Neutralizing antibodies against respiratory syncytial virus and uses thereof

Abstract

Disclosed are an antibody against respiratory syncytial virus (RSV), a nucleic acid molecule encoding the antibody, an expression vector comprising the nucleic acid molecule, a host cell comprising the nucleic acid molecule or vector, and a pharmaceutical composition comprising the antibody. The invention also provides a method for preparing and purifying the antibody and application of the antibody.

Description

[0001] This application is a divisional application of the invention application with an application date of July 22, 2022, application number 202210871810.9, and invention name “Anti-respiratory syncytial virus neutralizing antibodies and their uses”. Field of the Invention

[0002] The present application generally relates to the fields of genetic engineering and antibody drugs; in particular, to antibodies against respiratory syncytial virus (RSV) and the use of the antibodies in preventing or treating respiratory syncytial virus (RSV)-related diseases. Background of the Invention

[0004] Large-capacity, high-quality antibody libraries provide an important source of molecules for screening high-affinity antibodies for any antigen, and have high commercial and application value. At present, most of the high-quality antibody libraries reported at home and abroad are constructed using electroporation technology. However, the size of the antibody library constructed using electroporation technology is limited by the conversion efficiency, and multiple batches of antibody library preparation are required, which takes a long time and consumes a lot of manpower and material resources.

[0005] Respiratory syncytial virus (RSV) is the most important viral pathogen causing acute lower respiratory tract infections (ALRTI) in children under 5 years old worldwide. 1 RSV infection is the leading cause of hospitalization for viral respiratory tract infection in infants and young children, which seriously endangers children's health, especially premature infants, infants with congenital heart disease or primary immunodeficiency. The five countries with the highest incidence of RSV infection contribute nearly half of the global disease burden of RSV-ALRTI. 2 .

[0006] Respiratory syncytial virus (RSV) belongs to the Pneumoviridae family, the Orthopneumovirus genus, and is a non-segmented single-stranded negative-sense RNA virus. 3 According to the difference in surface antigens, it can be divided into two subtypes, A and B. 4 The RSV genome consists of 10 genes encoding 11 proteins. Among them, adhesion protein G and fusion protein F are the main protective antigens on the surface of the virus, which can stimulate the body to produce neutralizing antibodies. 5 G protein has great differences among subtypes, so most antibodies against G protein are subtype-specific antibodies. 6 The F protein is highly conserved among subtypes, and the antibodies induced by the F protein can simultaneously inhibit the infection of RSV A / B viruses.

[0007] The F protein is a type I transmembrane protein, and its inactive precursor (F0) consists of 574 amino acids. 7 , three F0s form a trimer, and when transported through the Golgi apparatus, the host's furin protease cuts F0 between amino acids 109 and 110 and between amino acids 136 and 137. After cutting, the middle 27 amino acid short peptide (P27) is released, and the remaining two segments F2 and F1 are connected by disulfide bonds (Cys69–Cys212 and Cys37–Cys439) to form an active F protein 8 There is a highly hydrophobic fusion peptide (FP) 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 surface of the virus or the cell, its structure is not stable, but in a high-energy metastable pre-fusion conformation (Prefusion glyprotein, Pre-F). 9 Subsequently, the N-terminus of the F1 protein undergoes a series of dramatic structural changes, which induces membrane fusion, thereby achieving the purpose of viral infection of cells. At the same time, this process also causes the F protein to transform from the high-energy metastable Pre-F structure to the stable post-fusion F protein structure (Postfusion glyprotein, Post-F).

[0008] There is currently no vaccine to prevent RSV. The U.S. Food and Drug Administration has approved Palivizumab (trade name Synagis) for the prevention of RSV infection in high-risk infants and young children. Up to 5 injections are required to cover the typical RSV season. Due to its high cost, it cannot be widely used in infants and young children.

[0009] Based on clinical needs, the development of anti-respiratory syncytial virus antibodies has important medical significance for preventing related diseases caused by RSV infection. SUMMARY OF THE INVENTION

[0011] In a first aspect, the present application provides an antibody against respiratory syncytial virus (RSV), comprising a heavy chain variable region comprising an amino acid sequence of HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising an amino acid sequence of LCDR1, LCDR2 and LCDR3, wherein

[0012] The amino acid sequence of the HCDR1 is shown in SEQ ID NO:32, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:33, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:34, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:35, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:36, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:37; or

[0013] The amino acid sequence of the HCDR1 is shown in SEQ ID NO:38, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:39, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:40, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:41, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:42, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:43;

[0014] The HCDR and LCDR amino acid sequences are based on the Kabat definition.

[0015] In some embodiments of the first aspect, the amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO: 28 or 30.

[0016] In some embodiments of the first aspect, the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO: 29 or 31.

[0017] In some embodiments of the first aspect, the amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO: 28, and the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO: 29; or

[0018] The amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO:30, and the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO:31.

[0019] In a second aspect, the present application provides an antibody against respiratory syncytial virus (RSV), wherein the amino acid sequence of the heavy chain variable region of the antibody is at least 90% identical to SEQ ID NO: 28 or 30, and the amino acid sequence of the light chain variable region of the antibody is at least 90% identical to SEQ ID NO: 29 or 31.

[0020] In some embodiments of either of the first and second aspects, the antibody is a neutralizing antibody.

[0021] In some embodiments of either of the first and second aspects, the antibody is capable of binding to the F protein of human respiratory syncytial virus (RSV).

[0022] In some embodiments of either of the first and second aspects, the antibody is a Fab fragment, a whole antibody, a F(ab')2 fragment, or a single-chain Fv fragment (scFv).

[0023] In some embodiments of either of the first and second aspects, the antibody is a monoclonal antibody.

[0024] In some embodiments of either of the first and second aspects, the antibody comprises a heavy chain constant region selected from the IgG1 subtype, IgG2 subtype, or IgG4 subtype.

[0025] In some embodiments of either of the first aspect and the second aspect, the heavy chain constant region comprises the Fc segment sequence of the IgG1 subtype heavy chain constant region and the amino acid sequences at positions 252, 254, and 256 of the Fc segment sequence are Y, T, and E, respectively, wherein the amino acid sequence of the antibody constant region is determined according to EU numbering.

[0026] In some embodiments of either of the first and second aspects, the antibody comprises a light chain constant region selected from the kappa subtype or the lambda subtype.

[0027] In a third aspect, the present application provides a nucleic acid molecule encoding the antibody described in the first aspect or the second aspect.

[0028] In a fourth aspect, the present application provides a pharmaceutical composition comprising the antibody described in the first aspect or the second aspect and a pharmaceutically acceptable excipient, diluent or carrier.

[0029] In a fifth aspect, the present application provides 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 preparation of a drug for preventing or treating RSV-related diseases.

[0030] In a sixth aspect, the present application provides a method for preventing or treating RSV-related diseases, comprising administering the antibody described in the first aspect or the second aspect, or the pharmaceutical composition described in the fourth aspect to an individual in need thereof.

[0031] In a seventh aspect, the present application provides a plasmid combination for expressing an antibody Fab fragment library, comprising a first plasmid and a second plasmid, wherein

[0032] The first plasmid comprises an antibody heavy chain VH-CH1 expression unit and a first recombination site;

[0033] The second plasmid comprises an antibody light chain VL-CL expression unit and a second recombination site; and

[0034] The first recombination site is different from the second recombination site.

[0035] In some embodiments of the seventh aspect, the plasmid combination comprises a plurality of the first plasmids, and the plurality of the first plasmids carry different antibody heavy chain VH-CH1 expression units and have the same first recombination site.

[0036] In some embodiments of the seventh aspect, the plasmid combination comprises a plurality of the second plasmids, and the plurality of the second plasmids carry different antibody light chain VL-CL expression units and have the same second recombination site.

[0037] In some embodiments of the seventh aspect, the first plasmid and / or the second plasmid is a phagemid.

[0038] In some embodiments of the seventh aspect, the first plasmid and the second plasmid comprise different origins of replication.

[0039] In some embodiments of the seventh aspect, the replication origin is selected from one or more of the following: pBRori, CDF ori, the replication origin of filamentous bacteriophage M13 (f1 ori) and p15Aori.

[0040] In some embodiments of the seventh 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 bacteria, respectively.

[0041] In some embodiments of the seventh aspect, the first recombination site is attP and the second recombination site is attB.

[0042] In some embodiments of the seventh aspect, the first recombination site and the second recombination site are located at the multiple cloning restriction sites of the first plasmid and the second plasmid, respectively.

[0043] In some embodiments of the seventh aspect, the antibody light chain constant region CL is a human κ light chain constant region or a human λ light chain constant region.

[0044] In some embodiments of the seventh aspect, the antibody heavy chain constant region CH1 segment is selected from IgG1, IgG2, IgG3 or IgG4 subtype.

[0045] In some embodiments of the seventh aspect, the first plasmid and / or the second plasmid comprises a resistance gene coding region.

[0046] In some embodiments of the seventh aspect, the 3' end of the nucleic acid molecule encoding the CH1 segment of the antibody heavy chain constant region is fused to the 5' end of the nucleic acid molecule encoding the gIII protein of the filamentous phage M13.

[0047] In some embodiments of the seventh aspect, the resistance gene is an antibiotic resistance gene.

[0048] In some embodiments of the seventh aspect, the resistance gene is selected from the group consisting of: a chloramphenicol resistance gene (CmR), an ampicillin resistance gene (Ampr), a kanamycin resistance gene (KaR), and a tetracycline resistance gene (TetR).

[0049] In an eighth aspect, the present application provides a recombinant system for expressing an antibody Fab fragment library, comprising a first plasmid, a second plasmid and a cell expressing a phage integrase; wherein

[0050] The first plasmid comprises an antibody heavy chain VH-CH1 expression unit and a first recombination site;

[0051] The second plasmid comprises an antibody light chain VL-CL expression unit and a second recombination site; and

[0052] The first recombination site is different from the second recombination site.

[0053] In some embodiments of the eighth aspect, the recombination system comprises a plurality of the first plasmids, and the plurality of the first plasmids carry different antibody heavy chain VH-CH1 expression units and have the same first recombination site.

[0054] In some embodiments of the eighth aspect, the recombination system comprises a plurality of the second plasmids, and the plurality of the second plasmids carry different antibody light chain VL-CL expression units and have the same second recombination sites.

[0055] In some embodiments of the eighth aspect, the first plasmid and / or the second plasmid is a phagemid.

[0056] In some embodiments of the eighth aspect, the first plasmid and the second plasmid comprise different origins of replication.

[0057] In some embodiments of the eighth aspect, the replication origin is selected from one or more of the following: pBRori, CDF ori, the replication origin of filamentous phage M13 (f1 ori) and p15Aori.

[0058] 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 bacteria, respectively.

[0059] In some embodiments of the eighth aspect, the first recombination site is attP and the second recombination site is attB.

[0060] In some embodiments of the eighth aspect, the first recombination site and the second recombination site are located at the multiple cloning restriction sites of the first plasmid and the second plasmid, respectively.

[0061] In some embodiments of the eighth aspect, the antibody light chain constant region CL is a human κ light chain constant region or a human λ light chain constant region.

[0062] In some embodiments of the eighth aspect, the antibody heavy chain constant region CH1 segment is selected from IgG1, IgG2, IgG3 or IgG4 subtype.

[0063] In some embodiments of the eighth aspect, the first plasmid and / or the second plasmid comprises a resistance gene coding region.

[0064] In some embodiments of the eighth aspect, the 3' end of the nucleic acid molecule encoding the CH1 segment of the antibody heavy chain constant region is fused to the 5' end of the nucleic acid molecule encoding the gIII protein of the filamentous phage M13.

[0065] In some embodiments of the eighth aspect, the resistance gene is an antibiotic resistance gene.

[0066] In some embodiments of the eighth aspect, the resistance gene is selected from the group consisting of: a chloramphenicol resistance gene (CmR), an ampicillin resistance gene (Ampr), a kanamycin resistance gene (KaR), and a tetracycline resistance gene (TetR).

[0067] In some embodiments of the eighth aspect, the bacteriophage integrase is a tyrosine integrase.

[0068] In some embodiments of the eighth aspect, the bacteriophage integrase is a phage lambda integrase.

[0069] In some embodiments of the eighth aspect, the bacteriophage integrase is inducibly expressed or constitutively expressed.

[0070] In some embodiments of the eighth aspect, the bacteriophage integrase is expressed inducibly.

[0071] In some embodiments of the eighth aspect, the cell expressing bacteriophage integrase is a prokaryotic cell.

[0072] In some embodiments of the eighth aspect, the cell expressing bacteriophage integrase is Escherichia coli.

[0073] In some embodiments of the eighth aspect, the cell expressing bacteriophage integrase is a genetically engineered bacterium.

[0074] In some embodiments of the eighth aspect, the first plasmid, the second plasmid and the cell expressing bacteriophage integrase each exist independently, or at least one of the first plasmid and the second plasmid has been introduced into the cell expressing bacteriophage integrase.

[0075] In a ninth aspect, the present application provides a recombinant cell for expressing an antibody Fab fragment library, which comprises a first plasmid, a second plasmid and expresses a phage integrase; wherein

[0076] The first plasmid comprises an antibody heavy chain VH-CH1 expression unit and a first recombination site;

[0077] The second plasmid comprises an antibody light chain VL-CL expression unit and a second recombination site; and

[0078] The first recombination site is different from the second recombination site.

[0079] In some embodiments of the ninth aspect, the recombinant cell comprises a plurality of the first plasmids, and the plurality of the first plasmids carry different antibody heavy chain VH-CH1 expression units and have the same first recombination site.

[0080] In some embodiments of the ninth aspect, the recombinant cell comprises a plurality of the second plasmids, and the plurality of the second plasmids carry different antibody light chain VL-CL expression units and have the same second recombination sites.

[0081] In some embodiments of the ninth aspect, the first plasmid and / or the second plasmid is a phagemid.

[0082] In some embodiments of the ninth aspect, the first plasmid and the second plasmid comprise different origins of replication.

[0083] In some embodiments of the ninth aspect, the replication origin is selected from one or more of the following: pBRori, CDF ori, the replication origin of filamentous bacteriophage M13 (f1 ori) and p15Aori.

[0084] In some embodiments of the ninth 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.

[0085] In some embodiments of the ninth aspect, the first recombination site is attP and the second recombination site is attB.

[0086] In some embodiments of the ninth aspect, the first recombination site and the second recombination site are located at the multiple cloning restriction sites of the first plasmid and the second plasmid, respectively.

[0087] In some embodiments of the ninth aspect, the antibody light chain constant region CL is a human kappa light chain constant region or a human lambda light chain constant region.

[0088] In some embodiments of the ninth aspect, the antibody heavy chain constant region CH1 segment is selected from IgG1, IgG2, IgG3 or IgG4 subtype.

[0089] In some embodiments of the ninth aspect, the first plasmid and / or the second plasmid comprises a resistance gene coding region.

[0090] In some embodiments of the ninth aspect, the 3' end of the nucleic acid molecule encoding the CH1 segment of the antibody heavy chain constant region is fused to the 5' end of the nucleic acid molecule encoding the gIII protein of the filamentous phage M13.

[0091] In some embodiments of the ninth aspect, the resistance gene is an antibiotic resistance gene.

[0092] In some embodiments of the ninth aspect, the resistance gene is selected from the group consisting of: a chloramphenicol resistance gene (CmR), an ampicillin resistance gene (Ampr), a kanamycin resistance gene (KaR), and a tetracycline resistance gene (TetR).

[0093] In some embodiments of the ninth aspect, the bacteriophage integrase is a tyrosine integrase.

[0094] In some embodiments of the ninth aspect, the bacteriophage integrase is a phage lambda integrase.

[0095] In some embodiments of the ninth aspect, the bacteriophage integrase is inducibly expressed or constitutively expressed.

[0096] In some embodiments of the ninth aspect, the bacteriophage integrase is expressed inducibly.

[0097] In some embodiments of the ninth aspect, the recombinant cell is a prokaryotic cell.

[0098] In some embodiments of the ninth aspect, the recombinant cell is Escherichia coli.

[0099] In some embodiments of the ninth aspect, the recombinant cell is a genetically engineered bacterium.

[0100] In a tenth aspect, the present application provides a method for preparing an antibody Fab fragment library, which comprises transducing a first plasmid and a second plasmid into a cell expressing a phage integrase; wherein

[0101] The first plasmid comprises an antibody heavy chain VH-CH1 expression unit and a first recombination site;

[0102] The second plasmid comprises an antibody light chain VL-CL expression unit and a second recombination site; and

[0103] The first recombination site is different from the second recombination site.

[0104] In some embodiments of the tenth aspect, a plurality of the first plasmids are transduced into the cell expressing phage integrase, wherein the plurality of the first plasmids carry different antibody heavy chain VH-CH1 expression units and have the same first recombination site.

[0105] In some embodiments of the tenth aspect, a plurality of the second plasmids are transduced into the cell expressing phage integrase, wherein the plurality of the second plasmids carry different antibody light chain VL-CL expression units and have the same second recombination site.

[0106] In some embodiments of the tenth aspect, transducing the first plasmid and / or the second plasmid into the cell expressing phage integrase comprises the following steps:

[0107] (1) transducing the first plasmid and / or the second plasmid into competent cells;

[0108] (2) using the helper phage to infect the competent cells obtained in step (1) to construct a phage library; and

[0109] (3) Transducing the phage library obtained in step (2) into the cells expressing phage integrase.

[0110] In some embodiments of the tenth aspect, the competent cells are prokaryotic cells.

[0111] In some embodiments of the tenth aspect, the competent cells do not express bacteriophage integrase.

[0112] In some embodiments of the tenth aspect, the helper phage is M13 helper phage.

[0113] In some embodiments of the tenth aspect, the first plasmid and / or the second plasmid is a phagemid.

[0114] In some embodiments of the tenth aspect, the first plasmid and the second plasmid comprise different origins of replication.

[0115] In some embodiments of the tenth aspect, the replication origin is selected from one or more of the following: pBRori, CDF ori, the replication origin of filamentous bacteriophage M13 (f1 ori) and p15A ori.

[0116] In some embodiments of the tenth 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 bacteria, respectively.

[0117] In some embodiments of the tenth aspect, the first recombination site is attP and the second recombination site is attB.

[0118] In some embodiments of the tenth aspect, the first recombination site and the second recombination site are located at the multiple cloning restriction sites of the first plasmid and the second plasmid, respectively.

[0119] In some embodiments of the tenth aspect, the antibody light chain constant region CL is a human κ light chain constant region or a human λ light chain constant region.

[0120] In some embodiments of the tenth aspect, the antibody heavy chain constant region CH1 segment is selected from IgG1, IgG2, IgG3 or IgG4 subtype.

[0121] In some embodiments of the tenth aspect, the first plasmid and / or the second plasmid comprises a resistance gene coding region.

[0122] In some embodiments of the tenth aspect, the resistance gene is selected from the group consisting of: a chloramphenicol resistance gene (CmR), an ampicillin resistance gene (Ampr), a kanamycin resistance gene (KaR), and a tetracycline resistance gene (TetR).

[0123] In some embodiments of the tenth aspect, the bacteriophage integrase is a tyrosine integrase.

[0124] In some embodiments of the tenth aspect, the bacteriophage integrase is a phage lambda integrase.

[0125] In some embodiments of the tenth aspect, the bacteriophage integrase is inducibly expressed or constitutively expressed.

[0126] In some embodiments of the tenth aspect, the bacteriophage integrase is expressed inducibly.

[0127] In some embodiments of the tenth aspect, the cell expressing bacteriophage integrase is a prokaryotic cell.

[0128] In some embodiments of the tenth aspect, the cell expressing bacteriophage integrase is Escherichia coli.

[0129] In some embodiments of the tenth aspect, the cell expressing bacteriophage integrase is a genetically engineered bacterium.

[0130] In the eleventh aspect, the present application provides use of the plasmid combination described in the seventh aspect, the recombination system described in the eighth aspect, or the recombinant cell described in the ninth aspect in preparing an antibody Fab fragment library. BRIEF DESCRIPTION OF THE DRAWINGS

[0132] Figure 1 A schematic diagram of the structure of the phagemid vector pHGDisn-attP-new is shown.

[0133] Figure 2 A schematic diagram of the structure of the prokaryotic expression vector pHKb-attB-new is shown.

[0134] Figure 3 The results of ELISA analysis of the binding activity of recombinant anti-RSV F protein monoclonal antibodies (R3B1h1, R22B1 and positive control antibody) to RSV-DS-Cav1-A (Figure A) and RSV-DS-Cav1-B (Figure B) are shown.

[0135] Figure 4 The blocking results of anti-RSV F protein monoclonal antibodies (R3B1h1, R22B1 and Clesrovimab) against RSV F protein phage binding to RSV-DS-Cav1-B are shown by ELISA analysis; wherein, AC are the blocking results of R3B1h1, R22B1 and Clesrovimab against RSV F protein purified phage binding to RSV-DS-Cav1-B, respectively.

[0136] Figure 5 The results of the Rapid Fluorescent Focus Inhibition Test (RFFIT) detecting the inhibition of RSV / A2 strain infection of Hep-2 cells by anti-RSV F protein monoclonal antibodies are shown.

[0137] Figure 6 The results of RFFIT detection of anti-RSV F protein monoclonal antibodies inhibiting RSV / 18537 strain infection of Hep-2 cells are shown.

[0138] Figure 7 The results show that anti-RSV F protein monoclonal antibodies inhibited the infection of Hep-2 cells by RSV isolated from clinical samples.

[0139] Sequence Description

[0140] SEQ ID NO: 1 shows a nucleotide sequence encoding a chloramphenicol resistance gene promoter (Cat promoter) derived from Escherichia coli.

[0141] SEQ ID NO: 2 shows a nucleotide sequence encoding an attP attachment site derived from Escherichia phage Lambda.

[0142] SEQ ID NO: 3 shows a nucleotide sequence encoding CDF ori derived from Escherichia coli.

[0143] SEQ ID NO: 4 shows a nucleotide sequence encoding an attB attachment site derived from Escherichia coli.

[0144] SEQ ID NO: 5 shows the nucleotide sequence of a chloramphenicol resistance gene derived from Escherichia coli.

[0145] SEQ ID NO: 6 shows the nucleotide sequence of the complete ampicillin resistance gene expression element derived from Escherichia coli.

[0146] SEQ ID NO: 7 shows the nucleotide sequence of the λ phage integrase expression element derived from the lactose operon-induced λ phage of Escherichia.

[0147] SEQ ID NO:8 shows the nucleotide sequence encoding the attL recombination site.

[0148] SEQ ID NO:9 shows the nucleotide sequence encoding the attR recombination site.

[0149] SEQ ID NO: 10 shows the amino acid sequence of the F protein of RSV subtype A strain A2.

[0150] SEQ ID NO:11 shows the amino acid sequence of the F protein of RSV subtype B strain 18537.

[0151] SEQ ID NO: 12 shows the amino acid sequence of the pre-fusion F protein mutant RSV-DS-Cav1-A of the DS-Cav1 structure.

[0152] SEQ ID NO: 13 shows the amino acid sequence of the pre-fusion F protein mutant RSV-DS-Cav1-B of the DS-Cav1 structure.

[0153] SEQ ID NO: 14 shows the amino acid sequence of the His tag.

[0154] SEQ ID NO: 15 shows the amino acid sequence of the heavy chain constant region of homo sapiens IgG1 subtype.

[0155] SEQ ID NO: 16 shows the amino acid sequence of the mutant IgG1-YTE of the heavy chain constant region of homo sapiens IgG1 subtype.

[0156] SEQ ID NO: 17 shows the amino acid sequence of the mouse (mus musculus) IgG2a subtype heavy chain constant region.

[0157] SEQ ID NO: 18 shows the amino acid sequence of the human (homo sapiens) kappa subtype light chain constant region.

[0158] SEQ ID NO: 19 shows the amino acid sequence of the human (homo sapiens) lambda subtype light chain constant region.

[0159] SEQ ID NO: 20 shows the amino acid sequence of the mouse (mus musculus) kappa subtype light chain constant region.

[0160] SEQ ID NO: 21 shows the amino acid sequence of the mouse (mus musculus) lambda subtype light chain constant region.

[0161] SEQ ID NO: 22 shows the amino acid sequence of the heavy chain variable region of the humanized anti-RSV monoclonal antibody palivizumab (Hu1129).

[0162] SEQ ID NO: 23 shows the amino acid sequence of the light chain variable region of the humanized anti-RSV monoclonal antibody palivizumab (Hu1129).

[0163] SEQ ID NO: 24 shows the amino acid sequence of the heavy chain variable region of the fully human anti-RSV monoclonal antibody Nirsevimab (MEDI8897).

[0164] SEQ ID NO: 25 shows the amino acid sequence of the light chain variable region of the fully human anti-RSV monoclonal antibody Nirsevimab (MEDI8897).

[0165] SEQ ID NO: 26 shows the amino acid sequence of the heavy chain variable region of the fully human anti-RSV monoclonal antibody Clesrovimab (RB1).

[0166] SEQ ID NO: 27 shows the amino acid sequence of the light chain variable region of the fully human anti-RSV monoclonal antibody Clesrovimab (RB1).

[0167] SEQ ID NO: 28 shows the amino acid sequence of the heavy chain variable region of the anti-RSV F protein Fab antibody R3B1h1.

[0168] SEQ ID NO: 29 shows the amino acid sequence of the light chain variable region of the anti-RSV F protein Fab antibody R3B1h1.

[0169] SEQ ID NO: 30 shows the amino acid sequence of the heavy chain variable region of the anti-RSV F protein Fab antibody R22B1.

[0170] SEQ ID NO: 31 shows the amino acid sequence of the light chain variable region of the anti-RSV F protein Fab antibody R22B1.

[0171] SEQ ID NOs: 32-34 respectively show the amino acid sequences of the heavy chain variable regions HCDR1, HCDR2 and HCDR3 of the anti-RSV F protein Fab antibody R3B1h1.

[0172] SEQ ID NOs: 35-37 respectively show the amino acid sequences of the light chain variable regions LCDR1, LCDR2 and LCDR3 of the anti-RSV F protein Fab antibody R3B1h1.

[0173] SEQ ID NOs: 38-40 respectively show the amino acid sequences of the heavy chain variable regions HCDR1, HCDR2 and HCDR3 of the anti-RSV F protein Fab antibody R22B1.

[0174] SEQ ID NOs: 41-43 respectively show the amino acid sequences of the light chain variable regions LCDR1, LCDR2 and LCDR3 of the anti-RSV F protein Fab antibody R22B1.

[0175] SEQ ID NO: 44 shows a nucleotide sequence encoding pBR ori derived from Escherichia coli.

[0176] SEQ ID NO:45 shows a nucleotide sequence encoding f1 ori derived from f1 phage.

[0177] SEQ ID NO: 46 shows a nucleotide sequence encoding the gIII protein of the filamentous phage M13 derived from the M13 phage. DETAILED DESCRIPTION OF THE INVENTION

[0179] By using the specific recombination system or recombinant cells of λ phage integrase (Int), the specific recombination integration and excision of the target nucleotide sequence in vivo and in vitro can be achieved through genetic engineering. It has been reported that the use of heat-induced genetically engineered bacteria expressing Int integrase can achieve specific recombination of antibody light chain expression plasmids and heavy chain expression plasmids in cells to produce a complete plasmid expressing functional Fab. 10 Thermo Fisher's Gateway homologous recombination technology uses the specific recombination principle of λ phage integrase, eliminating the need for classic gene cloning steps such as enzyme digestion and ligation. The in vitro recombination efficiency is as high as 95%. The Gateway kit used to construct cDNA libraries can achieve a library diversity of 1.0E+7.

[0180] The present application utilizes the lambda phage integrase recombination system or recombinant cells to overcome the disadvantage of insufficient DNA conversion efficiency during the construction of the antibody library through efficient phage infection. Phages packaged with antibody heavy chain expression units (e.g., heavy chain VH-CH1 expression units) are used to infect Escherichia coli that can induce the expression of Int integrase and contain a plasmid of antibody light chain expression units, and specific recombination of antibody heavy chain expression units (e.g., heavy chain VH-CH1 expression units) and light chain expression units is achieved in vivo through Int integrase, and a large-capacity Fab antibody library (more than 1.0E+12) is obtained by antibiotic screening.

[0181] The inventor of the present application utilizes the Fab antibody library screening of the above-mentioned structure to obtain new Fab antibodies for RSV. In many aspects of the present application, provide new antibodies for RSV, nucleic acid molecules encoding the antibody, vectors comprising the nucleic acid molecules, host cells comprising the nucleic acid molecules or vectors, compositions comprising the antibody, methods for preparing and purifying the antibody, and medical and biological applications of the antibody. According to the amino acid sequence of the variable region of the antibody provided by the present application, full-length antibody molecules can be constructed to prevent or treat the disease relevant to RSV as medicine.

[0182] The practice of the present application employs, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, and immunology within the skill of the art.

[0183] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0184] definition

[0185] As used herein, the term "recombination site" refers to a site in a phage genome and a bacterial genome for phage integration. In some embodiments of the present application, the first recombination site and the second recombination site may be sites for phage attachment in the respective genomes of the phage and its host bacteria, including the following situations: the first recombination site is a site in the phage genome for phage attachment and the second recombination site is a site in the genome of the host bacteria of the phage for phage attachment; or the second recombination site is a site in the phage genome for phage attachment and the first recombination site is a site in the genome of the host bacteria of the phage for phage attachment.

[0186] As used herein, the term "plasmid" refers to a DNA molecule other than chromosomes (or nucleoids) in organisms such as bacteria, yeast and actinomycetes, which exists in the cytoplasm (except for yeast, whose 2μm plasmid exists in the nucleus), has the ability to replicate autonomously, so that it can also maintain a constant copy number in daughter cells and express the genetic information it carries, and is a closed circular double-stranded DNA molecule. The term "plasmid" used herein covers bacterial plasmids, phagemids, etc. "Bacterial plasmid" is a commonly used vector in DNA recombination technology, and is a tool that can deliver a useful exogenous gene into a recipient cell for proliferation and expression through genetic engineering. "Phagemid" is a vector formed by the combination of a plasmid vector and a single-stranded phage vector, containing a single-stranded phage packaging sequence, a replicon, and a plasmid replicon, a cloning site, a marker gene, etc. In host cells such as Escherichia coli, it can be replicated in the form of a normal double-stranded plasmid DNA molecule, and when a helper phage is present, it is replicated according to the rolling circle model to produce single-stranded DNA and packaged into phage particles.

[0187] As used herein, the term "attP attachment site" refers to a special sequence on phage DNA that contains a 15 bp core region that is identical to the host chromosome sequence.

[0188] As used herein, the term "attB attachment site" refers to an attachment site in the bacterial genome, which contains a 15 bp core region that is identical to the phage genome sequence.

[0189] As used herein, the term "bacteriophage integrase" refers to a protein that has type I topoisomerase activity, can specifically bind to attP and attB attachment sites, and is essential for integrating bacteriophage into the host genome through site-specific recombination.

[0190] As used herein, the term "origin of replication" refers to the starting site of plasmid replication, consisting of the origin replicon (ORI) and its regulatory elements.

[0191] 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, polypeptides, and the like. "Antibodies" as used herein include not only complete (i.e., full-length) antibodies, but also antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), variants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, double antibodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configurations of immunoglobulin molecules comprising antigen recognition sites of desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0192] Typically, a complete or full-length antibody comprises two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and the 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). A full-length antibody can be any type of antibody, such as IgD, IgE, IgG, IgA or IgM (or subclasses thereof), but the antibody need not belong to any particular class. Immunoglobulins can be assigned to different classes based on the antibody amino acid sequence of the heavy chain constant domain. Typically, there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided 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 structure and three-dimensional structure of different classes of immunoglobulins are well known.

[0193] As used herein, the term "antigen binding fragment or antigen binding portion" refers to a portion or region of a complete antibody molecule that is responsible for binding to an antigen. The antigen binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of VH and VL typically contains three complementary determining regions, CDR1, CDR2, and CDR3.

[0194] It is well known to those skilled in the art that the complementarity determining region (CDR, usually CDR1, CDR2 and CDR3) is the region in the variable region that has the greatest impact on the affinity and specificity of the antibody. There are two common ways to define the CDR amino acid sequence of VH or VL, namely the Chothia definition and the Kabat definition. 11-13For the variable region amino acid sequence of a given antibody, the CDR amino acid sequence in the VH and VL amino acid sequences can be determined according to the Chothia definition or the Kabat definition. In the embodiments of the present application, the Kabat definition of the CDR amino acid sequence is used.

[0195] For a given antibody variable region amino acid sequence, the CDR amino acid sequence in the variable region amino acid sequence can be analyzed in a variety of ways, for example, it can be determined using the online software Abysis (http: / / www.abysis.org / ).

[0196] 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) a F(ab')2 fragment, which may be a bivalent fragment having two Fab' fragments connected by a disulfide bridge at the hinge region (i.e., a dimer of Fab'); (3) an Fv fragment having the VL and VH domains of a single arm of an antibody; (4) a single-chain Fv (scFv), which may be a single polypeptide chain consisting of a VH domain and a VL domain via a peptide connector; and (5) (scFv)2, which may comprise two VH domains connected by a peptide connector and two VL domains, wherein the two VL domains are combined with the two VH domains via a disulfide bridge.

[0197] In some specific embodiments of the present application, the antibody is a Fab fragment, ie, a monovalent fragment having a VL-CL chain and a VH-CH1 chain.

[0198] As used herein, the term "Fd fragment" refers to a fragment consisting of the heavy chain variable region VH and the heavy chain first constant region CH1 of an antibody.

[0199] 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 antigen epitope.

[0200] As used herein, the term "neutralizing antibody" refers to an antibody that can bind to antigens on the surface of pathogenic microorganisms, thereby preventing the pathogenic microorganisms from adhering to target cell receptors, or preventing the fusion of viral envelopes and cell membranes to prevent invasion of cells.

[0201] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may occur in minor individuals.

[0202] In a first aspect, the present application provides an antibody against respiratory syncytial virus (RSV), comprising a heavy chain variable region comprising an amino acid sequence of HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising an amino acid sequence of LCDR1, LCDR2 and LCDR3, wherein

[0203] The amino acid sequence of the HCDR1 is shown in SEQ ID NO:32, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:33, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:34, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:35, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:36, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:37; or

[0204] The amino acid sequence of the HCDR1 is shown in SEQ ID NO:38, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:39, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:40, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:41, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:42, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:43;

[0205] The HCDR and LCDR amino acid sequences are based on the Kabat definition.

[0206] In some embodiments of the first aspect, the amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO: 28 or 30.

[0207] In some embodiments of the first aspect, the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO: 29 or 31.

[0208] In some embodiments of the first aspect, the amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO: 28, and the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO: 29; or

[0209] The amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO:30, and the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO:31.

[0210] In a second aspect, the present application provides an antibody against respiratory syncytial virus (RSV), wherein the amino acid sequence of the heavy chain variable region of the antibody is at least 90% identical to SEQ ID NO: 28 or 30, and the amino acid sequence of the light chain variable region of the antibody is at least 90% identical to SEQ ID NO: 29 or 31.

[0211] In some embodiments of the second aspect, the amino acid sequence of the antibody heavy chain variable region is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 28 or 30.

[0212] In some embodiments of the second aspect, the amino acid sequence of the antibody light chain variable region is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 29 or 31.

[0213] 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 NOs: 28 and 30 by about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions and / or additions.

[0214] 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 NOs: 29 and 31 by about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions and / or additions.

[0215] In some embodiments of the second aspect, the C-terminal or N-terminal region of the amino acid sequence shown in any one of SEQ ID NOs: 28 and 30 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still maintaining similar function of the heavy chain variable region of the antibody.

[0216] 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 can be added to the C-terminal or N-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 the function of the heavy chain variable region of the similar antibody.

[0217] 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 added or deleted in the region outside the C-terminus or N-terminus of the amino acid sequence shown in any one of SEQ ID NOs: 28 and 30, as long as the altered amino acid sequence substantially maintains the function of the heavy chain variable region of the similar antibody.

[0218] In some embodiments of the second aspect, the C-terminal or N-terminal region of the amino acid sequence shown in any one of SEQ ID NOs: 29 and 31 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still maintaining similar function of the light chain variable region of the antibody.

[0219] 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 can be added to the C-terminal or N-terminal region of the amino acid sequence shown in any one of SEQ ID NOs: 29 and 31, and the resulting amino acid sequence still maintains the function of the light chain variable region of the similar antibody.

[0220] 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 added or deleted in the region other than the C-terminus or N-terminus of the amino acid sequence shown in any one of SEQ ID NOs: 29 and 31, as long as the altered amino acid sequence substantially maintains the function of the light chain variable region of the similar antibody.

[0221] In some embodiments of either of the first and second aspects, the antibody is a neutralizing antibody.

[0222] In some embodiments of either of the first and second aspects, the antibody is capable of binding to the F protein of human respiratory syncytial virus (RSV).

[0223] In some specific embodiments of any one of the first and second aspects, the F protein of human respiratory syncytial virus (RSV) is a recombinant human RSV F protein, such as the recombinant human RSV F protein shown in SEQ ID NO: 12 or 13.

[0224] In some embodiments of either of the first and second aspects, the antibody is a Fab fragment, a whole antibody, a F(ab')2 fragment, or a single-chain Fv fragment (scFv).

[0225] In some specific embodiments of any one of the first and second aspects, the antibody is a Fab fragment.

[0226] In some embodiments of either of the first and second aspects, the antibody is a monoclonal antibody.

[0227] In some embodiments of either of the first and second aspects, the antibody comprises a heavy chain constant region selected from the IgG1 subtype, IgG2 subtype, or IgG4 subtype.

[0228] In some embodiments of either of the first aspect and the second aspect, the heavy chain constant region comprises the Fc segment sequence of the IgG1 subtype heavy chain constant region and the amino acid sequences at positions 252, 254, and 256 of the Fc segment sequence are Y, T, and E, respectively, wherein the amino acid sequence of the antibody constant region is determined according to EU numbering.

[0229] In some embodiments of either of the first and second aspects, the antibody comprises a light chain constant region selected from the kappa subtype or the lambda subtype.

[0230] In a third aspect, the present application provides a nucleic acid molecule encoding the antibody described in the first aspect or the second aspect.

[0231] In some embodiments of the third aspect, the nucleic acid molecule is operably linked to a regulatory amino acid sequence that can be recognized by a host cell transformed with the vector.

[0232] In a fourth aspect, the present application provides a pharmaceutical composition comprising the antibody described in the first aspect or the second aspect and a pharmaceutically acceptable excipient, diluent or carrier.

[0233] In some embodiments of the fourth aspect, the pharmaceutical composition is used to prevent or treat RSV-related diseases.

[0234] In some embodiments of the fourth aspect, the RSV-related disease is a respiratory tract infection, such as bronchiolitis and pneumonia.

[0235] In some embodiments of the fourth aspect, the pharmaceutical composition may also include one or more of the following: lubricants, such as talc, magnesium stearate and mineral oil; wetting agents; emulsifiers; suspending agents; preservatives, such as benzoic acid, sorbic acid and calcium propionate; sweeteners and / or flavoring agents, etc.

[0236] In some embodiments of the fourth aspect, the pharmaceutical composition of the present application can be formulated into the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories or capsules.

[0237] In some embodiments of the fourth aspect, the pharmaceutical composition of the present application can be delivered by any physiologically acceptable mode of administration, including but not limited to oral administration, parenteral administration, nasal administration, rectal administration, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, etc.

[0238] In some embodiments of the fourth aspect, pharmaceutical compositions for therapeutic use can be formulated for storage in the form of lyophilized preparations or aqueous solutions by mixing reagents having the desired purity with pharmaceutically acceptable carriers, excipients, etc. as appropriate.

[0239] In a fifth aspect, the present application provides 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 preparation of a drug for preventing or treating RSV-related diseases.

[0240] In some embodiments of the fifth aspect, the RSV-related disease is a respiratory tract infection, such as bronchiolitis and pneumonia.

[0241] In a sixth aspect, the present application provides a method for preventing or treating RSV-related diseases, comprising administering the antibody described in the first aspect or the second aspect, or the pharmaceutical composition described in the fourth aspect to an individual in need thereof.

[0242] In some embodiments of the sixth aspect, the RSV-related disease is a respiratory tract infection, such as bronchiolitis and pneumonia.

[0243] In a seventh aspect, the present application provides a plasmid combination for expressing an antibody Fab fragment, comprising a first plasmid and a second plasmid, wherein

[0244] The first plasmid comprises an antibody heavy chain VH-CH1 expression unit and a first recombination site;

[0245] The second plasmid comprises an antibody light chain VL-CL expression unit and a second recombination site; and

[0246] The first recombination site is different from the second recombination site.

[0247] In some embodiments of the seventh aspect, the plasmid combination comprises a plurality of the first plasmids, and the plurality of the first plasmids carry different antibody heavy chain VH-CH1 expression units and have the same first recombination site.

[0248] In some embodiments of the seventh aspect, the plasmid combination comprises a plurality of the second plasmids, and the plurality of the second plasmids carry different antibody light chain VL-CL expression units and have the same second recombination site.

[0249] In an eighth aspect, the present application provides a recombinant system for expressing an antibody Fab fragment library, comprising a first plasmid, a second plasmid and a cell expressing a phage integrase; wherein

[0250] The first plasmid comprises an antibody heavy chain VH-CH1 expression unit and a first recombination site;

[0251] The second plasmid comprises an antibody light chain VL-CL expression unit and a second recombination site; and

[0252] The first recombination site is different from the second recombination site.

[0253] In some embodiments of the eighth aspect, the recombination system comprises a plurality of the first plasmids, and the plurality of the first plasmids carry different antibody heavy chain VH-CH1 expression units and have the same first recombination site.

[0254] In some embodiments of the eighth aspect, the recombination system comprises a plurality of the second plasmids, and the plurality of the second plasmids carry different antibody light chain VL-CL expression units and have the same second recombination sites.

[0255] In some embodiments of the eighth aspect, the first plasmid, the second plasmid and the cell expressing bacteriophage integrase each exist independently, or at least one of the first plasmid and the second plasmid has been introduced into the cell expressing bacteriophage integrase.

[0256] In some specific embodiments of the eighth aspect, the second plasmid has been introduced into the cell expressing bacteriophage integrase.

[0257] In a ninth aspect, the present application provides a recombinant cell for expressing an antibody Fab fragment library, which comprises a first plasmid, a second plasmid and expresses a phage integrase; wherein

[0258] The first plasmid comprises an antibody heavy chain VH-CH1 expression unit and a first recombination site;

[0259] The second plasmid comprises an antibody light chain VL-CL expression unit and a second recombination site; and

[0260] The first recombination site is different from the second recombination site.

[0261] In some embodiments of the ninth aspect, the recombinant cell comprises a plurality of the first plasmids, and the plurality of the first plasmids carry different antibody heavy chain VH-CH1 expression units and have the same first recombination site.

[0262] In some embodiments of the ninth aspect, the recombinant cell comprises a plurality of the second plasmids, and the plurality of the second plasmids carry different antibody light chain VL-CL expression units and have the same second recombination sites.

[0263] In some embodiments of the ninth aspect, the recombinant cell is a prokaryotic cell.

[0264] In some embodiments of the ninth aspect, the recombinant cell is Escherichia coli.

[0265] In some embodiments of the ninth aspect, the recombinant cell is a genetically engineered bacterium.

[0266] In some specific embodiments of the ninth aspect, the recombinant cell is a male Escherichia coli strain having an F factor, such as a TG1 strain.

[0267] In a tenth aspect, the present application provides a method for preparing an antibody Fab fragment library, which comprises transducing a first plasmid and a second plasmid into a cell expressing a phage integrase; wherein

[0268] The first plasmid comprises an antibody heavy chain VH-CH1 expression unit and a first recombination site;

[0269] The second plasmid comprises an antibody light chain VL-CL expression unit and a second recombination site; and

[0270] The first recombination site is different from the second recombination site.

[0271] In some embodiments of the tenth aspect, a plurality of the first plasmids are transduced into the cell expressing phage integrase, wherein the plurality of the first plasmids carry different antibody heavy chain VH-CH1 expression units and have the same first recombination site.

[0272] In some embodiments of the tenth aspect, a plurality of the second plasmids are transduced into the cell expressing phage integrase, wherein the plurality of the second plasmids carry different antibody light chain VL-CL expression units and have the same second recombination site.

[0273] In some embodiments of the tenth aspect, transducing the first plasmid and / or the second plasmid into the cell expressing phage integrase comprises the following steps:

[0274] (1) transducing the first plasmid and / or the second plasmid into competent cells;

[0275] (2) using the helper phage to infect the competent cells obtained in step (1) to construct a phage library; and

[0276] (3) Transducing the phage library obtained in step (2) into the cells expressing phage integrase.

[0277] In some embodiments of the tenth aspect, the competent cells are prokaryotic cells.

[0278] In some embodiments of the tenth aspect, the competent cells are Escherichia coli.

[0279] In some embodiments of the tenth aspect, the competent cells are genetically engineered bacteria.

[0280] In some specific embodiments of the tenth aspect, the competent cell is a male Escherichia coli strain having an F factor, such as a TG1 strain.

[0281] In some embodiments of the tenth aspect, the competent cells do not express bacteriophage integrase.

[0282] In some embodiments of the tenth aspect, the helper phage is M13 helper phage.

[0283] In some embodiments of any one of the seventh to tenth aspects, the first plasmid is a phagemid.

[0284] In some embodiments of any one of the seventh to tenth aspects, the second plasmid is a phagemid.

[0285] In some embodiments of any one of the seventh to tenth aspects, the first plasmid and the second plasmid comprise different origins of replication.

[0286] In some embodiments of any one of the seventh to tenth aspects, the replication origin is selected from one or more of the following: pBR ori, CDF ori, the replication origin of filamentous bacteriophage M13 (f1 ori), and p15A ori.

[0287] In some specific embodiments of any one of the seventh to tenth aspects, the first plasmid comprises pBR ori and fi ori.

[0288] In some specific embodiments of any one of the seventh to tenth aspects, the second plasmid comprises a CDF ori.

[0289] In some embodiments of any one of the seventh to tenth aspects, the first recombination site and the second recombination site are sites for phage attachment in the respective genomes of the phage and its host bacteria, respectively.

[0290] In some embodiments of any one of the seventh to tenth aspects, the first recombination site is attP.

[0291] In some embodiments of any one of the seventh to tenth aspects, the second recombination site is attB.

[0292] In some specific embodiments of any one of aspects 7 to 10, the first recombination site comprises the nucleotide sequence shown in SEQ ID NO:2 or a nucleotide sequence obtained by subjecting the nucleotide sequence shown in SEQ ID NO:2 to one or more substitutions, deletions or additions that do not substantially change the function of the first recombination site.

[0293] In some specific embodiments of any one of aspects 7 to 10, the second recombination site comprises the nucleotide sequence shown in SEQ ID NO:4 or a nucleotide sequence obtained by subjecting the nucleotide sequence shown in SEQ ID NO:4 to one or more substitutions, deletions or additions that do not substantially change the function of the second recombination site.

[0294] In some embodiments of any one of aspects 7 to 10, the number of the 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 1-30, preferably 1-20, and more preferably 1-10, wherein the obtained nucleotide sequence substantially maintains the function of the first recombination site or the second recombination site unchanged.

[0295] In some embodiments of any one of the seventh to tenth aspects, the first plasmid and the second plasmid are different.

[0296] In some specific 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 (eg, a bacterial plasmid).

[0297] In some specific embodiments of any one of the seventh to tenth aspects, the first plasmid is pHGDisn-attP-new.

[0298] In some specific embodiments of any one of the seventh to tenth aspects, the second plasmid is pHKb-attB-new.

[0299] In some embodiments of any one of the seventh to tenth aspects, the first recombination site is located at the multiple cloning restriction site of the first plasmid.

[0300] In some embodiments of any one of the seventh to tenth aspects, the second recombination site is located at the multiple cloning restriction site of the second plasmid.

[0301] In some specific embodiments of any one of the seventh to tenth aspects, the first recombination site is located between the XbaⅠ and KpnⅠ restriction sites of the pHGDisn-attP-new plasmid.

[0302] In some specific embodiments of any one of the seventh to tenth aspects, the second recombination site is located between the HindⅢ and BamHI restriction sites of the pHKb-attB-new plasmid.

[0303] In some embodiments of any one of the seventh to tenth aspects, the antibody light chain constant region CL is a human kappa light chain constant region or a human lambda light chain constant region.

[0304] In some embodiments of any one of aspects 7 to 10, the antibody heavy chain constant region CH1 segment is selected from IgG1, IgG2, IgG3 or IgG4 subtype.

[0305] In some embodiments of any one of the seventh to tenth aspects, the first plasmid and / or the second plasmid comprises a resistance gene coding region.

[0306] In some embodiments of any one of aspects 7 to 10, the 3' end of the nucleic acid molecule encoding the CH1 segment of the antibody heavy chain constant region is fused to the 5' end of the nucleic acid molecule encoding the gIII protein of the filamentous phage M13.

[0307] In some embodiments of any one of the seventh to tenth aspects, the presence of the resistance gene facilitates the selection of the recombinant system or recombinant cells.

[0308] In some embodiments of any one of the seventh to tenth aspects, the resistance gene is an antibiotic resistance gene.

[0309] In some embodiments of any one of the seventh to tenth aspects, the resistance gene is selected from the group consisting of: chloramphenicol resistance gene (CmR), ampicillin resistance gene (Ampr), kanamycin resistance gene (KaR) and tetracycline resistance gene (TetR).

[0310] In some embodiments of any one of the eighth to tenth aspects, the bacteriophage integrase is a tyrosine integrase.

[0311] In some embodiments of any one of the eighth to tenth aspects, the tyrosine integrase is a phage lambda integrase.

[0312] In some embodiments of any one of the eighth to tenth aspects, the bacteriophage integrase is inducibly expressed or constitutively expressed.

[0313] In some embodiments of any one of the eighth to tenth aspects, the bacteriophage integrase is expressed inducibly.

[0314] In some embodiments of any one of the eighth to tenth aspects, the inducible expression is by utilizing an inducible promoter, such as a lactose promoter (Plac) or an arabinose promoter (Para).

[0315] In some embodiments of the eighth aspect or the tenth aspect, the cell expressing bacteriophage integrase is a prokaryotic cell.

[0316] In some embodiments of the eighth aspect or the tenth aspect, the cell expressing bacteriophage integrase is Escherichia coli.

[0317] In some embodiments of the eighth aspect or the tenth aspect, the cell expressing bacteriophage integrase is a genetically engineered bacterium.

[0318] In some embodiments of the eighth aspect or the tenth aspect, the cell expressing bacteriophage integrase is a male Escherichia coli strain having an F factor.

[0319] In some specific embodiments of the eighth aspect or the tenth aspect, the cell expressing bacteriophage integrase is a TG1 strain.

[0320] In some specific embodiments of any one of aspects 7 to 10, the first plasmid comprises the antibody heavy chain VH-CH1 expression unit and the attP attachment site; and the second plasmid comprises the antibody light chain VL-CL expression unit and the attB attachment site.

[0321] 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.

[0322] In some embodiments of the eighth aspect or the 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.

[0323] In some embodiments of the eighth aspect or the tenth aspect, after the first recombination site is determined, the second recombination site, the type of the phage integrase and the cell type expressing the phage integrase applicable to the present application can be determined.

[0324] In some embodiments of the eighth aspect or the tenth aspect, once the second recombination site is determined, the first recombination site, the type of the phage integrase and the cell type expressing the phage integrase applicable to the present application can be determined.

[0325] In some embodiments of the eighth aspect or the tenth aspect, once the type of the phage integrase is determined, the type of cells expressing the phage integrase suitable for the present application can be determined.

[0326] In some embodiments of the eighth aspect or the 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 phage integrase is TG1 Escherichia coli.

[0327] 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.

[0328] In some embodiments of the ninth aspect, once 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 application can be determined.

[0329] In some embodiments of the ninth aspect, once 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 application can be determined.

[0330] In some embodiments of the ninth aspect, once the type of the bacteriophage integrase is determined, the type of the recombinant cell suitable for the present application can be determined.

[0331] 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.

[0332] In some specific embodiments of the tenth aspect, the method for preparing an antibody Fab fragment library may include the following steps:

[0333] (1) transducing a first plasmid (e.g., multiple first plasmids carrying different antibody heavy chain VH-CH1 expression units and the same attP attachment site, such as multiple phagemids) into a competent cell (e.g., a TG1 competent cell);

[0334] (2) using a helper phage (e.g., M13 helper phage) to infect the competent cells obtained in step (1) to construct a phage library (e.g., a phage library comprising multiple phages carrying different antibody heavy chain VH-CH1 expression units and the same attP attachment site);

[0335] (3) transducing a second plasmid (e.g., multiple second plasmids carrying different antibody light chain VL-CL expression units and the same attB attachment site) into cells expressing bacteriophage integrase (e.g., TG1 competent cells expressing λ phage integrase);

[0336] (4) inducing the cells obtained in step (3) to express bacteriophage integrase (e.g., λ phage integrase);

[0337] (5) using the phage library (e.g., a phage library comprising a plurality of phages carrying different antibody heavy chain VH-CH1 expression units and the same attP attachment site) to infect the cells expressing the phage integrase of step (4); and

[0338] (6) Using a helper phage (eg, M13 helper phage) to infect the cells obtained in step (5) to construct the antibody Fab fragment library.

[0339] In the eleventh aspect, the present application provides use of the plasmid combination described in the seventh aspect, the recombination system described in the eighth aspect, or the recombinant cell described in the ninth aspect in preparing an antibody Fab fragment library.

[0340] In other aspects, the application also provides a vector comprising a nucleic acid molecule encoding an antibody of the present invention or its light chain or heavy chain, 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 that can be recognized by a host cell transformed with the vector. In some embodiments, the method for producing the antibody comprises cultivating a host cell. In some embodiments, the method for producing the antibody also comprises recovering the antibody from a host cell culture medium.

[0341] In addition, antibodies specific for RSV 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.

[0342] It should be understood that the above detailed description is only for the purpose of enabling those skilled in the art to more clearly understand the content of the present application, and is not intended to be limiting in any aspect. Those skilled in the art can make various modifications and changes to the embodiments. Example

[0343] The following examples are for illustration purposes only and are not intended to limit the scope of this application.

[0344] Example 1: Preparation of a fully human recombinant Fab library

[0345] The content of this embodiment refers to Chinese invention patent No. CN 108251431B, which is incorporated herein by reference.

[0346] 1.1 Construction of recombinant expression plasmid containing attB and attP attachment sites

[0347] Based on the pADG-S plasmid, the synthesized recombinant gene fragment containing the chloramphenicol resistance gene promoter (Cat promoter, whose coding sequence is shown in SEQ ID NO: 1) and the attP attachment site (attP1, whose coding sequence is shown in SEQ ID NO: 2) was cloned into the vector pADG-S using double restriction enzymes XbaⅠ and KpnⅠ, and the phagemid vector pHGDisn-attP-new ( Figure 1 ).

[0348] Based on the pADK-S plasmid, the synthetic recombinant gene fragment containing the CDF replication origin (CDF ori, whose coding sequence is shown in SEQ ID NO: 3), the attB attachment site (attB1, whose coding sequence is shown in SEQ ID NO: 4), the chloramphenicol resistance gene (CmR, whose coding sequence is shown in SEQ ID NO: 5) and the complete ampicillin resistance gene expression element (AmpR, whose coding sequence is shown in SEQ ID NO: 6) was cloned into the vector pADK-S by double restriction digestion with HindⅢ and BamHI, and the prokaryotic expression vector pHKb-attB-new ( Figure 2 ).

[0349] pHGDisn-attP-new and pHKb-attB-new contain different replication origins and can replicate and coexist in the same E. coli cell. After the lambda phage integrase (λInt) induces specific recombination of the attP and attB attachment sites, the new plasmid formed contains a complete chloramphenicol promoter and coding gene, which can express chloramphenicol acetyltransferase and show chloramphenicol resistance, facilitating the screening of recombinant plasmids.

[0350] 1.2 Preparation of λInt gene-edited TG1 E. coli

[0351] The attB attachment site and attP attachment site undergo specific recombination in E. coli cells to generate attL site and attR site. This reaction must be catalyzed by λ phage integrase. 14 Through gene editing, the gene encoding the lactose operon-induced λ phage integrase expression element (its nucleotide sequence is shown in SEQ ID NO: 7) was inserted into the TG1 Escherichia coli genome, and the TG1-λInt recombinant engineering bacteria was commissioned to GenScript Biotech Co., Ltd.

[0352] 1.3 Construction of a fully human heavy chain antibody library

[0353] Referring to Chinese invention patent No. CN 108251431B, frozen natural human heavy chain variable region cDNA and PCR products of introduced mutant VH1, VH3 and VH5 were used as templates, PCR primers were designed to amplify the target fragments, and then cloned into the vector pHGDisn-attP-new using NcoⅠ and PmlⅠ double restriction enzymes. The ligation products were electroporated into TG1 competent cells to construct a fully human heavy chain antibody library with a diversity of 1.73E+9. The constructed antibody library was sent for sequencing analysis, with an average accuracy rate of over 85%.

[0354] 1.4 Construction of a fully human light chain antibody library

[0355] Referring to Chinese invention patent No. CN 108251431B, frozen natural human light chain variable region cDNA and mutated VK1 and VL3 PCR products were used as templates to design PCR primers to amplify the target fragments, and then cloned into the vector pHKb-attB-new using NcoⅠ and PmlⅠ double restriction enzymes. The ligation products were electroporated into TG1-λInt competent cells to construct a fully human light chain antibody library with a diversity of 1.95E+8. The constructed antibody library was sent for sequencing analysis, with an average accuracy rate of over 90%.

[0356] 1.5 Preparation of fully human recombinant Fab library

[0357] The heavy chain library constructed above was inoculated into 200 mL of liquid culture medium, cultured at 37°C and 220 rpm to the logarithmic growth phase, infected with M13 helper phage, and cultured overnight at 28°C and 220 rpm to amplify the phage. The purified heavy chain phage library was then prepared using the PEG / NaCl precipitation method, and the titer was determined and frozen in a -80°C refrigerator for later use.

[0358] The fully human light chain antibody library prepared above was inoculated into 50 mL of liquid culture medium, IPTG was added to a final concentration of 0.1 mM, and cultured at 37°C and 220 rpm until the logarithmic growth phase. The heavy chain phage library was then added for infection. After the infection, the bacterial liquid was evenly spread on the bacterial culture dish and cultured overnight in a constant temperature incubator at 32°C.

[0359] The bacteria growing on the above culture dish were counted and sent for sequencing to identify the recombination sites of the monoclones and the light chain sequences and heavy chain sequences. The recombination capacity was counted by the dilution method, and the total number of recombinant colonies was estimated to be 3.80E+12. The results of monoclonal sequencing showed that the clones growing on the plate were all recombinant plasmids, containing 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 and complete light chain genes and heavy chain Fd genes, and the accuracy of antibody genes exceeded 76.5%.

[0360] The colonies on the above bacterial culture dishes were collected, inoculated into liquid culture medium, cultured at 37° C. and 220 rpm until the logarithmic growth phase, and then infected with M13 helper phage to prepare a purified human Fab phage library.

[0361] Example 2: Preparation of RSV F protein and preparation of recombinant antibodies

[0362] 2.1 Preparation of RSV F protein

[0363] In the process of preparing RSV F protein monoclonal antibodies, F recombinant proteins of different subtypes of viruses are needed, including 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 two different subtypes of RSV F proteins, the inventors constructed the DS-Cav1 structure. 15 The pre-fusion F protein mutants were named 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). Since the F protein has post-translational modifications (such as glycosylation and disulfide bonds), the use of a mammalian cell expression system will be more conducive to maintaining the structure and function of the recombinant protein. In addition, a His tag (His, whose amino acid sequence is shown in SEQ ID NO: 14) is added to the C-terminus of this recombinant protein, which will be more conducive to the purification of the recombinant protein and the identification of the function of the monoclonal antibody.

[0364] Synthesize RSV-DS-Cav1-A and RSV-DS-Cav1-B genes, clone the synthesized genes into suitable eukaryotic expression vectors (such as pcDNA3.1 of Invitrogen, etc.) using conventional molecular biology techniques, and then transfect the prepared recombinant protein expression plasmid into HEK293 cells (such as HEK293F of Invitrogen) using liposomes (such as 293fectin of Invitrogen, etc.) or other cationic transfection reagents (such as PEI, etc.), and culture them under serum-free suspension culture conditions for 3-4 days. Then harvest the culture supernatant by centrifugation or the like. Use a metal chelate affinity chromatography column (such as HisTrapFF of GE, etc.) to purify the recombinant protein in the supernatant in one step. Then use a desalting column (such as Hitrapdesaulting of GE, etc.) to replace the recombinant protein storage buffer with PBS (pH7.0) or other suitable buffer. If necessary, the sample can be filtered and sterilized, and then stored in aliquots at -20°C.

[0365] 2.2 Preparation of recombinant antibodies

[0366] By conventional molecular biological methods, the nucleotide sequences encoding the heavy chain variable region and the light chain variable region of the antibody are cloned into a eukaryotic expression vector (such as pcDNA3.1 of Invitrogen) fused with the nucleotide sequences encoding the heavy chain constant region and the light chain constant region, and the whole antibody is expressed in combination. The heavy chain constant region of the antibody can 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 can 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).

[0367] The prepared recombinant antibody expression plasmid is transfected into HEK293 cells (such as Invitrogen's HEK293F) using liposomes (such as Invitrogen's 293fectin, etc.) or other transfection reagents (such as PEI, etc.), and cultured for 3-5 days under serum-free suspension culture conditions. Then the culture supernatant is harvested by centrifugation and purified in one step using a ProteinA / G affinity chromatography column (such as GE's Mabselect SURE, etc.). Then, the recombinant protein storage buffer is replaced with PBS (pH7.0) or other suitable buffer using a desalting column (such as GE's Hitrapdesaulting, etc.). If necessary, the antibody sample can be filtered and sterilized, and then stored in aliquots at -20°C.

[0368] Example 3: Screening of a fully human recombinant Fab library using RSV F protein

[0369] With reference to the literature (the experimental technical process can be found in Chinese patent application No. 201510097117.0, the entire contents of the above patent application are incorporated herein by reference), the recombinant RSV F protein (RSV-DS-Cav1-A / RSV-DS-Cav1-B) prepared in Example 2 was used, and the solid phase screening strategy (experimental scheme refers to phage display: General Experimental Guide / Clackson, T., Lowman, HB, ed.; Ma Lan et al. Translated. Chemical Industry Press, 2008.5) was used to screen the fully human recombinant Fab library constructed in Example 1, and a total of 4-6 rounds of screening were performed by binding, elution, neutralization, infection, and amplification, and finally two Fab antibodies that specifically bind to RSV F protein were obtained: R3B1h1 and R22B1.

[0370] Example 4: Identification of recombinant anti-RSV F protein monoclonal antibodies

[0371] By using conventional molecular biological methods, 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 prepare recombinant human IgG1-κ monoclonal antibodies. At the same time, the human anti-RSV monoclonal antibody Palivizumab (Hu1129) was prepared with reference to patent US 7704505B2 (the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 22; the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 23), the human anti-RSV monoclonal antibody Nirsevimab (MEDI8897) was prepared with reference to patent US 11186628B2 (the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 24; the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 25), and the human anti-RSV monoclonal antibody Clesrovimab (RB1) was prepared with reference to patent US 9963500 B2 (the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 26; the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 27) as a positive control antibody.

[0372] 4.1 Verification of binding activity of recombinant anti-RSV F protein monoclonal antibody

[0373] The prepared RSV-DS-Cav1-A and RSV-DS-Cav1-B were coated on 96-well ELISA plates, 3 μg / mL, 100 μL / well, and coated overnight at 4°C. After blocking with blocking solution PBS-0.1% Tween 20-3% milk (PBST-3% milk) at 37°C for 1 hour, each recombinant anti-RSV F protein monoclonal antibody was added and bound at 37°C for 1 hour. The ELISA plate was washed with PBS-0.1% Tween 20 (PBST) buffer, and HRP-mouse anti-human IgG (Bioss, bsm-0297M-HRP) was added and bound at 37°C for 1 hour. The ELISA plate was washed with PBST buffer, and OPD substrate colorimetric solution was added. After 5-10 minutes, 1M H2SO4 was used to stop the color development, and the optical density value was measured by a dual wavelength of 492nm / 630nm using an enzyme reader. ELISA analysis results ( Figure 3 ) showed that the recombinant anti-RSV F protein monoclonal antibodies R3B1h1 and R22B1 had comparable activity in binding to the two recombinant proteins RSV-DS-Cav1-A and RSV-DS-Cav1-B.

[0374] 4.2 Epitope analysis of recombinant anti-RSV virus F protein monoclonal antibodies

[0375] The recombinant protein RSV-DS-Cav1-B was coated on a 96-well ELISA plate (1 μg / mL, 100 μL / well) and coated overnight at 4°C. The plate was blocked with blocking solution PBST-3% milk at 37°C for 1 hour. A fixed concentration (1×10 11 cfu / mL) of each anti-RSV F protein purified phage (R3B1h1 / R22B1 / Clesrovimab / Nirsevimab) was gradiently diluted with the recombinant anti-RSV F protein monoclonal antibody (R3B1h1 / R22B1 / Clesrovimab), with a starting concentration of 200μg / mL, 3-fold gradient dilution, 10 concentration gradients, 100μL / well added to the blocked 96-well ELISA plate, and incubated at 37°C for 1 hour. The ELISA plate was washed with PBST, and then HRP anti-M13 secondary antibody (Beijing Yiqiao Shenzhou Technology Co., Ltd., 11973-MM05T-H) was added and incubated at 37°C for 1 hour. The ELISA plate was washed with PBST, and the OPD substrate color development solution was added. After 5-10 minutes, 1M H2SO4 was used to stop the color development, and the dual-wavelength optical density value of 492nm / 630nm was measured using an enzyme reader. The ELISA analysis results are as follows. Figure 4 As shown, the R3B1h1 monoclonal antibody can block the binding signal between the R22B1 / Nirsevimab phage and the recombinant protein RSV-DS-Cav1-B, but has no effect on the binding between the Clesrovimab phage and the recombinant protein RSV-DS-Cav1-B ( Figure 4 A); R22B1 monoclonal antibody can block the binding signal between R3B1h1 / Nirsevimab phage and recombinant protein RSV-DS-Cav1-B, but has no effect on the binding between Clesrovimab phage and recombinant protein RSV-DS-Cav1-B ( Figure 4 B)); Clesrovimab monoclonal antibody cannot block the binding of R3B1h1 / R22B1 / Nirsevimab phage to recombinant protein RSV-DS-Cav1-B ( Figure 4 C).

[0376] 4.3 Affinity analysis of recombinant anti-RSV F protein monoclonal antibodies

[0377] The affinity of anti-RSV F protein monoclonal antibody was determined by surface plasmon resonance using Biacore T200. The amino coupling kit (BR-1000-50), human antibody capture kit (BR-1008-39), CM5 chip (BR100012) and 10×HBS-EP at pH 7.4 (BR100669) and other related reagents and consumables were purchased from GE healthcare. According to the instructions in the kit, the carboxylated CM5 chip surface was activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, EDC) and N-hydroxysuccinimide (N-Hydroxysuccinimide, NHS), and the anti-human IgG (Fc) antibody (capture antibody) was diluted to 25 μg / mL with 10mM sodium acetate, pH 5.0, and then injected at a flow rate of 10 μL / min to achieve a coupling amount of approximately 10,000 response units (RU). After the capture antibody was injected, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, anti-RSV F protein monoclonal antibodies were diluted to 0.5-1 μg / mL and injected at 10 μL / min to ensure that about 80RU of antibodies were captured by anti-human Fc antibodies. RSV-DS-Cav1-A or RSV-DS-Cav1-B was then set up with a series of concentration gradients (e.g., 6.17 nM, 18.5 nM, 55.6 nM, 166.7 nM, 500 nM) and injected from low to high concentrations at 30 μL / min at 25°C, with an association time of 90 s and a dissociation time of 3600 s. 3 M MgCl2 was injected at 10 μL / min for 30 s to regenerate the chip surface. The binding rate (K) was calculated by fitting the binding and dissociation sensorgrams with a 1:1 binding model using Biacore T200 Evaluation Software Version 3.0. on ) and dissociation rate (K off ). With the ratio K off / K on Calculate the dissociation equilibrium constant (K D ). The fitting results are shown in Table 1 and Table 2.

[0378] Table 1. Affinity constants of recombinant anti-RSV F protein monoclonal antibodies binding to RSV-DS-Cav1-A

[0379] <![CDATA[K on (1 / MS)]]> <![CDATA[K off (1 / S)]]> <![CDATA[K D (M)]]> Palivizumab 3.158E+4 8.197E-5 2.595E-9 Clesrovimab 2.001E+4 3.515E-4 1.756E-8 Nirsevimab 3.149E+4 1.502E-5 4.770E-10 R3B1h1 3.184E+4 2.370E-5 6.213E-10 R22B1 3.991E+4 1.411E-5 6.213E-10

[0380] Table 2. Affinity constants of recombinant anti-RSV F protein monoclonal antibodies binding to RSV-DS-Cav1-B

[0381] <![CDATA[K on (1 / MS)]]> <![CDATA[K off (1 / S)]]> <![CDATA[K D (M)]]> Palivizumab 1.360E+5 2.001E-4 1.471E-9 Clesrovimab 9.171E+6 8.208E-5 8.951E-12 Nirsevimab 1.082E+5 3.327E-4 3.075E-9 R3B1h1 2.286E+5 8.093E-5 3.541E-10 R22B1 3.577E+5 1.010E-4 2.824E-10

[0382] 4.4 Recombinant anti-RSV F protein monoclonal antibody inhibits RSV infection of Hep-2 cells

[0383] 4.4.1 Recombinant anti-RSV F protein monoclonal antibody inhibits RSV / A2 infection of Hep-2 cells

[0384] Anti-RSV F protein monoclonal antibodies (R3B1h1, R22B1, Nirsevimab, Clesrovimab and Palivizumab) and negative isotype control antibodies were diluted with Hep-2 cell maintenance medium (DMEM + 2% FBS + 1% P / S + 2mM Glu) with a starting concentration of 66.7nM, 5-fold gradient dilution, a total of 10 concentration gradients, and 70μL / well was added to a 96-well sterile fully permeable cell culture plate. Take a frozen virus liquid RSV / A2, place it in a P2 laboratory biosafety cabinet and slowly thaw it, and dilute it to 1.5×10 3 PFU / mL, take 70μL / well of the diluted virus solution and add it to the above diluted antibody and mix thoroughly. At the same time, set up separate cell control wells (Hep-2 cell maintenance medium 140μL / well) and cell + RSV / A2 wells (Hep-2 cell maintenance medium 70μL / well + RSV / A2 dilution solution 70μL / well). After all samples are added, cover the culture plate and place it in a cell culture incubator (37±1℃, 5±1% CO2) for 1h. Take Hep-2 cells in the logarithmic growth phase, digest and centrifuge, and dilute the counted cell suspension to 4.3×10 5 70 μL / well of single cell suspension of cells / mL was inoculated into a neutralized 96-well cell culture plate and incubated in a cell culture incubator (37±1°C, 5±1% CO2) for 60-72h. After the virus infection, wash twice with PBS, add 80% acetone, and move to a 2-8°C refrigerator for fixation for 30min. Discard 80% acetone, wash twice with PBS, add detection antibody palivizumab diluted to 10μg / mL with PBS (100μL / well), and incubate at 37°C in the dark for 60min. Discard the primary antibody, wash twice with PBS, add FITC-labeled goat anti-human IgG (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., ZF-0308) diluted with PBS (1:400), 100μL / well, and incubate at 37°C in the dark for 40min. The secondary antibody was discarded, the cells were washed twice with PBS, and the chemiluminescence value was detected by Rapid Fluorescent Focus Inhibition Test (RFFIT) using a multi-function microplate reader (SpectraMax I3). The results showed ( Figure 5), R3B1h1 has the best activity, according to IC 50 The activity of nirsevimab was about 5 times higher than that of clesrovimab, about 12 times higher than that of palivizumab, and about 410 times higher than that of palivizumab.

[0385] Table 3. Half inhibitory concentration of anti-RSV F protein monoclonal antibodies to inhibit RSV / A2 strain infection of Hep-2 cells

[0386] Antibody Name <![CDATA[IC 50 (nM)]]> R3B1h1 0.005162 R22B1 0.07067 Nirsevimab 0.02306 Clesrovimab 0.06219 Palivizumab 2.12 Isotype antibody control /

[0387] 4.4.2 Recombinant anti-RSV F protein monoclonal antibody inhibits RSV / 18537 strain infection of Hep-2 cells

[0388] The neutralizing activity of anti-RSV F protein monoclonal antibodies (R3B1h1, R22B1, Nirsevimab, Clesrovimab and Palivizumab) against RSV / 18537 was tested according to the protocol of 4.4.1. Figure 6 ), R3B1h1 has the best activity, according to IC 50 The activity of nirsevimab was about 18 times higher than that of clesrovimab, about 12 times higher than that of palivizumab, and about 545 times higher than that of palivizumab.

[0389] Table 4. Half inhibitory concentration of anti-RSV F protein monoclonal antibodies to inhibit RSV / 18537 strain infection of Hep-2 cells

[0390]

[0391]

[0392] 4.4.3 Recombinant anti-RSV F protein monoclonal antibody inhibits clinically isolated RSV infection of Hep-2 cells

[0393] The neutralizing activity of anti-RSV F protein monoclonal antibodies (R3B1h1, R22B1, Nirsevimab, Clesrovimab and Palivizumab) against clinically isolated RSV was detected according to the protocol in 4.4.1.

[0394] A total of 11 virus strains isolated from clinical samples during the 2021 RSV epidemic season were obtained from the Guangzhou Women and Children's Medical Center. Among them, 9 strains were subtype A (numbered 2033, 2064, 2066, 2406, 2410, 2416, 2425, 2430 and 2438), and 2 strains were subtype B (numbered 2063 and 2427). The results showed that ( Figure 7), R3B1h1 and R22B1 can neutralize RSV clinical isolates well.

[0395] Sequence information

[0396] SEQ ID NO:1

[0397]

[0398] SEQ ID NO:2

[0399]

[0400] SEQ ID NO:3

[0401]

[0402]

[0403] SEQ ID NO:4

[0404]

[0405] SEQ ID NO:5

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[0409] SEQ ID NO:7

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[0418] SEQ ID NO:11

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[0487] SEQ ID NO:45

[0488]

[0489] SEQ ID NO:46

[0490]

[0491] References

[0492] 1.JAMA Pediatr.2016Mar;170(3):267-87.

[0493] 2.Lancet Glob Health.2017Oct;5(10):e984-e991.

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[0502] 11.Kabat,“Sequences of Proteins of Immunological Interest”,NationalInstitutes of Health,Bethesda,Md.(1991).

[0503] 12.A1-Lazikani,et al.,J.Mol.Biol.273:927-948(1997).

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Claims

1. An antibody against respiratory syncytial virus (RSV), comprising a heavy chain variable region comprising an amino acid sequence of HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising an amino acid sequence of LCDR1, LCDR2 and LCDR3, wherein The amino acid sequence of the HCDR1 is shown in SEQ ID NO:38, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:39, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:40, the amino acid sequence of the LCDR1 is shown in SEQ ID NO:41, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:42, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:43; in, The HCDR and LCDR amino acid sequences are according to the Kabat definition.

2. The antibody according to claim 1, wherein the amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO:

30.

3. The antibody according to claim 1, wherein the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO:

31.

4. The antibody according to any one of claims 1 to 3, wherein The amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO:30, and the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO:

31.

5. The antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region of the antibody is at least 90% identical to SEQ ID NO: 30, and the amino acid sequence of the light chain variable region of the antibody is at least 90% identical to SEQ ID NO:

31.

6. The antibody of any one of claims 1 to 5, wherein The antibody is a neutralizing antibody; and / or The antibody is capable of binding to the F protein of human respiratory syncytial virus (RSV); preferably, the antibody is capable of binding to the recombinant human RSV F protein as shown in SEQ ID NO: 12 or 13; and / or The antibody is a Fab fragment, a whole antibody, a 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.

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

8. A nucleic acid molecule encoding the antibody of 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. 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 in the preparation of a medicament for preventing or treating RSV infection; preferably, the RSV infection is RSV respiratory tract infection; more preferably, the medicament is used to prevent or treat bronchiolitis and pneumonia caused by the RSV infection.

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