Molecules that specifically bind to respiratory syncytial virus

Abstract

The present disclosure relates to a molecule that specifically binds to respiratory syncytial virus and its applications. The present disclosure also provides a method for preparing the molecule and applications of the molecule in preparing a product that specifically binds to a fusion protein, which is a surface glycoprotein of respiratory syncytial virus, and in preparing a respiratory syncytial virus vaccine, etc.

Description

Technical Field

[0001] The present disclosure relates to the fields of medicine and immunology, and specifically, to a molecule that specifically binds to the respiratory syncytial virus, a method for preparing the molecule, and its use for preventing and treating respiratory syncytial virus infection.

Background Art

[0002] The human respiratory syncytial virus (RSV) is widely distributed worldwide and is one of the most common viral pathogens causing lower respiratory illness (LRI) in infants, the elderly, and immunocompromised adults. Almost all children experience at least one infection by the age of 2, and the peak age of infection is between 2 months and 8 months. RSV is the leading cause of lower respiratory tract infections in infants and young children and is also the leading cause of hospitalization due to respiratory diseases in children. Among infant hospitalizations, 40 - 50% of bronchiolitis and 25% of pneumonia are due to RSV infection. Furthermore, according to some studies, severe infection in infants is a risk factor for future asthma, and it has been shown that its severity is much higher than that of other microbial pathogens. The elderly also suffer from severe RSV infections, but due to the persistent lack of specific treatment methods and safe and effective vaccines, it remains difficult worldwide to reduce the morbidity and mortality of RSV infections.

[0003] The immunity generated by natural RSV infection is insufficient and cannot generate permanent immunity. Therefore, as a major feature of RSV infection, antibodies produced in the body by previous infections cannot provide permanent protection, and in the same epidemic period, even if a person is reinfected with different subtypes of RSV, that is, even if multiple natural RSV infections occur, upper respiratory immune protection against viral infections cannot be induced throughout life, so repeated infections are very common.

[0004] Currently, methods for preventing and treating RSV infection include vaccine development, antiviral compounds (ribavirin), antisense drugs, RNA interference technology, and antibody products such as immunoglobulins and intravenous monoclonal antibodies. Palivizumab (Synagis (R) ) is a monoclonal antibody that is approved for the prevention of RSV in high-risk children. However, palivizumab is an expensive humanized monoclonal antibody that can only be used as a preventive treatment by passive immunization, and ribavirin is a nucleoside antimetabolite with severe toxicity and teratogenic effects.

[0005] Therefore, the development of novel anti-RSV drugs, particularly drugs that can treat RSV infection, is desired.

Summary of the Invention

Problems to be Solved by the Invention

[0006] To solve the problems existing in the prior art, the present disclosure provides an isolated human antibody or humanized antibody or antigen-binding fragment thereof that specifically binds to the fusion protein, which is a surface glycoprotein of respiratory syncytial virus (RSV). The present disclosure also provides a nucleic acid sequence encoding the antibody and its antigen-binding fragment, a vector containing the nucleic acid sequence, a corresponding host cell, and the use of the neutralizing antibody in the prevention and treatment of RSV infection.

Means for Solving the Problems

[0007] In a first aspect, the present disclosure provides an isolated human antibody or humanized antibody or antigen-binding fragment thereof that specifically binds to the fusion protein, which is a surface glycoprotein of respiratory syncytial virus (RSV), and includes a light chain variable region and a heavy chain variable region, wherein The heavy chain variable region comprises (1) a functional activating CDR variant having CDR1 of the amino acid sequence shown in SEQ ID NO:1 or a functional equivalent, (2) a functional activating CDR variant having CDR2 of the amino acid sequence shown in SEQ ID NO:2 or a functional equivalent, (3) a functional activating CDR variant having CDR3 of the amino acid sequence shown in SEQ ID NO:3 or a functional equivalent, and / or The light chain variable region comprises (1) a functional activating CDR variant having CDR1 of the amino acid sequence shown in SEQ ID NO:4 or a functional equivalent, (2) a functional activating CDR variant having CDR2 of the amino acid sequence shown in SEQ ID NO:5 or a functional equivalent, (3) a functional activating CDR variant having CDR3 of the amino acid sequence shown in SEQ ID NO:6 or a functional equivalent, and provides an isolated human antibody or humanized antibody or an antigen-binding fragment thereof.

[0008] In one specific embodiment, the anti-RSV antibody and its antigenic fragments comprise one, two, or three CDRs (preferably three CDRs) selected from the VH region sequences of the antibodies shown in Table 1. In some other embodiments, the antibody according to the present disclosure comprises one, two, or three CDRs (preferably three CDRs) selected from the VL region sequences of the antibodies shown in Table 1. In some embodiments, the antibody according to the present disclosure comprises the six CDR region sequences of the antibodies shown in Table 1. In one preferred embodiment, the CDR sequence of the antibody is the CDR sequence shown in Table 2. In some embodiments, the anti-RSV antibody or its antigen-binding fragment according to the present disclosure comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises (i) CDR1 comprising the amino acid sequence of SEQ ID NO:1, or a sequence containing one or more and no more than five amino acid substitutions (e.g., conservative substitutions), deletions, or insertions with respect to the said sequence, (ii) CDR2 comprising the amino acid sequence of SEQ ID NO:2, or a sequence containing one or more and no more than five amino acid substitutions (e.g., conservative substitutions), deletions, or insertions with respect to the said sequence, and (iii) CDR3 comprising the amino acid sequence of SEQ ID NO:3, or a sequence containing one or more and no more than five amino acid substitutions (e.g., conservative substitutions), deletions, or insertions with respect to the said sequence, and the light chain variable region comprises (i) CDR1 comprising the amino acid sequence of SEQ ID NO:4, or a sequence containing one or more and no more than five amino acid substitutions (e.g., conservative substitutions), deletions, or insertions with respect to the said sequence, (ii) CDR2 comprising the amino acid sequence of SEQ ID NO:5, or a sequence containing one or more and no more than two amino acid substitutions (e.g., conservative substitutions), deletions, or insertions with respect to the said sequence, and (iii) CDR3 comprising the amino acid sequence shown in SEQ ID NO:6, or a sequence containing one or more and no more than five amino acid substitutions (e.g., conservative substitutions), deletions, or insertions with respect to the said sequence, and the anti-RSV antibody containing the modified CDR still has the ability to bind to RSV.

[0009] In some embodiments, the anti-RSV antibody or antigen-binding fragment thereof according to the present disclosure comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises (i) CDR1 consisting of the amino acid sequence of SEQ ID NO:1, (ii) CDR2 consisting of the amino acid sequence of SEQ ID NO:2, and (iii) CDR3 consisting of the amino acid sequence of SEQ ID NO:3. The light chain variable region comprises (i) CDR1 consisting of the amino acid sequence of SEQ ID NO:4, (ii) CDR2 consisting of the amino acid sequence of SEQ ID NO:5, and (iii) CDR3 consisting of the amino acid sequence shown in SEQ ID NO:6.

[0010] In some embodiments, the anti-RSV antibody or antigen-binding fragment thereof according to the present disclosure comprises a heavy chain variable region VH and / or a light chain variable region VL. The heavy chain variable region comprises 1) an amino acid sequence having 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity, or higher identity, with the amino acid sequence of SEQ ID NO:7, or consists of said sequence. The anti-RSV antibody comprising said VH has the ability to bind to RSV, or 2) comprises the three heavy chain variable region CDRs of the antibody shown in Table 2 and has 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the amino acid sequence of SEQ ID NO:7. The anti-RSV antibody comprising said VH has the ability to bind to RSV, or 3) comprises an amino acid sequence having one or more substitutions (e.g., conservative substitutions), insertions or deletions with respect to the amino acid sequence of SEQ ID NO:7. The anti-RSV antibody comprising said VH has the ability to bind to RSV. The light chain variable region comprises, consists of, or consists essentially of an amino acid sequence having 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the amino acid sequence of SEQ ID NO:8, and the anti-RSV antibody comprising the VL has the ability to bind to RSV, or (2) comprises the three CDRs of the light chain variable region of the antibody shown in Table 2 and has 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the amino acid sequence of SEQ ID NO:8, and the anti-RSV antibody comprising the VL has the ability to bind to RSV, or (3) comprises an amino acid sequence having one or more substitutions (e.g., conservative substitutions), insertions, or deletions with respect to the amino acid sequence of SEQ ID NO:8, and the anti-RSV antibody comprising the VH has the ability to bind to RSV.

[0011] In a preferred embodiment, the anti-RSV antibody according to the present disclosure is an anti-RSV neutralizing antibody or an antigen-binding fragment thereof.

[0012] In one preferred embodiment, the present disclosure provides an anti-RSV neutralizing antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region VH comprises or consists of the amino acid sequence shown in SEQ ID NO:7, and the light chain variable region VL comprises or consists of the amino acid sequence shown in SEQ ID NO:8.

[0013] In some embodiments, the present disclosure covers antigen-binding fragments of anti-F protein neutralizing antibodies and includes, but is not limited to, Fv, Fab, Fab’, Fab’-SH, F(ab’)2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed by antibody fragments.

[0014] In some embodiments, the anti-RSV neutralizing antibody or its antigen-binding fragment further includes a heavy-chain and / or light-chain constant region sequence derived from a common sequence of the human antibody germline. The light-chain constant region is preferably the human κ or λ chain constant region. The heavy-chain constant region may be a γ, μ, α, δ, or ε chain constant region, and in some embodiments, the heavy-chain constant region is of the human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, or IgE isotype. Each type of heavy chain and light chain is characterized by a specific constant region having a sequence well-known in the art.

[0015] In some embodiments, preferably, the constant region is a human IgG constant region, for example, the constant region of the human IgG1, IgG2, IgG3, or IgG4 isotype.

[0016] Preferably, the monoclonal antibody according to the present disclosure may have a κ type or λ type light chain.

[0017] In one preferred embodiment, the light chain is of the λ type. The light chain may be a naturally occurring chain, including a naturally rearranged light chain type, a genetically modified light chain type, or a synthetic light chain type.

[0018] The heavy chain of the monoclonal antibody according to the present disclosure is selected from the isotypes IgM, IgA, or IgG, preferably IgG. In one preferred embodiment, the heavy chain of the monoclonal antibody is of the IgG type.

[0019] For example, it should be understood that sequence variants of these constant region domains may be used that contain modifications of one or more amino acids where the amino acid modification sites are labeled according to the (1991) EU index system of Kabat et al. Said modification sites are selected from 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 279, 280, 281, 282D, 283, 284, 285, 286, 287, 288S, 305, 306, 307, 308, 309, 310, 311, 312, 313, 315, 317, 339, 340, 341, 374, 376, 378, 380, 382, 383, 384, 385, 386, 387, 389, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440 and 443. Optionally, the Fc region contains amino acid residues that are not naturally present at additional sites and / or alternative sites known to those skilled in the art (examples include U.S. Patent Nos. 5,624,821; 6,277,375; 6,586,207; 6,737,056; 7,083,784; 7,317,091; 7,217,797; 7,276,585; 7,355,008; 2002 / 0147311; 2004 / 0002587; 2005 / 0215768; 2007 / 0135620; 2007 / 0224188; 2008 / 0089892; WO94 / 29351; WO99 / 58572; WO 98 / 48032; WO03 / 073238; WO05 / 35727A2; WO05 / 74524A2; J.W. Chin et al., (2002), Journal of the American Chemical Society 124:9026-9027; J.W. Chin & P.G. Schultz, (2002), ChemBioChem 11:1135-1137; J.W. Chin et al., (2002), PNAS United States of America 99:11020-11024; and, L. Wang & P.G. Schultz, (2002), Chem. 1-10).

[0020] In some embodiments, in order to prevent glycosylation of the antibody, for example, modifications are made to the human IgG constant region, and such modifications may be N297A or N297Q (Sazinsky, PNAS (2008), 105(51):20167 - 20172).

[0021] In some embodiments, in order to alter the interaction with the Fc receptor, for example, modifications are made to the human IgG constant region, and such modifications may be L234A and / or L235E or L235A.

[0022] In some embodiments, in order to prevent or reduce chain exchange, for example, modifications are made to the human IgG constant region, and such modifications may be S228P (Angal, S. Mol Immunol (1993), 30:105 - 108).

[0023] In some embodiments, in order to alter antibody - dependent cell - mediated cytotoxicity (ADCC) and / or complement - dependent cytotoxicity (CDC), for example, modifications are made to the human IgG constant region, and such modifications are known (including but not limited to Natsume et al., Cancer Res (2008), 68(10):3863 - 72; Idusogie et al., J. Immunol (2001), 166(4):2571 - 5; Moore et al., mAbs (2010, 2(2):181 - 189; Lazar et al., PNAS (2006), 103(11):4005 - 4010; Shields et al., J. Biol. Chem. (2001), 276(9):6591 - 6604; Stavenhagen. Cancer Res (2007), 67(18):8882 - 8890; Alegre et al., J. Immunol (1992) 148:3461 - 3468.; Kaneko, Niwa et al., Biodrugs (2011), 25(1):1 - 11.).

[0024] In some embodiments, disulfide bonds can be introduced by the T366W modification and optionally further by the S354C and Y349C modifications in the corresponding CH3 domain to induce heterodimerization (Carter, Journal of Immunological Methods (2001), 248:7-15.).

[0025] In some embodiments, the anti-RSV neutralizing antibody or antigen-binding fragment thereof specifically binds to a fusion protein (F protein), which is a surface glycoprotein of respiratory syncytial virus (RSV), and the fusion protein, which is a surface glycoprotein of respiratory syncytial virus (RSV), may be a protein (Pre-F) having a pre-fusion conformation of the fusion protein that is a surface glycoprotein of respiratory syncytial virus (RSV). In one preferred embodiment, the neutralizing antibody binds to the RSV Pre-F protein with a KD of 1×10 -5 M or less, for example, 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, or 1×10 -10 M or smaller.

[0026] In particular, the respiratory syncytial virus neutralizing antibody provided by the present disclosure is a human-derived antibody, a human antibody, or a single-domain antibody.

[0027] In a second aspect, based on the above research results, the present disclosure further provides a nucleic acid molecule encoding any of the above antibodies or fragments thereof. Based on the amino acid sequences of the above antibody molecules, those skilled in the art can easily obtain the nucleic acid molecules provided in the present application according to common general knowledge well known in the art. In one embodiment, the nucleic acid molecule includes nucleic acid molecules obtained by codon degeneracy.

[0028] The nucleic acid molecule encoding the antibody may be a naturally occurring nucleic acid derived by rearrangement occurring in its own cells or B cells, or the nucleic acid may be synthetic. The synthetic nucleic acid has modified internucleoside linkages and includes nucleic acids containing phosphorothioates to enhance the resistance of the nucleic acid to degradation. The nucleic acid may be fully synthesized by genetic engineering or nucleotide synthesis.

[0029] In one preferred embodiment, the present disclosure provides a vector comprising a nucleic acid encoding the light chain of at least one monoclonal antibody according to the present disclosure and / or a nucleic acid encoding the heavy chain of at least one monoclonal antibody according to the present disclosure. The nucleic acids may be present in the same vector or in different vectors. Preferably, the vector comprises a promoter operably linked to the nucleic acid to facilitate the expression of the nucleic acid encoding the light chain and / or heavy chain. Preferably, the vector further comprises an origin for replication and maintenance in a host cell. The vector may further comprise a nucleotide sequence encoding a signal sequence located 5' to the nucleic acid encoding the light chain or heavy chain. The signal sequence facilitates the secretion of the encoded peptide chain into the medium.

[0030] Therefore, it should be understood that the recombinant expression vector, or expression cassette, or transgenic cell line, or recombinant bacterium, etc. containing the nucleic acid molecule also belongs to the protection scope of the present disclosure.

[0031] In the art, many prokaryotic and eukaryotic expression systems are known, including eukaryotic host cells such as yeast cells, insect cells, plant cells, and mammalian cells. Preferably, the mammalian cells are selected from HEK293 cells, PerC6 cells, CHO cells, COS cells, or HELa cells and their derivatives, etc. Particularly preferred is a production cell line derived from humans.

[0032] In one preferred embodiment, the human monoclonal antibody according to the present disclosure is derived from blood lymphocytes of a plasma sample having a high titer against the RSV protein (RSV Pre-F and / or RSV Post-F). The human antibody thus obtained has a high affinity and neutralizing effect against RSV, and can achieve effective protection against infection.

[0033] The present disclosure further provides a method for generating a monoclonal antibody. In one embodiment, a monoclonal antibody is generated by culturing a host cell containing the expression vector. In one embodiment, the generated monoclonal antibody is secreted into the supernatant and can be purified by conventional chromatography techniques.

[0034] In one embodiment, the present disclosure provides an anti-RSV antibody or an antigen-binding fragment thereof prepared by the method according to the present disclosure.

[0035] In a third aspect, the present disclosure provides the use of an antibody that specifically binds to the RSV in at least one of the following a) to d). a) Preparation of a product that specifically binds to the fusion protein, which is a surface glycoprotein of the respiratory syncytial virus b) Preparation of a product that specifically binds to the respiratory syncytial virus antigen c) Preparation of a product for preventing, treating or adjuvantly treating the respiratory syncytial virus d) Preparation of a respiratory syncytial virus vaccine

[0036] Preferably, the product is a drug.

[0037] As a preferred application form, the present disclosure further provides a drug for preventing, treating or adjuvantly treating the infection of the respiratory syncytial virus, which contains the anti-RSV antibody or an antigen-binding fragment thereof provided by the present disclosure as an active ingredient.

[0038] Preferably, the antibody provided by the present disclosure and a pharmaceutically acceptable carrier are prepared into a drug and can be administered by various methods known in the art. The administration route and / or form vary depending on the desired result.

[0039] In a fifth aspect, the present disclosure provides a composition comprising any one of the anti-RSV antibodies or antigen-binding fragments thereof provided in the present application, preferably, the composition is a pharmaceutical composition.

[0040] In one embodiment, the composition further comprises a pharmaceutical carrier. In one embodiment, the anti-RSV antibody and its antigen-binding fragment contained in the composition conjugate to a conjugation moiety. In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent.

[0041] In a sixth aspect, the present disclosure provides a method for preventing or treating a disease or symptom associated with RSV infection, comprising administering a prophylactically effective amount or a therapeutically effective amount of the anti-RSV antibody according to the present disclosure, or the pharmaceutical composition according to the present disclosure, to a subject.

[0042] The present disclosure also relates to a method for alleviating or improving a disease or symptom associated with RSV infection, comprising administering an effective amount of the anti-RSV antibody according to the present disclosure, or the pharmaceutical composition according to the present disclosure, to a subject.

[0043] The administration may be performed by any suitable route, including but not limited to topical, parenteral, local or systemic, such as intranasal, intramuscular, intradermal, intraperitoneal, intravenous, subcutaneous, oral administration, or administration by the lung. In some embodiments, the pharmaceutical composition provided in the present application is administered by a nebulizer or an inhaler. The anti-RSV antibody or the pharmaceutical composition provided by the present disclosure can be administered to any suitable subject, such as a mammal, such as a human.

[0044] In a seventh aspect, the present disclosure provides a method for detecting RSV in a subject or sample, comprising: (a) contacting the subject or sample with either an anti-RSV antibody or a fragment thereof provided by the present disclosure; and (b) detecting the formation of a complex between the anti-RSV antibody or fragment thereof and RSV. In one preferred embodiment, the anti-RSV antibody and antigen-binding fragment thereof according to the present disclosure further comprise a detectable tag.

[0045] In an eighth aspect, the present disclosure relates to the use of either an anti-RSV antibody or a fragment thereof provided by the present disclosure in the preparation of a drug or kit for preventing or treating a disease or symptom associated with RSV infection in a subject.

[0046] In one embodiment, the present disclosure provides the use of a composition comprising the anti-RSV antibody in the preparation of a drug or kit for preventing or treating a disease or symptom associated with RSV infection in a subject.

[0047] In a ninth aspect, the present disclosure provides a kit comprising an antibody or composition according to the present disclosure, such as a diagnostic kit, a detection kit, a treatment kit, etc.

[0048] To the extent that it meets the ordinary knowledge in the art, the above preferred conditions can be combined with each other to obtain specific embodiments.

Brief Description of the Drawings

[0049]

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Embodiments for Carrying Out the Invention

[0050] I. Definitions Before explaining the present disclosure in detail below, it should be understood that the present disclosure is not limited to the specific methodologies, aspects, and reagents described herein, as these can be changed. Also, the terms used in this specification are only used to describe specific embodiments and are not intended to limit the scope of the present invention. It should also be understood that the scope of the present invention is only limited by the appended claims. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which the present disclosure pertains.

[0051] For the purpose of interpreting this specification, the following definitions are used, and terms used in the singular may, where appropriate, include the plural, and vice versa. It should be understood that the terms used in this specification are only used to describe specific embodiments and are not intended to be limiting.

[0052] Unless otherwise indicated, as used herein, the terms "about" or "approximately" mean within ±10% of a given value or range. When an integer is required, the term means rounding up or down to the nearest integer within ±10% of the given value or range.

[0053] When used in combination with a numerical value, the term "about" means a value within a range having a lower limit that is 5% less than the specified value and an upper limit that is 5% greater than the specified value.

[0054] The term "and / or" should be understood to mean either or both of the alternatives.

[0055] As used herein, the terms "comprising" or "including" mean including the recited element, integer, or step, but not excluding any other element, integer, or step. When the terms "comprising" or "including" are used herein, unless otherwise indicated, they also include the case of consisting of the recited element, integer, or step.

[0056] For example, when referring to an antibody variable region "comprising" a specific sequence, it means including an antibody variable region consisting of that specific sequence.

[0057] Provided herein are antibodies (e.g., monoclonal antibodies) that specifically bind to respiratory syncytial virus (RSV) and antigen-binding fragments thereof. In a specific embodiment, provided herein is a monoclonal anti-RSV antibody that specifically binds to respiratory syncytial virus (RSV), and the RSV antibody also includes variants of the parental antibody. In a specific embodiment, provided herein is an antibody that specifically binds to respiratory syncytial virus (e.g., human respiratory syncytial virus). In a particular embodiment, provided herein is an anti-RSV antibody (e.g., substitution of one or more amino acids in the framework region of the heavy chain variable region) that includes modifications in one or more amino acid residues, and the affinity for its antigen is maintained as compared to the parental antibody without said modifications.

[0058] The term "respiratory syncytial virus" or "RSV" is a single-stranded negative-strand RNA virus belonging to the family Paramyxoviridae, genus Pneumovirus. Since it can cause a unique cell fusion effect in cultured cells, it is named respiratory syncytial virus (RSV). RSV can be divided into two subtypes, such as RSV-A and RSV-B, based on differences in surface antigens. The virus is transmitted by airborne droplets and close contact. Respiratory syncytial virus contains 10 genes encoding 11 proteins. The fusion protein (F protein) and attachment protein (G protein), which are surface glycoproteins, are the most important viral antigens that induce the production of protective antibodies in the body. The G protein varies greatly depending on the subtype, and the F protein is highly conserved among subtypes. Since the F protein directly mediates the fusion of the virus and the cell, virus entry, and the formation of syncytia, it is the main target protein that induces the production of protective antibodies in the body. As used herein, "respiratory syncytial virus" or "RSV" refers to any respiratory syncytial virus or RSV molecule known to those of ordinary skill in the art. For example, RSV may include any of the above subtypes. For example, RSV may be of mammalian origin. For example, RSV may be of human origin.

[0059] The F protein is an N-glycosylated type I transmembrane protein with a full length of 574 amino acids, having a signal peptide cleaved at the N-terminus and a membrane anchor near the C-terminus. The amino acid sequence of the F protein is provided in GenBank under the accession number AAX23994. The F protein has structures of a pre-fusion conformation (Pre-F) and a post-fusion conformation (Post-F) on the surface of the virus particle. Pre-F exists as a trimer. After undergoing a conformational change, the hydrophobic fusion peptide inserts into the host cell membrane, and the F protein refolds into a stable extended post-fusion conformation (Post-F), leading to the fusion of the virus and the host cell membrane.

[0060] The term "laboratory strain" refers to RSV strains that have been widely passaged in in vitro cell culture. A "laboratory strain" can acquire adaptive mutations that can affect its biological properties. The terms "clinical strain", "clinical isolate" or "clinical sample isolate" refer to isolates of RSV obtained from an infected individual, isolated, passaged a limited number of times, and grown in tissue culture, such as RSV subtype A or subtype B.

[0061] The term "antibody" is used herein in its broadest sense and encompasses many antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, provided that they exhibit the desired antigen-binding activity. A complete antibody typically includes at least two full-length heavy chains and two full-length light chains, but may in some cases include fewer chains. For example, antibodies naturally present in camels may include only heavy chains. Antibodies may be humanized or human antibodies and single-domain antibodies, such as VH, VHH or VL. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd’ fragments, and other fragments such as modified fragments (e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p3-25, Kipriyanov). The fragments may include multiple chains linked, for example, by disulfide bonds and / or peptide linkers. Antibody fragments generally include at least or about 50 amino acids and typically include at least or about 200 amino acids.

[0062] As used herein, the terms “monoclonal antibody” or “mAb” refer to single-copy or cloned antibodies, e.g., derived from eukaryotic, prokaryotic, or phage clones, i.e., each antibody constituting the population is identical and / or binds to the same epitope, excluding possible variant antibodies (e.g., including natural variants or variants generated in the manufacturing process of monoclonal antibody products) that are normally present in small amounts. The modifier “monoclonal” indicates that the antibody is obtained from a substantially homogeneous group of antibodies and should not be construed to require that the antibody be produced by any particular method. Monoclonal antibodies may be produced, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic techniques such as CDR grafting, or combinations of these techniques or other techniques known in the art.

[0063] The term “natural antibody” refers to naturally occurring immunoglobulin molecules having different structures. “Native sequence Fc domain” includes an amino acid sequence identical to the amino acid sequence of the Fc domain found in nature. Native sequence human Fc domains include, for example, native sequence human IgG1 Fc domain (non-A and A allotypes), native sequence human IgG2 Fc domain, native sequence human IgG3 Fc domain, and native sequence human IgG4 Fc domain, and their naturally occurring variants.

[0064] The term “human antibody” refers to an antibody having an amino acid sequence corresponding to an antibody that is produced in a human or human cell, or is derived from a non-human source and utilizes a human antibody library or other coding sequence of a human antibody. The term “human antibody” specifically excludes humanized antibodies containing non-human antigen-binding residues.

[0065] The term "neutralizing antibody" means an antibody for reducing or suppressing at least one biological activity of the F protein. For example, an antibody that blocks the fusion of RSV and host cells, prevents syncytium formation, and prevents primary diseases caused by RSV. Alternatively, the neutralizing antibody according to the present disclosure can improve at least one symptom of RSV infection. The reduction of biological activity may be partial reduction or complete reduction. The degree to which an antibody neutralizes RSV is referred to as the neutralizing potency of the antibody. The neutralizing potency of an antibody can be determined or measured by one or more tests known to those skilled in the art and / or referred to herein, including, but not limited to, competitive binding assays, direct and indirect sandwich assays, immunoprecipitation assays, enzyme-linked immunosorbent assays (ELISA), plaque reduction assays, micro-neutralization assays, fixed antiserum dilution virus methods, fixed virus dilution antiserum methods, and pseudovirus neutralization assays.

[0066] "Complementary determining region" or "CDR region" or "CDR" or "hypervariable region" is an amino acid region in the antibody variable region that is mainly responsible for binding to the antigen epitope. The CDRs of the heavy chain and the light chain are usually called CDR1, CDR2, and CDR3, and are numbered in order from the N-terminus.

[0067] Various schemes for determining CDR sequences in specific VH or VL amino acid sequences are known in the art. Kabat Complementary Determining Regions (CDRs) are determined based on sequence variability and are the most common (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), while Chothia refers to the positions of structural loops (Chothia et al., (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 877-883), AbM CDRs are a compromise between Kabat CDRs and Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software, and "Contact" CDRs are based on the analysis of available complex crystal structures. According to different CDR determination schemes, each residue in these CDRs is as follows.

[0068]

Table 1

[0069] In one embodiment, the CDRs of the antibodies according to the present disclosure are CDR sequences at the following Kabat residue positions according to the Kabat numbering system. Positions 26-33 (CDR1), positions 51-58 (CDR2), and positions 97-116 (CDR3) in VH, and positions 27-32 (CDR1), positions 50-52 (CDR2), and positions 89-98 (CDR3) in VL.

[0070] The CDRs can be determined based on having the same Kabat numbering positions as a reference CDR sequence (e.g., any of the CDRs exemplified in the present disclosure).

[0071] The term "functionally active CDR variant" retains the function of the parental CDR (i.e., can specifically bind to the corresponding fragment of respiratory syncytial virus), is different from the parental CDR sequence in the sequence, has a modification of at least one amino acid residue compared to the parental CDR, or means an amino acid sequence having a sequence identity of more than at least 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or higher with the parental CDR sequence.

[0072] The modification of an amino acid residue may be a chemical change or sequence change of the amino acid sequence, provided that the modification retains the biological properties of the parental sequence. The sequence change may be a deletion, substitution, insertion of one to a plurality of amino acid residues, for example, 1, 2, 3, 4 or 5 amino acid residues, or an addition of an amino acid sequence of a fragment, or a chemical derivatization of one to a plurality of amino acid residues, for example, 1, 2, 3, 4 or 5 amino acid residues, or a combination thereof. The substitution of an amino acid residue may be a conservative substitution.

[0073] The term "conservative substitution" refers to the substitution of one amino acid with another amino acid of the same type, for example, the substitution of one acidic amino acid with another acidic amino acid, the substitution of one basic amino acid with another basic amino acid, or the substitution of one neutral amino acid with another neutral amino acid.

[0074] As used herein, the term "variant" in relation to an antibody means an antibody comprising an antibody region of interest (e.g., a heavy chain variable region or a light chain variable region, or a heavy chain CDR region or a light chain CDR region) having amino acid modifications by substitution, deletion and / or insertion of at least one amino acid residue (e.g., 1 to 30, or 1 to 20, or 1 to 10 amino acid residues, such as 1 or 2 or 3 or 4 or 5 amino acid residues), or chemical derivatization of one or more amino acid residues, and the variant substantially retains the biological properties of the antibody molecule before modification. On the other hand, the present disclosure encompasses any variant of the antibodies referred to herein. In one embodiment, the antibody variant retains 60% or more, 70% or more, 80% or more, 90% or more, or 100% of the biological activity (e.g., antigen-binding ability) of the antibody before modification. The heavy chain variable region or light chain variable region of the antibody, or each CDR region, can be modified alone or in combination. In some embodiments, the amino acid modifications in one or more heavy chain CDRs or all of the three heavy chain CDRs are 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, or 10 or less. Preferably, the amino acid modification is an amino acid substitution, preferably a conservative substitution. In some embodiments, the antibody variant has a sequence identity of at least more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or higher with the parental antibody in the target antibody sequence region.

[0075] An "isolated" antibody is an antibody that has been isolated from the components of its natural environment. In some embodiments, the antibody is purified to a purity of 95% or more than 99%, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For an overview of methods for assessing the purity of an antibody, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0076] "Isolated" nucleic acid refers to a nucleic acid molecule that has been isolated from the components of its natural environment. An isolated nucleic acid typically includes nucleic acid molecules that are contained within a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present outside of the chromosome or at a chromosomal location different from its natural chromosomal location.

[0077] The terms "affinity" or "binding affinity" mean the specific binding affinity that reflects the interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for ligand Y can typically be expressed by the equilibrium dissociation constant (KD). The equilibrium dissociation constant is the ratio of the dissociation rate constant and the association rate constant (kdis and kon, respectively). The smaller the KD, the smaller the dissociation, meaning the stronger the affinity between the antibody and the antigen. Affinity can be measured by the KD measured using a BIACORE instrument using common methods known in the art, such as surface plasmon resonance (SPR). Typically, an antibody (e.g., the neutralizing antibody TRN1021 according to the present disclosure) dissociates from an antigen (e.g., the F protein of RSV) with an equilibrium dissociation constant (KD) of 1×10 -5 M or less, e.g., about 1×10 -6 M less than, about 1×10 -7 M less than, about 1×10 -8 M less than, about 1×10 -9 M less than, about 1×10 -10 M less than, or a smaller equilibrium dissociation constant (KD).

[0078] The term "immunoconjugation" means an antibody conjugated with one or more heterologous molecules including, but not limited to, a vector. The term "pharmaceutical composition" refers to a formulation in which the biological activity of the active ingredient contained therein is effective and which does not contain other ingredients having unacceptable toxicity to the subject to whom the drug is administered.

[0079] The term "pharmaceutical carrier" means one or more non-toxic materials that do not interfere with the biological activity of the active ingredient administered with the therapeutic agent, including, but not limited to, buffers, preservatives, acceptable carriers, diluents, adjuvants (e.g., Freund's adjuvant (complete and incomplete)), excipients, vehicles, and any other additives or encapsulating substances. Pharmaceutical carriers applicable to the present disclosure may be conventional pharmaceutical formulation aids, as well as compositions and formulations applicable to the delivery of the disclosed neutralizing antibodies.

[0080] The term "treatment" means delaying, improving, remitting, halting, or reducing the progression, severity, and / or duration of RSV infection of the upper and / or lower respiratory tract or symptoms or respiratory conditions associated therewith (e.g., asthma, wheezing, or combinations thereof). In some embodiments, the term means reducing or suppressing the replication of RSV, inhibiting or reducing the spread of RSV to other tissues or subjects (e.g., spread to the lower respiratory tract), suppressing or reducing the infection of cells with RSV, or improving one or more symptoms associated with upper and / or lower respiratory tract RSV infection.

[0081] The term "prevention" means preventing or suppressing the progression or onset of RSV infection of the upper and / or lower respiratory tract or respiratory conditions associated therewith in a subject, preventing or suppressing the progression from upper respiratory tract RSV infection to lower respiratory tract RSV infection or respiratory conditions associated therewith resulting from an administered therapy (e.g., a prophylactic or therapeutic formulation), preventing the symptoms of upper and / or lower respiratory tract RSV infection or respiratory conditions associated therewith, or a combination of administered therapies (e.g., a combination of prophylactic or therapeutic formulations).

[0082] The term "effective amount" refers to the amount or dosage that, upon administration in a single or multiple doses, results in the desired effect or a portion thereof, and a "therapeutically effective amount" is an amount that produces a desired effect in a subject being treated, such as improvement of the subject's symptoms (e.g., improvement of one or more symptoms) and / or delay of symptom progression. A prophylactically effective amount of a disease means an amount sufficient to prevent, inhibit, or delay the occurrence of the disease. Determination of the effective amount is within the ability of those skilled in the art and depends, for example, on the particular disease involved, the degree or severity of the disease, the response of the individual patient, the particular antibody administered, the dosing pattern, the bioavailability characteristics of the formulation administered, the selected dosing protocol, and the use of adjunctive therapies.

[0083] The term "vaccine" or "vaccine composition" refers to a composition comprising an immunogenic composition that induces an immune response in an animal.

[0084] The term "subject" or "individual" refers to a primate (e.g., non-human primates such as humans and monkeys). In some embodiments, the individual or subject is a human.

[0085] II. Specific Embodiments In one aspect, the present disclosure provides (a) CDRs comprising the following heavy chain variable regions: CDR1 comprising the sequence of SEQ ID NO:1; CDR2 comprising the sequence of SEQ ID NO:2; CDR3 comprising the sequence of SEQ ID NO:3; and (b) CDRs comprising the following light chain variable regions: CDR1 comprising the sequence of SEQ ID NO:4; CDR2 comprising the sequence of SEQ ID NO:5; CDR3 comprising the sequence of SEQ ID NO:6 and provides an isolated antibody or antigen-binding fragment that specifically binds to a fusion protein, which is a surface glycoprotein of respiratory syncytial virus (RSV).

[0086] In one aspect, the present disclosure provides an isolated antibody or an antigen-binding fragment thereof that specifically binds to a fusion protein, which is a surface glycoprotein of respiratory syncytial virus (RSV), and includes three CDRs of the heavy chain variable region shown in SEQ ID NO:7 and three CDRs of the light chain variable region shown in SEQ ID NO:8.

[0087] According to the antibody or the antigen-binding fragment thereof according to any of the above aspects, the heavy chain variable region is (1) a sequence that includes the heavy chain CDRs according to any of the above aspects and has 80% or more identity with SEQ ID NO:7, or (2) a sequence that includes the sequence shown in SEQ ID NO:7.

[0088] According to the antibody or the antigen-binding fragment thereof according to any of the above aspects, the light chain variable region is (1) a sequence that includes the light chain CDRs according to any of the above aspects and has 80% or more identity with SEQ ID NO:8, or (2) a sequence that includes the sequence shown in SEQ ID NO:8.

[0089] According to the isolated antibody according to any of the above aspects, it includes a heavy chain variable region that includes the sequence shown in SEQ ID NO:7 and a light chain variable region that includes the sequence shown in SEQ ID NO:8.

[0090] According to the isolated antibody according to any of the above aspects, the antibody is an IgG antibody.

[0091] According to the isolated antibody or the antigen-binding fragment thereof according to any of the above aspects, the antigen-binding fragment is selected from Fab, Fab’-SH, Fv, scFv or (Fab’)2 fragments.

[0092] According to the isolated antibody or the antigen-binding fragment thereof according to any of the above aspects, it includes a constant region sequence at least a part of which is a human common constant region sequence.

[0093] In one aspect, the present disclosure provides a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof according to any of the above aspects.

[0094] In one aspect, the present disclosure provides a vector comprising the nucleic acid molecule according to the above aspect.

[0095] In some preferred embodiments, the vector is an expression vector.

[0096] In one aspect, the present disclosure provides a host cell comprising the vector.

[0097] In some preferred embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0098] In some preferred embodiments, the host cell is selected from Escherichia coli cells, yeast cells, mammalian cells, or other cells suitable for the preparation of an antibody or an antigen-binding fragment thereof, and the mammalian cells are, for example, CHO cells, HEK293 cells or COS cells.

[0099] In one aspect, the present disclosure provides a method for producing an antibody or an antigen-binding fragment thereof that binds to a fusion protein, which is a surface glycoprotein of respiratory syncytial virus (RSV), comprising culturing the host cell under conditions suitable for the expression of a nucleic acid encoding the antibody or an antigen-binding fragment thereof according to any of the above aspects, optionally isolating the antibody or an antigen-binding fragment thereof, and optionally collecting the produced antibody or an antigen-binding fragment thereof.

[0100] Provided is an antibody or an antigen-binding fragment thereof prepared by the method according to the above aspect.

[0101] In one aspect, the present disclosure provides a pharmaceutical composition comprising the antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

[0102] According to the drug composition according to the above aspect, it further comprises another therapeutic agent. The neutralizing antibody or drug composition provided by the present disclosure can be incorporated into appropriate carriers, excipients, and other reagents for combined use, thereby providing improved mobility, delivery, resistance, etc. It should be understood.

[0103] In one aspect, the present disclosure provides the use of an antibody or an antigen-binding fragment thereof according to any of the above aspects in the preparation of a drug for preventing or treating an RSV-related disease or symptom.

[0104] The drug according to the present disclosure must be sterile and stable under the preparation and storage conditions. In the case of a sterile powder for preparing a sterile injection solution, the preferred method of preparation is vacuum drying and lyophilization to produce a powder of the active ingredient and other desired ingredients from a pre-sterilized and filtered solution of the active ingredient and other desired ingredients. Optionally, the drug according to the present disclosure may be present in solution and provide an injectable unit dosage form by adding and / or mixing appropriate pharmaceutically acceptable excipients before or at the time of delivery. Preferably, the pharmaceutically acceptable excipients used in the present disclosure are suitable for high-concentration drugs, maintain appropriate fluidity, and can delay absorption as required.

[0105] In one aspect, the present disclosure provides a method for preventing or treating an RSV-related disease, comprising administering an effective amount of the antibody or an antigen-binding fragment thereof to a subject in need thereof.

[0106] In one embodiment, the neutralizing antibody according to the present disclosure can produce a 100% inhibitory effect against the virus.

[0107] In one aspect, the present disclosure provides a method for detecting whether RSV is present in a sample, comprising contacting the sample with the antibody or an antigen-binding fragment thereof.

[0108] For the purpose of clear and concise description, although features are described herein as part of some embodiments where they are the same or distinct, it is understood that the scope of the present disclosure may include some embodiments having combinations of all or some of the described features.

Example

[0109] Example 1: Screening, Expression and Purification of Respiratory Syncytial Virus Neutralizing Antibodies 1. Expression and Identification of RSV Pre-F and RSV Post-F Proteins The sequences of the pre-fusion conformation (Pre-F) and post-fusion conformation (Post-F) of the F protein of the RSV A2 strain, the target gene, were found from the NCBI database, and their eukaryotic expression vectors were constructed, and the proteins were expressed and purified. It was known that RSV Pre-F protein and RSV Post-F protein, which are consistent with the naturally occurring trimeric form of RSV F protein, could be obtained by PAGE detection.

[0110] Next, when detected by ELISA, it was shown that both RSV Pre-F protein and RSV Post-F protein had positive binding to the fusion protein antibody of mouse anti-respiratory syncytial virus and did not bind to other irrelevant antibodies. Therefore, both the RSV Pre-F protein and RSV Post-F protein expressed in this example are natural trimeric structure proteins with specific binding activity.

[0111] 2. Screening of Positive Plasma Samples and Isolation of Memory B Cells Peripheral venous blood was collected from healthy adult volunteers, and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation. Plasma samples with high titers against RSV F protein (RSV Pre-F and RSV Post-F) were screened by ELISA. Plasma samples with high antibody titers were screened, and the PBMCs of the samples were flow sorted.

[0112] Sorting of Memory B Cells by Flow Cytometry: PBMCs revived in a 37°C water bath were filtered through a 40-μm filter membrane, and individual plasma cells were sorted using a flow cytometer (manufacturer: BD, model number: FACSria). Gate control was set and selection was performed according to CD3-PE-Cy5- / CD16-PE-Cy5- / CD235a-PE-Cy5- / CD14-FITC- / IgD-PE- / CD20-APC+ / CD27-APC-H7+RSV Post-BV421+ / RSV pre-PE-Cy7+ to select a cell population of memory B cells specific for RSV F protein (Pre-F and / or Post-F) from PBMCs. The morphologically intact single cells thus selected were placed in a 96-well PCR plate (containing 20 μL of single-cell lysate per well) so that each well contained one memory B cell and stored in a -80°C refrigerator.

[0113] The sorting results are shown in Figure 1. Memory B cells in the samples to be detected were sorted by flow cytometry, and memory B cells specifically binding to Pre-F protein and / or Post-F protein were screened.

[0114] 3. Isolation of Antibody Variable Region Genes (1) RT-PCR: To the 96-well plate containing single B cells, 0.5 μM of constant region primers for different subtypes of heavy and light chains (the primers were designed at specific sites by conventional methods, refer to CN107760690B) and Superscript III reverse transcriptase were added, incubated at 37°C for 1 hour, and PCR amplification was performed under the following conditions: first, pre-denaturation at 95°C for 15 min, then denaturation at 95°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min for 30 cycles, and finally extension at 72°C for 10 min and storage at 4°C for 5 min. The obtained product cDNA was stored at -20°C.

[0115] (2) PCR: In a 50 μL reaction system, add 5 μL of the reverse transcription product, HotStarTaq Plus enzyme, dNTPs, and specific primers for the variable regions of different subtypes of heavy and light chains at 0.5 μM (the primers were designed at specific sites by conventional methods, refer to CN107760690B). Perform PCR amplification under the following conditions, that is, pre-denature at 94°C for 5 min, then denature at 94°C for 30 s, anneal at 55°C for 30 s, extend at 72°C for 50 s, repeat for 35 cycles, and finally extend at 72°C for 7 min.

[0116] The obtained PCR product was detected by 1% agarose gel electrophoresis, and the remaining PCR product was purified using the Qiagen PCR Purification Kit (Qiagen).

[0117] 4. Construction of the expression vector of the recombinant antibody and antibody expression The PCR products of the antibody variable region genes that were identified as positive by gel electrophoresis and could pair the heavy and light chains were ligated into the pcDNA3.3 vector by the TA cloning method to construct an expression vector for the anti-respiratory syncytial virus fully human-derived neutralizing antibody. The expression vector was transformed into DH5α competent bacteria, cultured overnight at 37°C on a plate containing ampicillin, 10 single colonies were picked up, and PCR was performed with specific primers. The reaction conditions were as follows. That is, pre-denature at 94°C for 3 min, then denature at 94°C for 30 s, anneal at 55°C for 30 s, extend at 72°C for 100 s, repeat for 28 cycles, and finally extend at 72°C for 5 min. 5 μL of the PCR product was detected by 1% agarose gel electrophoresis. As a result, in the positive transformants, it was shown that transformants containing the genes of the heavy and light chains of the antibody were identified.

[0118] The expression vectors of the heavy and light chains of the antibody that showed positive binding to the antigen were co-transfected into HEK293 cells. After transfection, a large amount of fresh medium was exchanged 6 - 8 hours later, and the cells were cultured in an incubator at 37°C and 8% CO2 to express the antibody. As a result, the fully human-derived monoclonal antibody TRN1021 against respiratory syncytial virus was obtained, and its sequence is shown in Tables 1 - 2 below.

[0119]

Table 2

[0120]

Table 3

[0121] 5. Purification and Analysis of the Expressed Antibody The transfected cells were subsequently cultured for 96 hours, after which the transfection supernatant was collected and centrifuged at 4000 rpm for 1 hour at 4°C to remove cell debris. According to the manufacturer's instructions, Protein A Agarose affinity chromatography was performed overnight, and the collected supernatant was slowly allowed to bind sufficiently to the antibody using a Protein A Agarose affinity chromatography column. After washing with 60 mL of PBS, the bound antibody was eluted with an elution buffer (0.1 M Gly-HCl buffer, pH 2.5), collected in Amicon Ultra-30 Centrifugal Filters (MerckMillipore) containing 1 mL of 1 M Tris-HC buffer (pH 9.0), centrifuged at 5000 g for 20 min at 4°C to concentrate the protein, the PBS was added again to the Amicon Ultra-30 Centrifugal Filters, centrifuged at 3500 g for 20 min at 4°C, and then the fresh equilibration buffer was exchanged, repeating this 3 times to obtain the antibody protein (fully human-derived monoclonal antibody TRN1021) concentrated to 1 mL. The Mini-Protein cell III reaction system (Bio-Rad) was employed to perform discontinuous vertical electrophoresis. The mixing ratio of the antibody sample and the loading buffer was 5:1. After mixing, the reducing sample was boiled for 5 min and loaded at 10 μL / 1 well, and the electrophoresis time was approximately 60 min. Coomassie Flash Blue R-250 was used for staining for more than 30 min, and then decolorized with a decolorizing solution until the background became clean. The purification effect was observed by SDS-PAGE protein electrophoresis analysis.

[0122] The results are shown in Figure 2. After purification, RN1021 showed distinct bands in the SDS-PAGE profiles for both non-reducing and reducing antibody proteins. Lane 1 was the non-reducing antibody protein, Lane 2 was the Marker, and Lane 3 was the reducing antibody protein. The molecular weight of the non-reducing band (i.e., the antibody) was approximately 180 kD. The reducing sample was cleaved into a heavy chain of approximately 65 kD and a light chain of approximately 25 kD, and there were few other heterobands. Subsequently, the activity and function of the purified antibody TRN1021 were reconfirmed by ELISA and the like.

[0123] Example 2: Comparison of the Expression Levels of TRN1021 and TRN1022 Antibodies in HEK293 Cells 1. Construction and Expression of Recombinant Antibody Expression Vectors The steps were the same as those in "4. Construction of Recombinant Antibody Expression Vectors and Antibody Expression" in Example 1.

[0124] 2. Antibody Protein Purification and Analysis The steps were the same as those in "5. Antibody Protein Purification and Analysis" in Example 1.

[0125] 3. Calculation of the Average Antibody Expression Level Five lots of TRN1021 antibody and five lots of TRN1022 antibody (refer to CN110016079A for this antibody) were each produced. The protein concentration of the purified antibody was detected with a micro UV spectrophotometer, and based on the total amount of medium added, the average antibody expression level was calculated. The antibody production information and average expression level are shown in Table 3 and Figure 3.

[0126] As can be seen from the data in Table 3, the expression levels of antibody TRN1021 and antibody TRN1022 were significantly different. TRN1021 was more easily expressed, and the average expression level could reach 2.603 mg / L, while the average expression level of TRN1022 was only 0.588 mg / L.

[0127]

Table 4

[0128] Example 3: Measurement of the Activity and Function of an Anti-Respiratory Syncytial Virus Antibody 1. Binding Activity of the Antibody The binding activity of the antibody TRN1021 expressed and purified by the same method as the ELISA method mentioned above was detected: The RSV Pre-F and RSV Post-F proteins were coated on an ELISA 96-well plate with carbonate coating buffer respectively and left overnight at 4°C. The plate was washed with PBST buffer, blocking solution was added, and blocked at 37°C for 2 h or left overnight at 4°C. The RSV antibody sample TRN1021 (primary antibody) obtained in Example 1 was diluted in 12 gradients from 1 μg / well in a 3-fold gradient. The positive controls were 100 μL / 1 well of the undiluted positive plasma sample (1:50) and the RSV monoclonal antibody RSV3216 (B016, Abcam, #ab24011) (1:1000). The negative controls were 100 μL / 1 well of the undiluted negative plasma sample (1:50) and an irrelevant antibody (TRN006) at 0.5 μg / mL, and 100 μL of blocking solution was added to the blank wells and incubated at 37°C for 1 h. The plate was washed again with PBST buffer, and 100 μL of goat anti-human IgG-HRP and goat anti-mouse IgG-HRP (secondary antibodies) diluted 1:10000 with blocking solution were added to each well and incubated at 37°C for 1 h. The plate was washed with PBST buffer, light was blocked, 100 μL of TMB was added to each well, left at 37°C for 5 min, and the reaction was stopped with 2 M sulfuric acid. The OD values were detected and calculated at two wavelengths of 450 / 630 nm.

[0129] As can be seen from Figure 4, the fully human-derived anti-RSV-F protein monoclonal antibody TRN1021 after expression and purification binds to the purified RSV Pre-F protein, and the binding is dose-dependent. The binding of antibody TRN1021 to RSV Pre-F is specific, and the fully human-derived anti-RSV-F protein monoclonal antibody TRN1021 after expression and purification does not have specific binding activity to the purified RSV Post-F protein.

[0130] 2. In vitro microneutralizing activity of antibodies Three-fold gradient dilutions of antibody TRN1021 were added to HEp-2 cell culture media in a 96-well microtiter plate in a volume of 50 μL / well. Then, 50 μL of RSV A2 virus was added at a concentration of 2 × 10 4 The prepared Hep-2 cell suspension (2 × 10 5 0.1 mL / well of 1000 cells / mL of 100% acetone was added to each well, and the plates were cultured in an incubator at 37°C and 5% CO2. Cytopathic effect (CPE) was observed every day using an inverted microscope, and if the cell syncytial lesion area ratio in the virus control well in the field of view was 80% or more of the bottom area of the well, it could be judged. After discarding the supernatant and washing twice with PBST buffer, 100 μL of fixative containing 80% acetone was added to each well, and the wells were fixed for 15 to 30 min. The fixative was discarded, and the wells were washed twice with PBST buffer, after which 300 μL of blocking solution was added to each well, and the wells were blocked at 37°C for 2 h. After washing three times with PBST buffer, 100 μL of RSV050-HRP (diluted 1:5000 with blocking solution) was added to each well, and the wells were incubated at 37°C for 1 h. Wash five times with PBST buffer, add 100 μL of TMB to one well, leave at room temperature for 5 to 10 minutes, and then immediately stop the reaction with 450 μL of 2M H2SO. Read the plate with a microplate reader and detect the OD value at two wavelengths of 450-630 nm. The OD value of the diluted antibody sample is 100 CCID. 50 An antibody concentration was defined as having neutralizing activity if it was less than half the OD value of the virus control wells.

[0131] Response and variable slope curves were fitted with log(inhibitor) and IC values were calculated using a nonlinear fit algorithm in Graphpad Prism. 50 Calculate the IC value. 50 Values represent the concentration of antibody TRN1021 required for a 50% decrease in absorbance measured at 450-630 nm.

[0132] According to the results in Figure 5, both the antibody TRN1021 and palivizumab (Synagis (R) ) have neutralizing activity against RSV A2 strain, but the antibody TRN1021 has a higher neutralizing titer, and the minimum neutralizing concentration detected by in vitro micro-neutralization activity is shown to be only 0.02 μg / mL.

[0133] 3. Affinity of the antibody By surface plasmon resonance (SPR) test, anti-human IgG (Fc) was coupled to two channels of a CM5 chip by amino coupling, and the anti-RSV F protein antibody was captured as a ligand. Finally, 5485.4 RU was coupled to channel 1 and 5622.4 RU was coupled to channel 2. The captured TRN1021 had a concentration of 1 μg / mL and a binding time of 130 s. Different concentrations of RSV F protein (2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL) were used as analytes, and the detection channels were flowed at a binding time of 90 s and a dissociation time of 600 s. The chip regeneration solution was 3M MgCl2 and the regeneration time was 30 s. Dynamic analysis (calculation of dissociation constant (KD)) was performed with Biacore X100 Evaluation Software (2.0.1). The results are shown in Figure 6.

[0134] When two types of RSV F proteins (RSV Pre-F protein and RSV Post-F protein) were measured by surface plasmon resonance (SPR), the antibody TRN1021 did not bind to the post-fusion F protein (RSV Post-F), and it was shown to bind to the antigen, i.e., the pre-fusion F protein (RSV Pre-F), with a low average dissociation constant of 10 -9 M. The monoclonal antibody TRN1021 derived from natural whole human against respiratory syncytial virus was shown to specifically bind to Pre-F.

[0135] 4. Antigen competition analysis The antigen competition of a monoclonal antibody against respiratory syncytial virus (RSV) derived from natural full-length human against the RSV Pre-F protein was analyzed by surface plasmon resonance (SPR). The antibody was captured with the antigen Pre-F protein. First, antibody TRN1021 at 50 μg / mL was used as the analyte, and the detection channel was flowed for 180 s of binding time, and then Synagis (R) at the same concentration was flowed through the detection channel as the second analyte, and binding and dissociation were carried out. The chip regeneration solution was glycine at pH 1.5, and the regeneration time was 30 s. Then, similarly, the antibody was captured with the antigen Pre-F protein. First, Synagis (R) at 50 μg / mL was flowed through the detection channel as the analyte for 180 s of binding time, and then the antibody TRN1021 at the same concentration was flowed through the detection channel as the second analyte, and binding and dissociation were carried out.

[0136] As a result, when the second analyte was flowed through the detection channel, the binding-dissociation curve of the antibody to the antigen was not affected by the second analyte, indicating that TRN1021 recognizes an antigen epitope different from that of Synagis (R) .

[0137] Example 4: Broad-spectrum neutralizing activity of neutralizing antibodies Detect the inhibitory ability of the target antibody TRN1021 against the infection of HEp-2 cells by different RSV virus strains in the virus neutralization test. In this test, three standard laboratory strains such as RSV A2, RSV Long, and RSV 9320, and 12 RSV virus strains isolated from clinical samples such as CL9325, CL8879, CL9133, CL9574, CL6477, CL8938, CL6495, CL0007, CL0014, CL0041, CL0042, and CL00J3 were used. The collection period of clinical virus samples was over 5 years (provided by Chongqing Children's Hospital). A total of 15 RSV viruses, including 10 A-subtype and 5 B-subtype RSV viruses (two subtypes A and B), were used to conduct a broad-spectrum neutralization test of the virus. As a result (Table 4), the TRN1021 antibody can neutralize all RSV virus strains isolated from standard laboratory strains such as RSV A2, RSV Long, and RSV 9320, and 12 clinical samples such as CL9325, CL8879, CL9133, CL9574, CL6477, CL8938, CL6495, CL0007, CL0014, CL0041, CL0042, and CL00J3, and its IC 50 range is 0.0004 μg / mL - 0.0631 μg / mL, indicating that the TRN1021 antibody is an antibody with extremely strong broad-spectrum neutralizing activity.

[0138]

Table 5

[0139] Analyze the broad-spectrum neutralizing ability of the TRN1021 antibody. The antibody TRN1022 (refer to this antibody in CN110016079A) and the commercially available drug palivizumab (Synagis (R) ) were used as controls, and the results of the geometric mean values of their IC 50 are shown in Figure 7. As a result, the IC 50The geometric mean (indicated by the black horizontal line in the figure) is lower than that of palivizumab, a commercially available monoclonal antibody drug, indicating that the broad-spectrum neutralizing activities of antibody TRN1021 and antibody TRN1022 are superior to those of palivizumab. On the other hand, the IC 50 geometric mean of antibody TRN1021 is lower than the IC 50 geometric mean of antibody TRN1022, indicating that the broad-spectrum neutralizing activity of antibody TRN1021 is superior to that of antibody TRN1022.

[0140] Example 5: In Vivo Pharmacodynamic Study in Animals Cotton mice were used as a standard animal model to evaluate the efficacy of anti-RSV monoclonal drugs, and the in vivo efficacy of antibody TRN1021 was evaluated. In the multiple-dose administration study, for the experimental group, intramuscular injection of antibody TRN1021 was performed at different doses. One day after administration, RSV A2 virus and RSV9320 virus were inoculated into cotton mice via the nose. Four days after virus inoculation, the virus titers in the lungs and nasal tissues of cotton mice and the concentration of antibody TRN1021 in the blood were evaluated.

[0141] The results (shown in Figure 8) showed that as the antibody administration dose increased, the inhibitory effect on the virus titer gradually became stronger. When the antibody TRN1021 concentration reached 2 mg / kg, the virus infection in the lungs and nasal tissues of cotton mice inoculated with RSV A2 virus (Figure 8A, 8B) could be effectively inhibited by 100%. When the antibody TRN1021 concentration reached 1 mg / kg, the virus infection in the lungs and nasal tissues of cotton mice inoculated with RSV 9320 virus (Figure 8C, 8D) could be effectively inhibited by 100%. From the non-linear fitting of the dose-effect curve, the drug blood concentration (i.e., EC 90 ) when the RSV A2 virus inhibition rate in the lung tissue of cotton mice reached 90% was 1.86 μg / mL, and the drug blood concentration (i.e., EC 90 ) when the RSV 9320 virus inhibition rate in the lung tissue of cotton mice reached 90% was calculated to be 1.83 μg / mL.

[0142] As shown in the dose - effectiveness relationship of the above - mentioned efficacy study, when the drug blood concentration (i.e., EC 90 ) is 1.86 μg / mL, the RSV virus inhibition rate against cotton mouse tissue reaches 90%. That is, for individuals infected with the RSV virus, the antibody TRN1021 has a very excellent protective effect.

[0143] Although the present disclosure has been described in detail using the above general description and specific embodiments, based on the present disclosure, some modifications or alterations may be made, which are obvious to those skilled in the art. Therefore, these modifications or alterations made without departing from the gist of the present disclosure belong to the scope claimed by the present disclosure.

Claims

1. (a) CDRs comprising the following heavy chain variable regions, CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3, and (b) CDRs comprising the following light chain variable regions, CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6, An isolated antibody or antigen-binding fragment that specifically binds to a fusion protein, which is a surface glycoprotein of respiratory syncytial virus (RSV), comprising the same.

2. The isolated antibody or antigen-binding fragment according to claim 1, comprising three CDRs of the heavy chain variable region having the amino acid sequence represented by SEQ ID NO: 7 and three CDRs of the light chain variable region having the amino acid sequence represented by SEQ ID NO:

8.

3. The isolated antibody or antigen-binding fragment according to claim 1 or 2, wherein the heavy chain variable region comprises the heavy chain CDRs according to claim 1 and has an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO:

7.

4. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the heavy chain variable region is the amino acid sequence represented by SEQ ID NO:

7.

5. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the light chain variable region comprises the light chain CDRs according to claim 1 and has an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO:

8.

6. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the light chain variable region is the amino acid sequence represented by SEQ ID NO:

8.

7. The isolated antibody or antigen-binding fragment according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region is the amino acid sequence represented by SEQ ID NO: 7, and the light chain variable region is the amino acid sequence represented by SEQ ID NO:

8.

8. The isolated antibody or antigen-binding fragment according to any one of claims 1 to 7, wherein the antibody is an IgG antibody.

9. The antigen-binding fragment is selected from a Fab, a Fab' containing a free SH, an Fv, a scFv, or a (Fab')2 fragment, and is the isolated antibody or antigen-binding fragment according to any one of claims 1 to 7.

10. The antibody or its antigen-binding fragment contains a constant region sequence, and at least a part of the constant region sequence is a human common constant region sequence, and is the isolated antibody or antigen-binding fragment according to any one of claims 1 to 9.

11. A nucleic acid molecule encoding the antibody or its antigen-binding fragment according to any one of claims 1 to 10.

12. A vector containing the nucleic acid molecule according to claim 11.

13. The vector is an expression vector, and is the vector according to claim 12.

14. A host cell containing the vector according to claim 12 or 13.

15. The host cell is a prokaryotic cell or a eukaryotic cell, and is the host cell according to claim 14.

16. The host cell is selected from Escherichia coli cells, yeast cells, mammalian cells, or other cells suitable for the preparation of the antibody or its antigen-binding fragment, and is the host cell according to claim 15.

17. The mammalian cell is a CHO cell, a HEK293 cell or a COS cell, and is the host cell according to claim 16.

18. A method for preparing an antibody or its antigen-binding fragment that binds to a fusion protein, which is a surface glycoprotein of respiratory syncytial virus (RSV), including culturing the host cell according to any one of claims 14 to 16 under conditions suitable for the expression of the nucleic acid encoding the antibody or its antigen-binding fragment according to any one of claims 1 to 10.

19. The method further includes isolating the antibody or its antigen-binding fragment, and is the method according to claim 18.

20. The method further includes collecting the generated antibody or its antigen-binding fragment, and is the method according to claim 19.

21. An antibody or its antigen-binding fragment prepared by the method according to any one of claims 18 to 20.

22. A pharmaceutical composition containing the antibody or its antigen-binding fragment according to any one of claims 1 to 10 or claim 21, and a pharmaceutically acceptable carrier.

23. The pharmaceutical composition further includes another therapeutic agent, and is the pharmaceutical composition according to claim 22.

24. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 10 or claim 21 for use in the prevention or treatment of a disease or symptom associated with respiratory syncytial virus (RSV) infection.

25. The antibody or antigen-binding fragment thereof according to claim 24, wherein the respiratory syncytial virus (RSV) is one or more selected from RSV type A virus and RSV type B virus.

26. A method for detecting whether respiratory syncytial virus (RSV) is present in a sample, comprising the step of contacting the sample with an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 10 or claim 21.

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