Anti-respiratory syncytial virus antibody and its uses

Abstract

An antibody that specifically binds to the fusion (F) protein of respiratory syncytial virus (RSV) and an antigen-binding fragment thereof are provided herein. Additionally, a method for the prevention, treatment, and diagnosis of viral infection and / or the treatment of one or more symptoms of RSV-mediated disease is provided.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims priority to U.S. provisional application No. 63 / 598,807 filed on November 14, 2023, the entirety of which is incorporated by reference.

[0003] Reference to the electronically submitted sequence list

[0004] This application includes a sequence list submitted electronically through the Patent Center. The contents of the electronic sequence list (SPC6267WOPCT1SequenceListing.xml; size: 189 KB; and creation date: November 8, 2024) are incorporated herein by reference in their entirety.

[0005] Field of invention

[0006] The present disclosure generally relates to anti-respiratory syncytial virus (RSV) antibodies and functional fragments thereof, nucleic acid sequences encoding such antibodies, and methods and reagents for the production and use thereof. Background Technology

[0007] Respiratory Syncytial Virus (RSV) is a common cold virus belonging to the family Paramyxoviridae. RSV is virulent and easily transmissible, infecting both children and adults. Two RSV subtypes, subtype A and subtype B, have been identified. RSV possesses two major surface glycoproteins: the fusion protein (F protein) and the adhesion protein (G protein). The RSV F protein is a viral membrane protein responsible for the fusion of the host cell and the virion after adhesion. Additionally, the infection of neighboring cells through the formation of fusion bodies is facilitated by the F protein, and its function is believed to depend on the protein's original oligomeric structure. The G protein is an 89 kD protein also known as the adhesion protein. While the G protein differs significantly between the two RSV subtypes, the F protein is more conserved, as evidenced by the fact that, for example, only about 53% homology exists within the G proteins from subtypes A and B.

[0008] Although both RSV F and G proteins have been shown to induce protective neutralizing antibody responses, RSV F protein plays a crucial role in infection and is the target for most of the neutralizing activity in human serum (Literature [Magro, M., et al., Proc Natl Acad Sci USA 109, 3089-3094 (2012)]; Literature[Johnson, S., et al., J Infect Dis 176, 1215-1224 (1997)]; literature[Beeler, JA, et al., J Virol 63, 2941-2950 (1989)]; Literature[Karron, RA, Proc Natl Acad Sci USA 94, 13961-13966 (1997)]; literature[Ngwuta, JO, Sci Transl Med 7, 309ra162 (2015)]). RSV F protein is also a target of palivizumab, a monoclonal antibody used to passively protect high-risk infants from severe disease (Literature [TI-RS Group, Pediatrics102, 531-537 (1998)]).

[0009] Mature RSV F glycoproteins initially undergo a metastable pre-fusion conformation (preF) before undergoing the conformational changes that cause the insertion of hydrophobic fusion peptides into the host cell membrane (Literature [McLellan, JS, Science It exists as a stable, extended post-fusion stereomorphism (postF) (McLellan, JS, 340, 1113-1117 (2013)]). J Virol 85, 7788-7796 (2011)]; Literature[Swanson, KA, Proc Natl Acad Sci USA The subsequent refolding of the F protein into [108, 9619-9624 (2011)]) leads to the fusion of the viral membrane and the host cell membrane. Due to inherent instability, the preF protein has a tendency to trigger the early transition to postF both in solution and on the viral surface (Liljeroos, L., Proc Natl Acad Sci USA 110, 11133-11138 (2013)]). Recently, stabilization of preF was achieved by protein engineering (reference [Krarup, A., Nat Commun 6, 8143 (2015)]; Literature[McLellan, JS, Science 342, 592-598 (2013)]), in animal models, stabilized preF was shown to induce neutralizing antibodies of higher titers than postF (McLellan, JS, Science 342, 592-598 (2013)]).

[0010] Despite advancements in RSV research, established treatment options for RSV disease remain limited. Severe forms of lower respiratory tract disease often require significant supportive care, including humidified oxygen administration and respiratory support (Fields et al., eds, 1990, Fields Virology, 2nd supplement ed., Vol. 1, Raven Press, New York at pages 1045-1072).

[0011] Ribavirin, the only drug approved for the treatment of infection, has been shown to be effective in treating pneumonia and bronchiolitis associated with RSV infection and to alter the course of severe RSV disease in immunocompromised children (Literature [Smith et al., New Engl. J. Med. 325:24-29 (1991)]). The use of ribavirin is restricted due to concerns surrounding the potential risk to pregnant women who may be exposed to the aerosolized drug while it is administered in a hospital setting.

[0012] Although commercially available vaccines such as Arexvy for the elderly (GlaxoSmithKline; Middlesex, UK) and Abrysvo for maternal immunization (Pfizer; New York, NY) have been developed, several vaccine candidates have been discontinued and others are currently under development (Literature [Murphy et al., Virus Res. 32: 13-36 (1994)]). Vaccine development has proven to be a challenge. In particular, since the peak incidence of lower respiratory tract disease occurs between 2 and 5 months of age, immunization may be necessary during the neonatal period immediately after birth. However, the neonatal immune response is known to be immature at that time. Furthermore, infants at that point still possess high titers of maternally acquired RSV antibodies, which can reduce vaccine immunogenicity (Murphy et al., J. Virol. 62:3907-3910 (1988)]; and literature[Murphy et al., Vaccine 9:185-189 (1991)]).

[0013] Currently, the approved approach for the prevention of RSV disease is passive immunization. For example, palivizumab (SYNAGIS®), a humanized antibody specific to an epitope on the F protein, is approved for intramuscular administration to pediatric patients for the prevention of severe lower respiratory tract disease caused by RSV at a recommended monthly dose of 15 mg per kg of body weight throughout the RSV season (November to April in the Northern Hemisphere). SYNAGIS® is a complex of human (95%) and murine (5%) antibody sequences. (Reference [Johnson et al., J. Infect. Diseases 176:1215-1224 (1997)] and U.S. Patent No. 5,824,307).

[0014] Although SYNAGIS® has been successfully used to prevent RSV infection in pediatric patients, multiple intramuscular administrations of 15 mg / kg of SYNAGIS® are required to achieve a prophylactic effect. The need for multiple intramuscular doses of the antibody requires repeated hospital visits, which is not only inconvenient for patients but may also lead to missed doses.

[0015] Efforts were made to improve the therapeutic profile of anti-RSV-F antibodies, leading to the identification and development of motavizumab, also referred to as NUMAX™. However, clinical tests revealed that certain patients administered motavizumab experienced severe hypersensitivity reactions. Further development of this humanized anti-RSV-F antibody was subsequently discontinued.

[0016] Other antibodies against RSV-F protein have been described, as described in U.S. Patent Nos. 6,656,467; 5,824,307; 7,786,273; 7,670,600; 7,083,784; 6818216; 7700735; 7553489; 7,323,172; 7,229,619; 7,425,618; 7,740,851; 7,658,921; 7,704,505; 7,635,568; 6,855,493; 6,565,849; 7,582,297; 7,208,162; 7,700,720; 6,413,771; It can be found in 5,811,524; 6,537,809; 5,762,905; 7,070,786; 7,364,742; 7,879,329; 7,488,477; 7,867,497; 553,441; 6,835,372; 7,482,024; 7,691,603; 8,562,996; 8,568,726; 9,447,173; US20100015596; WO2009088159A1; and WO2014159822. To date, SYNAGIS® and BEYFORTUS® (MedImmmune LLC; Gaithersburg, MD) have been approved by regulatory authorities, and CLESROVIMAB® (Merck; Loway, NJ) is currently in Phase 3 clinical trials for use in preventing RSV infection.

[0017] Therefore, there is still a need for the provision of highly specific, high-affinity, and highly potent anti-RSV antibodies and their antigen-binding fragments that can be used to treat and / or neutralize symptoms associated with at least one of subtype A and subtype B RSV virus strains, but preferably both, or at least one of subtype A and subtype B RSV virus strains.

[0018] An isolated or recombinant anti-respiratory syncytial virus (RSV) protein (F) antibody or an antigen-binding fragment thereof that specifically binds to the respiratory syncytial virus (RSV) F protein (F) is provided herein. The anti-RSV F antibody or the antigen-binding fragment thereof comprises a variable heavy chain region (V) comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3. H ), and a variable light chain region (V) including light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3. L Each includes (1) SEQ ID NO: 1, 2, 3, 4, 5 and 6; each (2) SEQ ID NO: 19, 20, 21, 22, 23 and 24; each (3) SEQ ID NO: 27, 28, 29, 30, 31 and 32; each (4) SEQ ID NO: 35, 36, 37, 38, 39 and 40; each (5) SEQ ID NO: 43, 44, 45, 46, 47 and 48; each (6) SEQ ID NO: 51, 52, 53, 54, 55 and 56; each (7) SEQ ID NO: 59, 60, 61, 62, 63 and 64; Each has the amino acid sequence of (8) SEQ ID NO: 67, 68, 69, 70, 71 and 72; each (9) SEQ ID NO: 75, 76, 77, 78, 79 and 80; or each (10) SEQ ID NO: 83, 84, 85, 86, 87 and 88.

[0019] In a specific embodiment, the anti-RSV F antibody or its antigen-binding fragment is V having an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, 25, 33, 41, 49, 57, 65, 73, 81, or 89. H , or V having an amino acid sequence at least 90% identical to SEQ ID NO: 8, 26, 34, 42, 50, 58, 66, 74, 82, or 90 L Includes

[0020] The anti-RSV F antibody or its antigen-binding fragment is, for example, (1) V having the amino acid sequence of SEQ ID NO: 7 H and V having the amino acid sequence of SEQ ID NO: 8 L ; (2) V having the amino acid sequence of SEQ ID NO: 25 H and V having the amino acid sequence of SEQ ID NO: 26 L ; (3) V having the amino acid sequence of SEQ ID NO: 33 H and V having the amino acid sequence of SEQ ID NO: 34 L ; (4) V having the amino acid sequence of SEQ ID NO: 41 H and V having the amino acid sequence of SEQ ID NO: 42 L ; (5) V having the amino acid sequence of SEQ ID NO: 49 H and V having the amino acid sequence of SEQ ID NO: 50 L ; (6) V having the amino acid sequence of SEQ ID NO: 57 H and V having the amino acid sequence of SEQ ID NO: 58 L ; (7) V having the amino acid sequence of SEQ ID NO: 65 H and V having the amino acid sequence of SEQ ID NO: 66 L ; (8) V having the amino acid sequence of SEQ ID NO: 73 H and V having the amino acid sequence of SEQ ID NO: 74 L ; (9) V having the amino acid sequence of SEQ ID NO: 81 H and V having the amino acid sequence of SEQ ID NO: 82 L ; or (10) V having the amino acid sequence of SEQ ID NO: 89 H and V having the amino acid sequence of SEQ ID NO: 90 L It may include.

[0021] In certain embodiments, the antibody or antigen-binding fragment comprises an antigen-binding fragment comprising a heavy chain polypeptide sequence having at least 90% the same amino acid sequence as SEQ ID NO: 12, 91, 92, 93, 94, 95, 96, 97, 98, or 99. The antibody or antigen-binding fragment may comprise, for example, an antigen-binding fragment comprising a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 12, 91, 92, 93, 94, 95, 96, 97, 98, or 99.

[0022] In certain embodiments, the antibody or antigen-binding fragment comprises an antigen-binding fragment comprising a light chain polypeptide sequence having at least 90% the same amino acid sequence as SEQ ID NO: 13, 100, 101, 102, 103, 104, 105, 106, 107, or 108. The antibody or antigen-binding fragment may comprise, for example, an antigen-binding fragment comprising a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 13, 100, 101, 102, 103, 104, 105, 106, 107, or 108.

[0023] In a specific embodiment, the antibody or antigen-binding fragment comprises (1) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 12 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 13; (2) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 91 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 100; (3) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 92 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 101; (4) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 93 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 102; (5) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 94 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 103; (6) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 95 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 104; (7) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 96 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 105; (8) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 97 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 106; (9) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 98 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 107; or (10) an antigen-binding fragment comprising a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 99 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 108.

[0024] In certain embodiments, the antibody or antigen-binding fragment is (1) SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 15; (2) SEQ ID NO: 91, SEQ ID NO: 109 or SEQ ID NO: 118; (3) SEQ ID NO: 92, SEQ ID NO: 110 or SEQ ID NO: 119; (4) SEQ ID NO: 93, SEQ ID NO: 111 or SEQ ID NO: 120; (5) SEQ ID NO: 94, SEQ ID NO: 112 or SEQ ID NO: 121; (6) SEQ ID NO: 95, SEQ ID NO: 113 or SEQ ID NO: 122; (7) SEQ ID NO: 96, SEQ ID NO: 114 or SEQ ID NO: 123; (8) SEQ ID NO: 97, SEQ ID NO: 115 or SEQ ID NO: 124; (9) SEQ ID NO: 98, SEQ ID NO: 116 or SEQ ID NO: 125; or (10) SEQ ID NO: 99, SEQ ID NO: 117 or SEQ ID NO: 126, comprising a heavy chain polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 99, SEQ ID NO: 117 or SEQ ID NO: 126. In certain embodiments, the antibody or its antigen-binding fragment comprises a light chain polypeptide having an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107 or SEQ ID NO: 108. In a specific embodiment, the antibody or its antigen-binding fragment is (1) SEQ ID NO: 15 and SEQ ID NO: 10, SEQ ID NO: 9 and SEQ ID NO: 10, or SEQ ID NO: 11 and SEQ ID NO: 10;(2) SEQ ID NO: 91 and SEQ ID NO: 100, SEQ ID NO: 109 and SEQ ID NO: 100, SEQ ID NO: 118 and SEQ ID NO: 100; (3) SEQ ID NO: 92 and SEQ ID NO: 101, SEQ ID NO: 110 and SEQ ID NO: 101, or SEQ ID NO: 119 and SEQ ID NO: 101; (4) SEQ ID NO: 93 and SEQ ID NO: 102, SEQ ID NO: 111 and SEQ ID NO: 102, or SEQ ID NO: 120 and SEQ ID NO: 102; (5) SEQ ID NO: 94 and SEQ ID NO: 103, SEQ ID NO: 112 and SEQ ID NO: 103, or SEQ ID NO: 121 and SEQ ID NO: 103; (6) SEQ ID NO: 95 and SEQ ID NO: 104, SEQ ID NO: 113 and SEQ ID NO: 104, or SEQ ID NO: 122 and SEQ ID NO: 104; (7) SEQ ID NO: 96 and SEQ ID NO: 105, SEQ ID NO: 114 and SEQ ID NO: 105, or SEQ ID NO: 123 and SEQ ID NO: 105; (8) SEQ ID NO: 97 and SEQ ID NO: 106, SEQ ID NO: 115 and SEQ ID NO: 106, or SEQ ID NO: 124 and SEQ ID NO: 106; (9) SEQ ID NO: 98 and SEQ ID NO: 107, SEQ ID NO: 116 and SEQ ID NO: 107, or SEQ ID NO: 125 and SEQ ID NO: 107; or (10) SEQ ID NO: 99 and SEQ ID NO: 108, SEQ ID NO: 117 and SEQ ID NO: 108, or SEQ ID NO: 126 and SEQ ID NO: 108, each comprising a heavy chain polypeptide and a light chain polypeptide selected from;

[0025] In certain embodiments, the antibody or its antigen-binding fragment may neutralize or block RSV-F activity by inhibiting at least one biological activity of RSV-F. In certain embodiments, the antibody or its antigen-binding fragment may bind to the PreF conformation of RSV-F with a higher binding affinity compared to the PostF conformation of RSV-F.

[0026] In certain embodiments, the antibody or antigen-binding fragment is a human antibody, a humanized antibody, a chimeric antibody, or a bispecific or multispecific antibody. The antibody or antigen-binding fragment may be, for example, a human antibody.

[0027] In certain embodiments, the antibody or antigen-binding fragment is conjugated to polyethylene glycol (PEG) or a therapeutic or diagnostic drug.

[0028] Additionally, an isolated nucleic acid molecule comprising a nucleic acid sequence encoding an anti-RSV F antibody or an antigen-binding fragment thereof according to the present invention is provided.

[0029] In addition, an expression vector comprising an isolated nucleic acid sequence according to the present invention is provided.

[0030] In addition, a host cell comprising an expression vector according to the present invention is provided.

[0031] Additionally, a method for producing an anti-RSV F antibody or an antigen-binding fragment thereof is provided. The method comprises the steps of culturing a host cell of the present invention under conditions for producing an anti-RSV F antibody or an antigen-binding fragment thereof, and recovering the anti-RSV F antibody or an antigen-binding fragment thereof from the cell or cell culture.

[0032] Additionally, a pharmaceutical composition is provided comprising one or more of an anti-RSV F antibody or an antigen-binding fragment thereof according to the present invention, and a pharmaceutically acceptable carrier and / or excipient. Additionally, a pharmaceutical composition is provided comprising one or more nucleic acid sequences according to the present invention, or one or more expression vectors according to the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0033] Additionally, a method for inhibiting at least one biological activity of RSV-F is provided. The method comprises (a) one or more antibodies or antigen-binding fragments thereof according to the present invention; (b) a nucleic acid molecule according to the present invention; (c) an expression vector according to the present invention; (d) a host cell according to the present invention; or (e) a pharmaceutical composition according to the present invention.

[0034] The method includes the step of administering to a subject who requires or is suspected of requiring it. In certain embodiments, the subject requires treatment, prevention, or improvement of a respiratory syncytial virus (RSV) infection, or treatment, prevention, or improvement of at least one symptom associated with an RSV infection. In certain embodiments, the method reduces lung virus titers in the subject. In certain embodiments, any one of (a) to (e) is administered at a therapeutically effective amount.

[0035] Additionally, one or more of the anti-RSV F antibody or its antigen-binding fragment provided herein are used in the manufacture of a drug for inhibiting at least one biological activity of RSV-F in a subject.

[0036] Additionally, one or more of the anti-RSV F antibody or its antigen-binding fragment provided herein are used in the manufacture of a drug for treating, preventing, or resolving respiratory syncytial virus (RSV) infection in a subject, or for treating, preventing, or resolving at least one symptom associated with RSV infection.

[0037] Other aspects, features, and advantages of the present invention will become apparent from the following disclosure, which includes the detailed description of the invention, preferred embodiments thereof, and the appended claims. Brief explanation of the drawing

[0038] FIG. 1 shows RSV antibody 1 analyzed in a 3-day neutralization assay using 25 RSV A and 18 RSV B viruses according to Example 6 (4 of which (17-058221, MEP-OUT-13-D1, MEP-OUT-01-D1, and 18-002094) contain F mutations I206M and Q209R). In vitro This shows a graph representation of antiviral activity. In Figure 1, IC50 indicates an inhibitory concentration of 50%; RSV represents respiratory syncytial virus. FIGS. 2a to 2c show graph representations of data related to the prophylactic efficacy of RSV antibody 1 non-YTE in an RSV-infected cotton rat model according to Example 7. FIG. 2a shows the lung virus titer determined by plaque assay on day 4 after infection with RSV A Long. FIG. 2b shows the lung virus titer determined by plaque assay on day 4 after infection with RSV B9320. FIG. 2c shows the lung virus titer determined by plaque assay on day 4 after infection with RSV B 18537. In FIGS. 2a to 2c, IM represents intramuscular; RSV represents respiratory syncytial virus; SD represents standard deviation; error bars represent mean values ​​with SD (n = 5 to 6); and dashed lines represent lower detection limits; The asterisk indicates a statistically significant difference (alpha-level 0.05) compared to the vehicle control group based on Tobit regression analysis using Bonferroni multiplicity correction. Figures 3a to 3c show graph representations of the correlation between the serum concentration of RSV antibody 1 non-YTE according to Example 8 and the relative decrease in log lung virus titers on day 4 post-infection in RSV-infected cotton rats. Figure 3a shows the results associated with RSV A Long. Figure 3b shows the results associated with RSV B 9320. Figure 3c shows the results associated with RSV B 18537. In Figures 3a to 3c, EC90 is an effective concentration of 90%; RSV is respiratory syncytial virus. Figures 4a and 4b show graph representations of the activation of Jurkat / NFAT luc effector cells expressing FcγRIIa (Figure 4a) or FcγRIIIa (Figure 4b) by RSV antibody 1 Fc variant bound to RSV-infected A549 target cells according to Example 9. In Figures 4a and 4b, Fc represents a crystallizable fragment; FcγR represents the Fc gamma receptor; LALA represents L234A / L235A; RSV represents respiratory syncytial virus; mAb represents a monoclonal antibody; RLU represents the relative optical unit; SD represents the standard deviation; and YTE represents M252Y / S254T / T256E. FIGS. 5a to 5c show graph representations of the correlation between serum concentrations of RSV antibody 1 (Fig. 5a), RSV antibody 1 non-YTE (Fig. 5b), and RSV antibody 1 non-YTE LALA (Fig. 5c) according to Example 9 and the relative log-length lung viral titer reduction on day 4 post-infection in RSV A2-infected BALB / c mice. Referring to FIGS. 5a to 5c, EC90 represents an effective concentration of 90%; LALA represents L234A / L235A; RSV represents respiratory syncytial virus; mAb represents a monoclonal antibody; RSV represents respiratory syncytial virus; YTE represents M252Y / S254T / T256E; and in each graph, the dotted line represents the EC90 for viral reduction. 90 It represents. Figure 6 shows a graph of the PK fit of RSV antibody 1 (n = 4) and nirsevimab (n = 2) administered IV at 5 mg / kg in cynomolgus monkeys according to Example 11. Referring to Figure 5, IV is intravenous; PK is pharmacokinetic. Referring further to Figure 5, study data points are indicated by filled symbols, and model fits are indicated by solid lines. Specific details for implementing the invention

[0039] Various publications, papers, and patents are cited or described in the background art and throughout this specification; each of these references is incorporated herein by reference in its entirety. Discussions of documents, acts, materials, devices, articles, etc., included in this specification are intended to provide context for the invention. Such discussions are not to be acknowledged that any or all of these matters constitute part of the prior art in relation to any of the inventions disclosed or claimed.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Otherwise, specific terms used herein have the meaning as set forth in this specification. All patents, published patent applications, and publications cited herein are incorporated by reference as fully presented herein.

[0041] It should be noted that, as used in the present invention and the appended claims, the singular forms “a,” “an,” and “the” include multiple referents unless the context clearly indicates otherwise.

[0042] Unless otherwise specified, any numerical value, such as sequence identity % or sequence identity range % described herein, should be understood in all cases to be modified by the term “about.” Accordingly, numerical values ​​typically include ± 10% of the stated value. For example, a dose of 10 mg includes 9 mg to 11 mg. As used herein, the use of a numerical range explicitly includes all individual numerical values ​​within the range, including all possible sub-ranges, integers, and fractions of values ​​within such ranges, unless the context clearly indicates otherwise.

[0043] As used herein, the connecting term “and / or” between multiple mentioned elements is understood to encompass both individual options and combined options. For example, where two elements are combined by “and / or,” the first option refers to the possibility of the first element being applied without the second element. The second option refers to the possibility of the second element being applied without the first element. The third option refers to the possibility of the first and second elements being applied together. Any one of these options is understood to fall within the meaning and thus satisfies the requirements of the term “and / or” as used herein. The possibility of simultaneous application of more than one of the options is also understood to fall within the meaning and thus satisfies the requirements of the term “and / or”.

[0044] Unless otherwise indicated, the term “at least” preceding a series of elements should be understood to refer to all of the series of elements. Those skilled in the art will be able to recognize or identify many equivalents to the specific embodiments of the invention described herein using only routine experimentation. Such equivalents are intended to be encompassed by the invention.

[0045] Throughout this specification and the following claims, unless the context otherwise requires, variations of the word “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a specified integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term “comprising” may be substituted with the terms “containing” or “including,” or, from time to time, with the term “having” as used herein.

[0046] As used herein, “consisting of” excludes any element, step, or component not specified in the claim element. As used herein, “essentially consisting of” does not exclude a material or step that does not substantially affect the fundamental and novel nature of the claim. Whenever used herein in the context of an aspect or embodiment of the invention, any of the foregoing terms “comprising,” “containing,” “including,” and “having” may be replaced with the terms “consisting of” or “essentially consisting of” to change the scope of the disclosure.

[0047] As used interchangeably herein, “polynucleotide” or “nucleic acid” refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any substrate that may be incorporated into the polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may include modified nucleotides, such as methylated nucleotides and analogs thereof. As used herein, “oligonucleotide” generally, but not necessarily, refers to a short, generally single-stranded synthetic polynucleotide of a length of about 200 nucleotides or less. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides. Unless otherwise specified, the left end of any single-stranded polynucleotide sequence disclosed herein is a 5’ end; The left direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The additional direction of a nascent RNA transcript from 5' to 3' is referred to as the transcription direction; a sequence region on the DNA strand having the same sequence as the RNA transcript located 5' to the 5' end of the RNA transcript is referred to as the “upstream sequence”; and a sequence region on the DNA strand having the same sequence as the RNA transcript located 3' to the 3' end of the RNA transcript is referred to as the “downstream sequence”.

[0048] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. Polymers may be linear or branched, may contain modified amino acids, or may be interrupted by non-amino acids. The term also encompasses amino acid polymers modified naturally or by intervention; e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. Polypeptides containing one or more analogs of amino acids, including but not limited to non-natural amino acids, as well as other modifications known in the art, are also included within this definition. Because polypeptides in this disclosure may be based on other members of the antibody or immunoglobulin phase, in certain embodiments, it is understood that “polypeptide” may exist as a single chain or two or more assembled chains.

[0049] When used in relation to amino acid sequences, the phrases “sequence identity percentage (%),” “identity %,” or “~ and ~% identical” describe the number of matching (“hits”) of identical amino acids in two or more aligned amino acid sequences compared to the number of amino acid residues constituting the total length of the amino acid sequence. In other words, when sequences are compared and aligned for maximum correspondence, as measured using sequence comparison algorithms known in the art, or when manually aligned and visually inspected, using alignment for two or more sequences, the percentage of identical amino acid residues ( for example, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identity can be determined over the entire length of the amino acid sequence. The same determination can be made for nucleotide sequences. Therefore, the sequences compared to determine sequence identity may differ due to amino acid substitution(s), addition(s), or deletion(s). Suitable programs for aligning protein sequences are known to those skilled in the art. The percentage of sequence identity of a protein sequence is, for example , using the NCBI BLAST algorithm (reference [Altschul SF, et al (1997), Nucleic Acids Res. It can be determined by programs such as CLUSTALW, Clustal Omega, FASTA, or BLAST.

[0050] As used herein, in the context of administering two or more therapies or components to a subject, the terms and phrases “in combination,” “in combination with,” “co-delivery,” and “administered with” refer to the simultaneous administration of two or more therapies or components, or of a therapeutic composition and an adjuvant. “Simultaneous administration” may be the administration of two components within at least the same day. Where two components are “administered with” or “administered in combination with,” they may be administered sequentially as separate compositions within a short period, for example, 24, 20, 16, 12, 8, or 4 hours, or within 1 hour, or they may be administered simultaneously as a single composition. The use of the term “in combination with” does not limit the order in which the therapies or components are administered to the subject. For example, the first therapy or component may be administered before, in parallel with, simultaneously with, or after the administration of the second therapy or component. In some embodiments, the first therapy or component and the second therapy or component are administered as the same composition. In other embodiments, the first therapy or component and the second therapy or component are administered as separate compositions.

[0051] As used herein, “non-naturally occurring” nucleic acids or polypeptides refer to nucleic acids or polypeptides that do not occur in nature. “Non-naturally occurring” nucleic acids or polypeptides may be synthesized and / or processed and / or produced and / or otherwise manipulated in a laboratory and / or manufacturing environment. In some cases, non-naturally occurring nucleic acids or polypeptides may include naturally occurring nucleic acids or polypeptides that are processed, processed, or manipulated to exhibit characteristics that were not present in the naturally occurring nucleic acids or polypeptides prior to processing. As used herein, “non-naturally occurring” nucleic acids or polypeptides may be nucleic acids or polypeptides isolated or separated from a natural source in which they were found, and such nucleic acids or polypeptides lack covalent bonds to the sequences to which they were associated in the natural source. “Non-naturally occurring” nucleic acids or polypeptides may be produced recombinantly or through other methods such as chemical synthesis. “Non-naturally occurring” polypeptides may be “non-naturally occurring” antibodies, such as recombinantly produced antibodies.

[0052] As used herein, the term “operably linked” refers to a link or juxtaposition in a relationship that allows the component so described to function in an intended manner. For example, a regulatory sequence operably linked to a nucleic acid sequence of interest may induce transcription of the nucleic acid sequence of interest, or a signal sequence operably linked to an amino acid sequence of interest may secrete or translocate the amino acid sequence of interest across a membrane.

[0053] As used herein, the terms “subject” and “patient” are used interchangeably and refer to any animal, preferably mammal, most preferably human, that will be or has been treated by the method as described herein and / or with an anti-RSV F antibody or an antigen-binding fragment thereof. As used herein, the term “mammal” encompasses any mammal. Examples of mammals include, but are not limited to, non-human primates (NHP) such as cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys or apes, and humans, more preferably human. Human subjects may include patients.

[0054] “Optional” or “optional” means that the subsequently described event or situation may or may not occur, and that the described content includes cases where the event or situation occurs and cases where it does not occur.

[0055] The terms “pharmaceuticalally acceptable carrier” or “pharmaceutically acceptable excipient” include any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents, absorption retardants, etc. The use of such media and formulations for pharmaceutically active substances is well known in the art. Their use in pharmaceutical compositions is considered, except where any conventional media or formulation is incompatible with the active ingredient. Additional active ingredients may also be incorporated into the pharmaceutical composition.

[0056] The term “pharmaceuticalally acceptable salt” refers to a salt of any of the compounds of the present invention that is known to be nontoxic and is commonly used in the pharmaceutical literature. In some embodiments, the pharmaceutically acceptable salt of a compound retains the biological efficacy of the compound described herein and is not biologically or otherwise undesirable. Examples of pharmaceutically acceptable salts are [Berge et al., Pharmaceutical Salts, J. Pharmaceutical Sciences, [This can be found in January 1977, 66(1), 1-19]. Pharmaceutically acceptable acid addition salts may be formed from inorganic and organic acids. Inorganic acids from which salts may be derived include, for example, hydrochloric acid, hydrobromide, sulfuric acid, nitric acid, and phosphoric acid. Organic acids from which salts may be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvate, lactic acid, oxalic acid, malic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethylsulfonic acid, p-toluenesulfonic acid, stearic acid, and salicylic acid. Pharmaceutically acceptable base addition salts may be formed from inorganic and organic bases. Inorganic bases from which the salt may be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum. Organic bases from which the salt may be derived include, for example, primary, secondary, and tertiary amines; substituted amines including naturally occurring substituted amines; cyclic amines; and basic ion exchange resins. Examples of organic bases include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, pharmaceutically acceptable base addition salts are selected from ammonium, potassium, sodium, calcium, and magnesium salts.

[0057] As used herein, the term “antibody” is intended in a broad sense and includes polyclonal antibodies, monoclonal antibodies including murine, human, humanized, and chimeric monoclonal antibodies, antigen-binding fragments, bispecific or multispecific antibodies, dimeric, tetrameric, or multimeric antibodies, Fv, Fab, and F(ab')2, and difunctional hybrids (e.g., in the literature [Lanzavecchia et al., Eur. J. Immunol. 17:105 (1987)]), single chain (literature[Huston et al., Proc. Natl. Acad. Sci. USA 85:5879 (1988)]; Literature[Bird et al., Science Immunoglobulin molecules comprising any other modified configuration of an immunoglobulin molecule including an antibody having a modified constant domain (e.g., U.S. Patent No. 5,624,821), a domain antibody, and an antigen-binding site of the required specificity. Immunoglobulin molecules may be assigned to five major classes, IgA, IgD, IgE, IgG, and IgM, based on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The light chains of antibodies of any vertebrate species may be assigned to one of two distinctly different types, kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. General principles of antibody molecular structures and various techniques related to antibody production are provided in the literature, for example, [Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988)].

[0058] The isolated proteins or constructs of the present application may also comprise antibody derivatives. As used herein, the term “antibody derivative” refers to a molecule comprising a full-length antibody or an antigen-binding fragment thereof, wherein one or more amino acids are chemically modified or substituted. Chemical modifications that may be used on antibody derivatives include, for example, alkylation, PEGylation, acylation, esterification, or amide formation to link the antibody to a second molecule. Exemplary modifications include PEGylation (e.g., cysteine-PEGylation), biotinylation, radiolabeling, and conjugation with a second agent (e.g., a cytotoxic agent).

[0059] As used herein, the term “human antibody” is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human mAbs of this disclosure have, for example, in the CDR and / or framework region and / or Fc region, amino acid residues not encoded by the human germline immunoglobulin sequence (e.g., In vitro or by random or site-specific mutagenesis In vivo It may include mutations introduced by somatic mutations. However, as used herein, the term “human antibody” is not intended to include mAbs in which a CDR sequence derived from a germline of another mammalian species (e.g., mouse) is transplanted into a human FR sequence.

[0060] The term “humanized antibody” refers to a human antibody in which one or more CDRs of such antibody are replaced with one or more corresponding CDRs obtained from antibodies derived from non-humans (e.g., mice, rats, rabbits, primates). Humanized antibodies may also contain one or more non-human CDR sequences as well as specific non-CDR sequences or residues derived from such non-human antibodies. Such antibodies may also be referred to as “chimeric antibodies.”

[0061] The term “recombinant” generally refers to any protein, polypeptide, or cell expressing a gene of interest produced by genetic engineering methods. As used in relation to proteins or polypeptides, the term “recombinant” means a polypeptide produced by the expression of a recombinant polynucleotide. Proteins used in the immunogenic compositions of this disclosure may be isolated from natural sources or produced by genetic engineering methods.

[0062] The antibodies of the present disclosure may, in some embodiments, be recombinant human antibodies. As used herein, the term “recombinant human antibody” refers to an antibody expressed using a recombinant expression vector transfected into a host cell (as further described below), an antibody isolated from a library of recombinant, combinatorial human antibodies (as further described below), or an antibody isolated from an animal (e.g., mouse) genetically modified for a human immunoglobulin gene (e.g., in the literature [Taylor et al. Nucl. Acids Res. It is intended to include all antibodies comprising human or humanized antibodies produced, expressed, generated, or isolated by recombinant means, such as antibodies produced, expressed, generated, or isolated by any other means involving splicing a human immunoglobulin gene sequence to another DNA sequence [see 20:6287-6295 (1992)]), or antibodies produced, expressed, generated, or isolated by any other means involving splicing a human immunoglobulin gene sequence to another DNA sequence. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies In vitro Mutagenic (or when animals genetically modified for human Ig sequences are used, In vivo It undergoes somatic mutagenesis, and therefore the V of the recombinant antibody H and V L The amino acid sequence of the region is human germline V H and V LDerived from and related to the sequence, but In vivo It is a sequence that may not naturally exist within the repertoire of human antibody germlines.

[0063] The “Complementary Determining Region” (CDR) is the antibody region that binds to the antigen. VH has three CDRs (HCDR1, HCDR2, HCDR3), and VL has three CDRs (LCDR1, LCDR2, LCDR3). CDRs are [references] Kabat (Wu et al. J Exp Med 132:211:50 (1970); Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., (1991)]), Chothia (Chothia et al. J Mol Biol 196: 901-17 (1987)]), and IMGT (Lefranc et al. Dev Comp Immunol 27: 55-77 (2003)]), and AbM (literature [Martin and Thornton, J Bmol Biol It can be defined using various diagrams such as 263: 800-15 (1996)]). The correspondence between various diagrams and variable area numbering is described ( for example , literature[Lefranc et al., Dev Comp Immunol 27: 55-77 (2003)]; literature[Honegger and Pluckthun, J Mol Biol 309:657-70 (2001)]; International ImMunoGeneTics (IMGT) Database; Web Resources, http: / / www_imgt_org referenceCDRs can be distinguished using available programs such as abYsis of UCL Business PLC. As used herein, the terms “CDR”, “HCDR1”, “HCDR2”, “HCDR3”, “LCDR1”, “LCDR2”, and “LCDR3” are used unless explicitly otherwise stated in this specification, In the aforementioned literature Any of the described methods includes CDRs defined by Kabat, Chothia, IMGT, or AbM. For example, the correspondence between numbering systems, including Kabat numbering and the IMGT unique numbering system, is well known to those skilled in the art ( for example , Kabat, the aforementioned literature; Chothia, the aforementioned literature; Martin, the aforementioned literature; Lefranc et al., the aforementioned literature reference ).

[0064] [Table 1]

[0065]

[0066] As used herein, the terms “treat,” “treat,” and “treating” refer to a reduction or improvement in the progression, severity, and / or duration of upper and / or lower respiratory tract RSV infection, otitis media, or associated symptoms or respiratory disease (e.g., asthma, wheezing, or a combination thereof) resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents). In certain embodiments, these terms refer to a reduction or inhibition of RSV replication, inhibition or reduction of RSV spread to other tissues or subjects (e.g., spread to the lower respiratory tract), inhibition or reduction of RSV-containing cell infection, or improvement of one or more symptoms associated with upper and / or lower respiratory tract RSV infection or otitis media.

[0067] As used herein, the terms “prevent,” “preventing,” and “prevention” refer to the prevention or suppression of the occurrence or onset of upper and / or lower respiratory tract RSV infection, otitis media, or associated symptoms or respiratory disease in a subject; the prevention or suppression of progression from upper respiratory tract RSV infection to lower respiratory tract RSV infection, otitis media, or associated symptoms or respiratory disease due to the administration of a therapy (e.g., a prophylactic or therapeutic agent); the prevention of upper and / or lower respiratory tract RSV infection, otitis media, or associated respiratory disease symptoms; or the administration of a combination of therapies (e.g., a combination of a prophylactic or therapeutic agent). As used herein, the terms “relieve” and “alleviate” refer to a reduction or alleviation of the severity of the disease or any of its symptoms.

[0068] The term “reduce” is a relative term, and accordingly, the agent reduces the response or disease when the response or disease is quantitatively reduced after the administration of treatment, or when the response or disease is reduced after the administration of treatment compared to a reference treatment. Thus, a treatment that reduces or prevents an infection or response, such as a pathological response, such as a vaccine-enhanced viral disease, may, but does not necessarily, completely eliminate such infection or response, as long as the infection or response is measurably reduced by, for example, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or about 80%, or even about 90% (i.e., down to 10% or less) in the absence of treatment or compared to a reference treatment.

[0069] “Preventive” treatment is treatment administered to subjects who do not show signs of the disease or show only early signs, with the aim of reducing the risk of pathology developing.

[0070] The term “therapeutic effective dose” means an amount that produces a desired effect according to the purpose of administration. The exact amount will vary depending on the therapeutic purpose and can be determined by those skilled in the art using techniques known in light of this disclosure (e.g., see Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0071] As used herein in relation to anti-RSV antibodies or their antigen-binding fragments, a therapeutically effective dose means an amount of anti-RSV antibody or its antigen-binding fragment that brings about the treatment of a disease, disorder, or illness; prevents or slows the progression of a disease, disorder, or illness; or reduces or completely alleviates symptoms associated with a disease, disorder, or illness.

[0072] According to specific embodiments, a therapeutically effective dose refers to a therapeutic amount sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or improving the severity of the disease, disorder, or disease to be treated, or associated symptoms; (ii) reducing the duration of the disease, disorder, or disease to be treated, or associated symptoms; (iii) preventing the progression of the disease, disorder, or disease to be treated, or associated symptoms; (iv) causing remission of the disease, disorder, or disease to be treated, or associated symptoms; (v) preventing the onset or onset of the disease, disorder, or disease to be treated, or associated symptoms; (vi) preventing the recurrence of the disease, disorder, or disease to be treated, or associated symptoms; (vii) reducing the hospitalization of a subject with the disease, disorder, or disease to be treated, or associated symptoms; (viii) reducing the duration of hospitalization of a subject with the disease, disorder, or disease to be treated, or associated symptoms; (ix) increasing the survival of a subject with the disease, disorder, or disease to be treated, or associated symptoms; (xi) suppressing or reducing the disease, disorder, or illness to be treated, or associated symptoms in the subject; and / or (xii) enhancing or improving the preventive or therapeutic effect(s) of another therapy.

[0073] Terms such as “specifically bind” or “specifically bind to” mean that an antibody or its antigen-binding fragment forms a complex with a relatively stable antigen under physiological conditions. Specific binding is at least about 1 × 10⁻⁶ -6 Equilibrium dissociation constant less than or equal to M (e.g., K DIt may be characterized by (where a smaller value indicates a stronger binding). Methods for determining whether two molecules bind specifically are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. As described herein, antibodies that bind specifically to RSV-F were identified by biolayer interferometry using surface plasmon resonance, for example, BIACORE™, for example, the ForteBio Octet HTX instrument (Pall Life Sciences). Additionally, multispecific antibodies that bind to the RSV-F protein and one or more additional antigens, or bispecific antibodies that bind to two different regions of RSV-F, are nevertheless considered to be “specifically binding” antibodies as used herein. In some embodiments, the antibodies disclosed herein are approximately 1 × 10⁶ -6 M; approximately 1 × 10⁻⁶ -7 M; approximately 1×10 -8 M; approximately 1×10 -9 M; approximately 1×10 -10 M; approximately 1×10 -6 M to about 1×10 -7 M; approximately 1×10 -7 M to about 1×10 -8 M; approximately 1×10 -8 M to about 1×10 -9 M; or about 1 × 10⁻⁶ -9 M to about 1 × 10 -10 Displays the equilibrium dissociation constant of M (and the singularity thereof).

[0074] As used herein, terms such as “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, etc., include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. In certain embodiments, as used herein, the terms “antigen-binding portion” or “antibody fragment” of an antibody refer to one or more fragments of an antibody that retain the ability to bind to RSV-F.

[0075] As used herein, “isolated antibody” is intended to refer to an antibody that is substantially free of another antibody (Ab) having different antigen specificity (e.g., an isolated antibody that specifically binds to RSV-F, or a fragment thereof that is substantially free of Ab that specifically binds to an antigen other than RSV-F).

[0076] As used herein, “blocking antibody” or “neutralizing antibody” (or “antibody neutralizing RSV-F activity”) is intended to refer to an antibody whose binding to RSV-F results in the inhibition of at least one biological activity of RSV-F. For example, the antibody of this disclosure may help block the fusion of RSV into host cells, prevent the formation of fusion bodies, or prevent primary disease caused by RSV. Alternatively, the antibody of this disclosure may demonstrate the ability to improve at least one symptom of RSV infection. Such inhibition of the biological activity of RSV-F is, in light of this disclosure, several standard In vitro Tests (e.g., such as the neutralization test described herein) or known in the art In vivo It can be evaluated by measuring one or more indicators of RSV-F biological activity by one or more of the assays (e.g., an animal model to examine protection from an RSV challenge after administration of one or more of the antibodies described herein).

[0077] As used herein, “therapeutic antibody” refers to any antibody or antigen-binding fragment thereof administered for the treatment or prophylaxis of animals, including humans. Such antibodies may be produced by any known method for the production of polypeptides in light of the present disclosure and thus include, but are not limited to, recombinantly produced antibodies, synthetically produced antibodies, and therapeutic antibodies derived from cells or tissues and other sources. Therapeutic antibodies isolated or produced from any source may be heterogeneous in length or may differ in post-translational modifications such as glycosylation (i.e., carbohydrate content). The heterogeneity of therapeutic antibodies may also vary depending on the source of the therapeutic antibody. Accordingly, references to therapeutic antibodies refer to the heterogeneous population produced or isolated. References to therapeutic antibodies herein refer to their monomeric, dimeric, or other multimeric forms, where appropriate.

[0078] The term “epitope” refers to an epitope that interacts with a specific antigen-binding site within a variable region of an antibody molecule known as a paratope. A single antigen may possess more than one epitope. Therefore, different antibodies can bind to different regions on an antigen and have different biological effects. The term “epitope” also refers to a region on an antigen to which B cells and / or T cells respond. It also refers to the region of an antigen to which an antibody binds. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and possess residues that directly contribute to the affinity of the interaction. Epitopes can also be stereomorphic, meaning they can consist of non-linear amino acids. In certain embodiments, the epitope may include a crystallized group which is a chemically active surface grouping of a molecule, such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group, and in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics. In some embodiments, an anti-RSV F antibody or antigen-binding fragment as described herein binds to SEQ ID NO: 16.

[0079] The term “effective dose 90” or “ED 90 " refers to the dosage of the agent that produces the desired effect, for example, a 90% reduction in virus-forming plaques compared to the negative control group.

[0080] term “IC 50 “” refers to the “half-maximum inhibitory concentration,” and this value measures the efficacy of inhibition of a compound (e.g., anti-RSV F antibody) for biological or biochemical utility. This quantitative measure indicates the amount of a specific inhibitor required to inhibit a given biological process by half. In certain embodiments, the RSV virus neutralizing effect for the anti-RSV antibody disclosed herein is the neutralization IC 50 It is expressed as a value.

[0081] “Conservative amino acid substitution” is the substitution of an amino acid residue by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitution will not substantially alter the functional properties of the protein. If two or more amino acid sequences differ from each other due to conservative substitution, the percentage or degree of similarity may be upscaled to compensate for the conservative nature of the substitution. Means for performing such adjustment are well known to those skilled in the art in light of this disclosure (e.g., see Pearson (1994) Methods Mol. Biol. 24: 307-331). Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) Aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) Basic side chains: lysine, arginine, and histidine; 6) Acidic side chains: aspartate and glutamate; and 7) Sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in the literature [Gonnet et al. (1992) Science 256: 1443 45]. A “moderately conservative” substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0082] In an attempt to assist readers of the present disclosure, this description is divided into various paragraphs or sections or relates to various embodiments of the present disclosure. Such divisions should not be construed as separating the substantial content of one paragraph or section or embodiment from the substantial content of another paragraph or section or embodiment. On the contrary, those skilled in the art will understand that this description has broad applicability and encompasses all combinations of various sections, paragraphs, and sentences that may be considered. Discussion of any embodiments is merely illustrative and is not intended to imply that the scope of the present disclosure, including the claims, is limited to such examples.

[0083] RSV antibody and its antigen-binding fragment

[0084] Despite decades of research, the development of safe and effective vaccines, or therapeutic, preventive, protective, and / or blocking antibodies against RSV remains challenging, highlighting the need for novel strategies to induce or provide protective immune responses. (Reference [Rogovik, AL, et al., Can Fam Physician 56, 769-772 (2010)]; literature[Graham, BS, Immunol Rev 239, 149-166 (2011)]; Literature[JR Groothuis, EA Simoes, VG Hemming, Pediatrics 95, 463-467 (1995)]). In fact, to date, there are only two commercially available vaccines (Arexvy for the elderly and Abrysvo for maternal immunization), and passive prophylaxis using the monoclonal antibody palivizumab (marketed as Synagis®) is limited to high-risk infants, partly due to its limited efficacy.

[0085] As such, the present disclosure generally relates to an anti-RSV F antibody or an antigen-binding fragment thereof that specifically binds to a respiratory syncytial virus (RSV) F protein (F), and such anti-RSV F antibody and the antigen-binding fragment thereof may be used in a method for treating, / or preventing, / or improving, / or inhibiting, / or neutralizing RSV or at least one symptom associated with RSV. In one embodiment, the present disclosure generally relates to a variable heavy chain region (V) comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3. H ), and a variable light chain region (V) including light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3. LThe invention relates to an isolated or recombinant anti-RSV F antibody or its antigen-binding fragment that specifically binds to a respiratory syncytial virus (RSV) F protein (F), comprising ), and to an antibody or its antigen-binding fragment that competes for binding to the respiratory syncytial virus (RSV) F protein (F), wherein these CDRs are each (1) SEQ ID NO: 1, 2, 3, 4, 5 and 6; each (2) SEQ ID NO: 19, 20, 21, 22, 23 and 24; each (3) SEQ ID NO: 27, 28, 29, 30, 31 and 32; each (4) SEQ ID NO: 35, 36, 37, 38, 39 and 40; each (5) SEQ ID NO: 43, 44, 45, 46, 47 and 48; Each has the amino acid sequence of (6) SEQ ID NO: 51, 52, 53, 54, 55 and 56; each (7) SEQ ID NO: 59, 60, 61, 62, 63 and 64; each (8) SEQ ID NO: 67, 68, 69, 70, 71 and 72; each (9) SEQ ID NO: 75, 76, 77, 78, 79 and 80; or each (10) SEQ ID NO: 83, 84, 85, 86, 87 and 88. In one embodiment, the present disclosure relates to an isolated or recombinant anti-respiratory syncytial virus (RSV) F protein (F) antibody or an antigen-binding fragment thereof that specifically binds to the respiratory syncytial virus (RSV) F protein (F), wherein the anti-RSV F antibody or the antigen-binding fragment thereof comprises a variable heavy chain region (V) comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3. H ), and a variable light chain region (V) including light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3. LEach includes (1) SEQ ID NO: 1, 2, 3, 4, 5 and 6; each (2) SEQ ID NO: 19, 20, 21, 22, 23 and 24; each (3) SEQ ID NO: 27, 28, 29, 30, 31 and 32; each (4) SEQ ID NO: 35, 36, 37, 38, 39 and 40; each (5) SEQ ID NO: 43, 44, 45, 46, 47 and 48; each (6) SEQ ID NO: 51, 52, 53, 54, 55 and 56; each (7) SEQ ID NO: 59, 60, 61, 62, 63 and 64; Each has an amino acid sequence of (8) SEQ ID NO: 67, 68, 69, 70, 71 and 72; each (9) SEQ ID NO: 75, 76, 77, 78, 79 and 80; or each (10) SEQ ID NO: 83, 84, 85, 86, 87 and 88. Additionally, the present disclosure generally comprises a variable heavy chain region (V) comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3. H ), and a variable light chain region (V) including light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3. LThe invention relates to an isolated or recombinant anti-RSV F antibody that specifically binds to the respiratory syncytial virus (RSV) F protein (F), comprising, or an antibody or its antigen-binding fragment that binds to the same epitope of the respiratory syncytial virus (RSV) F protein (F) as the epitope of the antigen-binding fragment thereof, wherein these CDRs are each (1) SEQ ID NO: 1, 2, 3, 4, 5 and 6; each (2) SEQ ID NO: 19, 20, 21, 22, 23 and 24; each (3) SEQ ID NO: 27, 28, 29, 30, 31 and 32; each (4) SEQ ID NO: 35, 36, 37, 38, 39 and 40; each (5) SEQ ID NO: 43, 44, 45, 46, 47 and 48; Each has the amino acid sequence of (6) SEQ ID NO: 51, 52, 53, 54, 55 and 56; each (7) SEQ ID NO: 59, 60, 61, 62, 63 and 64; each (8) SEQ ID NO: 67, 68, 69, 70, 71 and 72; each (9) SEQ ID NO: 75, 76, 77, 78, 79 and 80; or each (10) SEQ ID NO: 83, 84, 85, 86, 87 and 88. In some cases, the epitope contains SEQ ID NO: 16. Additionally, the present disclosure generally relates to an antibody or its antigen-binding fragment having a complementary determining region (CDR) identical to that of an isolated or recombinant anti-RSV F antibody or its antigen-binding fragment that specifically binds to a respiratory syncytial virus (RSV) F protein (F), wherein such isolated or recombinant anti-RSV F antibody or its antigen-binding fragment comprises a variable heavy chain region (V) comprising the amino acid sequence of SEQ ID NO: 7, 25, 33, 41, 49, 57, 65, 73, 81, or 89. HA variable light chain region (V) containing the amino acid sequence of SEQ ID NO: 8, 26, 34, 42, 50, 58, 66, 74, 82, or 90 L Includes ).

[0086] RSV

[0087] Human RSV is a member of the Pneumovirus subfamily of the Paramyxoviridae family. Human RSV has two distinct subgroups: Group A and Group B. Additionally, each subtype is further subdivided into two strains: Al and A2, and B1 and B2. RSV is an enveloped, unsegmented, negative-sense RNA virus with a genome consisting of approximately 15,000 nucleotides encoding 11 viral proteins.

[0088] Respiratory syncytial virus (RSV) infection is a major cause of lower respiratory tract disease in infants and children. RSV infection is also the most common cause of bronchiolitis, or inflammation of the small airways within the lungs, and pneumonia in children under 1 year of age in the United States. Additionally, RSV infection is recognized as a significant cause of respiratory disease in the elderly. Symptoms and diseases associated with RSV infection include, for example, asthma, wheezing, reactive airway disease (RAD), and chronic obstructive pulmonary disease (COPD). Accordingly, as described herein, the anti-RSV F antibody and its antigen-binding fragment provided herein may be used for the prevention of RSV and / or one or more of its symptoms, for the treatment of RSV infection and / or one or more of its symptoms, and / or for the alleviation of one or more of these RSV-mediated diseases.

[0089] RSV F protein

[0090] RSV encodes two major surface glycoproteins, glycoprotein G and glycoprotein F. Glycoprotein G, or adhesion proteins, mediate viral binding to cell receptors, whereas glycoprotein F, or fusion proteins, facilitate the fusion of the viral membrane and the cell membrane, allowing viral ribonucleoproteins to penetrate into the cell cytoplasm (Lopez et al. J. Virology 72:6922-6928 (1998)]). Glycoprotein F also promotes the fusion of the membrane of an infected cell and the membrane of an adjacent cell, causing the formation of a fusion body.

[0091] RSV F protein exhibits more than 91% similarity across RSV A and B subgroups, whereas RSV G protein exhibits only 53% amino acid similarity between RSV A and RSV B subgroups (Reference [Sullender Clin. Microbiol. Rev. 13:1-15 (2000)]). Since A and B virus subtypes circulate together in most RSV outbreaks, antibodies that neutralize RSV A and B subtypes, such as the anti-RSV F antibody or antigen-binding fragment provided herein, are preferred.

[0092] The “Respiratory Syncytial Virus F protein,” also referred to as “RSV-F,” “RSV F,” “RSV fusion protein,” or “RSV F glycoprotein,” is a type I transmembrane surface protein that possesses a signal peptide with an N-terminus cleaved and a membrane anchor near the C-terminus (Reference [Collins, PL et al., PNAS (USA) 81:7683-7687 (1984)]). RSV-F protein is synthesized from an inactive 67 kDa precursor designated as F0 (Calder, LJ; et al., Virology277,122-131 (2000)]). The F0 protein is proteolytically activated at two sites by a purine-like protease in the Golgi complex, yielding two disulfide-linked polypeptides, F2 and F1, from the N and C ends, respectively. There is a released peptide consisting of 27 amino acids called “pep27.” Purine cleavage sites (FCS) are located on both sides of pep27 (Collins, PL; Mottet, G., J. Gen. Virol. 72: 3095-3101 (1991)]; Literature[Sugrue, R. J, et al., J. Gen. Virol., 82, 1375-1386 (2001)]). The F2 subunit consists of heptad repeat C (HRC), whereas F1 contains a fusion polypeptide (FP), heptad repeat A (HRA), domain I, domain II, heptad repeat B (HRB), transmembrane (TM), and cytoplasmic domain (CP) (Sun, Z. et al., Viruses [Refer to 5:21 1-225 (2013)]). The RSV-F protein plays a role in the fusion of viral particles to the cell membrane and is expressed on the surface of infected cells, playing a role in cell-to-cell propagation of the virus and fusion formation. The amino acid sequence of the RSV-F protein is available in GenBank under accession number AAX23994.

[0093] RSV F protein pre-fusion conformation: A structural conformation adopted by the RSV F protein prior to the triggering of fusionogenic events leading to the transition of RSV F to the post-fusion conformation, and after processing into a mature RSV F protein in the secretory system. In the pre-fusion state, the RSV F protein has an antigen site (“antigen site Ø”) at the distal apex of the membrane, e.g., Example 1 of WO2011020079 Referring toIt includes (the disclosure thereof is incorporated herein by reference in its entirety), which comprises RSV F residues 62 to 69 and 196 to 209. As used herein, the RSV F protein in its pre-fusion conformation may be specifically bound by an anti-RSV F antibody specific to the RSV F protein in its pre-fusion conformation or by an antigen-binding fragment thereof, such as an antibody that specifically binds to the antigen site Ø, for example, one or more of the anti-RSV F antibody and antigen-binding fragments described herein. In some embodiments, the anti-RSV F antibody or antigen-binding fragment as described herein binds to an epitope comprising SEQ ID NO: 16.

[0094] RSV F protein post-fusion conformation: A structural conformation adopted by the RSV F protein that is not the pre-fusion conformation, wherein the N-terminus and C-terminus of the RSV F protein are located close together within a stable coil. The post-fusion conformation of the RSV F protein has been described at the atomic level (e.g., with reference to [McLellan et al., J. Virol., 85, 7788, 2011]; [Swanson et al., Proc. Natl. Acad. Sci. USA, 108, 9619, 2011]; and structural coordinates deposited under PDB accession number 3RRR; each of which is incorporated herein by reference). In the post-fusion conformation, the RSV F protein does not contain the antigenic site Ø. The RSV post-fusion conformation occurs, for example, after the fusion of the cell membrane and the F protein.

[0095] Antibody and antigen binding fragment

[0096] As discussed above, an anti-RSV F antibody or an antigen-binding fragment thereof that may be used for therapeutic, prophylactic, and diagnostic purposes is provided herein. The anti-RSV F antibody or an antigen-binding fragment thereof provided herein may be used, for example, for passive immunization of a subject against RSV or for treatment of a subject having a viral infection. In some embodiments, the anti-RSV F antibody or an antigen-binding fragment thereof provided herein is used for prophylaxis, that is, for prevention of RSV infection. In some cases, the anti-RSV F antibody or an antigen-binding fragment thereof provided herein is used as a therapeutic antibody, that is, for treatment of RSV viral infection. In other cases, the anti-RSV F antibody or an antigen-binding fragment thereof provided herein is used for passive immunization of a subject against RSV. The provided anti-RSV F antibody or an antigen-binding fragment thereof is also used for the detection of RSV infection or In vitro and In vivo It can be used to monitor RSV infection.

[0097] As such, in one embodiment, the present disclosure generally comprises a variable heavy chain region (V) comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3. H ), and a variable light chain region (V) including light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3. LThe invention relates to an isolated or recombinant anti-RSV F antibody or its antigen-binding fragment that specifically binds to a respiratory syncytial virus (RSV) F protein (F), comprising ), and to an antibody or its antigen-binding fragment that competes for binding to the respiratory syncytial virus (RSV) F protein (F), wherein these CDRs are each (1) SEQ ID NO: 1, 2, 3, 4, 5 and 6; each (2) SEQ ID NO: 19, 20, 21, 22, 23 and 24; each (3) SEQ ID NO: 27, 28, 29, 30, 31 and 32; each (4) SEQ ID NO: 35, 36, 37, 38, 39 and 40; each (5) SEQ ID NO: 43, 44, 45, 46, 47 and 48; Each has the amino acid sequence of (6) SEQ ID NO: 51, 52, 53, 54, 55 and 56; each (7) SEQ ID NO: 59, 60, 61, 62, 63 and 64; each (8) SEQ ID NO: 67, 68, 69, 70, 71 and 72; each (9) SEQ ID NO: 75, 76, 77, 78, 79 and 80; or each (10) SEQ ID NO: 83, 84, 85, 86, 87 and 88. In one embodiment, the present disclosure relates to an isolated or recombinant anti-respiratory syncytial virus (RSV) F protein (F) antibody or an antigen-binding fragment thereof that specifically binds to the respiratory syncytial virus (RSV) F protein (F), wherein the anti-RSV F antibody or the antigen-binding fragment thereof comprises a variable heavy chain region (V) comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3. H ), and a variable light chain region (V) including light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3. LEach includes (1) SEQ ID NO: 1, 2, 3, 4, 5 and 6; each (2) SEQ ID NO: 19, 20, 21, 22, 23 and 24; each (3) SEQ ID NO: 27, 28, 29, 30, 31 and 32; each (4) SEQ ID NO: 35, 36, 37, 38, 39 and 40; each (5) SEQ ID NO: 43, 44, 45, 46, 47 and 48; each (6) SEQ ID NO: 51, 52, 53, 54, 55 and 56; each (7) SEQ ID NO: 59, 60, 61, 62, 63 and 64; Each has an amino acid sequence of (8) SEQ ID NO: 67, 68, 69, 70, 71 and 72; each (9) SEQ ID NO: 75, 76, 77, 78, 79 and 80; or each (10) SEQ ID NO: 83, 84, 85, 86, 87 and 88. Additionally, the present disclosure generally comprises a variable heavy chain region (V) comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3. H ), and a variable light chain region (V) including light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3. LThe invention relates to an isolated or recombinant anti-RSV F antibody that specifically binds to the respiratory syncytial virus (RSV) F protein (F), comprising, or an antibody or its antigen-binding fragment that binds to the same epitope of the respiratory syncytial virus (RSV) F protein (F) as the epitope of the antigen-binding fragment thereof, wherein these CDRs are each (1) SEQ ID NO: 1, 2, 3, 4, 5 and 6; each (2) SEQ ID NO: 19, 20, 21, 22, 23 and 24; each (3) SEQ ID NO: 27, 28, 29, 30, 31 and 32; each (4) SEQ ID NO: 35, 36, 37, 38, 39 and 40; each (5) SEQ ID NO: 43, 44, 45, 46, 47 and 48; Each has the amino acid sequence of (6) SEQ ID NO: 51, 52, 53, 54, 55 and 56; each (7) SEQ ID NO: 59, 60, 61, 62, 63 and 64; each (8) SEQ ID NO: 67, 68, 69, 70, 71 and 72; each (9) SEQ ID NO: 75, 76, 77, 78, 79 and 80; or each (10) SEQ ID NO: 83, 84, 85, 86, 87 and 88. In some cases, the epitope contains SEQ ID NO: 16. Additionally, the present disclosure generally relates to an antibody or its antigen-binding fragment having a complementary determining region (CDR) identical to that of an isolated or recombinant anti-RSV F antibody or its antigen-binding fragment that specifically binds to a respiratory syncytial virus (RSV) F protein (F), wherein such isolated or recombinant anti-RSV F antibody or its antigen-binding fragment comprises a variable heavy chain region (V) comprising the amino acid sequence of SEQ ID NO: 7, 25, 33, 41, 49, 57, 65, 73, 81, or 89. HA variable light chain region (V) containing the amino acid sequence of SEQ ID NO: 8, 26, 34, 42, 50, 58, 66, 74, 82, or 90 L Includes ).

[0098] In some embodiments, the anti-RSV F antibody or its antigen-binding fragment is V having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 94%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 7, 25, 33, 41, 49, 57, 65, 73, 81, or 89. H It includes. In some embodiments, the anti-RSV F antibody or its antigen-binding fragment is V having the amino acid sequence SEQ ID NO: 7, 25, 33, 41, 49, 57, 65, 73, 81, or 89. H It includes. In some embodiments, the anti-RSV F antibody or its antigen-binding fragment is V having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 94%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 8, 26, 34, 42, 50, 58, 66, 74, 82, or 90. L It includes. In some embodiments, the anti-RSV F antibody or its antigen-binding fragment is V having the amino acid sequence SEQ ID NO: 8, 26, 34, 42, 50, 58, 66, 74, 82, or 90. L Includes. In some embodiments, the anti-RSV F antibody or its antigen-binding fragment is (1) V having the amino acid sequence of SEQ ID NO: 7. H and V having the amino acid sequence of SEQ ID NO: 8 L ; (2) V having the amino acid sequence of SEQ ID NO: 25 Hand V having the amino acid sequence of SEQ ID NO: 26 L ; (3) V having the amino acid sequence of SEQ ID NO: 33 H and V having the amino acid sequence of SEQ ID NO: 34 L ; (4) V having the amino acid sequence of SEQ ID NO: 41 H and V having the amino acid sequence of SEQ ID NO: 42 L ; (5) V having the amino acid sequence of SEQ ID NO: 49 H and V having the amino acid sequence of SEQ ID NO: 50 L ; (6) V having the amino acid sequence of SEQ ID NO: 57 H and V having the amino acid sequence of SEQ ID NO: 58 L ; (7) V having the amino acid sequence of SEQ ID NO: 65 H and V having the amino acid sequence of SEQ ID NO: 66 L ; (8) V having the amino acid sequence of SEQ ID NO: 73 H and V having the amino acid sequence of SEQ ID NO: 74 L ; (9) V having the amino acid sequence of SEQ ID NO: 81 H and V having the amino acid sequence of SEQ ID NO: 82 L ; or (10) V having the amino acid sequence of SEQ ID NO: 89 H and V having the amino acid sequence of SEQ ID NO: 90 L Includes

[0099] In some embodiments, the isolated or recombinant anti-RSV F antibody or antigen-binding fragment comprises an antigen-binding fragment comprising a heavy chain polypeptide sequence having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 94%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12, 91, 92, 93, 94, 95, 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12, 91, 92, 93, 94, 95, 96, 97, 98, or 99. In some embodiments, the isolated or recombinant anti-RSV F antibody or antigen-binding fragment comprises an antigen-binding fragment comprising a heavy chain polypeptide sequence having an amino acid sequence of SEQ ID NO: 12, 91, 92, 93, 94, 95, 96, 97, 98, or 99. In some embodiments, the isolated or recombinant anti-RSV F antibody or antigen-binding fragment comprises an antigen-binding fragment comprising a light chain polypeptide sequence having at least 90%, at least 91%, at least 92%, at least 94%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical amino acid sequence to SEQ ID NO: 13, 100, 101, 102, 103, 104, 105, 106, 107, or 108. In some embodiments, the isolated or recombinant anti-RSV F antibody or antigen-binding fragment comprises an antigen-binding fragment comprising a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 13, 100, 101, 102, 103, 104, 105, 106, 107 or 109.In some embodiments, the isolated or recombinant anti-RSV F antibody or antigen-binding fragment comprises (1) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 12 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 13; (2) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 91 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 100; (3) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 92 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 101; (4) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 93 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 102; (5) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 94 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 103; (6) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 95 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 104; (7) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 96 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 105; (8) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 97 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 106; (9) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 98 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 107; or (10) an antigen-binding fragment comprising a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 99 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 108.

[0100] In some embodiments, the isolated or recombinant anti-RSV F antibody or antigen-binding fragment is (1) SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 15; (2) SEQ ID NO: 91, SEQ ID NO: 109 or SEQ ID NO: 118; (3) SEQ ID NO: 92, SEQ ID NO: 110 or SEQ ID NO: 119; (4) SEQ ID NO: 93, SEQ ID NO: 111 or SEQ ID NO: 120; (5) SEQ ID NO: 94, SEQ ID NO: 112 or SEQ ID NO: 121; (6) SEQ ID NO: 95, SEQ ID NO: 113 or SEQ ID NO: 122; (7) SEQ ID NO: 96, SEQ ID NO: 114 or SEQ ID NO: 123; (8) SEQ ID NO: 97, SEQ ID NO: 115 or SEQ ID NO: 124; (9) SEQ ID NO: 98, SEQ ID NO: 116 or SEQ ID NO: 125; or (10) a heavy chain polypeptide having an amino acid sequence selected from SEQ ID NO: 99, SEQ ID NO: 117 or SEQ ID NO: 126. In some embodiments, the anti-RSV F antibody or its antigen-binding fragment comprises a light chain polypeptide having an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107 or SEQ ID NO: 108. In some embodiments, the anti-RSV F antibody or its antigen-binding fragment is (1) SEQ ID NO: 15 and SEQ ID NO: 10, SEQ ID NO: 9 and SEQ ID NO: 10, or SEQ ID NO: 11 and SEQ ID NO: 10;(2) SEQ ID NO: 91 and SEQ ID NO: 100, SEQ ID NO: 109 and SEQ ID NO: 100, SEQ ID NO: 118 and SEQ ID NO: 100; (3) SEQ ID NO: 92 and SEQ ID NO: 101, SEQ ID NO: 110 and SEQ ID NO: 101, or SEQ ID NO: 119 and SEQ ID NO: 101; (4) SEQ ID NO: 93 and SEQ ID NO: 102, SEQ ID NO: 111 and SEQ ID NO: 102, or SEQ ID NO: 120 and SEQ ID NO: 102; (5) SEQ ID NO: 94 and SEQ ID NO: 103, SEQ ID NO: 112 and SEQ ID NO: 103, or SEQ ID NO: 121 and SEQ ID NO: 103; (6) SEQ ID NO: 95 and SEQ ID NO: 104, SEQ ID NO: 113 and SEQ ID NO: 104, or SEQ ID NO: 122 and SEQ ID NO: 104; (7) SEQ ID NO: 96 and SEQ ID NO: 105, SEQ ID NO: 114 and SEQ ID NO: 105, or SEQ ID NO: 123 and SEQ ID NO: 105; (8) SEQ ID NO: 97 and SEQ ID NO: 106, SEQ ID NO: 115 and SEQ ID NO: 106, or SEQ ID NO: 124 and SEQ ID NO: 106; (9) SEQ ID NO: 98 and SEQ ID NO: 107, SEQ ID NO: 116 and SEQ ID NO: 107, or SEQ ID NO: 125 and SEQ ID NO: 107; or (10) SEQ ID NO: 99 and SEQ ID NO: 108, SEQ ID NO: 117 and SEQ ID NO: 108, or SEQ ID NO: 126 and SEQ ID NO: 108, each comprising a heavy chain polypeptide and a light chain polypeptide selected from;

[0101] In some embodiments, the anti-RSV F antibody or its antigen-binding fragment may bind to the PreF form of RSV-F with a higher binding affinity compared to the PostF form of RSV-F. In some embodiments, the anti-RSV F antibody or its antigen-binding fragment may neutralize or block RSV-F activity by inhibiting at least one biological activity of RSV-F. In some embodiments, the anti-RSV F antibody or its antigen-binding fragment may be 1.00 x 10⁻¹⁰ -7 M or less, 1.00 x 10 -8 M or less, 1.00 x 10 -9 M or less, 7.50 x 10 -10 M or less, 5.00 x 10 -10 M or less, 2.50 x 10 -10 M or less, 2.00 x 10 -10 M or less, 1.75 x 10 -10 M or less, 1.50 x 10 -10 M or less, 1.25 x 10 -10 M or less, or 1.24 x 10 -10 K less than or equal to M D It can bind to the RSV A preF form of RSV-F. In some embodiments, the anti-RSV F antibody or its antigen-binding fragment is 1.00 x 10⁻⁶ -7 M or less, 1.00 x 10 -8 M or less, 1.00 x 10 -9 M or less, 7.50 x 10 -10 M or less, 5.00 x 10 -10 M or less, 2.50 x 10 -10 M or less, 2.00 x 10 -10 M or less, 1.75 x 10 -10 M or less, 1.70 x 10 -10 M or less, 1.65 x 10 -10 M or less, or 1.59 x 10 -10 K less than or equal to M D It can be combined with the RSV B preF form of RSV-F.

[0102] In some embodiments, the anti-RSV F antibody or its antigen-binding fragment can neutralize RSV A. In some embodiments, the anti-RSV F antibody or its antigen-binding fragment can neutralize RSV B. In some embodiments, the anti-RSV F antibody or its antigen-binding fragment can bind to the antigenic region Ø of the preF form of RSV-F. In some embodiments, the anti-RSV F antibody or its antigen-binding fragment has an IC in the range of 26 to 390 pM. 50 Neutralize RSV A with a value and IC in the range of 150 to 1000 pM 50 RSV B can be neutralized at a value of about 1500 pM or less, 1250 pM or less, 1000 pM or less, 900 pM or less, 800 pM or less, 700 pM or less, 600 pM or less, 500 pM or less, 400 pM or less, 300 pM or less, 200 pM or less, 100 pM or less, 75 pM or less, 50 pM or less, 40 pM or less, 30 pM or less, 25 pM or less, 20 pM or less, 15 pM or less, 10 pM or less, 9 pM or less, 8 pM or less, or 7 pM or less. 50 RSV A or RSV B can be neutralized at a value. In some embodiments, the anti-RSV F antibody or its antigen-binding fragment may be present at an IC of about 20 pM to about 1500 pM, about 40 pM to about 1250 pM, about 50 pM to about 1000 pM, about 75 pM to about 900 pM, about 100 pM to about 800 pM, about 200 pM to about 700 pM, about 300 pM to about 600 pM, about 400 pM to about 500 pM, or any range between these ranges. 50RSV A or RSV B can be neutralized by the value. In some embodiments, the anti-RSV F antibody or its antigen-binding fragment can reduce lung virus titers in subjects infected with RSV. In some embodiments, the anti-RSV F antibody or its antigen-binding fragment can reduce lung virus titers by about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, or about 88 times compared to a vehicle control. In some embodiments, the anti-RSV F antibody or its antigen-binding fragment is about 22.5 μg / mL or less, 22.0 μg / mL or less, 21.0 μg / mL or less, 20.0 μg / mL or less, 19.0 μg / mL or less, 18.0 μg / mL or less, 17.0 μg / mL or less, 16.0 μg / mL or less, 15.0 μg / mL or less, 14.0 μg / mL or less, 13.0 μg / mL or less, 12.0 μg / mL or less, 11.0 μg / mL or less, 10.0 μg / mL or less, 9.0 μg / mL or less, 8.0 μg / mL or less, 7.0 μg / mL or less, 6.0 μg / mL or less, 5.0 μg / mL or less, 4.0 μg / mL or less, 3.0 μg / mL or less, EC for RSV neutralization of 2.8 μg / mL or less, 2.6 μg / mL or less, 2.4 μg / mL or less, 2.2 μg / mL or less, 2.0 μg / mL or less, 1.9 or less, 1.8 μg / mL or less, 1.7 or less, 1.6 or less, 1.5 or less, 1.4 or less, 1.3 or less, or 1.2 or less. 90Values ​​may be expressed. In some embodiments, the anti-RSV F antibody or its antigen-binding fragment may activate FcγRIIa and / or FcγRIIIa. In some embodiments, the anti-RSV F antibody or its antigen-binding fragment may evade off-target interactions, as can be measured by protein array-based techniques such as the retrogenics technique of Example 10.

[0103] In some embodiments, the binding specificity or epitope of the anti-RSV F antibody or its antigen-binding fragment provided herein may be determined by any assay known to those skilled in the art in light of the present disclosure, including but not limited to surface plasmon resonance assays, competition assays, and virus neutralization assays. The epitope may be located within the isolated protein, i.e., the isolated F protein, or within a protein within the virus. The ability of two antibodies to bind to the same epitope may be determined by assays known in the art, such as surface plasmon resonance assays and antibody competition assays. Typically, antibodies that immunospecifically bind to the same epitope may compete for binding to the epitope, which may be determined, for example, using techniques known in the art. In vitroIt can be measured by a binding competition assay (e.g., a competitive ELISA). Typically, a first antibody that immunospecifically binds to the same epitope as the second antibody may compete for binding to the epitope by approximately 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, where the competition percentage is the measured ability of the second antibody to replace the binding of the first antibody to the epitope. In some competition assays, the antigen is incubated in the presence of a predetermined limit dilution of the labeled antibody (e.g., a saturation concentration of 50 to 70%) and serial dilutions of an unlabeled competing antibody. Competition is determined by measuring the binding of the labeled antibody to the antigen for any reduction in binding in the presence of the competing antibody.

[0104] In some embodiments, the anti-RSV F antibody and its antigen-binding fragment described herein have a subnanomolar affinity for the epitope of the antigenic site Ø of RSV. In some embodiments, the anti-RSV F antibody and its antigen-binding fragment described herein have broad and potent resistance against RSV A and B, including recent RSV B strains with I206M and Q209R mutations. In vitro In some embodiments, the anti-RSV F antibody and its antigen-binding fragment described herein also exhibit activity at 2.0 and 2.5 μg / mL, respectively, in cotton rats. In vivo EC 90 It has a value and shows potent prophylactic efficacy against RSV A and B. In some embodiments, the anti-RSV F antibody and its antigen-binding fragment described herein In a test tube Although they can bind to and activate FcγR, Fc-mediated effector function does not contribute to the prophylactic efficacy of these antibodies and fragments in mice infected with RSV.

[0105] The anti-RSV antibody F or its antigen-binding fragment provided herein may be analyzed by any suitable method known in the art for the detection of viral neutralization. Methods for the detection of viral neutralization include, but are not limited to, plaque assays and fusion formation inhibition assays. These assays may be used to evaluate, for example, inhibition of viral attachment, viral entry, and cell-to-cell spread of the virus (e.g., the literature [Burioni et al., Proc. Natl. Acad. Sci. USA 91 :355-359 (1994)]; literature[Sanna et al., Virology 270:386-3961 (2000)]; and literature[De Logu et al., J CHn Microbiol [See 36:3198-3204 (1998)]). A person skilled in the art can identify any test capable of measuring virus neutralization.

[0106] Standard plaque assays include, for example, plaque reduction assays, plaque size reduction assays, neutralization assays, and neutralization kinetics assays. The plaque reduction assay can be used to measure the ability of an anti-RSV antibody or its antigen-binding fragment to induce viral neutralization in solution. The plaque size reduction assay can be used to measure the ability of an anti-RSV antibody or its antigen-binding fragment to inhibit cell-to-cell viral spread.

[0107] A viral neutralization assay can be used to measure the ability of an anti-RSV antibody or its antigen-binding fragment to induce viral neutralization on the surface of a target cell by association with the target cell prior to viral exposure.

[0108] Using animal models to evaluate the efficacy of the anti-RSV F antibody or its antigen-binding fragment provided herein In vivo Research can be conducted using animal models. In vivoStudies may be conducted to evaluate any toxicity resulting from the administration of these antibodies or their antigen-binding fragments. To evaluate the ability of anti-RSV F antibodies to suppress or treat RSV virus infection and to test for any toxicity, In vivo Various assays, such as those using animal models, are available to those skilled in the art. The therapeutic effects of anti-RSV F antibodies can be evaluated using animal models of pathogenic infection, including animal models of viral infection. These animal models are known in the art and include, but are not limited to, RSV-infected animal models such as cotton rats, inbred mice, calves, ferrets, hamsters, guinea pigs, chimpanzees, owl monkeys, rhesus monkeys, African green monkeys, Sebus monkeys, squirrel monkeys, bonnet monkeys, and baboons (for exemplary RSV-infected models, for example, see the literature [Prince et al., Am. J. Pathol. 93:771-791 (1978)]; Literature[Prince et al. Infect. Immunol. 26:764-766 (1979)]; literature[Byrd and Prince, Clinical Infectious Diseases See 25:1363-1368 (1997)] (including the references cited therein). Regarding the toxicity of antibodies or antigen-binding fragments or compositions In vivo For testing, any animal model system known in the art, including but not limited to rats, mice, cows, monkeys, and rabbits, may be used.

[0109] In certain embodiments, the anti-RSV F antibody or antibody fragment for use in the method of the present invention may be monospecific, bispecific, or multispecific. A multispecific antibody may be specific to different epitopes of one target polypeptide or may contain antigen-binding domains specific to epitopes of more than one target polypeptide.

[0110] As used herein, the term “multispecific antibody” refers to an antibody comprising a plurality of immunoglobulin variable domain sequences, wherein among these plurality of sequences, a first immunoglobulin variable domain sequence has binding specificity for a first epitope, and among these plurality of sequences, a second immunoglobulin variable domain sequence has binding specificity for a second epitope. In one embodiment, the first and second epitopes are the same antigen, for example , are on the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap or substantially overlap. In one embodiment, the first and second epitopes do not overlap or substantially overlap. In one embodiment, the first and second epitopes are different antigens, for example , is on different proteins (or different subunits of multimeric proteins). In one embodiment, the multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain. In one embodiment, the multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a quadrupspecific antibody molecule.

[0111] As used herein, the term “bispecific antibody” refers to a multispecific antibody that binds to two or fewer epitopes or two antigens. A bispecific antibody is characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope, and a second immunoglobulin variable domain sequence having binding specificity for a second epitope. In one embodiment, the first and second epitopes are the same antigen, for example , are on the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap or substantially overlap. In one embodiment, the first and second epitopes are different antigens, for example, are on different proteins (or different subunits of multimeric proteins). In one embodiment, the bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a second epitope. In one embodiment, the bispecific antibody comprises a half antibody or a fragment thereof having binding specificity for a first epitope, and a half antibody or a fragment thereof having binding specificity for a second epitope. In one embodiment, the bispecific antibody comprises a scFv or a fragment thereof having binding specificity for a first epitope, and a scFv or a fragment thereof having binding specificity for a second epitope. In one embodiment, the first epitope is located on the RSV F glycoprotein of the present invention, and the second epitope is located on another RSV antigen or another respiratory antigen binding domain, e.g., a respiratory antigen binding domain derived from coronavirus or influenza. In one embodiment, the first and second epitopes are located on the same RSV F glycoprotein of the present invention.

[0112] In some embodiments, the anti-RSV F antibody or antigen-binding fragment is a human antibody, a humanized antibody, a chimeric antibody, or a bispecific or multispecific antibody. In some embodiments, the anti-RSV F antibody or antigen-binding fragment is a human antibody. In some embodiments, the anti-RSV F antibody or antigen-binding fragment is a recombinant human antibody or its antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is conjugated to polyethylene glycol (PEG) or a therapeutic agent or diagnostic agent.

[0113] Fc deformation

[0114] In some embodiments, the anti-RSV-F antibody and its antigen-binding fragment provided herein may comprise a wild-type Fc region or a modified Fc region. For example, in some embodiments, the anti-RSV-F antibody or antigen-binding fragment comprises a heavy-chain polypeptide having a heavy-chain constant region comprising substitutions M252Y, S254T, and T256E according to the EU numbering of the IgG1 heavy-chain polypeptide. For example, in some embodiments, the anti-RSV-F antibody or antigen-binding fragment comprises a heavy-chain polypeptide having a heavy-chain constant region comprising the amino acid sequence of SEQ ID NO: 14, where SEQ ID NO: 14 comprises the substitutions M252Y, S254T, and T256E to produce the heavy chain of SEQ ID NO: 15, i.e., SEQ ID NO: 171. In some embodiments, the anti-RSV F antibody or antigen-binding fragment comprises a heavy-chain polypeptide having a heavy-chain constant region comprising substitutions L234A and L235A according to the EU numbering of the IgG1 heavy-chain polypeptide. In some embodiments, the anti-RSV F antibody or antigen-binding fragment comprises the heavy-chain polypeptide of SEQ ID NO: 11, i.e., the heavy-chain polypeptide comprises substitutions L234A and L235A according to the EU numbering of the IgG1 heavy-chain polypeptide. Similar substitutions may be made in SEQ ID NO: 91 to 99 to produce SEQ ID NO: 118 to 126 and 109 to 117, respectively.

[0115] In some embodiments, the anti-RSV-F antibody may be modified to alter the heavy chain constant region by incorporating IgG homozygotes. For example, the anti-RSV-F antibody may be modified to contain IgG1_G1m(17) homozygotes (SEQ ID NO: 170). As another example, the anti-RSV-F antibody may be modified to contain any IgG homozygotes, including but not limited to IgG1_G1m(z,a) homozygotes, IgG1_G1m(f) homozygotes, IgG_G1m(f,a) homozygotes, IgG_G1m(z,a,x) homozygotes, IgG_G1m(z,a,v) homozygotes, IgG2_G2m homozygotes, IGG2_G2m(n) homozygotes, IgG_G4m(a) homozygotes, and IgG_G4m(b) homozygotes. IgG isomorphs are known in the art, for example, see the literature [Vidarsson et al., Front. Immunol. 5:1-17 (2014)] and the literature [Jefferis et al., MAbs 1(4):332-8 (2009)].

[0116] In some embodiments, the Fc region may be modified to alter one or more properties of the Fc polypeptide. For example, the Fc region may be modified to alter (i.e., increase or decrease) the effector function compared to the effector function of the Fc region of a wild-type immunoglobulin heavy chain. The Fc region of an antibody interacts with a number of Fc receptors and ligands to confer a series of important functional capabilities referred to as effector functions. Fc effector functions include, for example, Fc receptor binding, complement fixation, and T cell depletion activity (see, for example, U.S. Patent No. 6,136,310). Methods for assessing T cell depletion activity, Fc effector functions, and antibody stability are known in the art. For example, the Fc region of an IgG molecule interacts with FcγR. These receptors are expressed in various immune cells, including, for example, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granulocytes, Langerhans cells, natural killer (NK) cells, and γδ T cells. The formation of the Fc / FcγR complex mobilizes these effector cells to the site of the bound antigen, typically triggering critical subsequent immune responses such as intracellular signaling events, the release of inflammatory mediators, B cell activation, endocytosis, phagocytosis, and cytotoxic attack. The ability to mediate cytotoxic and phagocytic effector functions is a potential mechanism by which antibodies destroy targeted cells. Recognition and lysis of bound antibodies on target cells by cytotoxic cells expressing FcγR are referred to as antibody-dependent cell-mediated cytotoxicity (ADCC). Other Fc receptors for various antibody isoforms include FcεR (IgE), FcαR (IgA), and FcμR (IgM).

[0117] Accordingly, in some embodiments, the modified Fc domain may have altered affinities, which include, but are not limited to, increased affinity, low affinity, or no affinity for the Fc receptor. For example, different IgG subclasses have different affinities for FcγR, and IgG1 and IgG3 typically bind substantially better to the receptor than IgG2 and IgG4. Furthermore, different FcγR mediate different effector functions. FcγRI, FcγRIIa / c, and FcγRIIIa are positive regulators of immune complex-triggered activation, characterized by having an intracellular domain containing an immune receptor tyrosine-based activation motif (ITAM). However, FcγRIIb is inhibitory because it has an immune receptor tyrosine-based inhibition motif (ITIM). Therefore, altering the affinity of the Fc domain for the receptor can modulate the effector function induced by the Fc domain.

[0118] In some embodiments, an Fc region modified for optimized binding to a specific FcγR is used to better mediate effector functions, such as antibody-dependent cytotoxicity, ADCC. This modified Fc region may contain modifications at one or more amino acid residues, including but not limited to amino acid positions 249, 252, 259, 262, 268, 271, 273, 277, 280, 281, 285, 287, 296, 300, 317, 323, 343, 345, 346, 349, 351, 352, 353, and 424 (according to the Kabat numbering scheme, literature [Kabat et al. (1991) Sequences of Proteins of Immunological Interest, US Department of Health and Human Services]). For example, a modification of the Fc region is G119S, G119A, S122D, S122E, S122N, S122Q, S122T, K129H, K129Y, D132Y, R138Y, E141Y, T143H, V147I, S150E, H151D, E155Y, E155I, E155H, K157E, G164D, E166L, E166H, S181A, S181D, S187T, S207G, S307I, K209T, K209E, K209D, A210D of the exemplary IgGl sequence presented in WO 2011 / 020079 SEQ ID NO: 1601 (SEQ ID NO: 14 of this application), It may be formed corresponding to any one or more of A213Y, A213L, A213I, I215D, I215E, I215N, I215Q, E216Y, E216A, K217T, K217F, K217A, and P279L.Modified Fc containing such mutations may have enhanced binding to FcRs, such as the activating receptor FcγIIIa, and / or reduced binding to the inhibitory receptor FcγRIIb (see, e.g., US 2006 / 0024298). The modified Fc region having increased binding to FcRs may be more effective in promoting the destruction of virus (e.g., RSV) infected cells in patients.

[0119] In some examples, the anti-RSV F antibody or antigen-binding fragment provided herein is of the antibody or its antigen-binding fragment In vivo To increase half-life and pharmacokinetics, it may be further modified to improve the interaction between the antibody or its antigen-binding fragment and the FcRn receptor (see, e.g., U.S. Patent No. 7,217,797, U.S. Patent Publications No. 2006 / 0198840 and 2008 / 0287657). FcRn is a neonatal FcR, and its binding recirculates antibodies or their antigen-binding fragments that have been endocytosed from endosomes back into the bloodstream. Combined with the exclusion of renal filtration due to the large molecular size of full-length molecules, this process results in a favorable antibody serum half-life in the range of 1 to 3 weeks. The binding of Fc to FcRn also plays a role in antibody transport.

[0120] In some embodiments, modifications to the Fc region include, but are not limited to, mutations in one or more of the amino acid residues 251 to 256, 285 to 90, 308 to 314, in the CH2 domain of the Fc heavy chain constant region (Kabat numbering, literature [Kabat et al. (1991)]) 251 to 256, 285 to 90, 308 to 314, and / or amino acid residues 385 to 389 and 428 to 436 in the CH3 domain of the Fc heavy chain constant region, wherein the modification alters the Fc receptor binding affinity and / or serum half-life compared to an unmodified antibody or its antigen-binding fragment. In some examples, the IgG constant domain is modified in the Fc region at one or more of the amino acids Gln245, Val246, Ser247, Thr286, Phe288, and Met31 in the CH3 domain of the exemplary IgGl sequences presented in WO 2011 / 020079 SEQ ID NO: 1601 and SEQ ID NO: 14 of this application. In some examples, the modification is made at one or more surface-exposed residues, and the modification is to substitute a residue having a charge, polarity, or hydrophobicity similar to that of the substituted residue.

[0121] In some embodiments, the Fc heavy chain constant region is modified at one or more of amino acid positions 251, 252, 254, 255 and 256 (Kabat numbering), where position 251 is substituted with Leu or Arg and / or, position 252 is substituted with Tyr, Phe, Ser, Trp or Thr and / or, position 254 is substituted with Thr or Ser and / or, position 255 is substituted with Leu, Gly, He or Arg and / or, and position 256 is substituted with Ser, Arg, Gln, Glu, Asp, Ala, Asp or Thr. In some examples, the Fc heavy chain constant region is modified at one or more of amino acid positions 308, 309, 311, 312 and 314, where position 308 is substituted with Thr or He, position 309 is substituted with Pro or / or, position 311 is substituted with Serine or Glu or / or, position 312 is substituted with Asp or / or, and position 314 is substituted with Leu. In some examples, the Fc heavy chain constant region is modified at one or more of amino acid positions 428, 433, 434, and 436, where position 428 is substituted with Met, Thr, Leu, Phe, or Ser and / or, position 433 is substituted with Lys, Arg, Ser, He, Pro, Gln, or His and / or, position 434 is substituted with Phe, Tyr, or His and / or, and position 436 is substituted with His, Asn, Asp, Thr, Lys, Met, or Thr. In some examples, the Fc heavy chain constant region is modified at one or more of amino acid positions 263 and 459, where position 263 is substituted with Gln or Glu and / or, or position 459 is substituted with Leu or Phe.

[0122] In some embodiments, the Fc heavy chain constant region can be modified to enhance binding to the complement protein C1q. In addition to interacting with FcR, Fc also interacts with the complement protein C1q to mediate complement-dependent cytotoxicity (CDC). C1q forms a complex with serine proteases C1r and C1s to form the C1 complex. C1q can bind to six antibodies, but binding to two IgGs is sufficient to activate the complement cascade. Similar to the interaction of Fc with FcR, different IgG subclasses have different affinities for C1q, with IgG1 and IgG3 typically binding substantially better than IgG2 and IgG4. Therefore, modified Fc with increased binding to C1q can mediate enhanced CDC and enhance the destruction of virus-infected cells (e.g., RSV). Exemplary modifications in the Fc region that increase binding to C1q include, but are not limited to, amino acid modifications at positions 345 and 353 (Kabat numbering). Exemplary modifications include modifications corresponding to K209W, K209Y, and E216S.

[0123] In addition, various Fc mutants are known to have substitutions designed to reduce or eliminate binding to Fc)R. These mutaines are useful when the effector function mediated by Fc needs to be reduced or eliminated. This often occurs when antagonism of cells harboring a target antigen is desired, but cell death is not. An example of such an Fc is the Fc mutaine described in U.S. Patent No. 5,457,035, which is modified at amino acid positions 248, 249, and 251 (Kabat numbering). In the exemplary IgGl sequence, amino acid 117 is modified from Leu to Ala, amino acid 118 from Leu to Glu, and amino acid 120 from Gly to Ala. Similar mutations may occur at any Fc sequence, such as the exemplary Fc sequence. This mutaine exhibits reduced affinity for the Fc receptor.

[0124] In some embodiments, the anti-RSV F antibody or its antigen-binding fragment provided herein may be engineered to contain a modified Fc region. For example, methods of fusing or conjugating a polypeptide to an invariant region of an antibody (i.e., methods of preparing an Fc fusion protein) are known in the art, for example, U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, and 5,112,946; EP 307,434; EP 367,166; EP 394,827; It is described in PCT publications WO 91 / 06570, WO 96 / 04388, WO 96 / 22024, WO 97 / 34631, and WO 99 / 04813; in the literature [Ashkenazi et al. (1991) Proc. Natl. Acad. Sd. USA 88:10535-10539]; in the literature [Traunecker et al. (1988) Nature 331:84-86]; in the literature [Zheng et al. (1995) J. Immunol. 154:5590-5600]; and in the literature [ViI et al. (1992) Proc. Natl. Acad. Sci. USA 89:11337-1341 (1992)], and is described elsewhere in this application. In some examples, a modified Fc region having one or more modifications that increase FcRn binding affinity and / or improve half-life may be fused to the anti-RSV antibody provided herein or its antigen-binding fragment.

[0125] In some embodiments, the anti-RSV F antibody or its antigen-binding fragment provided herein may be engineered to contain a modified Fc region to enhance ADCP activity. In particular, the Fc substitution may include the substitutions G236A, A330L, and I332E, for example, see the literature [Yamin et al., Nature 599:465-70 (2021)] and the literature [Bournazos et al., Nature 588:485-490 (2020)]. Additional Fc substitutions for enhancing antiviral antibody responses and inducing vaccine effects considered for the anti-RSV F antibody and its antigen-binding fragment are disclosed in the literature [Nawab, Human Vaccines & Immunotherapeutics 17(12):5532-45 (2021)].

[0126] Additional variations

[0127] In some embodiments, the anti-RSV F antibody and antibody fragment provided herein may be further modified by conjugation to a detectable moiety. The detectable moiety may be detected directly or indirectly. Methods for labeling antibodies with detectable moiety are known in the art and include, for example, recombinant and chemical methods. In some embodiments, the anti-RSV antibody and antibody fragment provided herein may be further modified by conjugation to a therapeutic moiety.

[0128] Nucleic acid molecules, polynucleotides, and host cells

[0129] The present disclosure further generally relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding an anti-RSV F antibody or an antigen-binding fragment thereof as described herein.

[0130] As used herein, a “vector” is a nucleic acid molecule used to transport genetic material to another cell, and such genetic material may be replicated and / or expressed. Any vector known to those skilled in the art in light of this disclosure may be used. Examples of vectors include plasmids, viral vectors (bacteriophages, animal viruses, and plant viruses), cosmids, and artificial chromosomes ( for example It includes, but is not limited to, YAC. Preferably, the vector is a DNA plasmid. The vector may be a DNA vector or an RNA vector. Those skilled in the art may construct the vector of this application using standard recombination techniques in light of the present disclosure. The vector of this application may be an expression vector. As used herein, the term “expression vector” refers to any type of genetic construct comprising a nucleic acid encoding RNA that can be transcribed. Expression vectors include, but are not limited to, vectors for recombinant protein expression, such as DNA plasmids or viral vectors, and vectors for delivering nucleic acids into a subject for expression in the subject’s tissue, such as DNA plasmids or viral vectors. It will be recognized by those skilled in the art that the design of an expression vector may vary depending on factors such as the selection of the host cell to be transformed and the expression level of the desired protein.

[0131] The vectors of this application may contain various regulatory sequences. As used herein, the term “regulatory sequence” refers to any sequence that allows, contributes to, or regulates the functional regulation of a nucleic acid molecule, including the replication, duplication, transcription, splicing, translation, stability, and / or transport of the nucleic acid or its derivative (i.e., mRNA) into a host cell or organism. In the context of this disclosure, this term refers to promoters, enhancers, and other expression control elements ( for example It includes factors affecting polyadenylation signaling and mRNA stability.

[0132] In some embodiments of the present application, the vector is a nonviral vector. Examples of nonviral vectors include, but are not limited to, DNA plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, etc. Examples of nonviral vectors include RNA replicons, mRNA replicons, modified mRNA replicons or self-amplified mRNA, closed linear deoxyribonucleic acid, for example , linear shared closed DNA, for example, includes, but is not limited to, linearly shared closed double-stranded DNA molecules. Preferably, the nonviral vector is a DNA plasmid. “DNA plasmid,” used interchangeably with “DNA plasmid vector,” “plasmid DNA,” or “plasmid DNA vector,” refers to a double-stranded, generally circular DNA sequence capable of autonomous replication within a suitable host cell. The DNA plasmid used for the expression of the encoded polynucleotide typically includes a replication origin, a multiple cloning site, and a selectable marker, such that the marker may be, for example, an antibiotic resistance gene. Examples of suitable DNA plasmids that may be used include, but are not limited to, commercially available expression vectors for use in well-known expression systems (including both prokaryotic and eukaryotic systems), such as pSE420 (Invitrogen, San Diego, Calif.), which may be used for the production and / or expression of proteins in Escherichia coli; pYES2 (Invitrogen, Thermo Fisher Scientific), which may be used for the production and / or expression in Saccharomyces cerevisiae strains of yeast; MAXBAC® full baculovirus expression system (Thermo Fisher Scientific), which may be used for the production and / or expression in insect cells; pcDNA™ or pcDNA3™ (Life Technologies, Thermo Fisher Scientific), which may be used for high-level constitutive protein expression in mammalian cells; and pVAX or pVAX-1 (Life Technologies, Thermo Fisher Scientific), which may be used for high-level transient expression of proteins of interest in most mammalian cells.The backbone of any commercially available DNA plasmid, using routine techniques and readily available starting materials, contains specific elements (. for example , reversing the orientation of replication origins and / or antibiotic resistance cassettes, or endogenous promoters in the plasmid ( for example , replacing the promoter in the antibiotic resistance cassette, or a polynucleotide sequence encoding the transcribed protein ( for example It can be modified to optimize protein expression in host cells, such as by replacing the coding sequence of the antibiotic resistance gene. for example , Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989)].

[0133] Vector of the present application, for exampleDNA plasmids or viral vectors may include any regulatory elements to establish the vector's usual function(s). Regulatory elements include, but are not limited to, promoters, enhancers, polyadenylation signals, translation stop codons, ribosome binding elements, transcription terminators, selection markers, replication origins, etc. A vector may include one or more expression cassettes. An “expression cassette” is a part of a vector that directs cellular machinery to produce RNA and proteins. An expression cassette typically includes three components: a promoter sequence, an open reading frame, and a 3'-untranslated region (UTR) that optionally contains a polyadenylation signal. An open reading frame (ORF) is a reading frame containing the coding sequence of the protein of interest from the start codon to the stop codon. As used herein, the term “operably linked” should be accepted in its broadest reasonable context and refers to the linkage of polynucleotide elements in a functional relationship. Polynucleotides are “operably linked” when they are placed in a functional relationship with another polynucleotide. For example, a promoter is operably linked to a coding sequence if it affects the transcription of the coding sequence. Any component suitable for use in the expression cassettes described herein may be used in any combination and any order to manufacture the vector of this application.

[0134] A vector may include a promoter sequence to control expression, preferably within an expression cassette. The term “promoter” is used in its ordinary sense and refers to a nucleotide sequence that initiates the transcription of an operantly linked nucleotide sequence. The promoter is located on the same strand near the nucleotide sequence it transcribes. The promoter may be constitutive, inducible, or repressive. The promoter may be a naturally occurring or synthetic promoter. The promoter may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. The promoter may be a homologous promoter (i.e., derived from the same genetic source as the vector) or a heterologous promoter (i.e., derived from a different vector or genetic source). For example, if the vector to be used is a DNA plasmid, the promoter may be endogenous to the plasmid (homologous) or derived from a different source (heterologous).

[0135] Examples of promoters that may be used include, but are not limited to, promoters derived from primate virus 40 (SV40), mouse mammary tumor virus (MMTV) promoters, human immunodeficiency virus (HIV) promoters, such as bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoters, Moloney virus promoters, avian leukemia virus (ALV) promoters, cytomegalovirus (CMV) promoters, such as CMV outpost promoters (CMV-IE), Epstein-Barr virus (EBV) promoters, or Rous sarcoma virus (RSV) promoters. Promoters may also be promoters derived from human genes, such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. Promoters may also be tissue-specific promoters, such as muscle or skin-specific promoters, or natural or synthetic promoters.

[0136] In addition, the present disclosure relates to a host cell comprising a nucleic acid molecule, plasmid, and / or vector as generally described herein. The host cell of the present disclosure is a bacterial cell (e.g., Esquericia Colai , Bacillus subtilis It may be any prokaryotic or eukaryotic cell, including but not limited to insect cells (e.g., using a baculovirus expression system), yeast or mammalian cells (e.g., CHO or BHK cell lines). Other suitable host cells are known to those skilled in the art.

[0137] Additionally, the present disclosure generally relates to a method for producing an anti-RSV F antibody or an antigen-binding fragment thereof as described herein, the method comprising the steps of culturing a host cell as described herein under conditions for producing an anti-RSV F antibody or an antigen-binding fragment thereof, and recovering the anti-RSV F antibody or an antigen-binding fragment thereof from the cell or cell culture. Additionally, the present disclosure generally relates to a genetically modified organism comprising a nucleic acid sequence as described herein; or an expression vector as described herein.

[0138] Pharmaceutical composition

[0139] The present disclosure further generally relates to a pharmaceutical composition comprising one or more isolated anti-RSV F antibodies or antigen-binding fragments thereof as described herein; and a pharmaceutically acceptable carrier and / or excipient. Additionally, the present disclosure further generally relates to a pharmaceutical composition comprising one or more nucleic acid sequences as described herein; or one or more expression vectors as described herein; and a pharmaceutically acceptable carrier and / or excipient, wherein said nucleic acid sequence and / or expression vector encode one or more anti-RSV F antibodies as described herein.

[0140] Typically, administration of the pharmaceutical compositions and therapeutic combinations of the present disclosure will have a therapeutic purpose of treating or preventing a disease, disorder, or illness (e.g., RSV infection) in subjects requiring it. The therapeutically effective dose may be an amount sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or improving the severity of the disease, disorder, or illness, or associated symptoms to be treated; (ii) reducing the duration of the disease, disorder, or illness, or associated symptoms to be treated; (iii) preventing the progression of the disease, disorder, or illness, or associated symptoms to be treated; (iv) causing remission of the disease, disorder, or illness, or associated symptoms to be treated; (v) preventing the onset or onset of the disease, disorder, or illness, or associated symptoms to be treated; (vi) preventing the recurrence of the disease, disorder, or illness, or associated symptoms to be treated; (vii) reducing hospitalization of subjects having the disease, disorder, or illness, or associated symptoms to be treated; (viii) reducing the length of hospitalization for a subject with a disease, disorder, or illness, or associated symptoms to be treated; (ix) increasing the survival of a subject with a disease, disorder, or illness, or associated symptoms to be treated; (xi) suppressing or reducing a disease, disorder, or illness, or associated symptoms to be treated in a subject; and / or (xii) enhancing or improving the prophylactic or therapeutic effect(s) of another therapy.

[0141] The therapeutically effective dose or dosage may vary depending on various factors such as the disease, disorder, or illness to be treated, the means of administration, the target site, the physiological status of the subject (including, e.g., age, weight, and health), whether the subject is a human or an animal, other medicines administered, and whether the treatment is prophylactic or therapeutic. The therapeutic dosage is optimally titrated to optimize safety and efficacy.

[0142] Compositions and therapeutic combinations of the present disclosure, such as anti-RSV F antibodies or antigen-binding fragments thereof combined with one or more therapeutic agents, may also comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier must be non-toxic and must not interfere with the efficacy of the active ingredient. The pharmaceutically acceptable carrier may comprise one or more excipients, such as binders, disintegrants, swelling agents, suspending agents, emulsifiers, wetting agents, lubricants, flavoring agents, sweeteners, preservatives, dyes, solubilizers, and coating agents. The pharmaceutically acceptable carrier may comprise a vehicle, such as lipid (nano)particles. The precise properties of the carrier or other material are related to the route of administration, for example , intramuscular, intradermal, subcutaneous, oral, intravenous, skin, mucosal ( for example It may vary depending on the route (e.g., , and), intranasal or intraperitoneal. For liquid injectable formulations, e.g., suspensions and solutions, suitable carriers and additives include water, glycol, oil, alcohol, preservatives, coloring agents, etc. For solid oral formulations, e.g., powders, capsules, caplets, gel caps, and tablets, suitable carriers and additives include starch, sugar, diluent, granulator, lubricant, binder, disintegrant, etc. For nasal spray / inhaler mixtures, aqueous solutions / suspensions may include water, glycol, oil, emollients, stabilizers, humectants, preservatives, fragrances, flavoring agents, etc. as suitable carriers and additives.

[0143] The compositions and therapeutic combinations of the present application, namely compositions and therapeutic combinations comprising an anti-RSV F antibody or an antigen-binding fragment thereof, may be formulated in any manner suitable for administration to a subject to facilitate administration and improve efficacy, such manner includes, but is not limited to, oral (enteral) administration and parenteral injection. Parenteral injection includes intravenous injection or infusion, subcutaneous injection, intradermal injection, and intramuscular injection. The compositions of the present application may also be formulated for other routes of administration, including transmucosal, ocular, rectal, long-acting implants, sublingual (under the tongue) administration, administration from the oral mucosa bypassing the portal circulation, inhalation, or nasal.

[0144] According to an aspect of the present disclosure, compositions and therapeutic combinations for administration typically include a pharmaceutically acceptable carrier, for example Buffer saline solution, etc. for example It will include a buffer solution in an aqueous carrier, such as phosphate-buffered saline (PBS). The composition and therapeutic combination may also contain pharmaceutically acceptable substances necessary to approximate physiological conditions, such as pH adjusters and buffers. For example, the composition or therapeutic combination of the present application comprising plasmid DNA may contain phosphate-buffered saline (PBS) as a pharmaceutically acceptable carrier.

[0145] The pharmaceutical compositions and therapeutic combinations of the present disclosure may be administered to a subject by any method known in the art in light of the present disclosure, and such method is parenteral administration ( for example , intramuscular, subcutaneous, intravenous, or intradermal injection), oral administration, transdermal administration, and nasal administration, including but not limited to these. Preferably, the pharmaceutical composition and therapeutic combination are administered parenterally ( for example It is administered (by intramuscular injection or intradermal injection) or transdermally.

[0146] How to use

[0147] The present disclosure generally relates to a method for inhibiting at least one biological activity of RSV-F, the method comprising: (a) one or more antibodies or antigen-binding fragments thereof as described herein; (b) nucleic acid molecules as described herein; (c) expression vectors as described herein; (d) host cells as described herein; or (e) pharmaceutical compositions as described herein.

[0148] The method includes the step of administering to a subject who requires it or is suspected of requiring it. Additionally, the present disclosure further generally relates to a method for treating, preventing, or resolving a respiratory syncytial virus (RSV) infection, or treating, preventing, or resolving at least one symptom associated with an RSV infection, wherein the method comprises: (a) one or more anti-RSV F antibodies or antigen-binding fragments thereof as described herein; (b) a nucleic acid molecule as described herein; (c) an expression vector as described herein; (d) a host cell as described herein; or (e) a pharmaceutical composition as described herein.

[0149] Including the step of administering to a patient who requires or is suspected of requiring it; the RSV infection is treated, prevented, or improved and / or the severity of at least one symptom associated with the RSV infection is treated, improved, or reduced, or such symptoms are prevented. Examples of RSV symptoms may include, but are not limited to, mild cold-like signs and symptoms, nasal congestion or runny nose, dry cough, mild fever, sore throat, sneezing, and headache. In severe cases, symptoms may include, but are not limited to, spread to the lower respiratory tract causing pneumonia or bronchiolitis (inflammation of the small airways leading to the lungs), fever, severe cough, wheezing (a high-pitched noise heard mainly when exhaling), rapid breathing or difficulty breathing, and bluish discoloration of the skin due to oxygen deficiency. In infants, RSV symptoms may include, but are not limited to, short, shallow, rapid breathing; difficulty breathing; cough; poor feeding; unusual fatigue (sedation); and irritability. In some embodiments, any one of (a) to (e) is administered in a therapeutically effective amount.

[0150] In some embodiments, the method inhibits at least one biological activity of RSV-F. In some embodiments, the method reduces lung virus titers in subjects infected with RSV. In some embodiments, the method reduces lung virus titers by about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, or about 88 times compared to a vehicle control. In some embodiments, the method activates FcγRIIa and / or FcγRIIIa. In some embodiments, the subject requires treatment, prevention, or remission of respiratory syncytial virus (RSV) infection, or treatment, prevention, or remission of at least one symptom associated with RSV infection. In some embodiments, the method neutralizes or blocks RSV-F activity by inhibiting at least one biological activity of RSV-F. In some embodiments, the method neutralizes RSV A. In some embodiments, the method neutralizes RSV B.

[0151] Diagnostic use

[0152] The anti-RSV F antibody or its antigen-binding fragment provided herein may be used in diagnostic assays for the detection, purification, and / or neutralization of RSV. An exemplary diagnostic assay of RSV In vitro and In vivo Includes detection. For example, from isolated biological samples (e.g., sputum) or In vivoA assay is provided for using the anti-RSV F antibody or its antigen-binding fragment provided herein to qualitatively and quantitatively measure levels of RSV. In some embodiments, the anti-RSV F antibody or its antigen-binding fragment may be used as a companion diagnostic, that is, they may be used to qualitatively and quantitatively measure levels of RSV in biological samples isolated from subjects being treated for RSV to determine the efficacy of a drug. In some embodiments, the RSV F protein is detected using the anti-RSV F antibody or antigen-binding fragment as described herein. In some embodiments, RSV is detected during diagnostic use by detecting the RSV preF conformation of the RSV F protein using the anti-RSV F antibody or antigen-binding fragment as described herein.

[0153] Combination therapy

[0154] The anti-RSV F antibody or its antigen-binding fragment provided herein may be administered alone or in combination with one or more therapeutic agents or therapies for the prevention and / or treatment of a disease or illness. For example, the provided anti-RSV F antibody or its antigen-binding fragment may be administered in combination with one or more antiviral agents for the prevention and / or treatment of RSV infection. Antiviral agents may include agents for reducing and / or eliminating pathogenic infection, or agents for alleviating one or more symptoms of pathogenic infection. In some examples, a plurality of antibodies or their antigen-binding fragments (e.g., one or more antiviral antibodies) may also be administered in combination, wherein at least one of the antibodies is the anti-RSV F antibody or its antigen-binding fragment provided herein. In some examples, a plurality of antibodies may be administered in combination for the prevention and / or treatment of RSV infection or multiple viral infections, wherein at least one of the antibodies is the anti-RSV F antibody or its antigen-binding fragment provided herein. In some examples, the provided anti-RSV F antibody may be administered in combination with one or more antiviral antibodies that bind to and neutralize the same virus, namely RSV, or different respiratory viruses such as influenza or SARS-CoV-2. In some examples, the provided anti-RSV F antibody or its antigen-binding fragment may be administered in combination with one or more antibodies that can suppress or alleviate one or more symptoms of RSV infection. In some examples, two or more of the anti-RSV F antibody or its antigen-binding fragments provided herein are administered in combination.

[0155] One or more additional agents may be administered simultaneously, sequentially, or intermittently with the anti-RSV F antibody or its antigen-binding fragment.

[0156] Any therapy known to be useful, or used or used for the prevention, management, treatment, or remission of RSV infection or one or more of its symptoms, may be used in combination with the anti-RSV F antibody or its antigen-binding fragment provided herein (for information regarding therapies that have been used or are used to prevent, treat, manage, or remission of RSV infection or one or more of its symptoms {e.g., prophylactic agents or therapeutic agents}, see, for example, the literature [Gilman et al., Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, 2001]; the literature [The Merck Manual of Diagnosis and Therapy, Berkow, MD et al. (eds.), 17th Ed., Merck Sharp & Dohme Research Laboratories, Rahway, NJ., 1999]; Refer to the literature [Cecil Textbook of Medicine, 20th Ed., Bennett and Plum (eds.), WB Saunders, Philadelphia, 1996]).

[0157] Detection kit

[0158] The present disclosure also generally relates to a detection kit comprising an anti-RSV F antibody or antigen-binding fragment as described herein, a nucleic acid molecule as described herein, a plasmid as described herein, a host cell as described herein, or a pharmaceutical composition as described herein, wherein the detection kit is optionally used to detect RSV. In some cases, RSV-F can be detected using the detection kit, such as by detecting the RSV preF form of RSV F.

[0159] embodiments

[0160] Embodiment 1. A variable heavy chain region (V) comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3. H ), and a variable light chain region (V) including light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3. L An isolated or recombinant anti-RSV F antibody or its antigen-binding fragment that specifically binds to the respiratory syncytial virus (RSV) F protein (F), comprising ), and an antibody or its antigen-binding fragment that competes for binding to the respiratory syncytial virus (RSV) F protein (F), wherein these CDRs are each (1) SEQ ID NO: 1, 2, 3, 4, 5 and 6; each (2) SEQ ID NO: 19, 20, 21, 22, 23 and 24; each (3) SEQ ID NO: 27, 28, 29, 30, 31 and 32; each (4) SEQ ID NO: 35, 36, 37, 38, 39 and 40; each (5) SEQ ID NO: 43, 44, 45, 46, 47 and 48; An antibody or its antigen-binding fragment having amino acid sequences of (6) SEQ ID NO: 51, 52, 53, 54, 55 and 56; (7) SEQ ID NO: 59, 60, 61, 62, 63 and 64; (8) SEQ ID NO: 67, 68, 69, 70, 71 and 72; (9) SEQ ID NO: 75, 76, 77, 78, 79 and 80; or (10) SEQ ID NO: 83, 84, 85, 86, 87 and 88.

[0161] Embodiment 2. An isolated or recombinant anti-respiratory syncytial virus (RSV) F protein (F) antibody or an antigen-binding fragment thereof that specifically binds to respiratory syncytial virus (RSV) F protein (F), wherein the anti-RSV F antibody or the antigen-binding fragment thereof comprises a variable heavy chain region (V) comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3.H ), and a variable light chain region (V) including light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3. L Each includes (1) SEQ ID NO: 1, 2, 3, 4, 5 and 6; each (2) SEQ ID NO: 19, 20, 21, 22, 23 and 24; each (3) SEQ ID NO: 27, 28, 29, 30, 31 and 32; each (4) SEQ ID NO: 35, 36, 37, 38, 39 and 40; each (5) SEQ ID NO: 43, 44, 45, 46, 47 and 48; each (6) SEQ ID NO: 51, 52, 53, 54, 55 and 56; each (7) SEQ ID NO: 59, 60, 61, 62, 63 and 64; An anti-RSV F antibody or its antigen-binding fragment having amino acid sequences of (8) SEQ ID NO: 67, 68, 69, 70, 71 and 72, respectively; (9) SEQ ID NO: 75, 76, 77, 78, 79 and 80, respectively; or (10) SEQ ID NO: 83, 84, 85, 86, 87 and 88.

[0162] Example 3. In Example 1 or Example 2, the antibody or its antigen-binding fragment is V having an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, 25, 33, 41, 49, 57, 65, 73, 81 or 89. H An anti-RSV F antibody or its antigen-binding fragment comprising

[0163] Example 4. In Example 3, the antibody or its antigen-binding fragment is V having the amino acid sequence of SEQ ID NO: 7, 25, 33, 41, 49, 57, 65, 73, 81, or 89. H An anti-RSV F antibody or its antigen-binding fragment comprising

[0164] Example 5. In any one of Examples 1 to 4, the antibody or its antigen-binding fragment is V having an amino acid sequence that is at least 90% identical to SEQ ID NO: 8, 26, 34, 42, 50, 58, 66, 74, 82, or 90. L An anti-RSV F antibody or its antigen-binding fragment comprising

[0165] Example 6. In Example 5, the antibody or its antigen-binding fragment is V having the amino acid sequence of SEQ ID NO: 8, 26, 34, 42, 50, 58, 66, 74, 82, or 90. L An anti-RSV F antibody or its antigen-binding fragment comprising

[0166] Example 7. In any one of Examples 1 to 6, the antibody or its antigen-binding fragment is

[0167] (1) V having the amino acid sequence of SEQ ID NO: 7 H and V having the amino acid sequence of SEQ ID NO: 8 L ;

[0168] (2) V having the amino acid sequence of SEQ ID NO: 25 H and V having the amino acid sequence of SEQ ID NO: 26 L ;

[0169] (3) V having the amino acid sequence of SEQ ID NO: 33 H and V having the amino acid sequence of SEQ ID NO: 34 L ;

[0170] (4) V having the amino acid sequence of SEQ ID NO: 41 H and V having the amino acid sequence of SEQ ID NO: 42 L ;

[0171] (5) V having the amino acid sequence of SEQ ID NO: 49 H and V having the amino acid sequence of SEQ ID NO: 50L ;

[0172] (6) V having the amino acid sequence of SEQ ID NO: 57 H and V having the amino acid sequence of SEQ ID NO: 58 L ;

[0173] (7) V having the amino acid sequence of SEQ ID NO: 65 H and V having the amino acid sequence of SEQ ID NO: 66 L ;

[0174] (8) V having the amino acid sequence of SEQ ID NO: 73 H and V having the amino acid sequence of SEQ ID NO: 74 L ;

[0175] (9) V having the amino acid sequence of SEQ ID NO: 81 H and V having the amino acid sequence of SEQ ID NO: 82 L ; or

[0176] (10) V having the amino acid sequence of SEQ ID NO: 89 H and V having the amino acid sequence of SEQ ID NO: 90 L An anti-RSV F antibody or its antigen-binding fragment comprising

[0177] Example 8. An anti-RSV F antibody or its antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises an antigen-binding fragment comprising a heavy chain polypeptide sequence having at least 90% identical amino acid sequence to SEQ ID NO: 12, 91, 92, 93, 94, 95, 96, 97, 98 or 99.

[0178] Example 9. The anti-RSV F antibody or its antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises an antigen-binding fragment comprising a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 12, 91, 92, 93, 94, 95, 96, 97, 98 or 99.

[0179] Example 10. An anti-RSV F antibody or its antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises an antigen-binding fragment comprising a light chain polypeptide sequence having at least 90% identical amino acid sequence to SEQ ID NO: 13, 100, 101, 102, 103, 104, 105, 106, 107 or 108.

[0180] Example 11. The anti-RSV F antibody or its antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises an antigen-binding fragment comprising a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 13, 100, 101, 102, 103, 104, 105, 106, 107 or 108.

[0181] Embodiment 12. In Embodiment 1 or Embodiment 2, the antibody or antigen-binding fragment is

[0182] (1) A heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 12 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 13;

[0183] (2) A heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 91 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 100;

[0184] (3) A heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 92 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 101;

[0185] (4) A heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 93 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 102;

[0186] (5) A heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 94 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 103;

[0187] (6) A heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 95 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 104;

[0188] (7) A heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 96 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 105;

[0189] (8) A heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 97 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 106;

[0190] (9) A heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 98 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 107; or

[0191] (10) Anti-RSV F antibody or its antigen-binding fragment comprising a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 99 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 108.

[0192] Example 13. In any one of Examples 1 to 12, the antibody or antigen-binding fragment is (1) SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 15; (2) SEQ ID NO: 91, SEQ ID NO: 109 or SEQ ID NO: 118; (3) SEQ ID NO: 92, SEQ ID NO: 110 or SEQ ID NO: 119; (4) SEQ ID NO: 93, SEQ ID NO: 111 or SEQ ID NO: 120; (5) SEQ ID NO: 94, SEQ ID NO: 112 or SEQ ID NO: 121; (6) SEQ ID NO: 95, SEQ ID NO: 113 or SEQ ID NO: 122; (7) SEQ ID NO: 96, SEQ ID NO: 114 or SEQ ID NO: 123; (8) SEQ ID NO: 97, SEQ ID NO: 115 or SEQ ID NO: 124; (9) SEQ ID NO: 98, SEQ ID NO: 116 or SEQ ID NO: 125; or (10) SEQ ID NO: 99, SEQ ID NO: 117 or SEQ ID NO: 126, comprising an anti-RSV F antibody or an antigen-binding fragment thereof having a heavy chain polypeptide having an amino acid sequence selected from SEQ ID NO: 99, SEQ ID NO: 117 or SEQ ID NO: 126.

[0193] Example 14. An anti-RSV F antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment comprises a light chain polypeptide having the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, or SEQ ID NO: 108.

[0194] Example 15. In any one of Examples 1 to 14, the antibody or its antigen-binding fragment is (1) SEQ ID NO: 15 and SEQ ID NO: 10, SEQ ID NO: 9 and SEQ ID NO: 10, or SEQ ID NO: 11 and SEQ ID NO: 10; (2) SEQ ID NO: 91 and SEQ ID NO: 100, SEQ ID NO: 109 and SEQ ID NO: 100, SEQ ID NO: 118 and SEQ ID NO: 100; (3) SEQ ID NO: 92 and SEQ ID NO: 101, SEQ ID NO: 110 and SEQ ID NO: 101, or SEQ ID NO: 119 and SEQ ID NO: 101; (4) SEQ ID NO: 93 and SEQ ID NO: 102, SEQ ID NO: 111 and SEQ ID NO: 102, or SEQ ID NO: 120 and SEQ ID NO: 102; (5) SEQ ID NO: 94 and SEQ ID NO: 103, SEQ ID NO: 112 and SEQ ID NO: 103, or SEQ ID NO: 121 and SEQ ID NO: 103; (6) SEQ ID NO: 95 and SEQ ID NO: 104, SEQ ID NO: 113 and SEQ ID NO: 104, or SEQ ID NO: 122 and SEQ ID NO: 104; (7) SEQ ID NO: 96 and SEQ ID NO: 105, SEQ ID NO: 114 and SEQ ID NO: 105, or SEQ ID NO: 123 and SEQ ID NO: 105; (8) SEQ ID NO: 97 and SEQ ID NO: 106, SEQ ID NO: 115 and SEQ ID NO: 106, or SEQ ID NO: 124 and SEQ ID NO: 106; (9) SEQ ID NO: 98 and SEQ ID NO: 107, SEQ ID NO: 116 and SEQ ID NO: 107, or SEQ ID NO: 125 and SEQ ID NO: 107;or (10) an anti-RSV F antibody or its antigen-binding fragment comprising a heavy chain polypeptide and a light chain polypeptide, each selected from SEQ ID NO: 99 and SEQ ID NO: 108, SEQ ID NO: 117 and SEQ ID NO: 108, or SEQ ID NO: 126 and SEQ ID NO: 108.;

[0195] Example 16. In any one of Examples 1 to 15, the antibody or the antigen-binding fragment thereof is an anti-RSV F antibody or the antigen-binding fragment thereof capable of neutralizing or blocking RSV-F activity by inhibiting at least one biological activity of RSV-F.

[0196] Example 17. An anti-RSV F antibody or its antigen-binding fragment, wherein in any one of Examples 1 to 16, the antibody or its antigen-binding fragment is capable of binding to the PreF conformation of RSV-F with a higher binding affinity than the PostF conformation of RSV-F.

[0197] Example 18. In any one of Examples 1 to 17, the antibody or its antigen-binding fragment is 1.00 x 10 -7 M or less, 1.00 x 10 -8 M or less, 1.00 x 10 -9 M or less, 7.50 x 10 -10 M or less, 5.00 x 10 -10 M or less, 2.50 x 10 -10 M or less, 2.00 x 10 -10 M or less, 1.75 x 10 -10 M or less, 1.50 x 10 -10 M or less, 1.25 x 10 -10 M or less, or 1.24 x 10 -10 K less than or equal to M D An anti-RSV F antibody or its antigen-binding fragment capable of binding to the RSV A preF conformation of RSV-F.

[0198] Example 19. In any one of Examples 1 to 18, the antibody or its antigen-binding fragment is 1.00 x 10 -7 M or less, 1.00 x 10 -8 M or less, 1.00 x 10 -9 M or less, 7.50 x 10 -10 M or less, 5.00 x 10 -10 M or less, 2.50 x 10 -10 M or less, 2.00 x 10 -10 M or less, 1.75 x 10 -10 M or less, 1.70 x 10 -10 M or less, 1.65 x 10 -10 M or less, or 1.59 x 10 -10 K less than or equal to M D An anti-RSV F antibody or its antigen-binding fragment capable of binding to the RSV B preF conformation of RSV-F.

[0199] Example 20. In any one of Examples 1 to 19, the antibody or its antigen-binding fragment is an anti-RSV F antibody or its antigen-binding fragment capable of neutralizing RSV A.

[0200] Example 21. In any one of Examples 1 to 20, the antibody or its antigen-binding fragment is an anti-RSV F antibody or its antigen-binding fragment capable of neutralizing RSV B.

[0201] Example 22. In any one of Examples 1 to 21, the antibody or the antigen-binding fragment thereof is an anti-RSV F antibody or the antigen-binding fragment thereof capable of binding to the antigen site Ø of the preF form of RSV-F.

[0202] Example 23. In any one of Examples 1 to 22, the antibody or its antigen-binding fragment has an IC in the range of about 26 to about 1000 pM. 50 An anti-RSV F antibody or its antigen-binding fragment capable of neutralizing RSV by value.

[0203] Example 24. In any one of Examples 1 to 23, the antibody or its antigen-binding fragment has an IC of about 400 pM or less, 300 pM or less, 200 pM or less, 100 pM or less, 75 pM or less, 50 pM or less, 40 pM or less, 30 pM or less, 25 pM or less, or 20 pM or less. 50 An anti-RSV F antibody or its antigen-binding fragment capable of neutralizing RSV A by value.

[0204] Example 24a. In any one of Examples 1 to 23, the antibody or the antigen-binding fragment thereof is an anti-RSV F antibody or the antigen-binding fragment thereof capable of neutralizing RSV B with an IC50 value of 1250 pM or less, 1000 pM or less, 900 pM or less, 800 pM or less, 700 pM or less, 600 pM or less, 500 pM or less, 400 pM or less, 300 pM or less, 200 pM or less, or 150 pM or less.

[0205] Example 25. In any one of Examples 1 to 24, the antibody or the antigen-binding fragment thereof is an anti-RSV F antibody or the antigen-binding fragment thereof capable of reducing lung virus titers in a subject infected with RSV.

[0206] Example 26. In any one of Examples 1 to 25, the antibody or the antigen-binding fragment thereof is an anti-RSV F antibody or the antigen-binding fragment thereof capable of reducing lung virus titer by about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, or about 88 times compared to a vehicle control.

[0207] Example 27. In any one of Examples 1 to 26, the antibody or its antigen-binding fragment is about 22.5 μg / mL or less, 22.0 μg / mL or less, 21.0 μg / mL or less, 20.0 μg / mL or less, 19.0 μg / mL or less, 18.0 μg / mL or less, 17.0 μg / mL or less, 16.0 μg / mL or less, 15.0 μg / mL or less, 14.0 μg / mL or less, 13.0 μg / mL or less, 12.0 μg / mL or less, 11.0 μg / mL or less, 10.0 μg / mL or less, 9.0 μg / mL or less, 8.0 μg / mL or less, 7.0 μg / mL or less, 6.0 μg / mL or less, 5.0 μg / mL or less, 4.0 μg / mL or less, 3.0 An anti-RSV F antibody or its antigen-binding fragment capable of exhibiting an EC90 value for the neutralization of RSV of μg / mL or less, 2.8 μg / mL or less, 2.6 μg / mL or less, 2.4 μg / mL or less, 2.2 μg / mL or less, 2.0 μg / mL or less, 1.9 or less, 1.8 μg / mL or less, 1.7 or less, 1.6 or less, 1.5 or less, 1.4 or less, 1.3 or less, or 1.2 or less.

[0208] Example 28. In any one of Examples 1 to 27, the antibody or its antigen-binding fragment is an anti-RSV F antibody or its antigen-binding fragment capable of activating FcγRIIa and / or FcγRIIIa.

[0209] Example 29. In any one of Examples 1 to 28, the antibody or its antigen-binding fragment is an anti-RSV F antibody or its antigen-binding fragment capable of avoiding off-target interactions.

[0210] Example 30. Any one of Examples 1 to 29, wherein the antibody or antigen-binding fragment is an anti-RSV F antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment is a human antibody, a humanized antibody, a chimeric antibody, or a bispecific or multispecific antibody.

[0211] Example 31. Any one of Examples 1 to 30, wherein the antibody or antigen-binding fragment is an anti-RSV F antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment is a human antibody.

[0212] Example 32. In any one of Examples 1 to 31, the antibody or antigen-binding fragment is an anti-RSV F antibody or its antigen-binding fragment conjugated to polyethylene glycol (PEG) or a therapeutic agent or diagnostic drug.

[0213] Example 33. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding an anti-RSV F antibody or an antigen-binding fragment thereof according to any one of Examples 1 to 32.

[0214] Example 34. An expression vector comprising an isolated nucleic acid sequence according to Example 33.

[0215] Example 35. A host cell that is transfected, transformed, or transduced with a nucleic acid sequence according to Example 33 or an expression vector according to Example 34.

[0216] Example 36. A method for producing an anti-RSV F antibody or an antigen-binding fragment thereof according to any one of Examples 1 to 32, comprising the steps of: culturing the host cells of Example 35 under conditions for producing the anti-RSV F antibody or the antigen-binding fragment thereof; and recovering the anti-RSV F antibody or the antigen-binding fragment thereof from the cells or cell culture.

[0217] Example 37. A pharmaceutical composition comprising one or more of an anti-RSV F antibody or an antigen-binding fragment thereof according to any one of Examples 1 to 32; and a pharmaceutically acceptable carrier and / or excipient.

[0218] Example 38. A pharmaceutical composition comprising one or more nucleic acid sequences according to Example 33; or one or more expression vectors according to Example 34; and a pharmaceutically acceptable carrier and / or excipient.

[0219] Embodiment 39. A genetically modified organism comprising a nucleic acid sequence according to Embodiment 33; or an expression vector according to Embodiment 34.

[0220] Embodiment 40. A method for inhibiting at least one biological activity of RSV-F, comprising: (a) one or more antibodies or antigen-binding fragments thereof according to any one of Embodiments 1 to 32; (b) a nucleic acid molecule according to Embodiment 33; (c) an expression vector according to Embodiment 34; (d) a host cell according to Embodiment 35; or (e) a pharmaceutical composition according to Embodiment 37 or Embodiment 38.

[0221] A method comprising the step of administering to a subject who requires it or is suspected of requiring it.

[0222] Embodiment 41. In any one of Embodiments 40, the subject needs to treat, prevent, or improve respiratory syncytial virus (RSV) infection, or needs to treat, prevent, or improve at least one symptom associated with RSV infection.

[0223] Embodiment 42. The method of Embodiment 40 or Embodiment 41, wherein the method reduces the lung virus titer in a subject.

[0224] Example 43. The method of Example 42, wherein the method reduces the lung virus titer by about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, or about 88 times compared to the vehicle control group.

[0225] Embodiment 44. Any one of Embodiments 40 to 43, wherein the method activates FcγRIIa and / or FcγRIIIa.

[0226] Embodiment 45. A method in any one of embodiments 40 to 44, wherein any one of a) to e) of embodiment 40 is administered in a therapeutically effective amount.

[0227] Sequence list

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239] Examples

[0240] Example 1: Preparation of Anti-RSV F Antibody

[0241] An anti-RSV F antibody was prepared as generally described in US 20200223906, the entirety of which is incorporated herein by reference. Briefly, peripheral blood mononuclear cells were obtained from adult donors approximately 20 to 35 years of age, and a monoclonal antibody derived from F-reactive memory B cells prior to RSV fusion was isolated therefrom. Detailed methods are described below.

[0242] PMBC Donor Sample: Heparin-treated blood samples (50 to 100 cc) were collected from four donors identified as IML #3512, 3483, 2992, and 3529. The samples were processed to obtain plasma, and peripheral blood-derived B cells were isolated. The isolated cells and plasma were stored frozen at -80°C as aliquots.

[0243] Antigens and antibodies : As described in the literature [Rappazzo et al., Immunity 55(9): 1710-24 (2022)], the production of recombinant hMPV preF (A + B) and RSV preF (A) was produced.

[0244] Single B cell classification of memory B cells:B cells were purified using the MACS B cell isolation kit (Miltenyi Biotec; cat #130-091-151; Miltenyi Biotec; Bergischgladbach, Germany) and stained with anti-human CD19 (PE-Cy7), CD3 (PerCP-Cy5.5), CD8 (PerCP-Cy5.5), CD14 (PerCP-Cy5.5), CD16 (PerCP-Cy5.5), IgM (BV711), IgD (APC-Cy7), IgA (AF488), IgG (BV605), and a mixture (25 nM) of labeled (APC or PE) hMPV preF (A + B) and RSV preF (A) protein tetramers. B cells exhibiting reactivity to hMPV and / or RSV preF protein tetramers were classified as single cells. Single cells were sorted into 96-well PCR plates containing 20 μL of lysis buffer [5 μL of 5× 1st strand cDNA buffer (Invitrogen), 0.625 μL of NP-40 (New England Biolabs; Ipswich, MA), 0.25 μL of RNaseOUT (Invitrogen; Waltham, MA), 1.25 μL of dithiothreitol (Invitrogen), and 12.6 μL of dH2O] per well using a BD FACS Aria II (BD Biosciences; Franklin Lakes, NJ). The plates were immediately stored at -80°C until cloning.

[0245] Amplification and cloning of antibody variable genes As previously described, antibody variable genes (IgH, IgK, and IgL) were amplified by reverse transcription PCR and nested PCR using a cocktail of IgG- and IgM-specific primers (reference [Tiller et al, J Immunol 2008]). The primers used in the second PCR round were S. cerevisiae To allow cloning by homologous recombination within the PCR product, it contained 40 base pairs of 5' and 3' homology to the digested expression vector. S. cerevisiaeThe lithium acetate chemical transformation method was used for intracloning (reference [Gietz and Schiestl, Nat Protoc 2007]). Unpurified heavy and light chain PCR products were combined with restriction-digested expression vectors in each transformation reaction. After transformation, individual yeast colonies were selected for sequencing and characterization.

[0246] Expression and purification of IgG and Fab fragments As previously described, grown in a 24-well plate S. cerevisiae IgG was expressed in the cultures (Reference [Bornholdt et al, Science 2016, PMID:26912366]). After 6 days, the cultures were harvested by centrifugation, and the IgG was purified by protein A-affinity chromatography. IgG was digested with papain to produce the Fab fragment, and the Fab and Fc mixture was passed through protein A agarose to remove the Fc fragment and undigested IgG. Then, the permeate of the protein A resin was passed through CaptureSelect™ IgG-CH1 affinity resin (ThermoFischer Scientific) to purify Fab from unwanted species. All IgG and Fab samples were prepared in PBS buffer.

[0247] Multiple Response Test: Multispecific reagent (PSR) binding was evaluated as previously described (reference [Xu et al. 2013 Protein Eng Des 26:663-70]). Briefly, soluble membrane protein (SMP) and soluble cytoplasmic protein (SCP) fractions were prepared from CHO cells and biotinylated with NHS-LC-biotin reagent (Pierce, ThermoFisher Cat#21336). Two million IgG-presenting yeast cells were transferred to a 96-well assay plate, pelleted to remove the supernatant, and then the pellet was resuspended in 50 μL of a 1:10 diluted stock of biotinylated SCP and SMP and incubated on ice for 20 minutes. Cells were washed twice with ice-cold PBSF, and samples were incubated on ice for 20 minutes in 50 μL of a secondary labeling mixture (Extravadin-R-PE, goat F(ab') 2-anti-human kappa-FITC, and propidium iodide). Multispecific reagent binding of the samples was analyzed using a FACSCanto II (BD Biosciences) equipped with an HTS sample injector. Flow cytometry data were analyzed for the mean fluorescence intensity (MFI) of the R-PE channel and normalized by three control antibodies representing low, medium, and high MFI values.

[0248] Example 2: Characterization of anti-RSV F antibody

[0249] Anti-RSV F antibodies were first analyzed in a 3-day viral neutralization assay using HeLa cells and the RSV laboratory strain rgRSV224 (RSV A A2-eGFP). For this purpose, 10 μL of antibody was added to a black 384-well clear-bottom microtitration plate at a 4-fold serial dilution. Subsequently, 1.5 x 10 5After adding 20 μL of HeLa cells (ATCC, CCL-2tm) at a cell / mL density (i.e., 3000 cells / well), 10 μL of rgRSV224 virus was added with an infection multiplicity (MOI) of 1. Cell controls (no virus added) and virus controls (cells infected with the virus but without antibodies) were placed together on each plate. After incubation for 3 days at 37°C and 5% CO2, viral replication was quantified by measuring eGFP expression using an Acumen Cellista instrument. IC50 was calculated from the dose-response curve. 50 The values ​​are listed in Table 2.

[0250] Next, anti-RSV F antibodies were tested for the neutralization of RSV B clinical isolate 05-036549 mCherry. In this assay, 10 μL of each antibody was added to a black 384-well clear-bottom microtitration plate at a 4-fold serial dilution. Subsequently, 1.5 x 10 5 After adding 20 μL of HeLa cells (ATCC, CCL-2tm) at a cell / mL density (i.e., 3000 cells / well), 10 μL of RSV B 05-036549 mCherry virus was added at an infection multiplicity (MOI) of 1.5. Cell controls (no virus added) and virus controls (cells infected with the virus but without antibodies) were placed together on each plate. After incubation for 3 days at 37°C and 5% CO2, viral replication was quantified by measuring the RFP signal using an Envision instrument. The IC50 was calculated from the dose-response curve. 50 The values ​​are listed in Table 2.

[0251] A subset of anti-RSV F antibodies was tested for their ability to inhibit the associated paramyxovirus hMPV in a 3-day viral neutralization assay using LLC-MK2 cells and the hMPV virus strain CAN97-83 (hMPV-eGFP). Briefly, 10 μL of antibody was added to a black 384-well clear-bottom microtitration plate at a 4-fold serial dilution. Subsequently, 3.0 x 10 5 After adding 20 μL of LLC-MK2 cells at a cell / mL density (i.e., 6000 cells / well), 10 μL of CAN97-83 virus was added with an infection multiplicity (MOI) of 0.1. Cell controls (no virus added) and virus controls (cells infected with the virus but without antibodies) were placed together on each plate. After incubation for 3 days at 37°C and 5% CO2, viral replication was quantified by measuring eGFP expression using an Acumen Cellista instrument. IC50 was calculated from the dose-response curve. 50 The values ​​are listed in Table 2.

[0252] [Table 2]

[0253]

[0254] Example 3: Further development of the anti-RSV F antibody ADI-59811

[0255] The anti-RSV F antibody ADI-59811 of Example 2 was further developed and analyzed for its use in methods to treat, prevent, or inhibit RSV infection. In particular, variants of the anti-RSV F antibody comprising heavy chain CDRs with SEQ ID NO: 1 to 3 and light chain CDRs with SEQ ID NO: 4 to 6 were further developed and analyzed for their use in methods to treat, prevent, or inhibit RSV infection.

[0256] In particular, an anti-RSV F antibody designated as RSV antibody 1 non-YTE was developed as a human IgG1 monoclonal antibody. Anti-RSV antibody 1 non-YTE was constructed using a heavy chain constant region based on IgG1-G1m (17) homozygous (SEQ ID NO: 170). Anti-RSV antibody 1 non-YTE included a variable heavy chain of SEQ ID NO: 7 and a variable light chain of SEQ ID NO: 8, and a heavy chain of SEQ ID NO: 9 and a light chain of SEQ ID NO: 10.

[0257] An anti-RSV F antibody designated as RSV antibody 1 was developed as a human IgG1 monoclonal antibody containing a variable heavy chain of SEQ ID NO: 7 and a variable light chain of SEQ ID NO: 8. RSV antibody 1 was constructed by swapping the heavy chain constant region of SEQ ID NO: 9 with the YTE heavy chain constant region (SEQ ID NO: 171), and accordingly, RSV antibody 1 contained a heavy chain of SEQ ID NO: 15 and a light chain of SEQ ID NO: 10. The YTE heavy chain constant region contained EU numbering substitutions M252Y, S254T, and T256E in the crystallizable fragment (Fc) domain (SEQ ID NO: 171).

[0258] RSV antibody 1, an anti-RSV F antibody designated as non-YTE LALA, was developed as a human IgG1 monoclonal antibody. This anti-RSV F antibody contains a variable heavy chain of SEQ ID NO: 7 and a variable light chain of SEQ ID NO: 8, and additionally contains the heavy chain and light chain sequences of SEQ ID NO: 9 and the light chain of SEQ ID NO: 10, respectively, and the sequence of SEQ ID NO: 9 was modified to incorporate substitutions L234A and L235A according to EU numbering into the crystallizable fragment (Fc) domain.

[0259] Example 4: Binding of anti-RSV F antibody against recombinant RSV A and BF proteins

[0260] The binding affinity of RSV antibody 1 non-YTE to recombinant RSV F protein stabilized in the pre-fusion conformation (sometimes referred to as the pre-fusion conformation) was evaluated by surface plasmon resonance (SPR) using a standard protocol (Table 3). Binding to the F protein after fusion could not be detected.

[0261] Antibody binding kinetics by Octet Antibody kinetics measurements for hMPV preF and RSV preF antigens were performed using the Octet BLI assay. Affinity measurements were performed on Octet HTX as previously generally described (e.g., see literature [Estep et al, High throughput solution-based measurement of antibody-antigen affinity and epitope binning. Mabs 5(2), 270-278 (2013)]). Briefly, Octet measurements were performed by loading IgG samples onto an AHC sensor, equilibrating the sensor offline in the assay buffer for 30 minutes, and then monitoring it online for 60 seconds to establish a baseline. The sensor with the loaded IgG was exposed to 100 nM of antigen in solution for 3 minutes, and then transferred to the assay buffer for 3 minutes for off-velocity measurements. All kinetics were analyzed using a 1:1 binding model.

[0262] [Table 3]

[0263]

[0264] Example 5: Anti-RSV F antibody binding site

[0265] The structure of the antigen-binding (Fab) fragment of RSV antibody 1 bound to the preF of RSV A2 was determined by cryo-electron microscopy (cryo-EM) at a resolution of 3.2 Å (data not presented). RSV antibody 1 It was observed that both the heavy chain and light chain interacted with the antigen site Ø.

[0266] The sequence conservation of the RSV antibody 1 binding site was evaluated by reviewing 5,719 F protein sequences derived from clinical and laboratory RSV isolates obtained from GenBank, the Global Influenza Data Sharing Initiative (GISAID), and internal databases (collection period: 1956–2022). The percentage of conservation of each amino acid within the binding site was calculated for different subtypes (i.e., 2,911 RSV A sequences and 2,808 RSV B sequences). Sequence analysis revealed that the RSV antibody 1 binding site is highly conserved (> 99%) at all 13 positions in RSV A and at 11 of the 13 positions in RSV B (Table 4). It was found that the remaining two positions (i.e., 206 and 209) were less conserved due to the mutations I206M and Q209R that emerged in RSV B between 2016 and 2018. Recent RSV B strains containing M206 and R209 were included in the strain panel used for the virus neutralization assay.

[0267] [Table 4]

[0268]

[0269] Example 6: In vitro neutralization of RSV A and B strains by anti-RSV F antibody

[0270] The neutralizing efficacy of RSV antibody 1 against RSV A and B was evaluated in a 3-day viral neutralization assay. The RSV F protein sequences for each tested virus are provided in Table 5 below. For each tested virus, a 9-point 4-fold serial dilution of the antibody was added to separate black 96-well clear-bottom tissue culture-treated microtitration plates (50 μL / well). Subsequently, 50 μL of RSV virus (final volume 200 μL) was added at an appropriate dilution in RPMI-1640 culture medium supplemented with 100 μL of HeLa cell suspension (5000 cells / well) and 10% (v / v) fetal bovine serum (FCS), 25 mM HEPES, 2 mM L-alanyl-glutamine, and 20 μg / mL gentamicin. Viral dilution was determined by viral titration and RT-qPCR readings. Cell controls (no virus added) and virus controls (virus-infected cells added to antibodies) were taken together from each plate. After incubation for 3 days at 37°C and 5% CO2, the supernatant was removed from the wells. The plates were washed twice with ice-cold PBS (100 μL / well), sealed, and stored overnight at -80°C. Next, 50 μL of 1x lysis solution supplemented with DNAse was added to the wells and incubated at RT for 5 minutes, followed by the addition of 5 μL of stop solution (Ambion Cell-to-CT bulk lysis buffer). Finally, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed for negative strand (genome) RSV RNA. Briefly, cell lysates were added to 0.27 μM (final concentration) of RSV A, RSV B, or β-actin forward primers. After the rapid denaturation step (for 5 minutes at 75°C), for cDNA synthesis based on anti-genome RNA (replication intermediate), 10x PCR buffer BI (containing 15 mM MgCl2), MgCl2 (3.5 mM, dNTPs (1 mM), RNAse inhibitor (1 U / μL), and Expand reverse transcriptase (0.33 U / μL; Roche) were added. After performing reverse transcription at 42°C for 30 minutes, denaturation was performed at 99°C for 5 minutes. A PCR reaction mixture was prepared by adding 2x Roche LightCycler 480 Probes Master, 0.3 μM RSV A or RSV B forward and reverse primers, 0.1 μM RSV A or RSV B probe, 0.3 μM β-actin forward and reverse primers, and 0.1 μM β-actin probe to the cDNA product. The PCR reaction consisted of a denaturation step (600 seconds at 95°C), followed by 45 cycles of denaturation (15 seconds at 95°C), annealing / extension (60 seconds at 60°C), and a final cooling step (10 seconds at 40°C). Using the delta Cp value normalized for β-actin, the antibody concentration (IC) required to reduce viral RNA by 50% compared to the untreated control was determined. 50 ...was decided. Data analysis was performed using Graphpad Prism and Excel.

[0271] Referring to FIG. 1, RSV antibody 1 has an IC in the range of 2 to 139 pM. 50 value, and median IC of 7 pM or 1.0 ng / mL 50 It was able to neutralize all 28 RSV viruses. RSV antibody 1 had a median IC50 of 6.2 pM (range 2.4 to 21.6). 50 RSV A, and a median IC of 12.5 pM (range 3.1 to 138.9). 50 RSV B was neutralized with... RSV B strain 18537 (also known as B Washington) at an IC10 of 139 pM. 50Neutralization with RSV antibody 1 was performed at a value that is approximately 20 times higher compared to the median value calculated for all strains. This reduction in efficacy can be explained by the presence of an RSV B 18537-specific mutation at the RSV antibody 1 binding site on RSV preF. These data demonstrate that RSV antibody 1 non-YTE possesses broad and potent neutralizing activity against both RSV A and B subtypes.

[0272] In Figure 1, each symbol represents the average of 3 to 12 replicate experiments measured in at least 2 independent experiments. RSV antibody 1 or RSV antibody 1 non-YTE was used in each viral neutralization assay. Note that RSV B strains 17-058221, MEP-OUT-13-D1, MEP-OUT-01-D1 and 18-002094 contain F mutations I206M and Q209R.

[0273] [Table 5]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281] Example 7: Prophylactic efficacy of RSV antibody 1 non-YTE in RSV A and B infected cotton rat models

[0282] The prophylactic efficacy of RSV antibody 1 without half-life-extended mutations (i.e., RSV antibody 1 non-YTE) is 10 5Plaque-forming units (pfu) were evaluated in cotton rats infected with RSV A Long, RSV B 9320, or RSV B 18537. Briefly, 6 to 8-week-old male sigmodon hispidus cotton rats (source: Sigmovir Biosystems, Inc., Rockville Md) (n = 5 to 6 animals / group) were administered six different doses of RSV antibody 1 non-YTE via intramuscular injection. Another group of cotton rats (n = 5 / 6) that received only the buffer solution served as the vehicle control group. At 24 hours after administration, the animals were 10 5 Test infections were administered intranasally with pfu's RSV-A Long, RSV B 9320, or RSV B 18537. On the fourth day after test infection, prophylactic efficacy was evaluated by measuring RSV virus titers in the lung tissue of the animals using a plaque assay. For the analysis of RSV A Long data, data from two independent studies were combined.

[0283] Now, referring to Figures 2a through 2c, prophylactic treatment with RSV antibody 1 non-YTE resulted in a dose-dependent reduction in lung viral titers in all three cotton rat models. The mean reduction compared to the vehicle control group was statistically significant in the dose groups of 2.5 to 0.03 mg / kg (RSV A Long), 3.0 to 0.1 mg / kg (RSV B 9320), and 3.0 to 0.3 mg / kg (RSV B 18537) (alpha = 0.05).

[0284] Example 8: In vivo EC in cotton rats infected with RSV A Long, RSV B 9320, and RSV B 18537 (Washington) 90 decision

[0285] On day 4 post-infection, serum concentrations of RSV antibody 1 non-YTE in each dose group (described in Example 7) were quantified using a Meso-Scale Discovery (MSD) based assay. The assay method is briefly described as follows: MSD Gold Small-spot streptavidin plates (Cat: L45SA-1) were blocked with 1x PBS + 1% BSA buffer for 30 minutes and tapped dry. Next, 30 μL of the final working concentration of the capture and detection reagent master mix (biotinylated and ruthenium-labeled anti-human Fc mAb; R10Z8E9) was added to each well combined with 20 μL of diluted standard, QC, or sample (50 μL / well), and incubated at RT for 60 minutes with shaking. The MSD plates were washed, and 1x MSD-T read buffer was added to all wells. The plates were read immediately on an MSD Sector S600 imager (Meso Scale Discovery; Rockville, MD). The MSD output file containing the raw ECL coefficients was swept into Cyberlab Content Manager (Agilent; Santa Clara, CA) and imported into Watson LIMS (Thermo Fisher Scientific; Waltham, MA) regression software for analysis. During assay qualification, the Watson study regression was predefined, and a 5-parameter logistic (automatically estimated) fit with 1 / Y2 weighted coefficients was used.

[0286] By plotting antibody serum concentration versus log 10 lung virus titer reduction (protective %), the effective concentration (EC) that yields a 90% reduction in log 10 lung virus titer compared to the vehicle control is identified. 90 ) was determined (Figs. 3a to 3c). From these plots, for RSV A Long, RSV B 9320, and RSV B 18537 using non-linear regression analysis In vivo EC 90The values ​​were calculated (Table 6). RSV antibody 1 had similar EC values ​​of 2.0 and 2.5 μg / ml against RSV A Long and RSV B 9320, respectively. 90 Indicates a value 6 to 7.5 times higher EC of 15 μg / ml for RSV B 18537 90 The lower activity against RSV B 18537 is consistent with in vitro neutralization data and can be explained by the presence of a mutation in the RSV antibody 1 binding site unique to RSV B 18537.

[0287] In conclusion, RSV antibody 1 non-YTE showed similar ECs for the two original strains RSV A Long or RSV B 9320. 90 It is effective in reducing lung viral load in cotton rats.

[0288] [Table 6]

[0289]

[0290] Example 9: In vitro and in vivo evaluation of Fc-mediated effector function of RSV antibody 1

[0291] Fc-mediated effector functions, such as virus clearance by ADCP and death of infected cells by ADCC, of ​​antiviral antibodies including RSV mAbs In vivo It is well established that it can contribute to efficacy (Literature [Tanget al.. Nat Commun. 10(1) :4153 (2019)]; Literature[DiLillo et al., Nat Med. 20(2):143-151 (2014)]; literature[van Erp et al., Front Immunol.10:548 (2019)]). To investigate whether Fc-mediated effector function plays a role in the efficacy of RSV antibody 1, the activity of RSV antibody 1 was compared with two Fc variants in ADCC and ADCP reporter bioassays and RSV-infected mouse models. The first variant, RSV antibody 1 non-YTE, contains a wild-type Fc domain without half-life extension mutations as described above, which is based on findings in other mAbs (Reference [Borrok et al., J Pharm Sci. [106(4): 1008-1017 (2017)]) It is expected to have a higher affinity for both FcγRIIa and FcγRIIIa. The second variant, RSV antibody 1 non-YTE LALA, contains the Fc-silencing mutant L234A / L235A on a wild-type Fc background as described above. Both variants are indistinguishable from RSV antibody 1 in direct antiviral activity.

[0292] Evaluation of RSV antibody 1 Fc variant in human ADCC and ADCP reporter bioassays: The potential of RSV antibody 1, RSV antibody 1 non-YTE, and RSV antibody 1 non-YTE LALA to induce ADCP and ADCC using Jurkat / NFAT-luc effector cells expressing FcγRIIa or FcγRIIIa and A549 target cells infected with RSVA2, respectively In a test tube It was evaluated. Briefly, the setup for the FcγRIIIa-V ADCC reporter bioassay was as follows: on day -1, 2 x 10 in a 96-well plate (Corning, 3903). 4A549 cells were pre-seeded. On Day 0, the cells were infected with RSV-GFP5 at an infection multiplicity (MOI) of 1 and incubated overnight at 37°C in 5% CO2. At 24 hours post-infection, the cells were washed, and the ADCC FcγRIIIa-V (V158), high-affinity variant, reporter bioassay (Promega) was performed as described by the manufacturer. Briefly; 25 μl of a 7-point 4-fold antibody dilution series was prepared in assay buffer and transferred to RSV-infected A549 cells in 25 μl of assay buffer. Subsequently, 25 μl of ADCC effector cells (630 μl in 3.6 ml of assay buffer) were added to the wells. The plates were incubated at 37°C and 5% CO2 for 6 hours, assay reagents were added to each well, and luminescence was measured on a Viewlux microplate imager with 0.3-second exposure at 3x binning. For the FcγRIIa-H ADCP reporter bioassay, A549 RSV-infected cells were set up as described for the ADCC reporter bioassay. The high-affinity variant (H131) ADCP reporter bioassay (Promega) was performed essentially according to the manufacturer's instructions. Briefly; 25 μl of a 2x antibody dilution was prepared in assay buffer and transferred to RSV-infected A549 cells in 25 μl of assay buffer. The antibody was allowed to bind to the cells for 15 minutes at 37°C. Subsequently, 25 μl of ADCP effector cells (700 μl in 3.4 ml of assay buffer) were added to the wells. The plates were incubated at 37°C and 5% CO2 for 6 hours, the assay reagent was added to each well, and luminescence was measured on a Viewlux microplate imager with 1 second exposure at 2x binning.

[0293] The ability of increasing concentrations of RSV antibody 1, RSV antibody 1 non-YTE, RSV antibody 1 non-YTE LALA, and negative control mAb to activate FcγRIIa and FcγRIIIa, respectively, expressed in Jurkat cells using RSVA2-infected A549 cells was tested in ADCP and ADCC reporter bioassays. Referring to Figures 4a and 4b, each data point is the mean of three independent replicate experiments, and error bars represent the standard deviation (SD).

[0294] Referring further to Figures 4a and 4b, the results indicated that RSV antibody 1 could bind to and activate both human FcγRs, albeit to different degrees (Figures 4a and 4b). RSV antibody 1 non-YTE was more effective than RSV antibody 1 in activating both FcγRIIa and FcγRIIIa, whereas RSV antibody 1 non-YTE LALA showed virtually no activity in the corresponding assay.

[0295] Evaluation of RSV antibody 1 Fc variant in RSV A infected mouse model : 1.7 x 10 6 The prophylactic efficacy of RSV antibody 1, RSV antibody 1 non-YTE, and RSV antibody 1 non-YTE LALA was compared in BALB / c mice (n = 6 / group) infected with pfu RSV A2. RSV antibody 1 and its Fc variants were administered intramuscularly at four different doses one day prior to intranasal RSV infection. Pulmonary viral titers were determined by the plaque assay on day 4 post-infection. Serum antibody concentrations on day 4 post-infection were quantified using an MSD-based assay and plotted against the relative decrease in log pulmonary viral titers (Figs. 5a to 5c). From these plots, nonlinear regression analysis was used In vivo EC 90The values ​​were calculated (Table 7). The analyzed data were derived from two independent studies. In each graph of Figures 5a through 5c, the dotted line represents the EC for virus reduction. 90 It indicates. In conclusion, all three RSV antibody 1 Fc variants have similar ECs against RSV A2. 90 The value was shown, indicating that Fc-mediated effector function did not contribute to the preventive efficacy of RSV antibody 1 in mice infected with RSV.

[0296] [Table 7]

[0297]

[0298] Example 10: Binding specificity of human plasma membrane proteins in cell arrays (Retrogenix cell microarray technology)

[0299] In vitro The human plasma membrane protein array is a research-grade screening tool for evaluating the potential off-target binding of antibody therapeutics. This screening was performed at Charles River, High Peak Chinley, Derbyshire, UK. Briefly, to generate the microarray, an expression vector encoding a full-length human protein was spotted on a glass slide. Subsequently, human embryonic kidney (HEK) 293 cells were applied to the slide and refracted to express the individual proteins. The cells were then fixed prior to the application of the test substance.

[0300] In the protein array assay described above, the off-target binding potential of RSV antibody 1 was screened against a library of 6,018 human plasma membrane proteins (secreted, cell surface-tethered secreted proteins) and 397 human heterodimers expressed in human HEK293 cells. RSV antibody 1 was selective for RSV preF, as indicated by specific interactions with the soluble recombinant target protein RSVF (RSV190373-preF) incorporated into the test system (data not presented). No off-target interactions with the tested human proteins were observed.

[0301] Example 11: Pharmacokinetics in Synomolgus Monkeys

[0302] The pharmacokinetics of RSV antibody 1 were characterized in a non-GLP PK study in four female cynomolgus monkeys following a single IV dose of 5 mg / kg. Additionally, a control molecule (i.e., nirsevimab; an anti-RSV mAb containing the YTE mutation) was included in two female monkeys of the study. Serum drug exposure analyses for RSV antibody 1 and nirsevimab were performed using a fit-for-purpose electrochemiluminescence immunoassay (ECLIA) method readable on an MSD Sector Imager S600. For both assays, anti-human Fc-specific (CH2 domain) mouse mAbs were applied as capture and detection reagents in a homogeneous sandwich format. Serum RSV antibody 1 and nirsevimab concentration-time data from both groups were fitted to a linear two-compartment PK model for PK parameter estimation.

[0303] The mean observed concentration-time points of serum RSV antibody 1 and nirsevimab in cynomolgus monkeys, along with the PK model fit, are shown in Figure 6. Referring to Figure 6, the lines represent model predictions, and the symbols represent the mean observed data from each animal at each sampling point over time. One animal in the RSV antibody 1 group had an abnormally poor elimination rate. This animal did not exhibit clinical symptoms that could affect PK and was not ADA positive throughout the study. This animal was considered an outlier and was removed from the analysis of the RSV antibody 1 group.

[0304] Example 12: Sequence Optimization

[0305] EpiVax Epimatrix In silicoThe sequence of RSV antibody 1 was analyzed for potential immunogenicity using the regulatory T (Treg) adjustment score of an immunogenicity prediction program (De Groot, AS & Martin, W. Reducing risk, improving outcomes: bioengineering less immunogenic protein therapeutics. Clin Imm, 2009. 131:189-201). EpiVax scores of -27.98 and 45.78 were obtained for the heavy chain (SEQ ID 9) and light chain (SEQ ID 10), respectively. Two variants of RSV antibody 1 were generated by reverting residues 3, 39, 46, and 74 (SEQ ID NO: 17) or residues 3 and 74 (SEQ ID NO: 18), which are found in germline IGK1-27, which produced Treg-adjusted Epivax scores of -2.79 and 8.24, respectively. As described in Example 4, the binding affinity of these variants to the RSV F protein was determined by SPR. The binding affinity of each variant was indistinguishable from the binding affinity of RSV antibody 1, which indicates the maintenance of activity against the designed mutants.

[0306] It will be obvious to those skilled in the art that the present disclosure is not limited to the exemplary embodiments described above and may be implemented in other specific forms without departing from the essential attributes thereof. Accordingly, the embodiments should be considered exemplary and non-limiting in all respects, and reference should be made to the appended claims rather than to the embodiments described above; thus, it is desirable that all modifications within the meaning and scope of the equivalence of the claims be included therein.

[0307] It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0308] All patents and publications mentioned in this specification represent the level of a person skilled in the art to which the present invention pertains.

[0309] The invention described herein by way of example may be appropriately practiced in the absence of any elements or elements, limitations or restrictions not specifically disclosed herein. Accordingly, for example, in each case of the invention, any of the terms “comprising,” “essentially consisting of,” and “consisting of” may be replaced with one of the other two terms. The terms and expressions used are for descriptive rather than limiting purposes, and the use of such terms and expressions is not intended to exclude any equivalents of the features or parts thereof shown and described, but it is recognized that various modifications are possible within the scope of the claimed invention. Additionally, where a feature or aspect of the invention is described in terms of Markush groups, those skilled in the art will recognize that the invention is also described in terms of any individual member of the Markush group or a subgroup of the member. For example, where X is described as being selected from the group consisting of bromine, chlorine, and iodine, the claim where X is bromine and the claim where X is bromine and chlorine are fully described. Other embodiments are within the following claims.

Claims

Claim 1 An isolated or recombinant anti-respiratory syncytial virus (RSV) F protein (F) antibody or an antigen-binding fragment thereof that specifically binds to the respiratory syncytial virus (RSV) F protein (F), wherein the anti-RSV F antibody or the antigen-binding fragment thereof comprises a variable heavy chain region (V) comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3. H ), and a variable light chain region (V) including light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3. L Each includes (1) SEQ ID NO: 1, 2, 3, 4, 5 and 6; each (2) SEQ ID NO: 19, 20, 21, 22, 23 and 24; each (3) SEQ ID NO: 27, 28, 29, 30, 31 and 32; each (4) SEQ ID NO: 35, 36, 37, 38, 39 and 40; each (5) SEQ ID NO: 43, 44, 45, 46, 47 and 48; each (6) SEQ ID NO: 51, 52, 53, 54, 55 and 56; each (7) SEQ ID NO: 59, 60, 61, 62, 63 and 64; each (8) SEQ ID NO: 67, 68, 69, 70, 71 and 72; (9) SEQ ID NO: 75, 76, 77, 78, 79 and 80, respectively; or (10) SEQ ID NO: 83, 84, 85, 86, 87 and 88, an anti-RSV F antibody or its antigen-binding fragment having amino acid sequences. Claim 2 In claim 1, the antibody or its antigen-binding fragment is V having an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, 25, 33, 41, 49, 57, 65, 73, 81, or 89. H , or V having an amino acid sequence at least 90% identical to SEQ ID NO: 8, 26, 34, 42, 50, 58, 66, 74, 82, or 90 L An anti-RSV F antibody or its antigen-binding fragment comprising Claim 3 In paragraph 2, (1) V having the amino acid sequence of SEQ ID NO: 7 H and V having the amino acid sequence of SEQ ID NO: 8 L ;(2) V having the amino acid sequence of SEQ ID NO: 25 H and V having the amino acid sequence of SEQ ID NO: 26 L ;(3) V having the amino acid sequence of SEQ ID NO: 33 H and V having the amino acid sequence of SEQ ID NO: 34 L ;(4) V having the amino acid sequence of SEQ ID NO: 41 H and V having the amino acid sequence of SEQ ID NO: 42 L ;(5) V having the amino acid sequence of SEQ ID NO: 49 H and V having the amino acid sequence of SEQ ID NO: 50 L ;(6) V having the amino acid sequence of SEQ ID NO: 57 H and V having the amino acid sequence of SEQ ID NO: 58 L ;(7) V having the amino acid sequence of SEQ ID NO: 65 H and V having the amino acid sequence of SEQ ID NO: 66 L ;(8) V having the amino acid sequence of SEQ ID NO: 73 H and V having the amino acid sequence of SEQ ID NO: 74 L ;(9) V having the amino acid sequence of SEQ ID NO: 81 H and V having the amino acid sequence of SEQ ID NO: 82 L ; or (10) V having the amino acid sequence of SEQ ID NO: 89 H and V having the amino acid sequence of SEQ ID NO: 90 L An anti-RSV F antibody or its antigen-binding fragment comprising Claim 4 An anti-RSV F antibody or its antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises an antigen-binding fragment comprising a heavy chain polypeptide sequence having at least 90% identical amino acid sequence to SEQ ID NO: 12, 91, 92, 93, 94, 95, 96, 97, 98 or 99. Claim 5 In paragraph 4, the anti-RSV F antibody or its antigen-binding fragment comprises an antigen-binding fragment comprising a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 12, 91, 92, 93, 94, 95, 96, 97, 98 or 99. Claim 6 An anti-RSV F antibody or its antigen-binding fragment according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment comprises an antigen-binding fragment comprising a light chain polypeptide sequence having at least 90% identical amino acid sequence to SEQ ID NO: 13, 100, 101, 102, 103, 104, 105, 106, 107 or 108. Claim 7 In claim 6, the antibody or antigen-binding fragment comprises an antigen-binding fragment comprising a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 13, 100, 101, 102, 103, 104, 105, 106, 107 or 108, an anti-RSV F antibody or its antigen-binding fragment. Claim 8 In claim 1, the antibody or antigen-binding fragment comprises: (1) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 12 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 13; (2) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 91 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 100; (3) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 92 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 101; (4) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 93 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 102; (5) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 94 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 103; and (6) an amino acid sequence having the amino acid sequence of SEQ ID NO: 95 An anti-RSV F antibody or an antigen-binding fragment comprising: (7) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 104 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 96 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 105; (8) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 97 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 106; (9) a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 98 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 107; or (10) an antigen-binding fragment comprising a heavy chain polypeptide sequence having the amino acid sequence of SEQ ID NO: 99 and a light chain polypeptide sequence having the amino acid sequence of SEQ ID NO:

108. Claim 9 In any one of claims 1 to 8, the anti-RSV F antibody or its antigen-binding fragment comprises a heavy chain polypeptide comprising the substitutions M252Y, S254T, and T256E. Claim 10 In any one of claims 1 to 9, the anti-RSV F antibody or its antigen-binding fragment comprises a heavy chain polypeptide comprising substitutions L234A and L235A. Claim 11 In any one of claims 1 to 10, the antibody or antigen-binding fragment is (1) SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 15; (2) SEQ ID NO: 91, SEQ ID NO: 109 or SEQ ID NO: 118; (3) SEQ ID NO: 92, SEQ ID NO: 110 or SEQ ID NO: 119; (4) SEQ ID NO: 93, SEQ ID NO: 111 or SEQ ID NO: 120; (5) SEQ ID NO: 94, SEQ ID NO: 112 or SEQ ID NO: 121; (6) SEQ ID NO: 95, SEQ ID NO: 113 or SEQ ID NO: 122; (7) SEQ ID NO: 96, SEQ ID NO: 114 or SEQ ID NO: 123; (8) SEQ An anti-RSV F antibody or an antigen-binding fragment thereof comprising a heavy chain polypeptide having an amino acid sequence selected from ID NO: 97, SEQ ID NO: 115 or SEQ ID NO: 124; (9) SEQ ID NO: 98, SEQ ID NO: 116 or SEQ ID NO: 125; or (10) SEQ ID NO: 99, SEQ ID NO: 117 or SEQ ID NO:

126. Claim 12 An anti-RSV F antibody or its antigen-binding fragment according to any one of claims 1 to 11, wherein the antibody or its antigen-binding fragment comprises a light chain polypeptide having the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, or SEQ ID NO:

108. Claim 13 In any one of claims 1 to 12, the antibody or its antigen-binding fragment is (1) SEQ ID NO: 15 and SEQ ID NO: 10, SEQ ID NO: 9 and SEQ ID NO: 10, or SEQ ID NO: 11 and SEQ ID NO: 10; (2) SEQ ID NO: 91 and SEQ ID NO: 100, SEQ ID NO: 109 and SEQ ID NO: 100, SEQ ID NO: 118 and SEQ ID NO: 100; (3) SEQ ID NO: 92 and SEQ ID NO: 101, SEQ ID NO: 110 and SEQ ID NO: 101, or SEQ ID NO: 119 and SEQ ID NO: 101; (4) SEQ ID NO: 93 and SEQ ID NO: 102, SEQ ID NO: 111 and SEQ ID NO: 102, or SEQ ID NO: 120 and SEQ ID NO: 102;(5) SEQ ID NO: 94 and SEQ ID NO: 103, SEQ ID NO: 112 and SEQ ID NO: 103, or SEQ ID NO: 121 and SEQ ID NO: 103;(6) SEQ ID NO: 95 and SEQ ID NO: 104, SEQ ID NO: 113 and SEQ ID NO: 104, or SEQ ID NO: 122 and SEQ ID NO: 104;(7) SEQ ID NO: 96 and SEQ ID NO: 105, SEQ ID NO: 114 and SEQ ID NO: 105, or SEQ ID NO: 123 and SEQ ID NO: 105;(8) SEQ ID NO: 97 and SEQ ID NO: 106, SEQ ID NO: 115 and SEQ ID NO: 106, or SEQ ID NO: 124 and SEQ ID NO: 106;(9) SEQ ID NO: 98 and SEQ ID NO: 107, SEQ ID NO: 116 and SEQ ID NO: 107, or SEQ ID NO: 125 and SEQ ID NO: 107;or (10) an anti-RSV F antibody or its antigen-binding fragment comprising a heavy chain polypeptide and a light chain polypeptide, each selected from SEQ ID NO: 99 and SEQ ID NO: 108, SEQ ID NO: 117 and SEQ ID NO: 108, or SEQ ID NO: 126 and SEQ ID NO: 108.; Claim 14 In any one of claims 1 to 13, the anti-RSV F antibody or the antigen-binding fragment thereof is capable of neutralizing or blocking RSV-F activity by inhibiting at least one biological activity of RSV-F. Claim 15 An anti-RSV F antibody or its antigen-binding fragment, wherein, in any one of claims 1 to 14, the antibody or its antigen-binding fragment is capable of binding to the PreF conformation of RSV-F with a higher binding affinity than the PostF conformation of RSV-F. Claim 16 An anti-RSV F antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment is a human antibody, a humanizing antibody, a chimeric antibody, a bispecific or multispecific antibody. Claim 17 In paragraph 16, the antibody or antigen-binding fragment is an anti-RSV F antibody or its antigen-binding fragment, which is a human antibody. Claim 18 In any one of claims 1 to 17, the antibody or antigen-binding fragment is an anti-RSV F antibody or its antigen-binding fragment conjugated to polyethylene glycol (PEG) or a therapeutic agent or diagnostic drug. Claim 19 An isolated nucleic acid molecule comprising a nucleic acid sequence encoding an anti-RSV F antibody or an antigen-binding fragment thereof according to any one of claims 1 to 18. Claim 20 An expression vector comprising an isolated nucleic acid sequence according to paragraph 19. Claim 21 A host cell containing an expression vector according to paragraph 20. Claim 22 A method for producing an anti-RSV F antibody or an antigen-binding fragment thereof according to any one of claims 1 to 17, comprising the steps of: culturing the host cells of claim 21 under conditions for producing the anti-RSV F antibody or the antigen-binding fragment thereof; and recovering the anti-RSV F antibody or the antigen-binding fragment thereof from the cells or cell culture. Claim 23 A pharmaceutical composition comprising one or more of an anti-RSV F antibody or an antigen-binding fragment thereof according to any one of claims 1 to 18, and a pharmaceutically acceptable carrier and / or excipient. Claim 24 A pharmaceutical composition comprising one or more nucleic acid sequences according to claim 19; or one or more expression vectors according to claim 20; and a pharmaceutically acceptable carrier and / or excipient. Claim 25 A method for inhibiting at least one biological activity of RSV-F, comprising the step of administering to a subject who requires or is suspected of requiring the same: a) one or more antibodies or antigen-binding fragments thereof according to any one of claims 1 to 18; b) a nucleic acid molecule according to claim 19; c) an expression vector according to claim 20; d) a host cell according to claim 21; or e) a pharmaceutical composition according to claim 23 or 24. Claim 26 In paragraph 25, a method for treating, preventing, or improving respiratory syncytial virus (RSV) infection, or treating, preventing, or improving at least one symptom associated with RSV infection. Claim 27 In paragraph 25 or 26, the method is a method for reducing lung virus titers within the subject. Claim 28 A method in which any one of paragraphs 25 to 27, any one of a) to e) is administered in a therapeutically effective amount. Claim 29 The use of one or more of an anti-RSV F antibody or its antigen-binding fragment according to any one of claims 1 to 18 in the manufacture of a drug for inhibiting at least one biological activity of RSV-F in a subject. Claim 30 The use of one or more of an anti-RSV F antibody or an antigen-binding fragment thereof according to any one of claims 1 to 18 in the manufacture of a drug for treating, preventing, or improving respiratory syncytial virus (RSV) infection in a subject, or for treating, preventing, or improving at least one symptom associated with RSV infection.

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