanized anti - RSV monoclonal antibody nirsevimab (MEDI8897).

[0084] SEQ ID NO:26 shows the amino acid sequence of the heavy chain variable region of the fully humanized anti - RSV monoclonal antibody clesrovimab (RB1).

[0085] SEQ ID NO:27 shows the amino acid sequence of the light chain variable region of the fully humanized anti-RSV monoclonal antibody Clesrovimab (RB1). SEQ ID NO:28 shows the amino acid sequence of the heavy chain variable region of the Fab antibody R3B1h1 against the RSV F protein. SEQ ID NO:29 shows the amino acid sequence of the light chain variable region of the Fab antibody R3B1h1 against the RSV F protein.

[0086] SEQ ID NO:30 shows the amino acid sequence of the heavy chain variable region of the Fab antibody R22B1 against the RSV F protein. SEQ ID NO:31 shows the amino acid sequence of the light chain variable region of the Fab antibody R22B1 against the RSV F protein. SEQ ID NOs: 32-34 show the amino acid sequences of the heavy chain complementarity-determining regions 1, 2, and 3 (HCDR1, HCDR2, and HCDR3), respectively, of the Fab antibody R3B1h1 against the RSV F protein.

[0087] SEQ ID NOs: 35-37 show the amino acid sequences of the light chain complementarity-determining regions 1, 2, and 3 (LCDR1, LCDR2, and LCDR3), respectively, of the Fab antibody R3B1h1 against the RSV F protein. SEQ ID NOs: 38-40 show the amino acid sequences of the heavy chain complementarity-determining regions 1, 2, and 3 (HCDR1, HCDR2, and HCDR3), respectively, of the Fab antibody R22B1 against the RSV F protein. SEQ ID NOs: 41-43 show the amino acid sequences of the light chain complementarity-determining regions 1, 2, and 3 (LCDR1, LCDR2, and LCDR3), respectively, of the Fab antibody R22B1 against the RSV F protein.

[0088] SEQ ID NO:44 shows the nucleotide sequence encoding the pBR ori derived from Escherichia coli. SEQ ID NO:45 shows the nucleotide sequence encoding the f1 ori derived from f1 phage. SEQ ID NO:46 shows the nucleotide sequence encoding the gIII protein of filamentous phage M13 derived from M13 phage. 〔Detailed Description of the Invention〕

[0089] Using the specific recombination system or recombinant cells of λ phage integrase (Int), specific recombinant integration and excision of target nucleotide sequences can be achieved in vivo and in vitro by genetic engineering means. It has been reported in the literature that when using genetically engineered bacteria in which Int integrase is induced to express by heat, specific recombination of the antibody light chain expression plasmid and the heavy chain expression plasmid can be achieved intracellularly to generate a complete plasmid expressing functional Fab. 10 . ThermoFisher's Gateway homologous recombination technology uses the specific recombination principle of λ phage integrase, does not require classical gene cloning steps such as enzyme cleavage and ligation, has a high in vitro recombination efficiency of 95%, is used to construct the Gatway kit of cDNA library, and the diversity to achieve the library reaches 1.0E+7.

[0090] In the present invention, the λ phage integrase recombination system or recombinant cells are used to overcome the drawback of insufficient DNA conversion efficiency during antibody library construction by efficient phage infection. Using phages packaged with the antibody heavy chain expression unit (for example, the heavy chain VH-CH1 expression unit), Int integrase can be induced to express, and Escherichia coli containing the plasmid of the antibody light chain expression unit is infected. By Int integrase, the antibody heavy chain expression unit (for example, the heavy chain VH-CH1 expression unit) and the light chain expression unit are specifically recombined in vivo, and a large-capacity Fab antibody library (exceeding 1.0E+12) is obtained by antibiotic screening.

[0091] The inventors of the present invention screen for novel Fab antibodies against RSV using the Fab antibody library constructed above. In various aspects of the present invention, novel antibodies against RSV, nucleic acid molecules encoding the antibodies, vectors containing the nucleic acid molecules, host cells containing the nucleic acid molecules or vectors, and compositions containing the antibodies, methods for producing and purifying the antibodies, and medical and biological uses of the antibodies are provided. According to the amino acid sequence of the variable region of the antibody according to the present invention, a full-length antibody molecule can be constructed as a drug for preventing or treating RSV-related diseases.

[0092] Unless otherwise specified, techniques of molecular biology, microbiology, cell biology, biochemistry, and immunology that are common in the art are used in the practice of the present invention. Unless otherwise specified, the terms used in the present invention have meanings commonly understood by those skilled in the art.

[0093] 〔Definition〕 As used herein, the term "recombinant site" refers to a site for phage integration in the phage genome and the bacterial genome. In some embodiments of the present invention, the first recombinant site and the second recombinant site may each be a site for phage attachment in the respective genomes of the phage and its host bacterium, including the following cases. The first recombinant site is a site for phage attachment in the phage genome, and the second recombinant site is a site for phage attachment in the genome of the host bacterium of the phage. Alternatively, the second recombinant site is a site for phage attachment in the phage genome, and the first recombinant site is a site for phage attachment in the genome of the host bacterium of the phage.

[0094] As used herein, the term "plasmid" refers to a DNA molecule other than a chromosome (or nucleoid) that exists within the cytoplasm of organisms such as bacteria, yeast, and actinomycetes (except for yeast with a 2μm plasmid in the nucleus). It has the ability to replicate autonomously, maintain a certain copy number in daughter cells, and express the genetic information it carries. It is a closed circular double-stranded DNA molecule. The term "plasmid" as used herein includes bacterial plasmids, phagemids, etc. A "bacterial plasmid" is a commonly used vector in DNA recombination technology and is a tool that can introduce foreign genes useful for growth and expression into recipient cells by genetic engineering means. A "phagemid" is a vector that combines a plasmid vector and a single-stranded phage vector. It contains a packaging sequence of a single-stranded phage, a replicon and a plasmid replicon, a cloning site, a marker gene, etc. In host cells such as Escherichia coli, it can replicate as a normal double-stranded plasmid DNA molecule. In the presence of a helper phage, it replicates according to the rolling circle model to generate single-stranded DNA, which is packaged into phage particles.

[0095] As used herein, the term "attP attachment site" refers to a specific sequence on phage DNA that contains a 15bp core region that is identical to the host chromosome sequence.

[0096] As used herein, the term "attB attachment site" refers to the attachment site in the bacterial genome that contains a 15bp core region that is identical to the phage genome sequence.

[0097] As used herein, the term "phage integrase" refers to a protein that can specifically bind to attP and attB attachment sites and has type I topoisomerase activity that is essential for integrating the phage into the host genome through site-specific recombination.

[0098] As used herein, the term "origin of replication" refers to the starting site of plasmid replication, which consists of an origin of replication (ORI) and its regulatory factors.

[0099] As used herein, the term "antibody" refers to an immunoglobulin molecule that can specifically bind to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, peptides, etc. As used herein, "antibodies" include not only intact antibodies (i.e., full-length antibodies), but also antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), variants thereof, fusion proteins containing antibody moieties, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and other modified constructs of immunoglobulin molecules containing antigen recognition sites with the required specificity, such as glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0100] Typically, an intact antibody or full-length antibody contains two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and first, second, and third constant regions (CH1, CH2, and CH3). Each light chain contains a light chain variable region (VL) and a constant region (CL). The full-length antibody may be an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), but the antibody does not have to belong to a specific class. Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the heavy chain constant domain of the antibody. Typically, there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Some of these classes can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are known.

[0101] As used herein, the term "antigen-binding fragment or antigen-binding portion" refers to a part or region of an intact antibody molecule that is involved in binding to an antigen. The antigen-binding domain may include the heavy-chain variable region (VH), the light-chain variable region (VL), or both. Each of VH and VL typically includes three complementarity-determining regions CDR1, CDR2, and CDR3.

[0102] Those skilled in the art know that the complementarity-determining regions (CDRs, usually having CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest influence on the affinity and specificity of the antibody for the antigen. There are two general methods for defining the CDR amino acid sequences of VH or VL, namely the Chothia definition and the Kabat definition. 11~13 For a given variable region amino acid sequence of an antibody, the CDR amino acid sequences in the VH and VL amino acid sequences can be determined according to the Chothia definition or the Kabat definition. In embodiments of the present invention, Kabat is used to define the CDR amino acid sequences.

[0103] For a given variable region amino acid sequence of an antibody, the CDR amino acid sequences of the variable region amino acid sequence can be analyzed in various ways, for example, the online software Abysis (http: / / www.abysis.org / ) can be used.

[0104] Examples of antigen-binding fragments include, but are not limited to: (1) A Fab fragment, which may be a monovalent fragment having a VL-CL chain and a VH-CH1 chain. (2) An F(ab’)2 fragment, which may be a divalent fragment having two Fab’ fragments that can be linked by a disulfide bridge in the hinge region (i.e., a dimer of Fab’). (3) An Fv fragment having the VL and VH domains of one arm of an antibody. (4) A single-chain Fv (scFv), which may be a single polypeptide chain composed of a VH domain and a VL domain via a peptide linker. (5) (scFv)2, which may include two VH domains linked by a peptide linker and two VL domains that bind to the two VH domains via disulfide bridges.

[0105] In some specific embodiments of the present invention, the antibody is a Fab fragment, i.e., a monovalent fragment having a VL-CL chain and a VH-CH1 chain.

[0106] As used herein, the term "Fd fragment" refers to a fragment consisting of the variable region VH of the heavy chain of an antibody and the first constant region CH1 of the heavy chain.

[0107] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigenic epitope.

[0108] As used herein, the term "neutralizing antibody" refers to an antibody that can bind to an antigen on the surface of a pathogenic microorganism, thereby preventing the pathogenic microorganism from attaching to a target cell receptor, preventing the fusion of the viral envelope and the cell membrane, and preventing entry into the cell.

[0109] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical except for naturally occurring mutations that may be present in a small number of individuals.

DETAILED DESCRIPTION OF THE INVENTION

[0110] According to a first aspect, the present invention provides an antibody against respiratory syncytial virus (RSV) comprising a heavy chain variable region comprising the amino acid sequences of HCDR1, HCDR2 and HCDR3, and a light chain variable region comprising the amino acid sequences of LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of said HCDR1 is represented by SEQ ID NO:32, the amino acid sequence of said HCDR2 is represented by SEQ ID NO:33, the amino acid sequence of said HCDR3 is represented by SEQ ID NO:34, the amino acid sequence of said LCDR1 is represented by SEQ ID NO:35, the amino acid sequence of said LCDR2 is represented by SEQ ID NO:36, the amino acid sequence of said LCDR3 is represented by SEQ ID NO:37, or the amino acid sequence of said HCDR1 is represented by SEQ ID NO:38, the amino acid sequence of said HCDR2 is represented by SEQ ID NO:39, the amino acid sequence of said HCDR3 is represented by SEQ ID NO:40, the amino acid sequence of said LCDR1 is represented by SEQ ID NO:41, the amino acid sequence of said LCDR2 is represented by SEQ ID NO:42, the amino acid sequence of said LCDR3 is represented by SEQ ID NO:43, wherein the HCDR and LCDR amino acid sequences are defined according to Kabat, provides an antibody.

[0111] In some embodiments according to the first aspect, the amino acid sequence of the heavy chain variable region of said antibody is represented by SEQ ID NO:28 or 30.

[0112] In some embodiments according to the first aspect, the amino acid sequence of the light chain variable region of said antibody is represented by SEQ ID NO:29 or 31.

[0113] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of said antibody is represented by SEQ ID NO:28, the amino acid sequence of the light chain variable region of said antibody is represented by SEQ ID NO:29, or The amino acid sequence of the heavy chain variable region of the antibody is shown by SEQ ID NO: 30, and the amino acid sequence of the light chain variable region of the antibody is shown by SEQ ID NO: 31.

[0114] According to a second aspect, the present invention provides an antibody against respiratory syncytial virus (RSV), wherein the amino acid sequence of the heavy chain variable region of the antibody has at least 90% identity with SEQ ID NO: 28 or 30, and the amino acid sequence of the light chain variable region of the antibody has at least 90% identity with SEQ ID NO: 29 or 31.

[0115] In some embodiments of the second aspect, the amino acid sequence of the heavy chain variable region of the antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 28 or 30.

[0116] In some embodiments of the second aspect, the amino acid sequence of the light chain variable region of the antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 29 or 31.

[0117] In some embodiments of the second aspect, the amino acid sequence of the heavy chain variable region of the antibody differs from the amino acid sequence shown in any one of SEQ ID NO: 28 and 30 by about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions and / or additions.

[0118] In some embodiments of the second aspect, the amino acid sequence of the light chain variable region of the antibody differs from the amino acid sequence shown in any one of SEQ ID NO: 29 and 31 by about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions and / or additions.

[0119] In some embodiments of the second aspect, the C-terminus or C-terminal region of the amino acid sequence shown in any one of SEQ ID NOs: 28 and 30 may be further truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, and a function similar to the variable heavy chain region of the antibody is still maintained.

[0120] In some embodiments of the second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be further added to the C-terminus or C-terminal region of the amino acid sequence shown in any one of SEQ ID NOs: 28 and 30, and the resulting amino acid sequence still maintains a function similar to the variable heavy chain region of the antibody.

[0121] In some embodiments of the second aspect, as long as the modified amino acid sequence substantially maintains a function similar to the variable heavy chain region of the antibody, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in the C-terminus or regions other than the C-terminus of the amino acid sequence shown in any one of SEQ ID NOs: 28 and 30.

[0122] In some embodiments of the second aspect, the C-terminus or C-terminal region of the amino acid sequence shown in any one of SEQ ID NOs: 29 and 31 may be further truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, and a function similar to the variable light chain region of the antibody is still maintained.

[0123] In some embodiments of the second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be further added to the C-terminal or C-terminal region of the amino acid sequence shown in any one of SEQ ID NO: 29 and 31, and the resulting amino acid sequence still maintains a function similar to the variable region of the light chain of the antibody.

[0124] In some embodiments of the second aspect, as long as the modified amino acid sequence substantially maintains a function similar to the variable region of the light chain of the antibody, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be further added or deleted from the C-terminal or a region other than the C-terminal of the amino acid sequence shown in any one of SEQ ID NO: 29 and 31.

[0125] In some embodiments of either the first aspect or the second aspect, the antibody is a neutralizing antibody.

[0126] In some embodiments of either the first aspect or the second aspect, the antibody can bind to the F protein of human respiratory syncytial virus (RSV).

[0127] In a specific embodiment of either the first aspect or the second aspect, the F protein of human respiratory syncytial virus (RSV) is a recombinant human RSV F protein, for example, the recombinant human RSV F protein shown in SEQ ID NO: 12 or 13.

[0128] In some embodiments of either the first aspect or the second aspect, the antibody is a Fab fragment, a complete antibody, an F(ab’)2 fragment, or a single-chain Fv fragment (scFv).

[0129] In a specific embodiment of either the first aspect or the second aspect, the antibody is a Fab fragment.

[0130] In some embodiments of either the first aspect or the second aspect, the antibody is a monoclonal antibody.

[0131] In some embodiments of either the first aspect or the second aspect, the antibody comprises a heavy chain constant region selected from the IgG1 subtype, IgG2 subtype, or IgG4 subtype.

[0132] In some embodiments of either the first aspect or the second aspect, the heavy chain constant region comprises the sequence of the Fc fragment of the IgG1 subtype heavy chain constant region, and the amino acid sequences at positions 252, 254, and 256 of the sequence of the Fc fragment are Y, T, and E, respectively, and the order of the constant region amino acids of the antibody is determined according to EU numbering.

[0133] In some embodiments of either the first aspect or the second aspect, the antibody comprises a light chain constant region selected from the κ subtype or the λ subtype.

[0134] According to a third aspect, the present invention provides a nucleic acid molecule encoding the antibody according to the first aspect or the second aspect.

[0135] In some embodiments of the third aspect, the nucleic acid molecule is operably linked to a regulatory amino acid sequence, and the regulatory amino acid sequence can be used for the identification of a host cell transformed with the vector.

[0136] According to a fourth aspect, the present invention provides a pharmaceutical composition comprising the antibody according to the first aspect or the second aspect and a pharmaceutically acceptable excipient, diluent, or carrier.

[0137] In some embodiments of the fourth aspect, the pharmaceutical composition is used for preventing or treating RSV-related diseases.

[0138] In some embodiments of the fourth aspect, the RSV-related disease is a respiratory tract infection such as bronchiolitis and pneumonia.

[0139] In some embodiments of the fourth aspect, the pharmaceutical composition may contain one or more of lubricants (e.g., talc, magnesium stearate, and mineral oil), wetting agents, emulsifiers, suspending agents, preservatives (e.g., benzoic acid, sorbic acid, calcium propionate), sweeteners and / or flavoring agents, etc.

[0140] In some embodiments of the fourth aspect, the pharmaceutical composition according to the present invention may be prepared in the form of tablets, pills, powders, troches, elixirs, suspensions, emulsions, solutions, syrups, suppositories or capsules.

[0141] In some embodiments of the fourth aspect, the pharmaceutical composition according to the present invention can be delivered by any physiologically acceptable administration method including, but not limited to, oral administration, parenteral administration, nasal administration, rectal administration, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, etc.

[0142] In some embodiments of the fourth aspect, the therapeutic pharmaceutical composition can be formulated for storage in the form of a lyophilized preparation or an aqueous solution by mixing a reagent of the required purity with any pharmaceutically acceptable carrier, excipient, etc.

[0143] According to the fifth aspect, the present invention provides the use of the antibody described in the first aspect or the second aspect, the nucleic acid molecule described in the third aspect, or the pharmaceutical composition described in the fourth aspect in the manufacture of a medicament for preventing or treating RSV-related diseases.

[0144] In some embodiments of the fifth aspect, the RSV-related disease is a respiratory tract infection such as bronchiolitis and pneumonia.

[0145] According to a sixth aspect, the present invention provides a method for preventing or treating an RSV-related disease, comprising administering to an individual in need thereof the antibody according to the first aspect or the second aspect, or the pharmaceutical composition according to the fourth aspect.

[0146] In some embodiments of the sixth aspect, the RSV-related disease is a respiratory tract infection such as bronchiolitis and pneumonia.

[0147] According to a seventh aspect, the present invention provides a combination of plasmids for expressing an antibody Fab fragment, comprising a first plasmid and a second plasmid, wherein the first plasmid comprises an antibody heavy chain VH-CH1 expression unit and a first recombination site, and the second plasmid comprises an antibody light chain VL-CL expression unit and a second recombination site, and the first recombination site and the second recombination site are different.

[0148] In some embodiments of the seventh aspect, the combination of plasmids comprises a plurality of the first plasmids, and the plurality of the first plasmids have different antibody heavy chain VH-CH1 expression units and the same first recombination site.

[0149] In some embodiments of the seventh aspect, the combination of plasmids comprises a plurality of the second plasmids, and the plurality of the second plasmids have different antibody light chain VL-CL expression units and the same second recombination site.

[0150] According to an eighth aspect, the present invention provides a recombination system for expressing an antibody Fab fragment library, comprising a first plasmid, a second plasmid, and a cell expressing a phage integrase, wherein the first plasmid comprises an antibody heavy chain VH-CH1 expression unit and a first recombination site, the second plasmid comprises an antibody light chain VL-CL expression unit and a second recombination site, and the first recombination site and the second recombination site are different.

[0151] In some embodiments of the eighth aspect, the recombinant system includes a plurality of the first plasmids, and the plurality of the first plasmids carry heavy chain VH-CH1 expression units of different antibodies and have the same first recombination site.

[0152] In some embodiments of the eighth aspect, the recombinant system includes a plurality of the second plasmids, and the plurality of the second plasmids carry light chain VL-CL expression units of different antibodies and have the same second recombination site.

[0153] In some embodiments of the eighth aspect, the cells expressing the first plasmid, the second plasmid, and the phage integrase exist independently of each other, or at least one of the first plasmid and the second plasmid is introduced into the cells expressing the phage integrase.

[0154] In some specific embodiments of the eighth aspect, the second plasmid is introduced into the cells expressing the phage integrase.

[0155] According to the ninth aspect, the present invention provides a recombinant cell for expressing an antibody Fab fragment library, which includes a first plasmid and a second plasmid, expresses a phage integrase, the first plasmid includes an antibody heavy chain VH-CH1 expression unit and a first recombination site, the second plasmid includes an antibody light chain VL-CL expression unit and a second recombination site, and the first recombination site and the second recombination site are different recombination sites.

[0156] In some embodiments of the ninth aspect, the recombinant cell includes a plurality of the first plasmids, and the plurality of the first plasmids carry heavy chain VH-CH1 expression units of different antibodies and have the same first recombination site.

[0157] In some embodiments of the ninth aspect, the recombinant cell contains a plurality of the second plasmids, and the plurality of the second plasmids have light chain VL-CL expression units of different antibodies and have the same second recombination site.

[0158] In some embodiments of the ninth aspect, the recombinant cell is a prokaryotic cell. In some embodiments of the ninth aspect, the recombinant cell is Escherichia coli. In some embodiments of the ninth aspect, the recombinant cell is a genetically engineered bacterium.

[0159] In some specific embodiments of the ninth aspect, the recombinant cell is a male Escherichia coli strain having an F factor such as the TG1 strain.

[0160] According to the tenth aspect, the present invention provides a method for producing an antibody Fab fragment library, which includes transducing a first plasmid and a second plasmid into a cell that expresses phage integrase. The first plasmid includes a heavy chain VH-CH1 expression unit of an antibody and a first recombination site, and the second plasmid includes a light chain VL-CL expression unit of an antibody and a second recombination site. The first recombination site and the second recombination site are different.

[0161] In some embodiments of the tenth aspect, a plurality of the first plasmids are transduced into the cell that expresses phage integrase. The plurality of the first plasmids have heavy chain VH-CH1 expression units of different antibodies and have the same first recombination site.

[0162] In some embodiments of the tenth aspect, a plurality of the second plasmids are transduced into the cell that expresses phage integrase. The plurality of the second plasmids have light chain VL-CL expression units of different antibodies and have the same second recombination site.

[0163] In some embodiments of the tenth aspect, transducing the first plasmid and / or the second plasmid into cells that express the phage integrase comprises step (1) of transducing the first plasmid and / or the second plasmid into competent cells, step (2) of infecting the competent cells obtained in step (1) with a helper phage to construct a phage library, and step (3) of transducing the phage library obtained in step (2) into cells that express the phage integrase.

[0164] In some embodiments of the tenth aspect, the competent cells are prokaryotic cells. In some embodiments of the tenth aspect, the competent cells are Escherichia coli. In some embodiments of the tenth aspect, the competent cells are genetically engineered bacteria.

[0165] In some specific embodiments of the tenth aspect, the competent cells are male Escherichia coli strains having an F factor such as the TG1 strain.

[0166] In some embodiments of the tenth aspect, the competent cells do not express the phage integrase. In some embodiments of the tenth aspect, the helper phage is an M13 helper phage.

[0167] In some embodiments of any one of the seventh to tenth aspects, the first plasmid is a phagemid. In some embodiments of any one of the seventh to tenth aspects, the second plasmid is a phagemid.

[0168] In some embodiments of any one of the seventh to tenth aspects, the first plasmid and the second plasmid include different origins of replication.

[0169] In some embodiments of any one of the seventh to tenth aspects, the origin of replication is one or more selected from pBR ori, CDF ori, the origin of replication of filamentous phage M13 (f1 ori), and p15A ori.

[0170] In some embodiments of any one of the seventh to tenth aspects, the first plasmid includes pBR ori and f1 ori.

[0171] In some embodiments of any one of the seventh to tenth aspects, the second plasmid includes CDF ori.

[0172] In some embodiments of any one of the seventh to tenth aspects, the first recombination site and the second recombination site are sites for the phage to attach in the genomes of the phage and its host bacterium, respectively.

[0173] In some embodiments of any one of the seventh to tenth aspects, the first recombination site is attP. In some embodiments of any one of the seventh to tenth aspects, the second recombination site is attB.

[0174] In some embodiments of any one of the seventh to tenth aspects, the first recombination site includes the nucleotide sequence shown in SEQ ID NO:2 or a nucleotide sequence obtained by adding one or more substitutions, deletions, or additions to the nucleotide sequence shown in SEQ ID NO:2 that do not substantially change the function of the first recombination site.

[0175] In some embodiments of any one of the seventh to tenth aspects, the second recombination site includes the nucleotide sequence represented by SEQ ID NO: 4 or a nucleotide sequence obtained by adding one or more substitutions, deletions, or additions at one or more positions that do not substantially change the function of the second recombination site to the nucleotide sequence represented by SEQ ID NO: 4.

[0176] In some embodiments of any one of the seventh to tenth aspects, the number of one or more substitutions, deletions, or additions that do not substantially change the function of the first recombination site or the second recombination site is 1 to 30, preferably 1 to 20, more preferably 1 to 10, and the obtained nucleotide sequence does not substantially change the function of the first recombination site or the second recombination site.

[0177] In some embodiments of any one of the seventh to tenth aspects, the first plasmid and the second plasmid are different.

[0178] In some embodiments of any one of the seventh to tenth aspects, the first plasmid is a phagemid, and the second plasmid is a prokaryotic expression vector (for example, a bacterial plasmid).

[0179] In some embodiments of any one of the seventh to tenth aspects, the first plasmid is pHGDisn-attP-new.

[0180] In some embodiments of any one of the seventh to tenth aspects, the second plasmid is pHKb-attB-new.

[0181] In some embodiments of any one of the seventh to tenth aspects, the first recombination site is located at the polyclonal cleavage site of the first plasmid.

[0182] In some embodiments of any one of the seventh to tenth aspects, the second recombination site is located at the polyclonal cleavage site of the second plasmid.

[0183] In some embodiments of any one of the seventh to tenth aspects, the first recombination site is located between the cleavage sites of XbaI and KpnI of the pHGDisn-attP-new plasmid.

[0184] In some embodiments of any one of the seventh to tenth aspects, the second recombination site is located between the cleavage sites of HindIII and BamHI of the pHKb-attB-new plasmid.

[0185] In some embodiments of any one of the seventh to tenth aspects, the light chain constant region CL of the antibody is a human κ light chain constant region or a human λ light chain constant region.

[0186] In some embodiments of any one of the seventh to tenth aspects, the CH1 fragment of the heavy chain constant region of the antibody is selected from IgG1, IgG2, IgG3, or IgG4 subtypes.

[0187] In some embodiments of any one of the seventh to tenth aspects, the first plasmid and / or the second plasmid contains a resistance gene coding region.

[0188] In some embodiments of any one of the seventh to tenth aspects, the 3' end containing the nucleic acid molecule encoding the CH1 fragment of the heavy chain constant region of the antibody is fused to the 5' end of the nucleic acid molecule encoding the gIII protein of filamentous phage M13.

[0189] In some embodiments of any one of the seventh to tenth aspects, the presence of the resistance gene is advantageous for screening the recombination system or recombinant cells.

[0190] In some embodiments of any one of the seventh to tenth aspects, the resistance gene is an antibiotic resistance gene.

[0191] In some embodiments of any one of the seventh to tenth aspects, the resistance gene is selected from a chloramphenicol resistance gene (CmR), an ampicillin resistance gene (Ampr), a kanamycin resistance gene (KaR), and a tetracycline resistance gene (TetR).

[0192] In some embodiments of any one of the eighth to tenth aspects, the phage integrase is a tyrosine integrase.

[0193] In some embodiments of any one of the eighth to tenth aspects, the tyrosine integrase is a λ phage integrase.

[0194] In some embodiments of any one of the eighth to tenth aspects, the phage integrase is inducible expression or constitutive expression.

[0195] In some embodiments of any one of the eighth to tenth aspects, the phage integrase is inducible expression.

[0196] In some embodiments of any one of the eighth to tenth aspects, the inducible expression utilizes an inducible promoter such as a lactose promoter (Plac) or an arabinose promoter (Para).

[0197] In some embodiments of the eighth or tenth aspect, the cell that expresses the phage integrase is a prokaryotic cell.

[0198] In some embodiments of the eighth or tenth aspect, the cell that expresses the phage integrase is Escherichia coli.

[0199] In some embodiments of the eighth or tenth aspect, the cell expressing the phage integrase is a genetically engineered bacterium.

[0200] In some embodiments of the eighth or tenth aspect, the cell expressing the phage integrase is a male Escherichia coli strain having an F factor.

[0201] In some specific embodiments of the eighth or tenth aspect, the cell expressing the phage integrase is the TG1 strain.

[0202] In some embodiments of any one of the seventh to tenth aspects, the first plasmid includes the heavy chain VH-CH1 expression unit of the antibody and the attP attachment site, and the second plasmid includes the light chain VL-CL expression unit of the antibody and the attB attachment site.

[0203] In some embodiments of the seventh aspect, the first recombination site and the second recombination site correspond to each other. For example, when the nucleotide sequence of the first recombination site is SEQ ID NO:2, the nucleotide sequence of the second recombination site is SEQ ID NO:4.

[0204] In some embodiments of the eighth or tenth aspect, the first recombination site, the second recombination site, the type of the phage integrase, and the type of the cell expressing the phage integrase correspond to each other.

[0205] In some embodiments of the eighth or tenth aspect, when the first recombination site is determined, the second recombination site, the type of the phage integrase, and the type of the cell expressing the phage integrase applicable to the present invention can be determined.

[0206] In some embodiments of the eighth or tenth aspect, when the second recombination site is determined, the first recombination site applicable to the present invention, the type of the phage integrase, and the type of the cell expressing the phage integrase can be determined.

[0207] In some embodiments of the eighth or tenth aspect, when the type of the phage integrase is determined, the type of the cell expressing the phage integrase applicable to the present invention can be determined.

[0208] In some embodiments of the eighth or tenth aspect, when the nucleotide sequence of the first recombination site is SEQ ID NO:2, the nucleotide sequence of the second recombination site is SEQ ID NO:4, the phage integrase is λ phage integrase, and the cell expressing the phage integrase is TG1 Escherichia coli.

[0209] In some embodiments of the ninth aspect, the first recombination site, the second recombination site, the type of the phage integrase, and the type of the recombinant cell correspond to each other.

[0210] In some embodiments of the ninth aspect, when the first recombination site is determined, the second recombination site, the type of the phage integrase, and the type of the recombinant cell applicable to the present invention can be determined.

[0211] In some embodiments of the ninth aspect, when the second recombination site is determined, the first recombination site, the type of the phage integrase, and the type of the recombinant cell applicable to the present invention can be determined.

[0212] In some embodiments of the ninth aspect, when the type of the phage integrase is determined, the type of the recombinant cell applicable to the present invention can be determined.

[0213] In some embodiments of the ninth aspect, when the nucleotide sequence of the first recombination site is SEQ ID NO:2, the nucleotide sequence of the second recombination site is SEQ ID NO:4, the phage integrase is λ phage integrase, and the recombinant cell is TG1 Escherichia coli.

[0214] In some specific embodiments of the tenth aspect, the method for producing an antibody Fab fragment library may include step (1) of transducing a first plasmid (for example, a plurality of first plasmids having the same attP attachment site as different heavy chain VH-CH1 expression units of an antibody, for example, a plurality of phagemids) into competent cells (for example, TG1 competent cells); step (2) of infecting the competent cells obtained in step (1) with a helper phage (for example, M13 helper phage) to construct a phage library (for example, a phage library containing a plurality of phages having the same attP attachment site as different heavy chain VH-CH1 expression units of an antibody); step (3) of transducing a second plasmid (for example, a plurality of second plasmids having the same attB attachment site as different light chain VL-CL expression units of an antibody) into cells expressing phage integrase (for example, TG1 competent cells expressing λ phage integrase); step (4) of inducing the cells obtained in step (3) to express phage integrase (for example, λ phage integrase); step (5) of infecting the cells expressing phage integrase in step (4) with the phage library (for example, a phage library containing a plurality of phages having the same attP attachment site as different heavy chain VH-CH1 expression units of an antibody); step (6) of infecting the cells obtained in step (5) with a helper phage (for example, M13 helper phage) to construct the antibody Fab fragment library. It may include.

[0215] According to the 11th aspect, the present invention provides the use of the plasmid combination according to the 7th aspect, the recombinant system according to the 8th aspect, or the recombinant cell according to the 9th aspect in the production of an antibody Fab fragment library.

[0216] According to other aspects, the present invention also provides a vector comprising a nucleic acid molecule encoding an antibody or a light or heavy chain thereof according to the present invention, a host cell comprising the vector, and a method for producing the antibody. In some embodiments, the nucleic acid molecule is operably linked to a regulatory sequence, and the regulatory sequence can be recognized by a host cell transformed with the vector. In some embodiments, the method for producing an antibody comprises culturing a host cell. In some embodiments, the method for producing an antibody also comprises recovering the antibody from the host cell culture medium.

[0217] Furthermore, the RSV-specific antibodies described herein can also be used to detect the presence of RSV in biological samples. Antibody-based detection methods are well known in the art and include, for example, ELISA, immunoblotting, radioimmunoassay, immunofluorescence, immunoprecipitation, and other related techniques.

[0218] It should be understood that the above detailed description is for the purpose of making those skilled in the art understand the content of the present invention more clearly and is not intended to limit the present invention in any way. Those skilled in the art can make various modifications and changes to these embodiments.

Examples

[0219] The following examples illustrate, but do not limit, the scope of the present invention.

[0220] [Example 1] Production of a fully humanized recombinant Fab library The content of this example refers to Chinese Invention Patent No. CN108251431B, which is incorporated herein by reference.

[0221] 1.1 Construction of a recombinant expression plasmid containing attB and attP attachment sites Based on the pADG-S plasmid, double digestion with XbaI and KpnI was performed, and a synthetic chloramphenicol resistance gene promoter (Cat promoter, whose coding sequence is shown in SEQ ID NO:1) and an attP attachment site (attP1, whose coding sequence is shown in SEQ ID NO:2) were cloned into the vector pADG-S, and a phagemid vector pHGDisn-attP-new (Figure 1) that contains the attP attachment site and expresses the heavy chain Fd (VH-CH1) structural domain of the antibody was constructed.

[0222] Based on the pADK-S plasmid, double digestion with HindIII and BamHI was performed, and a synthetic CDF replication origin (CDF ori, whose coding sequence is shown in SEQ ID NO:3), an attB attachment site (attB1, whose coding sequence is shown in SEQ ID NO:4), a chloramphenicol resistance gene (CmR, whose coding sequence is shown in SEQ ID NO:5), and a complete ampicillin resistance gene expression element (AmpR, whose coding sequence is shown in SEQ ID NO:6) were cloned into the vector pADK-S, and a prokaryotic expression vector pHKb-attB-new (Figure 2) that contains the attB attachment site and expresses the light chain of the antibody was constructed.

[0223] pHGDisn-attP-new and pHKb-attB-new contain different replication origins and replicate and coexist in the same Escherichia coli cells. Specific recombination at the attP and attB attachment sites is induced by λ phage integrase (λInt), and the newly formed plasmid contains a complete chloramphenicol promoter and coding genes, so it can express chloramphenicol acetyltransferase and show chloramphenicol resistance, facilitating the screening of recombinant plasmids.

[0224] 1.2 Production of TG1 Escherichia coli for λInt gene editing The attB attachment site and the attP attachment site underwent specific recombination within the E. coli cell, generating the attL site and the attR site. This reaction must be carried out under the catalysis of λ phage integrase. 14 By means of gene editing, a gene encoding a λ phage integrase expression element induced by the lactose operon (the nucleotide sequence thereof is shown in SEQ ID NO:7) was inserted into the TG1 E. coli genome. The TG1-λInt recombinant engineering strain was completed by commissioning to GenScript Biotechnology Co., Ltd.

[0225] 1.3 Construction of a fully humanized heavy chain antibody library Referring to Chinese Invention Patent No. CN108251431B, PCR primers were designed using cryopreserved natural human heavy chain variable region cDNA and PCR products of VH1, VH3, and VH5 with introduced mutations as templates. After amplifying the target fragment, double digestion with NcoI and PmlI was performed and cloned into the vector pHGDisn-attP-new. The ligation product was electrotransformed into TG1 competent cells to construct a fully humanized heavy chain antibody library with a diversity of 1.73E+9. When the constructed antibody library was subjected to sequencing analysis, the average correct rate exceeded 85%.

[0226] 1.4 Construction of a fully humanized light chain antibody library Referring to Chinese Invention Patent No. CN108251431B, PCR primers were designed using cryopreserved natural human light chain variable region cDNA and PCR products of VK1 and VL3 with introduced mutations as templates. After amplifying the target fragment, double digestion with NcoI and PmlI was performed and cloned into the vector pHKb-attB-new. The ligation product was electrotransformed into TG1-λInt competent cells to construct a fully humanized light chain antibody library with a diversity of 1.95E+8. When the constructed antibody library was subjected to sequencing, the average correct rate exceeded 90%.

[0227] 1.5 Production of a fully humanized recombinant Fab library The heavy chain library bacterial solution constructed above was inoculated into 200 mL of liquid medium, cultured at 37 °C and 220 rpm until the logarithmic growth phase, infected with M13 helper phage, cultured overnight at 28 °C and 220 rmp to amplify the phage. Then, the purified heavy chain phage library was prepared by the PEG / NaCl precipitation method. After measuring the titer, it was stored frozen at -80 °C until use.

[0228] The fully humanized light chain antibody library produced above was inoculated into 50 mL of liquid medium, IPTG was added until the final concentration reached 0.1 mM, cultured at 37 °C and 220 rpm until the logarithmic growth phase, then the heavy chain phage library was added and infected. After the infection ended, the bacterial solution was evenly spread on a bacterial culture dish and cultured overnight in a constant temperature incubator at 32 °C.

[0229] The bacteria grown on the above culture dish were counted and statistically analyzed. The recombination site and the light chain sequence and heavy chain sequence of a single clone were identified by sequencing. The size of the library after recombination was counted and calculated by the dilution method. It was estimated that the total number of colonies after recombination was 3.80E+12. As a result of sequencing a single clone, all the clones grown on the plate were plasmids after recombination, containing the newly generated attL (attL1, whose coding sequence is shown in SEQ ID NO:8) and attR (attR1, whose coding sequence is shown in SEQ ID NO:9) recombination sites, as well as the complete light chain gene and heavy chain Fd gene, and the correct rate of the antibody gene exceeded 76.5%.

[0230] The colonies on the above bacterial culture dish were collected, inoculated into liquid medium, cultured at 37 °C and 220 rpm until the logarithmic growth phase, then infected with M13 helper phage to produce a purified humanized Fab phage library.

[0231] [Example 2] Production of RSV F protein and production of recombinant antibody 2.1 Production of RSV F protein In the process of manufacturing an RSV F protein monoclonal antibody, F recombinant proteins of different subtypes of the virus are used. These F recombinant proteins include the F protein of RSV A subtype strain A2 (F-A2, whose amino acid sequence is shown in SEQ ID NO: 10) and the F protein of RSV B subtype strain 18537 (F-18537, whose amino acid sequence is shown in SEQ ID NO: 11). Based on the RSV F proteins of two different subtypes, the present inventors constructed pre-fusion F protein mutants of the DS-Cav1 structure 15 and named them RSV-DS-Cav1-A (whose amino acid sequence is shown in SEQ ID NO: 12) and RSV-DS-Cav1-B (whose amino acid sequence is shown in SEQ ID NO: 13), respectively. Since the F protein has post-translational modifications (e.g., glycosylation and disulfide bonds), using a mammalian cell expression system is more advantageous for maintaining the structure and function of the recombinant protein. Furthermore, adding a His tag (His, whose amino acid sequence is shown in SEQ ID NO: 14) to the C-terminus of this recombinant protein is more advantageous for the purification of the recombinant protein and the identification of monoclonal antibody function.

[0232] The RSV-DS-Cav1-A and RSV-DS-Cav1-B genes were synthesized and cloned into an appropriate eukaryotic expression vector (such as pcDNA3.1 from Invitrogen) using conventional molecular biology techniques. Next, the recombinant protein expression plasmid produced was transfected into HEK293 cells (such as HEK293F from Invitrogen) using liposomes (such as 293fectin from Invitrogen) or other cationic transfection reagents (such as PEI), and cultured for 3 to 4 days under serum-free suspension culture conditions. Subsequently, the culture supernatant was obtained by centrifugation or the like. The recombinant protein in the supernatant was further purified using a metal chelate affinity chromatography column (such as HisTrap FF from GE). Then, a desalting column (such as Hitrap desaulting from GE) was used to change the recombinant protein storage buffer to PBS (pH 7.0) or other appropriate buffers. If necessary, the sample was filtered and sterilized, and then aliquoted and stored at -20°C.

[0233] 2.2 Production of Recombinant Antibodies Using ordinary molecular biological means, the nucleotide sequences encoding the heavy-chain variable region and the light-chain variable region of the antibody were cloned into a eukaryotic expression vector (e.g., pcDNA3.1 from Invitrogen) that was fused with the nucleotide sequences encoding the heavy-chain constant region and the light-chain constant region, respectively, to express a combined complete antibody. The heavy-chain constant region of the antibody may be human IgG1 subtype (its amino acid sequence is shown in SEQ ID NO:15), human IgG1 subtype mutant IgG1-YTE (its amino acid sequence is shown in SEQ ID NO:16), mouse IgG2a subtype (its amino acid sequence is shown in SEQ ID NO:17), and the light-chain constant region may be human κ subtype (its amino acid sequence is shown in SEQ ID NO:18), human λ subtype (its amino acid sequence is shown in SEQ ID NO:19), mouse κ subtype (its amino acid sequence is shown in SEQ ID NO:20) or mouse λ subtype (its amino acid sequence is shown in SEQ ID NO:21).

[0234] Using liposomes (e.g., 293 fectin from Invitrogen) or other transfection reagents (e.g., PEI, etc.), the recombinant antibody expression plasmid produced was transfected into HEK293 cells (e.g., HEK293F from Invitrogen) and cultured for 3 to 5 days under serum-free suspension culture conditions. Then, the culture supernatant was obtained by centrifugation or the like. Further purification was performed using a Protein A / G affinity chromatography column (e.g., Mabselect SURE from GE). Then, using a desalting column (e.g., Hitrap desaulting from GE), the recombinant protein storage buffer was changed to PBS (pH 7.0) or other appropriate buffers. If necessary, the antibody sample was filtered and sterilized, and then aliquoted and stored at -20°C.

[0235] [Example 3] Screening of a fully humanized recombinant Fab library with RSV F protein Referring to the literature (the technical procedures of the experiment can be referred to in Chinese Patent Application No. 201510097117.0, and the entire content of this application is incorporated herein by reference), using the recombinant RSV F protein (RSV-DS-Cav1-A / RSV-DS-Cav1-B) produced in Example 2, a solid-phase screening strategy (for the experimental scheme, refer to "Phage Display: A Practical Approach" edited by (USA) Clackson, T. and (USA) Lowman, H.B., translated by Ma Lan et al., Chemical Industry Press, May 2008), the fully humanized recombinant Fab library constructed in Example 1 was screened, and a total of 4 to 6 screenings were performed by binding, elution, neutralization, infection, and amplification. Finally, two Fab antibodies, R3B1h1 and R22B1, that specifically bind to the RSV F protein were obtained.

[0236] [Example 4] Identification of Recombinant Anti-RSV F Protein Monoclonal Antibodies Using ordinary molecular biology methods, the nucleic acid molecules encoding the light and heavy chains of the two molecules of R3B1h1 and R22B1 were cloned into eukaryotic expression vectors respectively to produce recombinant human IgG1-κ format monoclonal antibodies. Also, referring to Patent US 7704505 B2, palivizumab (Hu1129), a human-derived anti-RSV monoclonal antibody (the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 22, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 23), was produced. Referring to Patent US 11186628 B2, nirsevimab (MEDI8897), a human-derived anti-RSV monoclonal antibody (the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 24, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 25), was produced. Referring to Patent US 9963500 B2, clesrovimab (RB1), a human-derived anti-RSV monoclonal antibody (the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 26, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 27), was produced as a positive control antibody.

[0237] 4.1 Verification of the Binding Activity of Recombinant Anti-RSV F Protein Monoclonal Antibodies The prepared RSV-DS-Cav1-A and RSV-DS-Cav1-B were each coated on a 96-well ELISA plate at 3 μg / mL, 100 μL / well and left overnight at 4°C. After blocking with a blocking solution (PBS-0.1%, Tween 20-3%, milk (PBST-3% milk)) at 37°C for 1 hour, each was added to each recombinant anti-RSV F protein monoclonal antibody and allowed to bind at 37°C for 1 hour. The ELISA plate was washed with PBS-0.1% Tween 20 (PBST) buffer, HRP-mouse anti-human IgG (Bioss, bsm-0297M-HRP) was added, and binding was allowed to occur at 37°C for 1 hour. The ELISA plate was washed with PBST buffer, OPD substrate chromogenic solution was added, and after 5 - 10 minutes, the color development was terminated with 1 M H2SO4, and the optical density value was measured at a dual wavelength of 492 nm / 630 nm using a microplate reader. As a result of the ELISA analysis (Figure 3), it was shown that the recombinant anti-RSV F protein monoclonal antibodies R3B1h1 and R22B1 had equivalent binding activities to the two recombinant proteins, RSV-DS-Cav1-A and RSV-DS-Cav1-B.

[0238] 4.2 Analysis of the Epitope of Recombinant Anti-RSV Virus F Protein Monoclonal Antibodies The recombinant protein RSV-DS-Cav1-B was coated on a 96-well ELISA plate (1 μg / mL, 100 μL / well) and left overnight in a refrigerator at 4°C. It was blocked at 37°C for 1 hour using the blocking solution PBST-3% milk. At a predetermined concentration (1×10 11Using each anti-RSV F protein purified phage (R3B1h1 / R22B1 / Clesrovimab / Nirsevimab) at cfu / mL, the recombinant anti-RSV F protein monoclonal antibodies (R3B1h1 / R22B1 / Clesrovimab) were each serially diluted at an initial concentration of 200 μg / mL in a 3-fold gradient with 10 concentration gradients and added to the blocked 96-well ELISA plates at 100 μL / well, and incubated at 37 °C for 1 hour. The ELISA plates were washed with PBST, and then HRP anti-M13 secondary antibody (Beijing Protein Innovation Co., Ltd., 11973-MM05T-H) was added and incubated at 37 °C for 1 hour. The ELISA plates were washed with PBST, OPD substrate chromogenic solution was added, and after 5 - 10 minutes, the color development was terminated with 1 M H2SO4, and the optical density values were measured at a dual wavelength of 492 nm / 630 nm using a microplate reader. As a result of the ELISA analysis shown in Figure 4, the R3B1h1 monoclonal antibody blocked the binding signal between the R22B1 / Nirsevimab phage and the recombinant protein RSV-DS-Cav1-B, and did not affect the binding between the Clesrovimab phage and the recombinant protein RSV-DS-Cav1-B (Figure 4A). The R22B1 monoclonal antibody blocked the binding signal between the R3B1h1 / Nirsevimab phage and the recombinant protein RSV-DS-Cav1-B, and did not affect the binding between the Clesrovimab phage and the recombinant protein RSV-DS-Cav1-B (Figure 4B). The Clesrovimab monoclonal antibody was unable to block the binding between the R3B1h1 / R22B1 / Nirsevimab phage and the recombinant protein RSV-DS-Cav1-B (Figure 4C).

[0239] 4.3 Analysis of the affinity of recombinant anti-RSV F protein monoclonal antibodies Using the Biacore T200, the affinity of anti-RSV F protein monoclonal antibody was measured by surface plasmon resonance technology. Related reagents and consumables such as the amino coupling kit (BR-1000-50), human antibody capture kit (BR-1008-39), CM5 chip (BR100012), and 10×HBS-EP (BR100669) at pH 7.4 were all purchased from GE healthcare. According to the kit instruction manual, the surface of the carboxylated CM5 chip was activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and the anti-human IgG (Fc) antibody (capture antibody) was diluted to 25 μg / mL with 10 mM sodium acetate at pH 5.0, and then injected at a flow rate of 10 μL / min to achieve a coupling amount of about 10,000 response units (RU). After injecting and capturing the antibody, unreacted groups were blocked by injecting 1 M ethanolamine. In the kinetic measurement, the anti-RSV F protein monoclonal antibody was diluted to 0.5 - 1 μg / mL and injected at 10 μL / min to ensure that about 80 RU of the antibody was captured by the anti-human Fc antibody. Then, a series of concentration gradients (e.g., 6.17 nM, 18.5 nM, 55.6 nM, 166.7 nM, 500 nM) were set for RSV-DS-Cav1-A or RSV-DS-Cav1-B, and injected from low concentration to high concentration at 25 °C and 30 μL / min. The binding time was set to 90 s and the dissociation time was set to 3600 s, and 3 M MgCl2 was injected at 10 μL / min for 30 s to regenerate the surface of the chip. Using the Biacore T20 evaluation software version 3.0, the binding and dissociation sensorgrams were fitted by a 1:1 binding model to calculate the association rate (K on ) and the dissociation rate (K off ). The dissociation equilibrium constant (K D ) was calculated by the ratio K off / K on . The fitting results are shown in Table 1 and Table 2.

[0240]

Table 1

[0241]

Table 2

[0242] 4.4 Inhibition of RSV Infection in Hep-2 Cells by Recombinant Anti-RSV F Protein Monoclonal Antibodies 4.4.1 Inhibition of RSV / A2 Infection in Hep-2 Cells by Recombinant Anti-RSV F Protein Monoclonal Antibodies Anti-RSV F protein monoclonal antibodies (R3B1h1, R22B1, Nirsevimab, Clesrovimab, and Palivizumab) and negative isotype control antibodies were diluted in Hep-2 cell maintenance medium (DMEM + 2% FBS + 1% P / S + 2 mM Glu) at an initial concentration of 66.7 nM with a 5-fold serial dilution, for a total of 10 concentration gradients, and added to 96-well sterile full-permeability cell culture plates at 70 μL / well. One frozen viral stock solution of RSV / A2 was placed in a biosafety cabinet for P2 laboratories and slowly thawed, then diluted to 1.5×10 3 PFU / mL in Hep-2 cell maintenance medium. The diluted viral solution was added to the above-diluted antibodies at 70 μL / well and mixed thoroughly until homogeneous. Also, cell control wells (140 μL / well of Hep-2 cell maintenance medium) and cell + RSV / A2 wells (70 μL / well of Hep-2 cell maintenance medium + 70 μL / well of RSV / A2 dilution) were set up separately. After adding all samples, the culture plates were covered and placed in a cell incubator (37 ± 1°C, 5 ± 1% CO2) for 1 h of neutralization. Hep-2 cells in the logarithmic growth phase were digested, centrifuged, and then the cell suspension counted in Hep-2 cell maintenance medium was 4.3×10 5Dilute it into a single-cell suspension of [[unit]] / mL, add 70 μL / well to a neutralized 96-well cell culture plate, place it in a cell incubator (37 ± 1 °C, 5 ± 1% CO2), and incubate for 60 - 72 h. After the end of the virus infection, wash it twice with PBS, add 80% acetone, transfer it to a refrigerator at 2 - 8 °C, and fix it for 30 min. Discard the 80% acetone, wash it twice with PBS, then add PBS and dilute it to the detection antibody palivizumab (100 μL / well) at 10 μg / mL, and incubate at 37 °C in the dark for 60 min. Discard the primary antibody, wash it twice with PBS, then add PBS, add FITC-labeled goat anti-human IgG (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., ZF-0308) at (1:400) at 100 μL / well, and incubate at 37 °C in the dark for 40 min. Discard the secondary antibody, wash it twice with PBS, and detect the chemiluminescence value using a multifunctional microplate reader (SpectraMax I3) by the rapid fluorescent focus inhibition test (RFFIT). As a result (Figure 5), R3B1h1 has the best activity, and IC 50 As a result of comparing the [[value]] values (Table 3), its activity was improved by about 5 times compared to nirsevimab, about 12 times compared to clesrovimab, and about 410 times compared to palivizumab.

[0243]

Table 3

[0244] 4.4.2 Inhibition of RSV / 18537 strain infection of Hep-2 cells by recombinant anti-RSV F protein monoclonal antibody Refer to the scheme of 4.4.1, and detect the neutralizing activity of RSV / 18537 by anti-RSV F protein monoclonal antibodies (R3B1h1, R22B1, nirsevimab, clesrovimab, and palivizumab). As a result (Figure 6), R3B1h1 has the best activity, and IC 50As a result of comparing the values (Table 4), the activity is improved by about 18 times compared to Nirsevimab, about 12 times compared to Clesrovimab, and about 545 times compared to Palivizumab.

[0245]

Table 4

[0246] 4.4.3 Inhibition of Clinically Isolated RSV Infection in Hep-2 Cells by Recombinant Anti-RSV F Protein Monoclonal Antibodies With reference to the scheme of 4.4.1, the neutralizing activities of clinically isolated RSV by anti-RSV F protein monoclonal antibodies (R3B1h1, R22B1, Nirsevimab, Clesrovimab, and Palivizumab) were detected.

[0247] A total of 11 strains were obtained from Guangzhou Women and Children's Medical Center as clinically isolated samples during the RSV virus season in 2021. Among them, 9 strains were of subtype A (numbers are 2033, 2064, 2066, 2406, 2410, 2416, 2425, 2430, and 2438 respectively), and 2 strains were of subtype B (numbers are 2063 and 2427 respectively). As a result (Figure 7), both R3B1h1 and R22B1 can well neutralize RSV clinical isolation samples.

[0248] 〔Sequence Information〕 SEQ ID NO:1 CGAATAAATACCTGTGACGGAAGATCACTTCGCAGAATAAATAAATCCTGGTGTCCCTGTTGATACCGGGAAGCCCTGGGCCAACTTTTGGCGAAAATGAGACGTTGATCGGCACGTAAGAGGTTCCAACTTTCACCATAATGAAATAAGATCACTACCGGGCGTATTTTTTGAGTTATCGAGATTTTCA SEQ ID NO:2 AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACACATTGATGAGCAATGCTTTTTTATAATGCCAACTTTGTACAAAAAAGCTGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATCCAGTCACTATGAATCAACTACTTAGATGGTATTAGTGACCTGTA SEQ ID NO:3 GATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTTTTGCCCTGTAAACGAAAAAACCACCTGGGGAGGTGGTTTGATCGAAGGTTAAGTCAGTTGGGGAACTGCTTAACCGTGGTAACTGGTTTTCGCAGAGCACAGCAACCAAATCTGTCCTTCCAGTGTAGCCGGACTTTGGCGCACACTTCAAGAGCAACCGCGTGTTTAGCTAAACAAATCCTCTGCGAACTCCCAGTTACCAATGGCTGCTGCCAGTGGCGTTTTACCGTGCTTTTCCGGGTTGGACTCAAGTGAACAGTTACCGGATAAGGCGCAGCAGTCGGGCTGAACGGGGAGTTCTTGCTTACAGCCCAGCTTGGAGCGAACGACCTACACCGAGCCGAGATACCAGTGTGTGAGCTATGAGAAAGCGCCACACTTCCCGTAAGGGAGAAAGGCGGAACAGGTATCCGGTAAACGGCAGGGTCGGAACAGGAGAGCGCAAGAGGGAGCGACCCGCCGGAAACGGTGGGGATCTTTAAGTCCTGTCGGGTTTCGCCCGTACTGTCAGATTCATGGTTGAGCCTCACGGCTCCCACAGATGCACCGGAAAAGCGTCTGTTTATGTGAACTCTGGCAGGAGGGCGGAGCCTATGGAAAAACGCCACCGGCGCGGCCCTGCTGTTTTGCCTCACATGTTAGTCCCCTGCTTATCCACGGAATCTGTGGGTAACTTTGTATGTGTCCGCAGCGC SEQ ID NO:4 ACAAGTTTGTACAAAAAAGCAGGCT SEQ ID NO:5 ATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACTATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTTTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAA SEQ ID NO:6 SEQ ID NO:7 SEQ ID NO:8 AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACACATTGATGAGCAATGCTTTTTTATAATGCCAACTTTGTACAAAAAAGCAGGCT SEQ ID NO:9 ACAAGTTTGTACAAAAAAGCTGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATCCAGTCACTATGAATCAACTACTTAGATGGTATTAGTGACCTGTA SEQ ID NO:10 MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN SEQ ID NO:11 MELLIHRSSAIFLTLAVNALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNRLLPIVNQQSCRISNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITTIIIVIIVVLLSLIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSK SEQ ID NO:12 MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO:13 MELLIHRSSAIFLTLAVNALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVCKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTFKVLDLKNYINNRLLPILNQQSCRISNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO:14 HHHHHH SEQ ID NO:15 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:16 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:17 AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO:18 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:19 GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO:20 RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO:21 GQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS SEQ ID NO:22 QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTATYYCARSMITNWYFDVWGAGTTVTVSS SEQ ID NO:23 DIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYQQKPGKAPKLWIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIK SEQ ID NO:24 QVQLVQSGAEVKKPGSSVMVSCQASGGLLEDYIINWVRQAPGQGPEWMGGIIPVLGTVHYGPKFQGRVTITADESTDTAYMELSSLRSEDTAMYYCATETALVVSETYLPHYFDNWGQGTLVTVSS SEQ ID NO:25 DIQMTQSPSSLSAAVGDRVTITCQASQDIVNYLNWYQQKPGKAPKLLIYVASNLETGVPSRFSGSGSGTDFSLTISSLQPEDVATYYCQQYDNLPLTFGGGTKVEIK SEQ ID NO:26 EVQLVESGGGLVRPGRSLRLSCTVSGFSFDDSAMSWVRQAPGKGLEWISFIKSKTYGGTKEYAASVKGRFTISRDDSKNIAYLQMNSLKTEDTAVYYCTRGAPYGGNSDYYYGLDVWGQGTTVTVSS SEQ ID NO:27 DIQMTQSPSSLSASVGDRVTITCRTSQDVRGALAWYQQKPGKAPKLLIFDASSLETGVPSRFSGSGSGTVFTLTISSLQPEDFAAYYCQQFLDFPFTFGQGTRLEIK SEQ ID NO:28 QVQLVQSGAEVKKPGSSVKVSCKASGGLLEDYIINWVRQAPGQGLEWMGGIIPVLGTVHYGPKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCATETAIVVTESYRPHFFDNWGQGTLVTVSS SEQ ID NO:29 DIQMTQSPSSLSAsVGDRVTITCQASQDIVNYLNWYQQKPGKAPKLLIYVASNLETGVPSRFSGSGSGTDFtfTISSLQPEDiATYYCQEFAGLALNFGGGTKVEIK SEQ ID NO:30 QVQLVQSGAEVKKPGSSVMVSCQASGDPLQNYIINWVRQAPGQGPEWMGAIIPALGSAHYGPKFQGRVTITADESTDTAYMELSSLRSEDTAMYYCATETAVIISESYLPHFFDNWGQGTLVTVSS SEQ ID NO:31 DIQMTQSPSSLSAAVGDRVTITCQASQDIVNYLNWYQQKPGKAPKLLIYVASNLETGVPSRFSGSGSGTDFSLTISSLQPEDVATYYCQEYDSLALAFGGGTKVEIK SEQ ID NO:32 DYIIN SEQ ID NO:33 GIIPVLGTVHYGPKFQG SEQ ID NO:34 ETAIVVTESYRPHFFDN SEQ ID NO:35 QASQDIVNYLN SEQ ID NO:36 VASNLET SEQ ID NO:37 QEFAGLALN SEQ ID NO:38 NYIIN SEQ ID NO:39 AIIPALGSAHYGPKFQG SEQ ID NO:40 ETAVIISESYLPHFFDN SEQ ID NO:41 QASQDIVNYLN SEQ ID NO:42 VASNLET SEQ ID NO:43 QEYDSLALA SEQ ID NO:44 TTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA SEQ ID NO:45 CGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTT SEQ ID NO:46 GGTGGCGGCTCCGGTTCCGGTGATTTTGATTATGAAAAAATGGCAAACGCTAATAAGGGGGCTATGACCGAAAATGCCGATGAAAACGCGCTACAGTCTGACGCTAAAGGCAAACTTGATTCTGTCGCTACTGATTACGGTGCTGCTATCGATGGTTTCATTGGTGACGTTTCCGGCCTTGCTAATGGTAATGGTGCTACTGGTGATTTTGCTGGCTCTAATTCCCAAATGGCTCAAGTCGGTGACGGTGATAATTCACCTTTAATGAATAATTTCCGTCAATATTTACCTTCCCTCCCTCAATCGGTTGAATGTCGCCCTTTTGTCTTTGGCGCTGGTAAACCATATGAATTTTCTATTGATTGTGACAAAATAAACTTATTCCGTGGTGTCTTTGCGTTTCTTTTATATGTTGCCACCTTTATGTATGTATTTTCTACGTTTGCTAACATACTGCGTAATAAGGAGTCTTGATAA

[0249] 〔References〕 1. JAMA Pediatr. 2016 Mar;170(3):267-87. 2. Lancet Glob Health. 2017 Oct;5(10):e984-e991. 3. J Gen Virol. 2017 Dec;98(12):2912-2913. 4. J Gen Virol. 1985 Oct;66 (Pt 10):2111-24. 5. J Virol. 2008 Mar;82(5):2040-55. 6. J Virol. 1987 Oct;61(10):3163-6. 7. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7683-7. 8. Virus Res. 2000 Jun;68(1):25-33. 9. Virology. 1993 Nov;197(1):1-11. 10. Gene. 1994 Dec 30;151(1-2):109-13. 11. Kabat, “Sequences of Proteins of Immunological Interest”, National Institutes of Health, Bethesda, Md. (1991). 12. A1-Lazikani, et al., J. Mol. Biol. 273:927-948 (1997). 13. Martin, et al., Proc. Natl. Acad. Sci.USA86:9268-9272 (1989). 14. Annu Rev Biochem. 1989; 58:913-49. 15. Science. 2013 Nov 1;342(6158):592-8.

Claims

1. An antibody against respiratory syncytial virus (RSV) comprising a heavy chain variable region comprising the amino acid sequences of HCDR1, HCDR2 and HCDR3, and a light chain variable region comprising the amino acid sequences of LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of said HCDR1 is represented by SEQ ID NO: 32, the amino acid sequence of said HCDR2 is represented by SEQ ID NO: 33, the amino acid sequence of said HCDR3 is represented by SEQ ID NO: 34, the amino acid sequence of said LCDR1 is represented by SEQ ID NO: 35, the amino acid sequence of said LCDR2 is represented by SEQ ID NO: 36, the amino acid sequence of said LCDR3 is represented by SEQ ID NO: 37, or the amino acid sequence of said HCDR1 is represented by SEQ ID NO: 38, the amino acid sequence of said HCDR2 is represented by SEQ ID NO: 39, the amino acid sequence of said HCDR3 is represented by SEQ ID NO: 40, the amino acid sequence of said LCDR1 is represented by SEQ ID NO: 41, the amino acid sequence of said LCDR2 is represented by SEQ ID NO: 42, the amino acid sequence of said LCDR3 is represented by SEQ ID NO: 43, wherein the HCDR and LCDR amino acid sequences are defined according to Kabat, an antibody.

2. The antibody according to claim 1, wherein the amino acid sequence of the heavy chain variable region of said antibody is represented by SEQ ID NO: 28 or 30.

3. The antibody according to claim 1, wherein the amino acid sequence of the light chain variable region of said antibody is represented by SEQ ID NO: 29 or 31.

4. The amino acid sequence of the heavy chain variable region of said antibody is represented by SEQ ID NO: 28, and the amino acid sequence of the light chain variable region of said antibody is represented by SEQ ID NO: 29, or the amino acid sequence of the heavy chain variable region of said antibody is represented by SEQ ID NO: 30, and the amino acid sequence of the light chain variable region of said antibody is represented by SEQ ID NO: 31, The antibody according to any one of claims 1 to 3.

5. An antibody against respiratory syncytial virus (RSV), wherein the amino acid sequence of the heavy chain variable region of said antibody has at least 90% identity with SEQ ID NO: 28 or 30, and the amino acid sequence of the light chain variable region of said antibody has at least 90% identity with SEQ ID NO: 29 or 31.

6. Said antibody is a neutralizing antibody, and / or The antibody can bind to the F protein of human respiratory syncytial virus (RSV), preferably, the antibody can bind to the recombinant human RSV F protein represented by SEQ ID NO: 12 or 13, and / or The antibody is a Fab fragment, a complete antibody, an F(ab') ２ fragment, or a single-chain Fv fragment (scFv), preferably, the antibody is a Fab fragment, and / or the antibody is a monoclonal antibody. The antibody according to any one of claims 1 to 5.

7. The antibody comprises a heavy chain constant region selected from the IgG1 subtype, IgG2 subtype, or IgG4 subtype, preferably, the heavy chain constant region comprises the sequence of the Fc fragment of the heavy chain constant region of the IgG1 subtype, and the amino acid sequences at positions 252, 254, and 256 of the sequence of the Fc fragment are Y, T, and E, respectively, and the order of the amino acids in the constant region of the antibody is determined according to EU numbering, and / or the antibody comprises a light chain constant region selected from the κ subtype or λ subtype. The antibody according to any one of claims 1 to 6.

8. A nucleic acid molecule encoding the antibody according to any one of claims 1 to 7.

9. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 7 and a pharmaceutically acceptable excipient, diluent, or carrier.

10. The pharmaceutical composition according to claim 9, which is used for preventing or treating RSV-related diseases, preferably, the RSV-related diseases are airway infections such as bronchiolitis and pneumonia.

11. Use of the antibody according to any one of claims 1 to 7, the nucleic acid molecule according to claim 8, or the pharmaceutical composition according to claim 9 or 10 in the manufacture of a medicament for preventing or treating RSV-related diseases, preferably, the RSV-related diseases are airway infections, such as bronchiolitis, and pneumonia.

12. A method for preventing or treating RSV-related diseases, comprising administering to an individual in need thereof the antibody according to any one of claims 1 to 7, the nucleic acid molecule according to claim 8, or the pharmaceutical composition according to claim 9 or 10, preferably, the RSV-related diseases are airway infections, such as bronchiolitis, and pneumonia.

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