Antibodies recognizing rsv pre-f protein and uses

Abstract

The present invention relates to the field of immunology and molecular virology, in particular the field of prevention and treatment of RSV. In particular, the present invention provides monoclonal antibodies capable of specifically binding to a new epitope between the pre-F protein and the V epitope or a new epitope between the II and the V epitope, and their use for the detection, prevention and / or treatment of RSV infection and / or diseases caused by said infection.

Description

Technical Field

[0001] This invention relates to the fields of immunology and molecular virology, particularly to the prevention and treatment of RSV. Specifically, this invention provides a method capable of specifically binding to the pre-F protein. Monoclonal antibodies containing novel epitopes between epitopes II and V, or between epitopes II and V, and their use for the detection, prevention, and / or treatment of RSV infection and / or disease caused by said infection. Background Technology

[0002] Human respiratory syncytial virus (RSV) is the most important pathogen causing acute lower respiratory tract infection in children under 5 years old, and is also the primary factor causing lower respiratory tract infection hospitalization in infants. Infants under 2 years old almost all have been infected with RSV, and infants under 6 months are the main population of RSV infection hospitalization, accounting for about 50%. Globally, there are more than 300 million cases of lower respiratory tract infection caused by RSV infection each year, of which there are more than 3 million cases of hospitalization and 66,000 to 239,000 cases of death (Ting Shi, David A McAllister, et al., Lancet, 390 (2017) 946-958; Geoghegan S, Erviti A, et al., Am J Respir Crit Care Med 195 (2017) 96-103). Compared with other respiratory viral infections, children infected with RSV have a higher disease burden, especially infants under 6 months, who have a higher disease burden than children aged 6-24 months, including higher ICU admission rates, hospitalization costs, and longer hospital stays. RSV infection increases the risk of severe pneumonia in children by 14 times (The Pneumonia Etiology Research for Child Health (PERCH) Study Group, Lancet, 394 (2019) 757-779). The RSV infection rate of infants with bronchial and pulmonary dysplasia, congenital heart disease, and immune deficiency is as high as 50-70% (A.C. Cooper, N.C. Banasiak, et al., Pediatr Nurs, 29 (2003) 452-456). In addition, the elderly are also susceptible to RSV, and the number of deaths caused by RSV infection each year exceeds 12,000. RSV is also the main pathogen of lower respiratory tract infection in Chinese children. In China, about 215,000 to 500,000 infants are hospitalized due to RSV infection each year (Li Y, Johnson EK, et al., Lancet Respir Med, 2021, 9(2): 175-185).

[0003] There is no safe and effective RSV vaccine on the market, only one strain of neutralizing antibody (Palivizumab, trade name: Synagis) that recognizes RSV fusion glycoprotein is approved by the US FDA for marketing, for preventing severe lower respiratory tract infection caused by RSV in high-risk infants and young children with preterm birth, chronic lung disease, bronchial and lung development abnormalities, congenital heart disease. However, due to the insufficient neutralization titer, high production cost and high price of the drug, it is only used for "high-risk infants and young children" and cannot be widely used. In addition, Sanofi Pasteur and AstraZeneca jointly developed a new generation of RSV preventive drug "Nirsevimab" based on human antibody D25. The drug is a long-acting preventive monoclonal antibody, and infants and young children only need one dose of intramuscular injection to provide long-lasting protection for up to 5 months, so that infants and young children can safely pass through the entire RSV epidemic season. The results of clinical phase IIb trial showed that compared with the control group, Nirsevimab reduced the visit rate and hospitalization rate by 70.1% and 78.4%, respectively (M Pamela Griffin, Yuan Yuan, et al., N Engl J Med, 383 (2020) 415-425). Currently, Nirsevimab has been approved by the US FDA as a "breakthrough therapy", and in January 2021, China included monoclonal antibody Nirsevimab in "breakthrough therapy drugs". Currently, there is no RSV-specific preventive or therapeutic drug on the market in China.

[0004] RSV is a single-stranded negative-sense non-segmented RNA virus of the family Pneumoviridae, with 15222 nucleotides, containing 10 genes, encoding 11 proteins. Among them, the full-length 574 amino acid fusion (F) protein of type I transmembrane protein is the main target protein for the development of RSV vaccine and antibody. RSV F protein exists in two conformations, pre-fusion (pre-F) and post-fusion (post-F), where pre-F is in a high-energy, metastable pre-fusion conformation, mediating fusion of the viral envelope with the cell membrane, and post-F is a highly stable post-fusion conformation. Compared with post-F protein, pre-F protein has more and stronger high neutralizing epitopes, which can induce the production of antibodies with high neutralizing activity.

[0005] In summary, RSV causes serious harm to children around the world and a heavy health care burden, and China urgently needs to develop a broad-spectrum high neutralizing activity antibody with independent intellectual property rights for RSV prevention and / or treatment for the prevention and / or treatment of RSV infection in children. SUMMARY

[0006] Six neutralizing sites have been found on pre-F and post-F I, II, III, IV and V, among which, site and site V are pre-F specific high neutralizing sites, and sites I, II, III and IV are located on both pre-F and post-F conformational F proteins. Site specifically located at the distal membrane end top of the pre-F trimer, which includes the a4 helix (196-209 amino acids) of the F1 subunit and part of the F2 region (62-69 amino acids), both of which undergo significant changes during the prefusion conformation to postfusion conformation. Antibodies D25, 5C4 and AM22 recognize this epitope, and the three antibodies are all pre-F specific high neutralizing antibodies, with a neutralization titer of more than 100 times that of Palivizumab. Site I exists in both pre-F and post-F conformational proteins, which is a linear epitope composed of 387-392 amino acids between the a8 helix and the b14. Antibody 131-2a recognizes this epitope, and the antibody has poor neutralization ability. Site II exists in both pre-F and post-F conformational proteins, which is composed of a6 helix, a7 helix and the loop therebetween (255-276 amino acids). Representative antibodies recognizing this epitope include murine antibody 1129, humanized Palivizumab and its second-generation antibody Motavizumab, which exhibit moderate neutralization ability. Site III also exists in both pre-F and post-F conformational proteins, and the representative antibody recognizing this epitope is MPE8, which is biased to bind to the pre-F conformational protein. MPE8 has broad-spectrum neutralizing activity and can simultaneously neutralize four types of viruses, hRSV, BRSV, hMPV and PVM. Site IV also exists in both pre-F and post-F conformational proteins, which is a linear structure composed of 422-438 amino acids. The representative antibody of this epitope is 101F, which exhibits moderate neutralization ability. Site V specifically exists in the pre-F conformational protein, which is a trimeric epitope composed of 146-194 and 287-300 amino acids. Antibody AM14 recognizes this epitope and can only bind to two monomer binding regions of pre-F, with high neutralization ability.

[0007] The inventors of the present application have unexpectedly developed a monoclonal antibody capable of specifically binding to the pre-F protein Monoclonal antibodies to neoepitopes between the A and V epitopes or neoepitopes between the II and V epitopes. Antibodies of the present application are capable of specifically recognizing the pre-F conformation and neutralize RSV efficiently, blocking or inhibiting RSV fusion with cells. The present application thus provides the following aspects.

[0008] Antibodies or antigen-binding fragments thereof

[0009] 5B11

[0010] In a first aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to an epitope of a respiratory syncytial virus (RSV) pre-F protein, the epitope comprising at least 6 amino acid residues (e.g., noncontiguous amino acid residues) within amino acid residues 160-182 of the pre-F protein and amino acid residues 294-295.

[0011] In certain embodiments, the epitope comprises at least amino acid residues 161, 165, 166, 169, 180, 182, 294, and 295.

[0012] In certain embodiments, the epitope further comprises amino acid residue 196.

[0013] In certain embodiments, the epitope further comprises amino acid residues 162 and 184.

[0014] In certain embodiments, the epitope comprises amino acid residues 161-182 (e.g., 161-184) and amino acid residues 294-295.

[0015] In certain embodiments, the epitope is a conformational epitope.

[0016] In certain embodiments, the amino acid positions are determined according to SEQ ID NO: 1.

[0017] In certain embodiments, the epitope comprises at least E161, N165, K166, S169, S180, S182, E294, and E295; in certain embodiments, the epitope further comprises K196; in certain embodiments, the epitope further comprises G162 and G184.

[0018] In a second aspect, the present application provides an antibody or antigen-binding fragment thereof, comprising:

[0019] (a) a heavy chain variable region (VH) comprising 3 CDRs: a VH CDR1 comprising a sequence set forth in SEQ ID NO: 5, or a variant thereof, a VH CDR2 comprising a sequence set forth in SEQ ID NO: 6, or a variant thereof, and a VH CDR3 comprising a sequence set forth in SEQ ID NO: 7, or a variant thereof; and / or,

[0020] (b) a light chain variable region (VL) comprising 3 CDRs: a VL CDR1 comprising a sequence set forth in SEQ ID NO: 8, or a variant thereof, a VL CDR2 comprising a sequence set forth in SEQ ID NO: 9, or a variant thereof, and a VL CDR3 comprising a sequence set forth in SEQ ID NO: 10, or a variant thereof;

[0021] wherein said variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or said variant has one or several (e.g. 1, 2 or 3) amino acid substitutions, deletions or additions compared to the sequence from which it is derived. In certain embodiments, said substitution is a conservative substitution.

[0022] In certain embodiments, the antibody or antigen-binding fragment thereof is capable of specifically binding to a RSV pre-F protein.

[0023] In certain embodiments, the antibody or antigen-binding fragment thereof competes for binding to a RSV pre-F protein with the antibody or antigen-binding fragment thereof of the first aspect. In certain embodiments, said competing binding means being capable of blocking at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% or preferably at least 99% of the binding of the antibody or antigen-binding fragment thereof of the first aspect to a RSV pre-F protein. Said competing binding can be determined by a competitive binding assay.

[0024] Competitive binding assays are well known to those skilled in the art and are immunological assays that detect and quantify an unknown by its ability to inhibit the binding of a labeled known antigen to its specific antibody, also known as competitive inhibition assays. One exemplary method involves pre-coating microtiter plates with antigen, then incubating serial dilutions of unlabeled test antibody with a specific concentration of labeled known mAb (i.e., the antibody or antigen binding fragment thereof of the first aspect) in the pre-coated microtiter plates, and then measuring the amount of known antibody bound to the plate after washing at different dilutions of the test antibody. The stronger the ability of the test antibody to compete with the known antibody for binding to the antigen, the weaker the ability of the known antibody to bind to the antigen, and the less known antibody bound to the plate. Typically, the antigen is pre-coated on 96-well microtiter plates and the ability of the test mAb to block the labeled known mAb is measured using a radioimmunoassay, an enzyme immunoassay such as ELISA, or a fluorescent immunoassay.

[0025] In certain embodiments, the antibody or antigen binding fragment thereof recognizes the same epitope as, or overlaps spatially with, the epitope recognized by the antibody or antigen binding fragment thereof of the first aspect, such that binding of the antibody or antigen binding fragment thereof of the first aspect to the RSV pre-F protein is blocked. In certain embodiments, the antibody or antigen binding fragment thereof binds to the same epitope of the RSV pre-F protein as the antibody or antigen binding fragment thereof of the first aspect.

[0026] In certain embodiments:

[0027] (i) the antibody or antigen binding fragment thereof comprises: 3 heavy chain CDRs as follows: VH CDR1 of SEQ ID NO: 5, VH CDR2 of SEQ ID NO: 6, and VH CDR3 of SEQ ID NO: 7; and / or, 3 light chain CDRs as follows: VL CDR1 of SEQ ID NO: 8, VL CDR2 of SEQ ID NO: 9, and VL CDR3 of SEQ ID NO: 10; and / or,

[0028] (ii) the antibody or antigen binding fragment thereof comprises: 3 CDRs contained within a heavy chain variable region (VH) as set forth in SEQ ID NO: 3 or 60; and / or, 3 CDRs contained within a light chain variable region (VL) as set forth in SEQ ID NO: 4 or 61. In certain embodiments, the 3 CDRs contained within the VH and / or the 3 CDRs contained within the VL are defined by the Kabat, IMGT, or Chothia numbering system.

[0029] In certain embodiments, the antibody or antigen binding fragment thereof comprises:

[0030] a VH comprising a sequence as set forth in SEQ ID NO: 3, or a variant thereof, and / or, a VL comprising a sequence as set forth in SEQ ID NO: 4, or a variant thereof;

[0031] wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it is derived, or the variant has one or more substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions) of amino acids compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

[0032] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH as set forth in SEQ ID NO: 3, and / or, a VL as set forth in SEQ ID NO: 4.

[0033] In certain embodiments, the antibody or antigen-binding fragment thereof is humanized, which comprises framework region sequences derived from human immunoglobulin. In certain embodiments, the antibody or antigen-binding fragment thereof comprises heavy chain framework region sequences derived from human heavy chain germline sequences, and light chain framework region sequences derived from human light chain germline sequences, which heavy chain and / or light chain framework regions optionally comprise back mutations from human-derived residues to murine-derived residues. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH as set forth in SEQ ID NO: 60, and / or, a VL as set forth in SEQ ID NO: 61.

[0034] In a third aspect, the present application also provides an antibody or antigen-binding fragment thereof which competes for binding to RSV pre-F protein with the antibody or antigen-binding fragment thereof of the second aspect (e.g., mAb 5B11 or an antigen-binding fragment thereof). In certain embodiments, the competing binding means being able to block at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95%, or preferably at least 99% of the binding of the antibody or antigen-binding fragment thereof of the second aspect (e.g., mAb 5B11 or an antigen-binding fragment thereof) to RSV pre-F protein. The competing binding can be determined by the competition binding assay described above.

[0035] In certain embodiments, the antibody or antigen-binding fragment thereof recognizes the same epitope as, or overlaps in space with, the antibody or antigen-binding fragment thereof of the second aspect (e.g., monoclonal antibody 5B11 or an antigen-binding fragment thereof), such that binding of the antibody or antigen-binding fragment thereof of the second aspect (e.g., monoclonal antibody 5B11 or an antigen-binding fragment thereof) to the RSV pre-F protein is blocked. In certain embodiments, the antibody or antigen-binding fragment thereof binds to the same epitope of the RSV pre-F protein as the antibody or antigen-binding fragment thereof of the second aspect (e.g., monoclonal antibody 5B11 or an antigen-binding fragment thereof).

[0036] 6B2

[0037] In a fourth aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to an epitope of a respiratory syncytial virus (RSV) pre-F protein, the epitope comprising at least 5 amino acid residues (e.g., non-contiguous amino acid residues) located within amino acid residues 160-185 and amino acid residues 290-295 of the pre-F protein.

[0038] In certain embodiments, the epitope comprises at least amino acid residues 161, 162, 184, 293, and 294.

[0039] In certain embodiments, the epitope comprises amino acid residues 161-184 and amino acid residues 293-294.

[0040] In certain embodiments, the epitope is a conformational epitope.

[0041] In certain embodiments, the amino acid positions are determined according to SEQ ID NO: 1.

[0042] In certain embodiments, the epitope comprises at least E161, G162, G184, K293, and E294.

[0043] In a fifth aspect, the present application provides an antibody or antigen-binding fragment thereof, comprising:

[0044] (a) a heavy chain variable region (VH) comprising a VH CDR1 comprising a sequence as set forth in SEQ ID NO: 13, or a variant thereof, a VH CDR2 comprising a sequence as set forth in SEQ ID NO: 14, or a variant thereof, and a VH CDR3 comprising a sequence as set forth in SEQ ID NO: 15, or a variant thereof; and / or,

[0045] (b) a light chain variable region (VL) comprising the following 3 CDRs: a VL CDR1 comprising a sequence set forth in SEQ ID NO: 16, or a variant thereof, a VL CDR2 comprising a sequence set forth in SEQ ID NO: 17, or a variant thereof, and a VL CDR3 comprising a sequence set forth in SEQ ID NO: 18, or a variant thereof;

[0046] wherein said variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or said variant has one or several (e.g. 1, 2 or 3) amino acid substitutions, deletions or additions when compared to the sequence from which it is derived. In certain embodiments, said substitution is a conservative substitution.

[0047] In certain embodiments, the antibody or antigen-binding fragment thereof is capable of specifically binding to a RSV pre-F protein.

[0048] In certain embodiments, the antibody or antigen-binding fragment thereof competes for binding to a RSV pre-F protein with the antibody or antigen-binding fragment thereof of the fourth aspect. In certain embodiments, said competition for binding means being capable of blocking the binding of the antibody or antigen-binding fragment thereof of the fourth aspect to the RSV pre-F protein by at least 50%, preferably by at least 60%, preferably by at least 70%, preferably by at least 80%, preferably by at least 90%, preferably by at least 95% or preferably by at least 99%. Said competition for binding can be determined by the competition binding assay described herein above.

[0049] In certain embodiments, the antibody or antigen-binding fragment thereof recognizes the same epitope as the antibody or antigen-binding fragment thereof of the fourth aspect, or there is a steric overlap such that the binding of the antibody or antigen-binding fragment thereof of the fourth aspect to the RSV pre-F protein is blocked.

[0050] In certain embodiments, the antibody or antigen-binding fragment thereof binds to the same epitope of the RSV pre-F protein as the antibody or antigen-binding fragment thereof of the fourth aspect.

[0051] In certain embodiments:

[0052] (i) the antibody or antigen-binding fragment thereof comprises: 3 heavy chain CDRs: a VH CDR1 of SEQ ID NO: 13, a VH CDR2 of SEQ ID NO: 14, a VH CDR3 of SEQ ID NO: 15; and / or, 3 light chain CDRs: a VL CDR1 of SEQ ID NO: 16, a VL CDR2 of SEQ ID NO: 17, a VL CDR3 of SEQ ID NO: 18; and / or,

[0053] (ii) the antibody or antigen-binding fragment thereof comprises: 3 CDRs contained within a heavy chain variable region (VH) as set forth in SEQ ID NO: 11; and / or, 3 CDRs contained within a light chain variable region (VL) as set forth in SEQ ID NO: 12. In certain embodiments, the 3 CDRs contained within the VH and / or the 3 CDRs contained within the VL are defined by the Kabat, IMGT, or Chothia numbering system.

[0054] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0055] a VH comprising a sequence as set forth in SEQ ID NO: 11, or a variant thereof, and / or, a VL comprising a sequence as set forth in SEQ ID NO: 12, or a variant thereof;

[0056] wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it is derived, or the variant has one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, deletions, or additions compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

[0057] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH as set forth in SEQ ID NO: 11, and / or, a VL as set forth in SEQ ID NO: 12.

[0058] In a sixth aspect, the present application provides an antibody or antigen-binding fragment thereof that competes for binding to RSV pre-F protein with the antibody or antigen-binding fragment thereof of the fifth aspect (e.g., mAb 6B2 or an antigen-binding fragment thereof). In certain embodiments, the competing binding refers to the ability to block binding of the antibody or antigen-binding fragment thereof of the fifth aspect (e.g., mAb 6B2 or an antigen-binding fragment thereof) to RSV pre-F protein by at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95%, or preferably at least 99%. The competing binding can be determined by the competition binding assay described above.

[0059] In certain embodiments, the antibody or antigen-binding fragment thereof recognizes the same epitope as, or overlaps spatially with, the epitope recognized by the antibody or antigen-binding fragment thereof of the fifth aspect (e.g., mAb 6B2 or an antigen-binding fragment thereof), such that binding of the antibody or antigen-binding fragment thereof of the fifth aspect (e.g., mAb 6B2 or an antigen-binding fragment thereof) to RSV pre-F protein is blocked. In certain embodiments, the antibody or antigen-binding fragment thereof binds to the same epitope of RSV pre-F protein as the antibody or antigen-binding fragment thereof of the fifth aspect (e.g., mAb 6B2 or an antigen-binding fragment thereof).

[0060] 7G5

[0061] In a seventh aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to an epitope of a respiratory syncytial virus (RSV) pre-F protein, the epitope comprising at least 3 amino acid residues (e.g., non-contiguous amino acid residues) located within amino acid residues 160-185 of the pre-F protein.

[0062] In certain embodiments, the epitope comprises at least amino acid residues 161, 162, and 184.

[0063] In certain embodiments, the epitope comprises amino acid residues 161-184.

[0064] In certain embodiments, the epitope is a conformational epitope.

[0065] In certain embodiments, the amino acid positions are determined according to SEQ ID NO: 1.

[0066] In certain embodiments, the epitope comprises at least E161, G162, and G184.

[0067] In an eighth aspect, the present application provides an antibody or antigen-binding fragment thereof, comprising:

[0068] (a) a heavy chain variable region (VH) comprising three CDRs: a VH CDR1 comprising a sequence as set forth in SEQ ID NO: 21, or a variant thereof, a VH CDR2 comprising a sequence as set forth in SEQ ID NO: 22, or a variant thereof, and a VH CDR3 comprising a sequence as set forth in SEQ ID NO: 23, or a variant thereof; and / or,

[0069] (b) a light chain variable region (VL) comprising three CDRs: a VL CDR1 comprising a sequence as set forth in SEQ ID NO: 24, or a variant thereof, a VL CDR2 comprising a sequence as set forth in SEQ ID NO: 25, or a variant thereof, and a VL CDR3 comprising a sequence as set forth in SEQ ID NO: 26, or a variant thereof;

[0070] wherein said variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or said variant has one or several (e.g. 1, 2 or 3) amino acid substitutions, deletions or additions compared to the sequence from which it is derived. In certain embodiments, said substitutions are conservative substitutions.

[0071] In certain embodiments, the antibody or antigen-binding fragment thereof is capable of specifically binding to a RSV pre-F protein.

[0072] In certain embodiments, the antibody or antigen-binding fragment thereof competes for binding to a RSV pre-F protein with the antibody or antigen-binding fragment thereof of the seventh aspect. In certain embodiments, said competition for binding means that the antibody or antigen-binding fragment thereof of the seventh aspect is capable of blocking the binding of the antibody or antigen-binding fragment thereof of the seventh aspect to the RSV pre-F protein by at least 50%, preferably by at least 60%, preferably by at least 70%, preferably by at least 80%, preferably by at least 90%, preferably by at least 95% or preferably by at least 99%. Said competition for binding can be determined by the competition binding assay described herein above.

[0073] In certain embodiments, the antibody or antigen-binding fragment thereof recognizes the same epitope as the antibody or antigen-binding fragment thereof of the seventh aspect, or there is a steric overlap such that the binding of the antibody or antigen-binding fragment thereof of the seventh aspect to the RSV pre-F protein is blocked.

[0074] In certain embodiments, the antibody or antigen-binding fragment thereof binds to the same epitope of the RSV pre-F protein as the antibody or antigen-binding fragment thereof of the seventh aspect.

[0075] In certain embodiments:

[0076] (i) the antibody or antigen-binding fragment thereof comprises: 3 heavy chain CDRs as follows: a VH CDR1 of SEQ ID NO: 21, a VH CDR2 of SEQ ID NO: 22, a VH CDR3 of SEQ ID NO: 23; and / or, 3 light chain CDRs as follows: a VL CDR1 of SEQ ID NO: 24, a VL CDR2 of SEQ ID NO: 25, a VL CDR3 of SEQ ID NO: 26; and / or,

[0077] (ii) the antibody or antigen-binding fragment thereof comprises: 3 CDRs contained within a heavy chain variable region (VH) as set forth in SEQ ID NO: 19; and / or, 3 CDRs contained within a light chain variable region (VL) as set forth in SEQ ID NO: 20. In certain embodiments, the 3 CDRs contained within the VH and / or the 3 CDRs contained within the VL are defined by the Kabat, IMGT, or Chothia numbering system.

[0078] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0079] a VH comprising a sequence as set forth in SEQ ID NO: 19, or a variant thereof, and / or, a VL comprising a sequence as set forth in SEQ ID NO: 20, or a variant thereof;

[0080] wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it is derived, or the variant has one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, deletions, or additions compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

[0081] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH as set forth in SEQ ID NO: 19, and / or, a VL as set forth in SEQ ID NO: 20.

[0082] In a ninth aspect, the present application also provides an antibody or antigen binding fragment thereof that competes for binding to RSV pre-F protein with the antibody or antigen binding fragment thereof of the eighth aspect (e.g., mAb 7G5 or an antigen binding fragment thereof). In certain embodiments, the competing binding refers to the ability to block binding of the antibody or antigen binding fragment thereof of the fifth aspect (e.g., mAb 7G5 or an antigen binding fragment thereof) to RSV pre-F protein by at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95%, or preferably at least 99%. The competing binding can be determined by the competition binding assay described above.

[0083] In certain embodiments, the antibody or antigen binding fragment thereof recognizes the same epitope as, or overlaps in space with, the epitope recognized by the antibody or antigen binding fragment thereof of the eighth aspect (e.g., mAb 7G5 or an antigen binding fragment thereof), such that binding of the antibody or antigen binding fragment thereof of the eighth aspect (e.g., mAb 7G5 or an antigen binding fragment thereof) to RSV pre-F protein is blocked. In certain embodiments, the antibody or antigen binding fragment thereof binds to the same epitope of RSV pre-F protein as the antibody or antigen binding fragment thereof of the eighth aspect (e.g., mAb 7G5 or an antigen binding fragment thereof).

[0084] In certain embodiments of any of the above aspects, the antibody or antigen binding fragment thereof is murine. In certain embodiments of any of the above aspects, the antibody or antigen binding fragment thereof comprises a framework region sequence derived from a murine immunoglobulin.

[0085] In certain embodiments of any of the above aspects, the antibody or antigen binding fragment thereof is humanized. In certain embodiments of any of the above aspects, the antibody or antigen binding fragment thereof comprises a framework region sequence derived from a human immunoglobulin. In certain embodiments of any of the above aspects, the antibody or antigen binding fragment thereof comprises a heavy chain framework region sequence derived from a human heavy chain germline sequence, and a light chain framework region sequence derived from a human light chain germline sequence. The heavy chain framework region and / or light chain framework region optionally comprises back-mutations from human-derived residues to murine-derived residues.

[0086] In certain embodiments of any of the above aspects, the antibody or antigen binding fragment thereof further comprises a constant region derived from a murine or human immunoglobulin.

[0087] In certain embodiments of any of the above aspects, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a murine or human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4), and the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a murine or human immunoglobulin (e.g., kappa or lambda).

[0088] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region as set forth in SEQ ID NO: 62, and the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region as set forth in SEQ ID NO: 63.

[0089] In certain embodiments of any of the above aspects, the antigen-binding fragment is selected from the group consisting of a Fab, Fab', (Fab')2, Fv, disulfide linked Fv, scFv, diabody, and single domain antibody (sdAb).

[0090] In certain embodiments of any of the above aspects, the antibody is a murine, chimeric, humanized, bispecific, or multispecific antibody.

[0091] In certain embodiments of any of the above aspects, the antibody or antigen-binding fragment thereof possesses one or more of the following characteristics:

[0092] (a) neutralizes RSV (e.g., RSV type A and / or type B) in vitro or in a subject (e.g., a human);

[0093] (b) blocks or inhibits fusion of RSV (e.g., RSV type A and / or type B) to a cell in vitro or in a subject (e.g., a human);

[0094] (c) prevents and / or treats RSV (e.g., RSV type A and / or type B) infection or a disease (e.g., pneumonia, such as pediatric pneumonia) associated with RSV (e.g., RSV type A and / or type B) infection.

[0095] In certain embodiments, exemplary strains of RSV type A include those set forth as GenBank: KT992094.1, GU591760.1, KJ627328.1, KJ723478.1, KU316139.1, KU316112.1, FJ614813.1, KJ627352.1, KJ627274.1, KU316092.1, and / or HQ317235.1.

[0096] In certain embodiments, exemplary strains of the RSV of type B include strains as set forth in GenBank: JN032119.1, KX765905.1, KY249659.1, KJ627302.1, JQ736675.1, KX765900.1, JF714712.1, AF013254.1, and / or AY353550.1, and / or, RSV 18537 strain.

[0097] Isolated nucleic acid molecules

[0098] In a tenth aspect, the present application provides an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof, or a heavy chain variable region and / or a light chain variable region thereof, as described in any of the above aspects.

[0099] In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain or a heavy chain variable region of an antibody or antigen-binding fragment thereof of any of the aspects of the present application and a second nucleotide sequence encoding a light chain or a light chain variable region of the antibody or antigen-binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different isolated nucleic acid molecules. When the first nucleotide sequence and the second nucleotide sequence are present on different isolated nucleic acid molecules, the isolated nucleic acid molecule described herein comprises a first nucleic acid molecule comprising the first nucleotide sequence and a second nucleic acid molecule comprising the second nucleotide sequence.

[0100] Vectors

[0101] In an eleventh aspect, the present application provides a vector comprising a nucleic acid molecule as described above. In certain embodiments, the vector is a cloning vector or an expression vector.

[0102] In certain embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain or a heavy chain variable region of an antibody or antigen-binding fragment thereof of any of the aspects of the present application and a second nucleotide sequence encoding a light chain or a light chain variable region of the antibody or antigen-binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector described herein comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

[0103] Host cells

[0104] In a twelfth aspect, the present application provides a host cell comprising the nucleic acid molecule or the vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., E. coli cells), and eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.).

[0105] Methods of manufacture

[0106] The antibodies of the present application can be produced in various methods known in the art, for example, by genetic engineering recombination techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present application are obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors, which are then transfected into host cells. Then, the transfected host cells are cultured under specific conditions, and the antibodies of the present application are expressed.

[0107] Antigen-binding fragments of the present application can be obtained by proteolytic hydrolysis of the intact antibody molecule (see Morimoto et al., J. Biochem. Biophys. Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). Alternatively, these antigen-binding fragments can be produced directly by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)). For example, Fab’ fragments can be directly obtained from host cells; Fab’ fragments can be chemically coupled to form F(ab’)2 fragments (Carter et al., Bio / Technology, 10: 163-167 (1992)). In addition, Fv, Fab, or F(ab’)2 fragments can be directly isolated from recombinant host cell culture solutions. Those of ordinary skill in the art are fully aware of other techniques for preparing these antigen-binding fragments.

[0108] In a thirteenth aspect, the present application provides a method of producing an antibody or an antigen-binding fragment thereof of any aspect of the present application, comprising culturing a host cell as described above under conditions that allow expression of the antibody or the antigen-binding fragment thereof, and recovering the antibody or the antigen-binding fragment thereof from the cultured host cell culture.

[0109] Pharmaceutical compositions

[0110] In a fourteenth aspect, the present application provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any aspect of the present application, and a pharmaceutically acceptable carrier and / or excipient.

[0111] In certain exemplary embodiments, the pharmaceutically acceptable carrier and / or excipient comprises a sterile injectable liquid, such as an aqueous or non-aqueous suspension or solution. In certain exemplary embodiments, such sterile injectable liquid is selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), dextrose solution (e.g., 5% dextrose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH-buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0112] Pharmaceutical uses

[0113] In a fifteenth aspect, the present application provides use of the antibody or antigen-binding fragment thereof of any aspect of the present application for the manufacture of a medicament for neutralizing the virulence of RSV, or for inhibiting or blocking RSV fusion with cells, or for preventing and / or treating RSV infection or a disease associated with RSV infection (e.g., pneumonia, such as pediatric pneumonia) in a subject.

[0114] In certain embodiments, the subject is a mammal, e.g., a human.

[0115] In certain embodiments, the antibody or antigen-binding fragment thereof is used alone or in combination with another pharmaceutically active agent.

[0116] Methods of disease prevention and / or treatment

[0117] In a sixteenth aspect, the present application provides a method for preventing and / or treating RSV infection or a disease associated with RSV infection (e.g., pneumonia, such as pediatric pneumonia) in a subject (e.g., a human), comprising: administering to the subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof of any aspect of the present application or the pharmaceutical composition of the present application.

[0118] The antibody or antigen-binding fragment thereof or pharmaceutical composition of the present application can be formulated into any dosage form known in the medical arts, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. The antibody or antigen-binding fragment thereof or pharmaceutical composition of the present application should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by incorporating the antibody or antigen-binding fragment thereof of the present application in the required amount in an appropriate solvent with one or more of the other ingredients enumerated above, as desired (including, but not limited to, pH adjusting agents, surfactants, adjuvants, ion strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtered sterilization. Furthermore, sterile injection solutions can be prepared as sterile lyophilized powders for reconstitution with a suitable vehicle, e.g., water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), dextrose solution (e.g., 5% dextrose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH-buffered solutions (e.g., phosphate-buffered saline), Ringer's solution, and any combination thereof, prior to use.

[0119] The antibody or antigen-binding fragment thereof of the present application, or the pharmaceutical composition of the present application, can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracerebrospinal, inguinal, intravesical, local (e.g., powder, salve, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / means of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and / or means of administration will vary depending on the intended purpose. In certain embodiments, the antibody or antigen-binding fragment thereof or pharmaceutical composition of the present application is administered by intravenous injection or bolus.

[0120] Conjugates

[0121] In a seventeenth aspect, the present application provides a conjugate comprising the antibody or antigen-binding fragment thereof of any aspect of the present application, and a detectable label linked to the antibody or antigen-binding fragment thereof.

[0122] In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin.

[0123] Kits

[0124] In a nineteenth aspect, the present application provides a method for detecting the presence or level of RSV in a sample, comprising using an antibody or antigen-binding fragment thereof of any aspect of the present application or a conjugate of the present application.

[0125] In certain embodiments, the kit comprises a conjugate of the present application.

[0126] In certain embodiments, the kit comprises an antibody or antigen-binding fragment thereof of any aspect of the present application, and optionally a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof. Optionally, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin.

[0127] Methods of detection

[0128] In a nineteenth aspect, the present application provides a method for detecting the presence or level of RSV in a sample, comprising using an antibody or antigen-binding fragment thereof of any aspect of the present application or a conjugate of the present application.

[0129] In certain embodiments, the method is for therapeutic purposes, diagnostic purposes, or non-therapeutic non-diagnostic purposes.

[0130] In certain embodiments, the method is an immunological detection, such as immunoblotting, enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay.

[0131] In certain embodiments, the method comprises using a conjugate of the present application.

[0132] In certain embodiments, the method comprises using an antibody or antigen-binding fragment thereof of any aspect of the present application, and the method further comprises using a second antibody carrying a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin, to detect the antibody or antigen-binding fragment thereof.

[0133] In certain embodiments, the method comprises: (1) contacting the sample with the antibody or antigen-binding fragment thereof or conjugate; (2) detecting the formation of an antigen-antibody immune complex or detecting the amount of the immune complex. In certain embodiments, the formation of the immune complex indicates the presence of RSV or cells infected with RSV.

[0134] Use of kits of parts

[0135] In a twentieth aspect, the present application provides use of the antibody or antigen-binding fragment thereof of any aspect of the present application or the conjugate of the present application in the manufacture of a kit for detecting the presence or level of RSV in a sample and / or for diagnosing whether a subject is infected with RSV.

[0136] In certain embodiments, the kit detects the presence or level of RSV in a sample by the method as described in the nineteenth aspect.

[0137] In certain embodiments, the sample is a body fluid sample (e.g., respiratory tract secretion) or a tissue sample (e.g., respiratory tract tissue sample) from a subject (e.g., a mammal, preferably a human).

[0138] Definitions of terms

[0139] In the present application, unless otherwise indicated, the scientific and technical terms used herein have the meanings that would be generally understood by one of ordinary skill in the art. Also, the viral, biochemical, immunological laboratory procedures used in the present application are those known to and conventionally used by those in the art. In order to better understand the present application, the following definitions and explanations of terms are provided.

[0140] When the terms "for example," "for instance," "such as," "including," "containing," or "comprising" or variations thereof are used herein, these terms are not to be interpreted in an exclusionary sense, but rather in an illustrative sense.

[0141] Unless otherwise indicated, the terms "a" and "an" and "the" and similar referents are to be construed as covering both the singular and the plural unless otherwise indicated by context.

[0142] As used herein, the term "RSV fusion protein" or "F protein" refers to the fusion protein (F protein) of respiratory syncytial virus (RSV), which is well known to those skilled in the art and exemplary amino acid sequences of which can be found, for example, at UniProtKB: P03420.1. In this document, when referring to the amino acid sequence of the F protein, the sequence shown in SEQ ID NO: 1 is used for description. For example, the expression "amino acid residues 160-182 of the F protein" refers to the amino acid residues 160-182 of the polypeptide shown in SEQ ID NO: 1. However, those skilled in the art understand that mutations or variations (including but not limited to substitutions, deletions and / or additions, such as F proteins of different genotypes or genetic subtypes) can be naturally occurring or artificially introduced in the amino acid sequence of the F protein without affecting its biological function. Therefore, in the present application, the term "F protein" shall include all such sequences, including, for example, the sequence shown in SEQ ID NO: 1 and its natural or artificial variants. Also, when describing a sequence fragment of the F protein, it includes not only the sequence fragment of SEQ ID NO: 1, but also the corresponding sequence fragment in its natural or artificial variants. For example, the expression "amino acid residues 160-182 of the F protein" includes the amino acid residues 160-182 of SEQ ID NO: 1, and the corresponding fragment in its variants (natural or artificial). According to the present application, the expression "corresponding sequence fragment" or "corresponding fragment" refers to the fragment located at the equivalent position in the compared sequence when the sequences are optimally aligned, i.e. when the sequences are aligned to obtain the highest percentage identity.

[0143] As used herein, the term "pre-F protein" refers to the F protein existing in the pre-F conformation. As used herein, the term "post-F protein" refers to the F protein existing in the post-F conformation. For more detailed description of the pre-F protein, post-F protein and their conformations, see McLellan et al. (2010), J Virol, 84: 12236-12244; McLellan et al. (2013), Science, 340: 1113-1117; McLellan et al. (2015), Curr Opin Virol, 11: 70-75; Chinese patent application 201480013927.7, and PCT international application PCT / CN2014 / 073505 (the entire contents of which are incorporated herein by reference for all purposes).

[0144] As used herein, the term "epitope" refers to the portion of an antigen that is recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, the epitope can be formed by contiguous amino acids or noncontiguous amino acids brought into proximity by the tertiary folding of the protein, referred to as linear or conformational epitopes, respectively. Epitopes formed from contiguous amino acids are typically retained upon denaturation of the protein, whereas epitopes formed by tertiary folding are typically lost upon denaturation of the protein. Epitopes usually comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or noncontiguous amino acids in a unique spatial conformation.

[0145] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions / total number of positions x 100%). In certain embodiments, the two sequences are the same length.

[0146] Determination of the percent identity between two sequences can also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated in the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.

[0147] As used herein, the term "antibody" refers to an immunoglobulin molecule that is generally composed of two pairs of polypeptide chains (each pair having one light (LC) and one heavy (HC) chain). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define a different class of antibody, IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids. The heavy chains also contain a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The constant domains exhibit less inter-chain variability than the variable domains, but still exhibit some inter-chain variability. The variable domains of the heavy and light chains, VH and VL, can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V H and V L consists of 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair, VH and VL, form the antigen binding site. Assignment of amino acids to each region or domain can follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0148] As used herein, the term "complementarity determining region" or "CDR" refers to amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs in each of the variable regions of the heavy and light chains, designated CDR1, CDR2 and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, e.g., as defined by the numbering system of Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or IMGT (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, one of skill in the art will readily be able to identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0149] In the present application, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present application can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present application are preferably determined by the Kabat, Chothia or IMGT numbering system. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present application are preferably determined by the IMGT numbering system.

[0150] As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0151] The term "antibody" is not limited by any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody can be an antibody of different isotype, e.g., an IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody.

[0152] As used herein, the term "antigen binding fragment" of an antibody refers to a polypeptide that comprises a fragment of a full-length antibody that retains the ability to specifically bind the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nded. Raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen binding fragments of an antibody can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementarity determining region (CDR) fragments, scFv, diabodies, single domain antibodies, chimeric antibodies, linear antibodies, nanobodies (technology from Domantis), probody, and polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding capacity of the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0153] As used herein, the term "full length antibody" means an antibody that is composed of two "full length heavy chains" and two "full length light chains". Wherein, a "full length heavy chain" refers to a polypeptide chain that is composed of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, a heavy chain constant region CH3 domain in the direction of N-terminal to C-terminal; and, optionally, a heavy chain constant region CH4 domain when the full length antibody is of IgE isotype. Preferably, a "full length heavy chain" is a polypeptide chain that is composed of VH, CH1, HR, CH2 and CH3 in the direction of N-terminal to C-terminal. A "full length light chain" is a polypeptide chain that is composed of a light chain variable region (VL) and a light chain constant region (CL) in the direction of N-terminal to C-terminal. The two pairs of full length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between the HR of the two full length heavy chains. The full length antibody of the present application can be from a single species, e.g., human; can also be a chimeric antibody or a humanized antibody. The full length antibody of the present application comprises two antigen binding sites formed by a pair of VH and VL, respectively, which specifically recognize / bind the same antigen.

[0154] As used herein, the term "Fd" means an antibody fragment consisting of a VH and CHI domain; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al., Nature 341 :544 546 (1989)); the term "Fab fragment" means an antibody fragment consisting of a VL, VH, CL and CHI domain; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means the fragment obtained by reducing the disulfide bonds of a F(ab')2 fragment, consisting of an intact light chain and a Fd fragment of a heavy chain (consisting of a VH and CHI domain).

[0155] As used herein, the term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is commonly held to be the minimum antibody fragment that is capable of forming a complete antigen binding site. It is generally considered that the six CDRs confer the antigen binding specificity to an antibody. However, even a single variable domain (e.g., a Fd fragment, which contains only three CDRs specific for an antigen) is capable of recognizing and binding antigen, although it may do so at a lower affinity than the entire binding site.

[0156] As used herein, the term "Fc" means an antibody fragment formed by disulfide bonds between the second, third constant regions of the first heavy chain and the second, third constant regions of the second heavy chain of an antibody. The Fc fragment of an antibody has a variety of different functions, but does not participate in antigen binding.

[0157] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and a VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present application are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond can also exist between the VH and VL of the scFv. In certain embodiments of the application, the scFv can form a di-scFv, which refers to two or more individual scFv linked in series to form an antibody. In certain embodiments of the application, the scFv can form a (scFv)2, which refers to two or more individual scFv linked in parallel to form an antibody.

[0158] As used herein, the term "diabodies" refers to antibody fragments with two VHand VLdomains on a single polypeptide chain, but using a too short linker such that it does not allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complement domains of another chain and creating two antigen binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. et al., Structure 2:1121-1123 (1994)).

[0159] As used herein, the term "single-domain antibody (sdAb)" has the meaning generally understood by those skilled in the art, and refers to an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind the same antigen to which a full-length antibody binds. Single-domain antibodies are also known as nanobodies.

[0160] Each of the above antibody fragments retains the ability to specifically bind the same antigen to which the full-length antibody binds and / or competes with the full-length antibody for specific binding to the antigen.

[0161] Antigen-binding fragments of antibodies (e.g., the above antibody fragments) can be obtained using conventional techniques known to those of skill in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage) from a given antibody (e.g., an antibody provided herein) and screened for specificity in the same manner as is done for whole antibodies.

[0162] Herein, unless the context clearly indicates otherwise, the term "antibody" includes not only intact antibodies, but also antigen-binding fragments of antibodies.

[0163] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the light or / and heavy chain is derived from one antibody (which can be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light or / and heavy chain is derived from another antibody (which can be derived from the same or a different species or belong to the same or a different antibody class or subclass), but which retains the binding activity for the target antigen (U.S. P 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851 6855 (1984)). In certain embodiments, the term "chimeric antibody" can include an antibody in which the variable regions of both the heavy and light chains are derived from a first antibody, and the constant regions of both the heavy and light chains are derived from a second antibody.

[0164] As used herein, the term "variant", in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by the introduction of an amino acid residue substitution, deletion, or addition. In certain instances, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to the polypeptide or peptide). For example, but not by way of limitation, a polypeptide can be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, attachment to a cellular ligand or other protein, etc. A derivatized polypeptide or peptide can be produced by chemical modification using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. Furthermore, a variant has similar, the same, or improved function as the polypeptide or peptide from which it is derived.

[0165] As used herein, the term "specifically binds" refers to a nonrandom binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. The strength or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (K D ) of the interaction. In the present application, the term "K D " refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.

[0166] The specific binding properties between two molecules can be determined using methods well known in the art. One method involves measuring the rate of antigen binding site / antigen complex formation and dissociation. Both the "association rate constant" (kaor kon) and the "dissociation rate constant" (kdisor koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361 : 186-187). The ratio of kdis / kon is equal to the dissociation constant K D (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). K D , kon, and kdisvalues can be measured using any effective method. In certain embodiments, the dissociation constant can be measured using surface plasmon resonance (SPR) in Biacore. In addition, the dissociation constant can be measured using bioluminescence interferometry or Kinexa.

[0167] As used herein, the detectable label described in the present application can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H、 125 I、 35 S、 14 C or 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or a cyanine dye derivative (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridinium esters, luminol and its derivatives, ruthenium derivatives such as terpyridine ruthenium), magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for use with avidin (e.g., streptavidin) modified versions of the above labels.

[0168] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of the inserted polynucleotide, the vector is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, and the host cell into which the vector has been introduced can express the genetic material elements carried by the vector. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 -derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), pox viruses, baculoviruses, papillomaviruses, papova viruses (e.g., SV40). A vector can contain a variety of elements that control expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, a vector can contain a replication origin.

[0169] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0170] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of an amino acid residue with an amino acid residue having a similar side chain, e.g., substitutions that take place within a family of amino acid residues that are physicochemically or functionally similar, e.g., have similar size, shape, charge, chemical properties, including ability to form covalent or hydrogen bonds, etc. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a conservative substitution preferably replaces a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods of identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0171] The writing of the twenty conventional amino acids referred to herein follows conventional usage. See, e.g., Immunology - A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present application, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also in the present application, amino acids are generally represented by their single and three letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0172] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are compatible, physiologically and / or pharmacologically, with the subject and the active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to retard absorption, preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents to maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents to retard absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thiomersal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning commonly understood by those skilled in the art, which are capable of stabilizing the desired activity of the active ingredient in the pharmaceutical, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried lactose, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), and the like. In certain exemplary embodiments, the pharmaceutically acceptable carriers or excipients include sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (such as 0.9% (w / v) NaCl), glucose solutions (such as 5% dextrose), solutions containing surfactants (such as 0.01% polysorbate 20), pH buffered solutions (such as phosphate buffered solutions), Ringer's solutions, and any combination thereof.

[0173] As used herein, the term "preventing" refers to an approach taken to stop or delay the occurrence of a disease or disorder or a symptom thereof in a subject. As used herein, the term "treating" refers to an approach taken to obtain a beneficial or desired clinical result. For the purposes of the present application, a beneficial or desired clinical result includes, but is not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of the state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Moreover, "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0174] As used herein, the term "subject" refers to a mammal, for example, a human. In certain embodiments, the subject (e.g., human) has an RSV infection or a disease associated with RSV infection (e.g., pneumonia, such as pediatric pneumonia), or is at risk of having the above-mentioned disease.

[0175] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve a desired effect. For example, an effective amount for preventing a disease (e.g., RSV infection or a disease associated with RSV infection (e.g., pneumonia, such as pediatric pneumonia)) refers to an amount sufficient to prevent, stop, or delay the occurrence of the disease; an effective amount for treating a disease refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already having the disease. Determining such effective amounts is well within the capability of those skilled in the art. For example, an amount effective for therapeutic uses will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, such as age, body weight, and sex, the mode of administration of the drug, and other therapies that the patient has or is concurrently receiving, and the like.

[0176] Advantages of the Invention

[0177] The monoclonal antibodies and antigen-binding fragments thereof of the present application have broad-spectrum RSV binding activity, and are capable of specifically binding to the F protein of a variety of RSV strains of type A and / or type B, and the monoclonal antibodies and antigen-binding fragments thereof of the present application specifically bind to the pre-F protein on the pre-F protein The novel epitope between the A and V epitopes or the novel epitope between the II and V epitopes has high RSV neutralization activity, and is effective in preventing RSV infection in vivo.

[0178] The monoclonal antibodies and antigen-binding fragments thereof of the present application can be used for detecting, preventing, and / or treating RSV infection or a disease caused by RSV infection. In addition, the antibodies of the present application can also be used for specifically detecting RSV or its F protein, and have clinical value for the diagnosis of RSV infection.

[0179] Embodiments of the present application will be described in detail below with reference to the attached drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are merely illustrative of the present application and are not intended to limit the scope of the present application. Various objects and advantageous aspects of the present application will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0180] Figure 1 Experimental results showing neutralization of RSV A representative strain (RSV A2) (A) and RSV B representative strain (18537) (B) by monoclonal antibodies 5B11, 6B2 and 7G5. Figure 1 Experimental results showing neutralization of RSV A representative strain (RSV A2) (A) and RSV B representative strain (18537) (B) by monoclonal antibodies 5B11, 6B2 and 7G5. Figure 1 Experimental results showing neutralization of RSV A representative strain (RSV A2) (A) and RSV B representative strain (18537) (B) by monoclonal antibodies 5B11, 6B2 and 7G5. Experimental results showing neutralization of RSV A representative strain (RSV A2) (A) and RSV B representative strain (18537) (B) by monoclonal antibodies 5B11, 6B2 and 7G5.

[0181] Experimental results showing neutralization of RSV A representative strain (RSV A2) (A) and RSV B representative strain (18537) (B) by monoclonal antibodies 5B11, 6B2 and 7G5. Figure 2 Experimental results showing neutralization of RSV A representative strain (RSV A2) (A) and RSV B representative strain (18537) (B) by monoclonal antibodies 5B11, 6B2 and 7G5. Figure 2 Experimental results showing neutralization of RSV A representative strain (RSV A2) (A) and RSV B representative strain (18537) (B) by monoclonal antibodies 5B11, 6B2 and 7G5. Figure 2 Experimental results showing neutralization of RSV A representative strain (RSV A2) (A) and RSV B representative strain (18537) (B) by monoclonal antibodies 5B11, 6B2 and 7G5. Experimental results showing neutralization of RSV A representative strain (RSV A2) (A) and RSV B representative strain (18537) (B) by monoclonal antibodies 5B11, 6B2 and 7G5.

[0182] Experimental results showing neutralization of RSV A representative strain (RSV A2) (A) and RSV B representative strain (18537) (B) by monoclonal antibodies 5B11, 6B2 and 7G5. Figure 3 Experimental results showing neutralization of RSV A representative strain (RSV A2) (A) and RSV B representative strain (18537) (B) by monoclonal antibodies 5B11, 6B2 and 7G5. Experimental results showing neutralization of RSV A representative strain (RSV A2) (A) and RSV B representative strain (18537) (B) by monoclonal antibodies 5B11, 6B2 and 7G5.

[0183] Experimental results showing neutralization of RSV A representative strain (RSV A2) (A) and RSV B representative strain (18537) (B) by monoclonal antibodies 5B11, 6B2 and 7G5. Figure 4 Experimental results showing neutralization of RSV A representative strain (RSV A2) (A) and RSV B representative strain (18537) (B) by monoclonal antibodies 5B11, 6B2 and 7G5. Experimental results showing neutralization of RSV A representative strain (RSV A2) (A) and RSV B representative strain (18537) (B) by monoclonal antibodies 5B11, 6B2 and 7G5.

[0184] Experimental results showing neutralization of RSV A representative strain (RSV A2) (A) and RSV B representative strain (18537) (B) by monoclonal antibodies 5B11, 6B2 and 7G5. Figure 5 Experimental results showing neutralization of RSV A representative strain (RSV A2) (A) and RSV B representative strain (18537) (B) by monoclonal antibodies 5B11, 6B2 and 7G5. Experimental results showing neutralization of RSV A representative strain (RSV A2) (A) and RSV B representative strain (18537) (B) by monoclonal antibodies 5B11, 6B2 and 7G5.

[0185] Experimental results showing neutralization of RSV A representative strain (RSV A2) (A) and RSV B representative strain (18537) (B) by monoclonal antibodies 5B11, 6B2 and 7G5. Figure 6 Experimental results showing neutralization of RSV A representative strain (RSV A2) (A) and RSV B representative strain (18537) (B) by monoclonal antibodies 5B11, 6B2 and 7G5. Experimental results showing neutralization of RSV A representative strain (RSV A2) (A) and RSV B representative strain (18537) (B) by monoclonal antibodies 5B11, 6B2 and 7G5.

[0186] Experimental results showing neutralization of RSV A representative strain (RSV A2) (A) and RSV B representative strain (18537) (B) by monoclonal antibodies 5B11, 6B2 and 7G5. Figure 7 Experimental results showing neutralization of RSV A representative strain (RSV A2) (A) and RSV B representative strain (18537) (B) by monoclonal antibodies 5B11, 6B2 and 7G5. Figure 7 Experimental results showing neutralization of RSV A representative strain (RSV A2) (A) and RSV B representative strain (18537) (B) by monoclonal antibodies 5B11, 6B2 and 7G5.Figure 7 B is the plaque results in lung tissue of each group of mice; Figure 7 C is the plaque results in nose tissue of each group of mice; Figure 7 D is the staining results of pathological sections of lung tissue of each group of mice.

[0187] Figure 8 Figure 6 shows the nasal and lung viral titer detection in prophylactic evaluation of 5B11 on cotton rat model against RSV A and B viruses (strains used for A and B types are RSV A2 and 18537, respectively). Among them, Figure 8 A is the lung viral titer detection results of each group of cotton rats in the evaluation experiment of 5B11 preventing RSV A virus; Figure 8 B is the nasal viral titer detection results of each group of cotton rats in the evaluation experiment of 5B11 preventing RSV A virus; Figure 8 C is the lung viral titer detection results of each group of cotton rats in the evaluation experiment of 5B11 preventing RSV B virus; Figure 8 D is the nasal viral titer detection results of each group of cotton rats in the evaluation experiment of 5B11 preventing RSV B virus.

[0188] Figure 9 Figure 7 shows the lung tissue pathological score of 5B11 in the prophylactic evaluation of 5B11 on cotton rat model against RSV A and B viruses (strains used for A and B types are RSV A2 and 18537, respectively). Among them, Figure 9 A-E are the lung tissue pathological scores of each group of cotton rats in the evaluation experiment of 5B11 preventing RSV A virus; Figure 9 F-J are the lung tissue pathological scores of each group of cotton rats in the evaluation experiment of 5B11 preventing RSV B virus.

[0189] Figure 10 Figure 8 shows the humanization design scheme of 5B11.

[0190] Figure 11 Figure 9 shows the neutralization and reactivity detection of 5B11 humanized antibodies; among them, Figure 11 A is the detection result of humanized antibody N5B11 neutralizing RSV A virus (RSV A2), Figure 11 B is the detection result of humanized antibody N5B11 neutralizing RSV B virus (18537), Figure 11 C is the detection result of humanized antibody N5B11 binding to RSV A (RSV A2) pre-F, Figure 11 D is the detection result of humanized antibody N5B11 binding to RSV B (18537) pre-F.

[0191] Sequence information

[0192] The description of the sequences involved in the present application is provided in the following table.

[0193] Table 1: Sequence information

[0194]

[0195]

[0196]

[0197] Note: K = G, or T; Y = C or T; S = C or G; W = A or T; B = C, G or T; R = A or G. DETAILED DESCRIPTION

[0198] The application will now be described with reference to the following examples which are intended to be illustrative, and not limiting, of the application.

[0199] Unless otherwise indicated, molecular biology and immunological techniques used in the present application are performed according to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Ed., John Wiley & Sons, Inc., 1995; the use of restriction enzymes is in accordance with the recommendations of the manufacturers. Where specific conditions are not indicated in the examples, they are performed according to the standard conditions or the conditions recommended by the manufacturer. Where the manufacturer of reagents or instruments is not indicated, it is only because such items are commonly available from a variety of suppliers. It will be appreciated by one of ordinary skill in the art that the examples describe the present application in terms of preferred embodiments, and that the application is not limited to the preferred embodiments described in the examples.

[0200] Example 1: Preparation of monoclonal antibodies (mAbs) against RSV-F protein

[0201] (1) Preparation of immunogen:

[0202] DNA-F was kindly provided by NIH Vaccine Center, USA. The plasmid was inserted with full-length gene of F protein of RSV A2 strain (GenBank: FJ614814.1, SEQ ID NO: 1) on VRC8400 vector. The DNA-F with low endotoxin at a concentration of 2 mg / mL was obtained by endotoxin-free plasmid maxi kit (purchased from TianGen, item number: DP117) as the immunogen for mice. At the same time, the present application also prepared recombinant adenovirus (rAd-F) containing full-length gene of F protein (SEQ ID NO: 1) for mouse immunization. The recombinant adenovirus was a virus that had been constructed and cryopreserved in the laboratory. In order to prepare for mouse immunization, the cryopreserved virus was quickly thawed, 293β5 cells (239 cells stably transfected with human β5 gene, constructed and preserved by the laboratory) were infected, and the virus was collected 48 h after infection, concentrated to 1×10 10 PFU / mL by density gradient centrifugation, and prepared for mouse immunization.

[0203] (2) Experimental mice:

[0204] 6-week-old SPF female Balb / C mice were purchased from Shanghai Slac Animal Limited Liability Company.

[0205] (3) Preparation of hybridoma:

[0206] Single antibody hybridoma cells were obtained by using standard in vivo immunization method and PEG fusion method. For details, see Ed Harlow et al., “Antibodies A Laboratory Manual”, Cold Spring Harbor Laboratory 1988. The brief process is as follows:

[0207] (4) Mouse immunization:

[0208] The immunogen DNA-F (100 μg per mouse) or rAd-F (5×10 7PFU / mouse) were injected into mice. The DNA-F immunization cycle was 4 weeks, and the rAd-F immunization cycle was 2 weeks. To enhance the immune response, the mice were subcutaneously injected with complete Freund's adjuvant when immunized with the DNA-F immunogen, and subcutaneously injected with incomplete Freund's adjuvant when immunized with the rAd-F. Subsequently, the mice were weekly subjected to orbital blood collection, and the serum of each group of mice was subjected to reactivity and serum neutralization titer detection, and the mice with higher serum neutralizing antibody titers were selected for spleen immunization. The HEp-2 cells infected with RSV A2 for 48 hours were scraped with a cell scraper, and after centrifugation, the MEM culture medium was removed, the cell pellet was resuspended with 500 μL of 1x PBS, and 50 μL of the suspension was taken for spleen immunization. After 3 days, the spleen was ground and the spleen cells were separated, and the spleen cells were fused with mouse myeloma cells (SP2 / 0) using PEG.

[0209] (5) Cell fusion:

[0210] First, the mouse spleen was ground to obtain a spleen cell suspension, which was then mixed with mouse myeloma cells SP2 / 0 in the logarithmic growth phase, and cell fusion was performed under the action of PEG1500. The fused cells were resuspended in 400 mL of fusion culture medium and cultured in 20 pieces of 96-well cell culture plates. The fusion culture medium was complete screening medium RPMI1640 containing HAT and 20% FBS.

[0211] (6) Screening of hybridoma:

[0212] After 10 days of culture of the fused cells in the 96-well cell culture plates, the cell supernatant was aspirated for virus neutralization experiment and ELISA detection, and the virus was RSV A2 mKate. In the virus neutralization experiment, the antibody secreted by the positive well was required to inhibit the infection of Hep2 cells by respiratory syncytial virus; in the ELISA detection, the antibody secreted by the positive well was required to specifically react with the respiratory syncytial virus infected Hep2 cells fixed on the cell plate. After 3 times of cloning of the positive clones, a monoclonal cell strain capable of stably secreting antibodies was obtained.

[0213] (7) Screening results of hybridoma monoclonal antibodies:

[0214] Nine anti-RSV prefusion specific neutralizing monoclonal antibodies, 5B11, 6B2, 7G5, etc., were obtained.

[0215] (8) Culture of hybridoma:

[0216] The 9 stable hybridoma monoclonal cell strains were first expanded in a carbon dioxide incubator, transferred to 24-well plates, and then transferred to 50 mL cell culture bottles for expansion culture. Then, the cells in the cell culture bottles were collected and injected into the abdominal cavity of mice, and 7-10 days later, the monoclonal ascites was aspirated from the abdominal cavity of the mice.

[0217] (9) Purification of mAbs:

[0218] The ascites of mAbs were first precipitated with 50% ammonium sulfate solution, then the precipitate was dissolved with PBS, and then purified by Protein A column under AKTA system to obtain purified mAbs, and the purity of the purified mAbs was identified by SDS-PAGE.

[0219] Example 2: Neutralizing activity of anti-RSV F protein mAbs

[0220] Neutralizing activity is an important indicator for evaluating the potential of mAbs for preventing and treating diseases. The neutralizing activity of mAbs 5B11, 6B2 and 7G5 obtained in Example 1 against respiratory syncytial virus A and B representative strains RSV A2 and 18537 was detected by microwell cell neutralization experiment (method referred to Yong-Peng Sun et al., Journal of Virological Methods. 2018, 260: 34-40). The control antibodies used were 5C4 (mouse antibody screened by the laboratory, PCT No: PCT / CN2014 / 073505), D25 (plasmid expressed by self, Patent No: US 8,568,726 B2) and 1129 (sequence donated by NIH, plasmid expressed by self).

[0221] Table 2: Summary of IC50 of each RSV neutralizing antibody

[0222] mAbs RSV A2 (ng / mL) 18537 (ng / mL) 4E10 785.5 101.9 5B11 7.4 59.3 6B2 94.9 97.2 6H7 175.2 241.3 11A12 101.8 178.4 11C1 525.1 233.9 12F8 132.2 91.9 7G5 31.6 103.8 10F3 43.1 125.7 5C4 9.4 — D25 22.2 167.2 1129 472.0 520.2

[0223] The results are shown in Table 2 and Figure 1 The results show that mAbs 5B11, 6B2 and 7G5 all showed better neutralizing ability against RSV A2 and 18537 than 1129, and the titer of 5B11 neutralizing RSV A2 was comparable to that of control antibody 5C4, which was more than 50 times the neutralizing ability of 1129, and the neutralizing ability against 18537 was about 10 times that of 1129.

[0224] Example 3: ELISA detection of anti-RSV F protein mAbs reactivity

[0225] The operation steps and methods of indirect ELISA detection of antigen-antibody binding are referred to (Min Zhao et al., J Biol Chem. 2015, 290(32): 19910-22). The detection results are shown in Figure 2 As shown, the three mAbs (5B11, 6B2 and 7G5) only bind to the pre-F conformational protein (DS-Cav1) of RSV A and B viruses, and do not bind to the post-F conformational protein, which are antibodies recognizing specific epitopes of RSV pre-F.

[0226] Example 4. Sequence analysis of the light chain gene and the heavy chain gene of the anti-RSV F protein monoclonal antibody

[0227] Semi-adherent culture about 10 7 The hybridoma cells were blown up to make them suspended, and the suspension was transferred to a new 4 mL centrifuge tube, which was centrifuged at 1500 rpm for 3 min, and the cell precipitate was collected and resuspended in 100 μL of sterile PBS (pH = 7.45) and transferred to a new 1.5 mL centrifuge tube. 800 μL of Trizol (purchased from Roche, Germany) was added, mixed gently, and allowed to stand for 10 min. 200 μL of chloroform was added, shaken vigorously for 15 s, allowed to stand for 10 min, and centrifuged at 12000 rpm at 4°C for 15 min. The upper liquid was transferred to a new 1.5 mL centrifuge tube, and an equal volume of isopropanol was added, mixed, and allowed to stand for 10 min. The mixture was centrifuged at 12000 rpm at 4°C for 10 min, and the supernatant was discarded. 600 μL of 75% ethanol was added for washing, and the mixture was centrifuged at 12000 rpm at 4°C for 5 min. The supernatant was discarded, and the precipitate was dried at 60°C under vacuum for 5 min. The transparent precipitate was dissolved in 70 μL of DEPC H2O, and divided into two tubes. 1 μL of reverse transcription primer was added to each tube, and MVJkR (SEQ ID NO: 27) was added to one tube for amplifying the light chain variable region gene, and MVDJhR (SEQ ID NO: 28) was added to the other tube for amplifying the heavy chain variable region gene. 1 μL of dNTP (purchased from Shanghai Shenguo) was added to each tube, which was placed in a 72°C water bath for 10 min, immediately placed in an ice bath for 5 min, and then 10 μL of 5x reverse transcription buffer, 1 μL of AMV (10 u / μL, purchased from Promega), and 1 μL of Rnasin (40 u / μL, purchased from Promega) were added. After mixing, the RNA was reverse transcribed into cDNA at 42°C.

[0228] The antibody variable region was isolated by polymerase chain reaction (PCR) using a primer set synthesized according to the Ig-Prime kits of Novagen Company and two additional downstream primers MVJkR and MVDJhR (synthesized by Shanghai Shenguo), wherein MVJkR is a downstream primer for amplifying the light chain variable region gene, and MVDJhR is a downstream primer for amplifying the heavy chain variable region gene. The template is the two cDNAs synthesized above. The PCR conditions are as follows: 94°C for 5 min, 94°C for 40 s, 53°C for 1 min, 72°C for 50 s for 35 cycles, and 72°C for 15 min. The PCR product was directly sent to Shanghai Shenguo for sequencing. After the sequence was compared by IMGT, the antibody variable region sequence was determined, and the corresponding amino acid sequence was determined.

[0229] The variable region genes of the antibodies were cloned from the 5B11, 6B2 and 7G5 monoclonal antibody hybridoma cell lines according to the above method, and the amino acid sequences of the CDR regions of the monoclonal antibodies were determined according to the IMGT method (Marie-Paule Lefranc and Gerard Lefranc. Immunoglobulins or Antibodies: Bridging Genes, Structures and Functions, Biomedicines 2020, 8, 319) according to the IMGT numbering system. The primer sequences used for amplifying the variable region genes of the 5B11, 6B2 and 7G5 monoclonal antibodies are shown in Table 3.

[0230] Table 3: Primer sequences used for amplifying the variable region genes of the 5B11, 6B2 and 7G5 monoclonal antibodies

[0231]

[0232]

[0233] Note: K = G or T; Y = C or T; S = C or G; W = A or T; B = C, G or T; R = A or G; M = A or C; H = A, C or T; V = A, C or G; I = inosine deoxyribonucleotide residue

[0234] Example 5: Detection of the broad-spectrum binding activity of the anti-RSV F protein monoclonal antibodies

[0235] More than 1000 nucleic acid sequences of RSV F proteins were downloaded from NCBI, and phylogenetic tree analysis of the RSV F proteins was performed using MEGA software. Twenty representative RSV F protein sequences were selected from the phylogenetic tree, and the twenty sequences were constructed into a VRC8400 vector (synthesized by Shanghai Generay Biotech Co., Ltd.). Then the plasmids were transfected into 293 cells to overexpress the proteins on the cell membrane, and flow cytometry was used to detect the binding of the 5B11, 6B2 and 7G5 antibodies to the 293 cells overexpressing the F proteins. The experimental results are shown in Figure 3 The results show that 5B11, 6B2 and 7G5 all have strong broad-spectrum binding activity and good binding to RSV A and B strain F proteins, and are potential RSV broad-spectrum neutralizing antibodies.

[0236] Example 6. Identification of the three-dimensional structure of the anti-RSV F protein monoclonal antibodies and key amino acid sites

[0237] To determine the binding epitopes of 5B11, 6B2 and 7G5 antibodies to RSV prefusion protein, we prepared antigen-antibody complexes of 5B11, 6B2 and 7G5 antibodies and RSV prefusion protein (DS-Cav1), and analyzed the structures of the antigen-antibody complexes by cryo-EM three-dimensional reconstruction technology. The experimental results are shown in Figure 4 Figure 1, which show that 5B11, 6B2 and 7G5 antibodies bind to a new epitope on RSV prefusion protein (DS-Cav1), wherein antibodies 5B11 and 6B2 can specifically bind to a new epitope between II epitope and V epitope on respiratory syncytial virus pre-F protein, and antibody 7G5 can specifically bind to a new epitope between II epitope and V epitope on respiratory syncytial virus pre-F protein. Figure 1, which show that 5B11, 6B2 and 7G5 antibodies bind to a new epitope on RSV prefusion protein (DS-Cav1), wherein antibodies 5B11 and 6B2 can specifically bind to a new epitope between II epitope and V epitope on respiratory syncytial virus pre-F protein, and antibody 7G5 can specifically bind to a new epitope between II epitope and V epitope on respiratory syncytial virus pre-F protein.

[0238] To further determine the key amino acid binding sites of the three antibodies, we performed alanine scanning on the binding regions of the three antibodies, i.e., mutating the amino acids on the surface of F protein to alanine (A), and constructing a mutant plasmid library based on the DNA-F plasmid. The mutant plasmid was transfected into 293 cells to overexpress F protein on the cell surface, and the binding of 5B11, 6B2 and 7G5 antibodies to these mutant proteins was detected by flow cytometry. The experimental results are shown in Figure 5 Figure 2, which show that the key amino acid sites for 5B11 binding are E161, G162, N165, K166 and G184, the key amino acid sites for 6B2 binding are E161, G162, G184, K293 and E294, and the key amino acid sites for 7G5 binding are E161, G162 and G184.

[0239] In addition, to further understand the structural basis of the interaction between antibody 5B11 and RSV F protein, we prepared 5B11 Fab-DS-Cav1 antigen-antibody complexes, and obtained a 3.29 angstrom high-resolution structure of 5B11 Fab-DS-Cav1 antigen-antibody complexes by cryo-EM three-dimensional reconstruction technology (Cryo-EM). The results are shown in Figure 6As shown. From the structure, it can be seen that 5B11 binds to the F1 subunit on the F protein, and the main binding region is aa.160-182 and the loop between β6 and β7 (aa.294-295). The heavy chain of 5B11 mainly interacts with the RSV F protein through CDR1 and CDR3, which is specifically manifested in that D31 on the heavy chain CDR1 of 5B11 forms a hydrogen bond with E295 on the F1 subunit, Y32 forms a hydrogen bond with E294 and E295, S33 forms a hydrogen bond with E161, and in addition, H35 on FR2 also interacts with E161 to form a hydrogen bond. Y101 on the heavy chain CDR3 of 5B11 forms a hydrogen bond with K196 on the F1 subunit, and G102 forms a hydrogen bond with N165. The light chain of 5B11 mainly interacts with the RSV F protein through CDR2 and CDR3, which is specifically manifested in that N50 on the heavy chain CDR2 of 5B11 interacts with S169 on the F1 subunit to form a hydrogen bond, and W92 on the heavy chain CDR3 of 5B11 interacts with K166, S180 and S182 on the F1 subunit to form a hydrogen bond.

[0240] Example 7. Preventive evaluation of anti-RSV F protein monoclonal antibodies on Balb / C model against RSV virus

[0241] Passive immunotherapy with antibodies for infectious diseases is a potentially effective antiviral treatment approach. It has been proved by in vitro micropore neutralization test that the monoclonal antibodies 5B11, 6B2 and 7G5 of the present application have strong neutralizing activity on RSV type A and type B strains, and are characterized by wide neutralization spectrum and high neutralization titer on respiratory syncytial virus type A and type B strains. In order to further verify the in vivo anti-respiratory syncytial virus effect of monoclonal antibodies 5B11, 6B2 and 7G5, the present application carries out in vivo verification experiment of the prevention of monoclonal antibodies against RSV A2 strain based on the animal model infected with respiratory syncytial virus A2 strain in a biosafety laboratory on Balb / C mice. The specific process is as follows:

[0242] (1) Materials and Methods

[0243] Animals: Balb / C mice, SPF level, 12 weeks old, female, body weight about 20g.

[0244] Monoclonal antibodies: 5B11, 6B2, 7G5 and 1129

[0245] Respiratory syncytial virus A2 strain, purchased from ATCC.

[0246] Anesthetics: Isoflorane (Isoflorane)

[0247] Animal grouping: One week in advance, mice were sent to the biosafety laboratory to adapt to the environment, a total of 50 mice, 10 mice in each group, 5 mice in each cage, and the weight of each mouse was recorded. The detailed scheme is shown in Table 4.

[0248] Virus infection: Respiratory syncytial virus A2 strain was diluted to 5x10 7 PFU / mL in advance, and the virus inoculation amount of mice was 100 μL per mouse. Before inoculation, the mice were anesthetized with isoflurane, and then the virus was inoculated into the mice through the nose.

[0249] Intervention of monoclonal antibody: 24 hours before virus infection, the mice in the antibody prevention group were given a low dose of antibody for prevention, and each mouse was injected intraperitoneally with 100 μL of antibody at a dose of 1.5 mg / kg.

[0250] Observation record: 1-12 days after virus infection, the weight change and the corresponding behavior characteristics of the mice were recorded every day. On the 5th day after infection, 5 mice were taken from each group for detection of virus titer in nasal and lung tissues and lung tissue pathological characteristics.

[0251] Table 4: Experimental scheme for preventive evaluation of anti-RSV F protein antibody animals

[0252]

[0253] (2) Results and Analysis

[0254] After infection with RSV A2, the body weight and the presence or absence of reverse hair of a total of 50 mice in the positive control group, 5B11, 6B2, 7G5 and 1129 were monitored every day. The weight monitoring results are shown in Figure 7 A, the body weight of the mice in the positive group began to decrease on the 4th day after infection, and decreased to the lowest on the 7th day, and then gradually recovered. Compared with the 1129 control group, the 5B11, 6B2 and 7G5 groups did not show obvious weight loss, and could protect the mice from weight loss to a certain extent, and the protection effect was better than that of the 1129 control group. During the experiment, it was found that the mice in the positive group and the 1129 control group had obvious reverse hair phenomenon on the 5th day after infection, while no obvious reverse hair phenomenon was found in the other groups.

[0255] The virus plaque results in the nasal and lung tissues are shown in Figure 7 C and 7B, respectively. From the plaque detection results in the lung tissue, we can see that the virus titers of the lung tissues of the mice in the positive group and the 1129 control group are higher, reaching 10 5.4 PFU / g and 10 4.9PFU / g; while no virus was detected in the 5B11, 6B2, and 7G5 groups, showing a significant statistical difference compared to the positive group and the 1129 control group (P<0.05). Furthermore, the three antibodies (5B11, 6B2, and 7G5) could also reduce viral infection in the upper respiratory tract nasal cavity to some extent, with better effects than the 1129 control group. Figure 7 C). In summary, the three antibodies (5B11, 6B2, and 7G5) can prevent upper and lower respiratory tract infections even at low doses.

[0256] Pathological characteristics of lung tissue Figure 7 D) We can see that in the positive group and the 1129 control group, the lung tissue of mice showed extensive inflammatory cell infiltration in the blood vessels, bronchioles, capillary walls, and alveolar walls, with significant swelling. In some severe cases, the cell cavities were compressed, and the alveolar walls ruptured. Pathological sections from the 6B2 and 7G5 antibody groups showed mild pathological signs in the lung tissue, with thickening of the blood vessel walls, bronchiole walls, and capillary walls, and mild inflammatory cell infiltration around them. However, the alveolar structure was clear and intact, without obvious inflammatory cell infiltration or thickening. In the 5B11 group of mice, no inflammatory cell infiltration was observed around the blood vessels, bronchioles, capillary walls, and alveolar walls; the morphology and structure were normal.

[0257] The above experimental results show that, under the same dose, the prevention effect of the three antibodies 5B11, 6B2 and 7G5 is better than that of the 1129 control group. They can effectively prevent RSV infection, reduce weight loss and symptoms in mice, inhibit viral replication in the upper and lower respiratory tracts, and reduce inflammatory response.

[0258] Example 8.5 Prophylactic evaluation of B11 monoclonal antibody against RSV in a rat model

[0259] (1) Prophylactic evaluation of 5B11 against RSV type A virus in a cotton rat model

[0260] To investigate the potential of 5B11 as a prophylactic agent against RSV, this invention further evaluated the prophylactic effect of 5B11 against RSV type A virus in a rat model. The experimental design and grouping are shown in Table 5, with a total of 5 groups and 5 rats in each group. The antibody groups were pre-diluted to 15 mg / mL and 1.5 mg / mL with PBS at high doses (15 mg / kg) and low doses (1.5 mg / kg), respectively. One day before challenge (Day 1), a certain volume of antibody was injected intramuscularly into the right hind limb of each rat, based on its body weight. Twenty-four hours later (Day 0), 100 μL of antibody at a titer of 2 × 10⁻⁶ was administered intranasally. 7PFU / mL RSV A2 virus. The PBS group was injected with 100 μL PBS in the hind leg muscle of each cotton rat one day before infection, and 100 μL of RSV A2 virus with a titer of 2 x 105 7 PFU / mL RSV A2 virus. On the 5th day after infection, the cotton rats in each group were euthanized with CO2, and the nasal and lung tissues were dissected for virus titration and lung pathological inflammation evaluation.

[0261] Table 5: Experimental grouping of the prophylactic evaluation of RSV F protein antibodies in cotton rats

[0262]

[0263]

[0264] Lung plaque detection results Figure 8 A) shows that the lung virus titers of the 5B11 high and low dose groups were not detected, indicating that 5B11 at a low dose can completely prevent RSV lung infection; the 1129 high dose group had no virus titer detected, and can completely prevent RSV lung infection, while the 1129 low dose group detected a higher virus titer, which was reduced by 25 times (1.4 Log) compared with the PBS group, and only can protect the virus lung infection to a certain extent.

[0265] Nasal virus plaque detection Figure 8 B) shows that the 5B11 high and low dose groups and the 1129 high dose group have significant statistical differences compared with the PBS group. Among them, the 5B11 high dose group can completely protect the nasal RSV infection, which is reduced by 500 times (2.7 log) compared with the PBS group. The 1129 low dose group is reduced by 5 times (0.7 log) compared with the PBS group, and the 1129 high dose group is reduced by 50 times (1.7 log) compared with the PBS group, which cannot completely protect the nasal virus infection of mice.

[0266] The lung tissue pathological inflammation of each group was quantified, and the inflammation cell infiltration degree of different parts was scored, and the variance analysis was used to statistically analyze the differences of inflammation scores between groups. Lung pathological score Figure 9A-E) show that the perivascular inflammation and interstitial inflammation in the lungs of each group of cotton rats are not obvious, and the lung inflammation in the PBS positive group is mainly manifested as severe tracheitis, bronchitis and alveolitis. No obvious vasculitis, tracheitis, bronchitis, alveolitis and interstitial pneumonia are observed in the 5B11 high and low dose groups. The 1129 high and low dose groups show tracheitis and bronchitis, and the tracheitis and bronchitis in the 1129 high dose group are relatively mild, while the tracheitis and bronchitis in the 1129 low dose group are relatively severe. The above results suggest that 5B11 has good potential to inhibit lung inflammation, and low-dose 5B11 can achieve or even better than high-dose 1129 to inhibit lung inflammation.

[0267] (2) Preventive evaluation of 5B11 on RSV B virus in a cotton rat model

[0268] The present application further performs preventive evaluation of 5B11 on RSV B virus in a cotton rat model. The experimental design groups are shown in Table 6, and there are a total of 5 groups, each group containing 4-5 cotton rats. The antibody groups are pre-diluted to 15 mg / mL and 1.5 mg / mL with PBS according to high dose (15 mg / kg) and low dose (1.5 mg / kg) of the antibody, and one day before the challenge (Day-1), a certain volume of antibody is injected into the right hind limb muscle of each cotton rat according to the body weight of each cotton rat. After 24 hours (Day 0), 100 μL of RSV B 18537 virus with a titer of 3.8 x 10 6 PFU / mL is given to the nose of each cotton rat. The PBS group is injected with 100 μL of PBS into the hind limb muscle of each cotton rat one day before the challenge, and 24 hours later, 100 μL of RSV B 18537 virus with a titer of 3.8 x 10 6 PFU / mL is given to the nose of each cotton rat. The detection index is the same as the preventive experiment of 5B11 on RSV A virus in a cotton rat model.

[0269] Table 6: Grouping of preventive evaluation experiment of RSV F protein antibody in a cotton rat

[0270]

[0271] Lung plaque detection results Figure 8 C) show that the PBS group can detect a higher virus titer, and the 5B11 high and low dose groups and the 1129 high and low dose groups have a significant statistical difference compared with the PBS group. No virus titer is detected in the 5B11 high and low dose groups, which indicates that 5B11 at a low dose can completely prevent RSV lung infection; only the high dose group of 1129 does not detect the virus titer, which can completely prevent RSV lung infection, while the 1129 low dose group detects a higher virus titer, which is 10 times (1 log) lower than the PBS group in virus infection, and can only inhibit the lung infection of the virus to a certain extent.

[0272] Virus plaque assay in the nose Figure 8 D) PBS group showed higher virus titers, 5B11 high and low dose groups and 1129 high dose group showed significant statistical difference compared with PBS group. Among them, 5B11 high dose group could completely protect the nasal RSV infection, and the low dose group reduced the virus infection by 63 times (1.8 log) compared with PBS group. 1129 low dose group reduced the virus infection by 4 times (0.6 log) compared with PBS group, and 1129 high dose group reduced the virus infection by 1995 times (3.3 log) compared with PBS group.

[0273] The lung tissue pathology inflammation of each group was quantified, and the difference of inflammation score between each group was analyzed by variance analysis. The lung pathology score Figure 9 F-J) showed that the perivascular inflammation, alveolitis and interstitial pneumonia of the lung of each group were not obvious, and the lung inflammation of PBS positive group was mainly severe tracheitis and bronchitis. 5B11 high dose group showed no obvious vascular inflammation, tracheitis, bronchitis, alveolitis and interstitial pneumonia. Compared with PBS group and 1129 low dose group, 5B11 low dose group showed a certain degree of tracheitis alleviation, but there was no obvious statistical difference. 1129 high dose group was similar to 5B11 high dose group, and also showed no obvious tracheitis and bronchitis. However, due to individual differences of experimental animals, 1129 high and low dose groups each had one cotton rat showing strong alveolitis and interstitial pneumonia.

[0274] In summary, high and low dose 5B11 can completely inhibit RSV A / B virus infection in the lower respiratory tract, and low dose 5B11 can also effectively reduce RSV A / B virus infection in the upper respiratory tract, with virus reduction of 500 times and 63 times, respectively. In addition, high and low dose 5B11 groups showed no obvious lung inflammation, suggesting that low dose 5B11 can effectively protect cotton rats from inflammation caused by viral infection, and 5B11 only needs one tenth of the dose of 1129 to achieve the desired protection effect.

[0275] Example 9. Humanization of 5B11 mAb and neutralization activity and reactivity evaluation

[0276] Since 5B11 is a murine antibody, it has a high immunogenicity, and therefore it is necessary to humanize 5B11 to reduce the immunogenicity of 5B11. The principles of humanization design are as follows: 1. The light and heavy chains of 5B11 antibody are respectively compared with the human embryonic gene sequence with the highest homology; 2. The amino acids in FR that are on the CDR contact surface or located close to the CDR are likely to play an important role in the binding of antigen and antibody, and these amino acids are usually retained (red marked amino acids), and the amino acids far away from the CDR and not hydrophobic amino acids are usually directly mutated; 3. The amino acids in FR that are on the interface of VH-VK are usually retained (blue marked amino acids); 4. The exposed and internal embedded amino acids are obtained by simulating the antibody structure, and since the internal embedded amino acids can affect the binding of antigen and antibody, they are selectively retained (green marked amino acids); 5. The exposed amino acids and the remaining amino acids that have little effect on the activity of the antibody are directly mutated (black marked amino acids). After humanization of 5B11, it is named N5B11, and the degree of humanization of N5B11 is 90%( Figure 10 ). The amino acid sequences of the light chain variable region and the heavy chain variable region of the humanized antibody N5B11 are shown in SEQ ID NO: 60 and 61, respectively.

[0277] The N5B11 light and heavy chain variable regions are constructed into the PTT5 eukaryotic expression vector containing human anti-constant region preserved in the laboratory, to construct plasmids PTT5-N5B11-heavy chain (containing a nucleotide sequence encoding the heavy chain constant region shown in SEQ ID NO: 62) and PTT5-N5B11-light chain (containing a nucleotide sequence encoding the light chain constant region shown in SEQ ID NO: 63), and the double plasmids are transiently transfected into expi-293 cells for expression. After 7 days, the supernatant is harvested, and the supernatant is purified using protein A to obtain the antibody N5B11. Subsequently, the neutralization activity and the binding activity of the purified N5B11 are detected (the detection methods of the neutralization activity and the binding activity are referred to embodiments 2 and 3, respectively), and the experimental results show that the neutralization activity( Figure 11 A-B) and the binding activity( Figure 11 C-D) of the humanized antibody N5B11 and the murine antibody 5B11 are consistent.

[0278] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details according to all the teachings disclosed herein, and these changes are within the scope of protection of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to the respiratory syncytial virus (RSV) pre-F protein, wherein, The antibody or its antigen-binding fragment comprises: As shown in SEQ ID NO: 3 or 60, the heavy chain variable region (VH) contains VH CDR1, VH CDR2, and VH CDR3; and, VL CDR1, VL CDR2 and VL CDR3 contained in the light chain variable region (VL) as shown in SEQ ID NO: 4 or 61; The three CDRs contained in the VH and the three CDRs contained in the VL are defined by the Kabat, IMGT or Chothia numbering system.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment comprises: The sequences are VH CDR1 of SEQ ID NO: 5, VH CDR2 of SEQ ID NO: 6, and VH CDR3 of SEQ ID NO: 7; and, VL CDR1 with sequence SEQ ID NO: 8, VL CDR2 with sequence SEQ ID NO: 9, and VL CDR3 with sequence SEQ ID NO:

10.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment comprises: VH containing a sequence as shown in SEQ ID NO: 3 or a variant thereof, and / or, VL containing a sequence as shown in SEQ ID NO: 4 or a variant thereof; The variant has at least 70% sequence identity with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions compared to the sequence from which it originates.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment comprises: VH as shown in SEQ ID NO: 3, and / or VL as shown in SEQ ID NO:

4.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein it is humanized.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment contains a framework region sequence derived from human immunoglobulin.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment comprises: a heavy chain framework region sequence derived from a human heavy chain germline sequence, and a light chain framework region sequence derived from a human light chain germline sequence.

8. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment comprises: VH as shown in SEQ ID NO: 60, and / or VL as shown in SEQ ID NO:

61.

9. The antibody or antigen-binding fragment thereof of claim 1, further comprising a constant region derived from mouse or human immunoglobulin.

10. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The heavy chain of the antibody or its antigen-binding fragment includes a heavy chain constant region derived from human immunoglobulin, and the light chain of the antibody or its antigen-binding fragment includes a light chain constant region derived from human immunoglobulin.

11. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The heavy chain of the antibody or its antigen-binding fragment includes a heavy chain constant region derived from human IgG1, IgG2, IgG3 or IgG4, and the light chain of the antibody or its antigen-binding fragment includes a light chain constant region derived from the κ chain or λ chain of human immunoglobulin.

12. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The heavy chain of the antibody or its antigen-binding fragment includes a heavy chain constant region as shown in SEQ ID NO: 62, and the light chain of the antibody or its antigen-binding fragment includes a light chain constant region as shown in SEQ ID NO:

63.

13. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, diabody; and / or, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

14. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody is a bispecific antibody or a multispecific antibody.

15. An isolated nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1-14.

16. A vector comprising the nucleic acid molecule of claim 15.

17. A host cell comprising the nucleic acid molecule of claim 15 or the vector of claim 16.

18. A method for preparing an antibody or antigen-binding fragment thereof according to any one of claims 1-14, comprising culturing a host cell according to claim 17 under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

19. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-14, and a pharmaceutically acceptable carrier and / or excipient.

20. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-14 for the preparation of a medicament for neutralizing the virulence of RSV, or for inhibiting or blocking RSV fusion with cells, or for preventing and / or treating RSV infection or RSV-related disease in a subject.

21. The use as described in claim 20, wherein, The disease associated with RSV infection is pneumonia.

22. The use as claimed in claim 21, wherein, The pneumonia mentioned is pediatric pneumonia.

23. The use as described in claim 20, wherein, The subjects were mammals.

24. The use as described in claim 23, wherein, The subjects were human.

25. The use as described in claim 20, wherein, The antibody or its antigen-binding fragment may be used alone or in combination with other pharmaceutically active agents.

26. A conjugate comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-14, and a detectable label linked to said antibody or antigen-binding fragment thereof.

27. The conjugate of claim 26, wherein, The detectable marker is selected from enzymes, chemiluminescent reagents, fluorescent dyes, radionuclides, or biotin.

28. A kit comprising an antibody or antigen-binding fragment thereof as claimed in any one of claims 1-14 or a conjugate as claimed in claim 26 or 27.

29. The kit of claim 28, wherein, The kit comprises the conjugate as described in claim 26 or 27.

30. The kit of claim 28, wherein, The kit comprises an antibody or an antigen-binding fragment thereof as described in any one of claims 1-14, and a second antibody that optionally specifically recognizes the antibody or the antigen-binding fragment thereof; optionally, the second antibody further comprises a detectable label.

31. The kit according to claim 30, wherein, The detectable marker is selected from enzymes, chemiluminescent reagents, fluorescent dyes, radionuclides, or biotin.

32. A method for detecting the presence or level of RSV in a sample for non-diagnostic purposes, comprising using an antibody or antigen-binding fragment thereof as described in any one of claims 1-14 or a conjugate as described in claim 26 or 27.

33. The method of claim 32, wherein, The method described is an immunological detection.

34. The method of claim 32, wherein, The methods described are immunoblotting, enzyme immunoassay, chemiluminescence immunoassay, fluorescence immunoassay, or radioimmunoassay.

35. The method of claim 32, wherein, The method includes using the conjugate as described in claim 26 or 27.

36. The method of claim 32, wherein, The method includes using an antibody or antigen-binding fragment thereof as described in any one of claims 1-14, and the method further includes using a second antibody carrying a detectable label to detect the antibody or antigen-binding fragment thereof.

37. The method of claim 36, wherein, The detectable marker is selected from enzymes, chemiluminescent reagents, fluorescent dyes, radionuclides, or biotin.

38. The method of claim 32, wherein, The method includes: (1) contacting the sample with the antibody or its antigen-binding fragment or conjugate; and (2) detecting the formation of antigen-antibody immune complexes or detecting the amount of the immune complexes.

39. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-14 or the conjugate of claim 26 or 27 in the preparation of a kit for detecting the presence or level of RSV in a sample, and / or for diagnosing whether a subject is infected with RSV.

40. The use as described in claim 39, wherein, The kit is used to detect the presence or level of RSV in a sample by means of the method described in any one of claims 32-38.

41. The use as described in claim 39, wherein, The sample is a bodily fluid or tissue sample from the subject.

42. The use as described in claim 39, wherein, The sample is a respiratory secretion or respiratory tissue sample from the subject.

43. The use as described in claim 39, wherein, The subjects were mammals.

44. The use as described in claim 43, wherein, The subjects were human.

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