Antibodies that specifically bind to RSV

Abstract

A set of antibodies or antigen-binding fragments thereof that specifically bind to RSV are provided, as well as corresponding polynucleotides, vectors, host cells, compositions comprising the antibodies, and methods of producing the antibodies.

Description

Antibodies that specifically bind to RSV

[0001] Incorporation by reference of related applications

[0002] This application claims priority to Chinese invention patent application CN202211571449.4 filed on December 8, 2022 and Chinese invention patent application CN202310007283.1 filed on January 4, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention provides a group of antibodies or antigen-binding fragments thereof that specifically bind to RSV. The present invention also provides polynucleotides encoding the antibodies or antigen-binding fragments thereof, vectors comprising the polynucleotides, host cells comprising the vectors, methods for generating the antibodies, and compositions comprising the antibodies. Background Art

[0004] Respiratory Syncytial Virus (RSV) causes approximately 33 million cases of acute lower respiratory tract infections and over 100,000 deaths annually in children under the age of 5 years; it causes approximately 7.2 deaths per 100,000 people annually in adults over the age of 65 years. Furthermore, RSV infection does not confer permanent immunity and does not protect children from reinfection. RSV has become one of the most important pathogens causing acute lower respiratory tract infections worldwide in children under the age of 5 years and in immunocompromised individuals over the age of 65 years, and has become a recognized global health issue.

[0005] Viral diseases are generally prevented through vaccines, but RSV has not had an approved vaccine for decades since its isolation in 1957. Previous vaccine development efforts have resulted in cases where the vaccine not only failed to provide protection but actually made pneumonia caused by RSV infection more severe, leading to deaths in children. This demonstrates the significant risks associated with vaccine development. Consequently, increasing attention is being focused on the development of RSV antibody drugs. Currently, the only preventive drug for the disease is the palivizumab monoclonal antibody, first approved by the US FDA in 1998. Therefore, there is an urgent need to develop new anti-RSV drugs, particularly those that can treat RSV infection.

[0006] RSV is a non-segmented, single-stranded, negative-sense RNA virus belonging to the Paramyxoviridae family and the Pneumovirus genus. RSV virus particles contain an envelope. The virus particle is composed of an envelope, a nucleocapsid, and a core. The RSV genome is 15.2 kb in length and transcribes 10 genes (NS1, NS2, N, P, M, SH, G, F, M2, and L from the 3' end to the 5' end), encoding 11 proteins. Among them, M2 contains two open reading frames, encoding two proteins, M2-1 and M2-2; each of the remaining genes encodes one protein. NS1 and NS2 are non-structural proteins; N (nucleocapsid protein), P (phosphoprotein), and L (polymerase subunit protein) are nucleocapsid proteins; M, M2-1, and M2-2 are matrix proteins; F (fusion protein), G (adhesion protein), and SH (small hydrophobic protein) are transmembrane glycoproteins. The G protein and F protein on the surface of RSV virus particles mediate the adsorption and fusion between the virus and host cells, virus-infected cells and uninfected cells, and are the main protective antigens of RSV, which can induce the body to produce protective neutralizing antibodies. Research results show that the G protein is highly variable between the two subtypes, and it induces the body to produce type-specific neutralizing antibodies, which do not have a broad protective effect. The F protein is a highly conserved protein, and the protective antibodies it induces are broad-spectrum neutralizing antibodies that can simultaneously inhibit the infection of RSV A and B. It is a potential target protein for RSV prevention and therapeutic drugs and vaccine research. The F protein is active only after being cut into two fragments, F1 (48kDa) and F2 (26kDa), by host proteases. The F protein generally forms a trimer structure of F1-F2 heterodimers to perform its fusion function. On the F protein, it is currently believed that there are 6 epitopes that can produce neutralizing antibodies, namely the epitopes I, II, III, IV and V.

[0007] Summary of the Invention

[0008] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to RSV, comprising:

[0009] (1) CDR-H1 as set forth in SEQ ID NO: 1, CDR-H2 as set forth in SEQ ID NO: 2, CDR-H3 as set forth in SEQ ID NO: 3, CDR-L1 as set forth in SEQ ID NO: 4, CDR-L2 as set forth in SEQ ID NO: 5, and CDR-L3 as set forth in SEQ ID NO: 6;

[0010] (2) CDR-H1 set forth in SEQ ID NO: 9, CDR-H2 set forth in SEQ ID NO: 10, CDR-H3 set forth in SEQ ID NO: 11, CDR-L1 set forth in SEQ ID NO: 12, CDR-L2 set forth in SEQ ID NO: 13, and CDR-L3 set forth in SEQ ID NO: 14;

[0011] (3) CDR-H1 as set forth in SEQ ID NO: 17, CDR-H2 as set forth in SEQ ID NO: 18, CDR-H3 as set forth in SEQ ID NO: 19, CDR-L1 as set forth in SEQ ID NO: 20, CDR-L2 as set forth in SEQ ID NO: 21, and CDR-L3 as set forth in SEQ ID NO: 22; or

[0012] (4) CDR-H1 as shown in SEQ ID NO: 25, CDR-H2 as shown in SEQ ID NO: 26, CDR-H3 as shown in SEQ ID NO: 27, CDR-L1 as shown in SEQ ID NO: 28, CDR-L2 as shown in SEQ ID NO: 29, and CDR-L3 as shown in SEQ ID NO: 30.

[0013] In one embodiment, the antibody or antigen-binding fragment thereof of the invention comprises:

[0014] (1) VH as shown in SEQ ID NO: 7 and VL as shown in SEQ ID NO: 8;

[0015] (2) VH as shown in SEQ ID NO: 15 and VL as shown in SEQ ID NO: 16;

[0016] (3) VH as shown in SEQ ID NO: 23 and VL as shown in SEQ ID NO: 24; or

[0017] (4) VH shown in SEQ ID NO: 31 and VL shown in SEQ ID NO: 32.

[0018] In one embodiment, the antibody or antigen-binding fragment thereof of the invention comprises:

[0019] (1) the heavy chain constant region as shown in SEQ ID NO: 35, and

[0020] (2) The light chain constant region shown in SEQ ID NO: 33 or 34.

[0021] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises an α heavy chain, a δ heavy chain, an ε heavy chain, a γ heavy chain or a μ heavy chain. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention belongs to the IgG1, IgG2, IgG3 or IgG4 subclass. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a λ light chain or a κ light chain. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is a full-length antibody. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is an antibody fragment selected from Fv, scFv, Fab, Fab', F(ab')2 and xFab. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is a chimeric antibody or a human antibody or antigen-binding fragment thereof.

[0022] In one embodiment, the antibodies or antigen-binding fragments thereof of the invention have one or more of the following properties:

[0023] (1) Ability to specifically bind to RSV A2 pre-F protein;

[0024] (2) is capable of specifically binding to RSV A2 pre-F protein with an EC50 value of less than 60, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 ng / mL, or with an EC50 value that is less than (e.g., 1% to 99%, such as at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) the EC50 value of MEDI8897;

[0025] (3) It can be less than 5.0, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.009, 0.008, 0.007, 0.006, 0.005, 0.004, 0.003, that specifically binds to RSV A2 pre-F protein with a KD value of 0.002 or 0.001 nM, or with a KD value that is less than (e.g., 1% to 99%, such as at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) the KD value of MEDI8897;

[0026] (4) being able to inhibit RSV (e.g., type A and / or type B) from infecting host cells;

[0027] (5) capable of inhibiting RSV type A strain (e.g., A2) infection of a host cell with an IC50 value of less than 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 ng / mL, or with an IC50 value that is less than the IC50 value of MEDI8897 (e.g., 1% to 99%, such as at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%);

[0028] (6) capable of inhibiting RSV B strain (e.g., B9320) infection of host cells with an IC50 value of less than 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, or 3 ng / mL, or with an IC50 value that is less than (e.g., 1% to 99%, such as at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) the IC50 value of MEDI8897;

[0029] (7) capable of inhibiting RSV B strain (e.g., B18537) infection of host cells with an IC50 value of less than 50, 45, 40, 35, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 ng / mL, or with an IC50 value that is less than the IC50 value of MEDI8897 (e.g., 1% to 99%, such as at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%);

[0030] (8) can compete with or inhibit the binding of MEDI8897 to RSV A2 pre-F protein;

[0031] (9) capable of inhibiting the binding of MEDI8897 to RSV A2 pre-F protein by 1% to 100%, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%;

[0032] (10) can compete with or inhibit the binding of MK-1654 to RSV A2 pre-F protein;

[0033] (11) capable of inhibiting the binding of MK-1654 to RSV A2 pre-F protein by 1% to 100%, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%;

[0034] (12) can compete with or inhibit the binding of Motavizumab to RSV A2 pre-F protein;

[0035] (13) capable of inhibiting the binding of Motavizumab to RSV A2 pre-F protein by 1% to 100%, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%;

[0036] (14) can compete with or inhibit the binding of MPE8 to RSV A2 pre-F protein;

[0037] (15) capable of inhibiting the binding of MPE8 to RSV A2 pre-F protein by 1% to 100%, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%;

[0038] (16) Capable of binding to RSV A2 pre-F protein epitope;

[0039] (17) can bind to the IV epitope of RSV A2 pre-F protein;

[0040] (18) Capable of binding to RSV A2 pre-F protein epitope and IV epitope;

[0041] (19) can bind to epitope II of RSV A2 pre-F protein;

[0042] (20) capable of binding to epitope III of RSV A2 pre-F protein; and / or

[0043] (21) It is able to bind to epitopes II and III of RSV A2 pre-F protein.

[0044] In one aspect, the present invention provides a polynucleotide encoding an antibody or antigen-binding fragment thereof of the present invention.

[0045] In one embodiment, a polynucleotide of the invention comprises:

[0046] (1) SEQ ID NOs: 36 and 37;

[0047] (2) SEQ ID NOs: 38 and 39;

[0048] (3) SEQ ID NOs: 40 and 41; or

[0049] (4) SEQ ID NOs: 42 and 43.

[0050] In one aspect, the present invention provides a vector comprising a polynucleotide of the present invention.

[0051] In one aspect, the present invention provides a host cell comprising a polynucleotide or vector of the present invention. In one embodiment, the host cell is a eukaryotic cell. In one embodiment, the host cell is a CHO cell.

[0052] In one aspect, the present invention provides a method for producing an antibody or an antigen-binding fragment thereof, comprising:

[0053] (a) culturing the host cell of the present invention under conditions suitable for expressing the antibody or antigen-binding fragment thereof, and

[0054] (b) optionally, recovering the antibody or antigen-binding fragment thereof.

[0055] In one aspect, the present invention provides a composition comprising an antibody or antigen-binding fragment thereof of the present invention.

[0056] In one aspect, the invention provides an antibody or antigen-binding fragment thereof or composition of the invention for use as a medicament.

[0057] On the one hand, the present invention provides an antibody or its antigen-binding fragment or composition of the present invention for use in treating a disease. In one embodiment, the disease is a lower respiratory tract infection. In one embodiment, the disease is a disease caused by RSV infection.

[0058] In one aspect, the present invention provides the use of an antibody or antigen-binding fragment thereof or a composition of the present invention in the manufacture of a medicament for treating a disease. In one embodiment, the disease is a lower respiratory tract infection.

[0059] In one aspect, the present invention provides a method of treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of an antibody or antigen-binding fragment thereof or composition of the present invention. In one embodiment, the disease is a lower respiratory tract infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1-1 and Figure 1-2 show the antigen binding curves (ELISA) of the antibodies of the present invention.

[0061] Figure 2-1 and Figure 2-2 show the antigen binding curves (BA) of the antibodies of the present invention.

[0062] Figure 3-1 and Figure 3-2 show the virus neutralization curves of the antibodies of the present invention.

[0063] FIG4 shows a schematic diagram of a competitive binding epitope assay for the antibodies of the present invention. DETAILED DESCRIPTION

[0064] For purposes herein, "acceptor human framework" refers to a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework as defined below. The acceptor human framework "derived" from a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence, or it can contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0065] "Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0066] An "affinity matured" antibody is one with one or more alterations in one or more hypervariable regions (HVRs) which result in improved affinity of the antibody for antigen, compared to a parent antibody which does not possess such alteration(s).

[0067] The terms "anti-RSV antibody" and "antibody that binds to RSV" refer to antibodies that can bind to RSV with sufficient affinity so that the antibody can be used as a diagnostic, prophylactic, and / or therapeutic agent for targeting RSV. In one embodiment, the extent to which an anti-RSV antibody binds to unrelated, non-RSV proteins is less than about 10% of the binding of the antibody to RSV, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to RSV has a binding affinity of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, such as 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13In certain embodiments, "anti-RSV antibodies" and "antibodies that bind to RSV" are "anti-RSV pre-F antibodies" and "antibodies that bind to RSV pre-F," particularly "anti-RSV A2 pre-F antibodies" and "antibodies that bind to RSV A2 pre-F." In certain embodiments, anti-RSV antibodies bind to a pre-F epitope that is conserved among different RSV strains. In a preferred embodiment, "anti-RSV antibodies," "antibodies that specifically bind to RSV," and "antibodies that bind to RSV" are antibodies with a Kd of 0. D The value is 1.0x 10 -8 mol / l or less, in one embodiment, K D The value is 1.0x 10 -9 mol / l or less, in one embodiment, K D The value is 1.0x 10 -9 mol / l to 1.0x 10 -13 In this context, binding affinity is determined using standard binding assays, such as surface plasmon resonance (SPR) technology. GE-Healthcare Uppsala, Sweden: SARTORIUS, R8), for example using RSV A2 pre-F protein.

[0068] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0069] An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'SH, F(ab')2, xFab; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0070] The term "epitope" refers to a site on an antigen (either proteinaceous or non-proteinaceous) that is bound by anti-RSV antibodies. An epitope can be formed from a continuous amino acid string (linear epitope) or comprise discontinuous amino acids (conformational epitope), for example, due to the folding of the antigen (i.e., by the tertiary folding of proteinaceous antigens) becoming spatially close. Linear epitopes typically remain bound by anti-RSV antibodies after proteinaceous antigens are exposed to denaturants, while conformational epitopes are typically destroyed after being treated with denaturants. The epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation.

[0071] Screening for antibodies that bind to a particular epitope (i.e., those that bind to the same epitope) can be performed using routine methods in the art, such as, for example, but not limited to, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see "Antibodies," Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).

[0072] Antigen structure-based antibody profiling (ASAP) (also known as modification-assisted profiling (MAP)) allows for the separation of a population of monoclonal antibodies that specifically bind to RSV into bins based on the binding profile of each antibody from the population to a chemically or enzymatically modified antigen surface (see, e.g., US 2004 / 0101920). The antibodies in each bin bind to the same epitope, which can be a unique epitope that is distinct from or partially overlaps with the epitope represented by another bin.

[0073] Competitive binding can also be used to easily identify whether an antibody binds to the same RSV epitope as, or competes for binding with, a reference anti-RSV antibody. For example, an "antibody that binds to the same epitope as a reference anti-RSV antibody" refers to an antibody that blocks the binding of the reference anti-RSV antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. For example, to determine whether an antibody binds to the same epitope as a reference anti-RSV antibody, the reference antibody is allowed to bind to RSV under saturating conditions. After removing excess reference anti-RSV antibody, the ability of the anti-RSV antibody in question to bind to RSV is assessed. If the anti-RSV antibody is able to bind to RSV after saturation binding of the reference anti-RSV antibody, it can be concluded that the anti-RSV antibody in question binds to a different epitope than the reference anti-RSV antibody. However, if the anti-RSV antibody in question is unable to bind to RSV after saturation binding of the reference anti-RSV antibody, then the anti-RSV antibody in question may bind to the same epitope as the reference anti-RSV antibody. In order to confirm whether the antibody in question is in conjunction with identical epi-position or only hindered by spatial reason, routine experiment (such as peptide mutation and use ELISA, RIA, surface plasmon resonance, flow cytometry or the available any other quantitative or qualitative antibody binding assay of this area) can be used.This assay method should be carried out with two kinds of settings, and i.e. two kinds of antibodies are all as saturated antibodies.If in two kinds of settings, all only have the first (saturated) antibody to be able to bind RSV, so can draw a conclusion, the anti-RSV antibody in question and with reference to anti-RSV antibody competition in conjunction with RSV.

[0074] In some embodiments, two antibodies are considered to bind to the same or overlapping epitope if a 1, 5, 10, 20, or 100-fold excess of one antibody inhibits binding of the other by at least 50%, at least 75%, at least 90%, or even 99% or more as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502).

[0075] In some embodiments, two antibodies are considered to bind to the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other. Two antibodies are considered to have "overlapping epitopes" if only a subset of amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0076] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0077] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is of the IgG4 isotype and has an S228P mutation in the hinge region to improve the stability of IgG4 antibodies. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0078] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 ,Bi 212 , P 32 , Pb 212 and radioisotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and various anti-tumor or anti-cancer agents disclosed below.

[0079] "Effector functions" refer to those biological activities attributable to the Fc region of an antibody and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0080] An "effective amount" of an agent (eg, a pharmaceutical formulation) refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0081] The term "Fc region" herein is used to define the C-terminal region containing at least a portion of the constant region in an immunoglobulin heavy chain. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226, or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may be present or absent. In one embodiment, the anti-RSV antibody as described herein is of the IgG1 isotype and comprises the constant heavy chain domain of SEQ ID NO: 35. In one embodiment, it additionally comprises a C-terminal lysine (Lys447). In one embodiment, the anti-RSV antibody as described herein is of the IgG4 isotype. In one embodiment, it additionally comprises a C-terminal lysine (Lys447). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0082] "Framework" or "FR" refers to the variable domain residues excluding the hypervariable region (HVR) residues. Generally, the FR of a variable domain is composed of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences in VH (or VL) generally appear in the following order: FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

[0083] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to a native antibody structure or has heavy chains that contain an Fc region as defined herein.

[0084] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the initial transformed cell are included herein.

[0085] "Human antibody" refers to an antibody that possesses an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source using a human antibody repertoire or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues. In certain embodiments, human antibodies are derived from non-human transgenic mammals, such as mice, rats, or rabbits. In certain embodiments, human antibodies are derived from hybridoma cell lines.

[0086] "Human consensus framework" refers to a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one embodiment, for VL, the subgroup is as described in Kabat et al., supra, subgroup kappa I. In one embodiment, for VH, the subgroup is as described in Kabat et al., supra, subgroup III.

[0087] "Humanized" antibodies refer to chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise at least one, typically two, substantially entire variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. Optionally, a humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0088] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contacts"). Generally, antibodies comprise six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:

[0089] (a) Hypervariable loops, present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0090] (b) CDRs, present at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0091] (c) antigenic contacts, present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)); and

[0092] (d) a combination of (a), (b), and / or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

[0093] In one embodiment, the HVR residues include those identified below in the description of the amino acid sequences.

[0094] Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0095] An "immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent.

[0096] "Individual" or "subject" refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0097] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99% as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC methods). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0098] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0099] "Isolated nucleic acid encoding an anti-RSV antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or different vectors, and such nucleic acid molecules present in one or more locations in a host cell.

[0100] When used herein, the term "monoclonal antibody" refers to an antibody obtained from a group of substantially homogeneous antibodies, i.e., the individual antibodies constituting the group are identical and / or bind to the same epitope, except for example, possible variant antibodies containing naturally occurring mutations or occurring during the generation of monoclonal antibody preparations, such variants generally being present in very small amounts. Unlike polyclonal antibody preparations that typically comprise different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates that the antibody is characterized by being obtained from a group of substantially homogeneous antibodies, and should not be interpreted as requiring antibodies to be generated by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention can be generated by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of human immunoglobulin loci, and such methods and other exemplary methods for generating monoclonal antibodies are described herein.

[0101] A "naked antibody" is an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in pharmaceutical formulations.

[0102] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains bonded by disulfide bonds. From N to C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N to C-terminus, each light chain has a variable region (VL), also known as a variable light domain or light chain variable domain, followed by a constant light (CL) domain. Depending on the amino acid sequence of its constant domain, antibody light chains can be classified into one of two types, called kappa (κ) and lambda (λ).

[0103] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0104] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm needed to achieve maximum alignment over the full length of the compared sequences. However, for purposes herein, % amino acid sequence identity values ​​are generated using the ggsearch program of the FASTA package, version 36.3.8c or later, and the BLOSUM50 comparison matrix. The FASTA program package was written by W. R. Pearson and DJ Lipman (1988) "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448; W. R. Pearson (1996) "Effective protein sequence comparison", Meth. Enzymol. 266:227-258; and Pearson et al. (1997) Genomics 46:24-36 and is publicly available at http: / / fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml. Alternatively, sequences can be compared using the public server available at http: / / fasta.bioch.virginia.edu / fasta_www2 / index.cgi using the ggsearch (global protein:protein) program and default options (BLOSUM50; open: -10; extend: -2; Ktup=2) to ensure that a global rather than a local alignment is performed. The percent amino acid identity is given in the output alignment header.

[0105] The term "pharmaceutical formulation" refers to a preparation that is in such form as to permit the biological activity of the active ingredient contained therein to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.

[0106] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0107] As used herein, "treatment" (and grammatical variations thereof) refers to clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, alleviating / reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and regression or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the development of disease or slow the progression of disease.

[0108] As used herein, the term "prevent" includes slowing the onset of a disease, reducing the risk of developing a disease, inhibiting or delaying the manifestation or development of symptoms associated with the disease, reducing the severity of subsequent contractions or the development of the disease, ameliorating associated symptoms, and inducing immunity to prevent the disease.

[0109] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in antibody binding to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated using a VH or VL domain from an antibody that binds to the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0110] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0111] I. Compositions and Methods

[0112] In one aspect, the invention provides isolated antibodies that bind RSV.

[0113] In certain embodiments, antibodies that bind RSV are provided. The antibodies of the invention are useful, for example, for diagnosing or treating RSV infection, such as a lower respiratory tract infection.

[0114] A. Exemplary anti-RSV antibodies

[0115] In certain embodiments, an anti-RSV antibody is provided, wherein the antibody:

[0116] i) binds to RSV, in particular RSV pre-F protein, more particularly RSV A2 pre-F protein; and / or

[0117] ii) inhibiting RSV, in particular RSV A1, RSV B9320 and / or RSV B18537, from infecting host cells, such as Hep2 cells.

[0118] In one aspect, the present invention provides an anti-RSV antibody comprising at least one, two, three, four, five, or six CDRs selected from: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0119] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3. In one embodiment, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3. In another embodiment, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In yet another embodiment, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, and a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2. In yet another embodiment, the antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3.

[0120] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL CDR sequences selected from the group consisting of: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In one embodiment, the antibody comprises (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0121] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from the group consisting of: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from the group consisting of: (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0122] In another aspect, the present invention provides an antibody comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f) a CDR-L3 comprising an amino acid sequence selected from SEQ ID NO: 6.

[0123] In any of the above embodiments, the anti-RSV antibody is human or humanized.In one embodiment, the anti-RSV antibody comprises the CDRs as in any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0124] In another aspect, the anti-RSV antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-RSV antibody comprising the sequence retains the ability to bind to RSV. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 7. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the CDRs (i.e., in the FRs). Optionally, the anti-RSV antibody comprises the VH sequence in SEQ ID NO: 7, including post-translational modifications of the sequence. In a specific embodiment, the VH comprises one, two or three CDRs selected from the following: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3.

[0125] In another aspect, an anti-RSV antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-RSV antibody comprising the sequence retains the ability to bind to RSV. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 8. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the CDR (i.e., in the FR). Optionally, the anti-RSV antibody comprises the VL sequence in SEQ ID NO: 8, including post-translational modifications of the sequence. In a specific embodiment, the VL comprises one, two or three CDRs selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0126] In another aspect, an anti-RSV antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 7 and SEQ ID NO: 8, respectively, including post-translational modifications of those sequences.

[0127] In another aspect, the present invention provides antibodies that bind to the same epitope as the anti-RSV antibodies provided herein. For example, in certain embodiments, antibodies that bind to the same epitope as an anti-RSV antibody comprising the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 8 are provided.

[0128] In one aspect, the present invention provides an anti-RSV antibody comprising at least one, two, three, four, five, or six CDRs selected from: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14.

[0129] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11. In one embodiment, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11. In another embodiment, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14. In yet another embodiment, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14, and a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10. In yet another embodiment, the antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:9; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:10; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:11.

[0130] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL CDR sequences selected from the group consisting of: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14. In one embodiment, the antibody comprises (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14.

[0131] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from the group consisting of: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from the group consisting of: (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14.

[0132] In another aspect, the present invention provides an antibody comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (f) a CDR-L3 comprising an amino acid sequence selected from SEQ ID NO: 14.

[0133] In any of the above embodiments, the anti-RSV antibody is human or humanized.In one embodiment, the anti-RSV antibody comprises the CDRs as in any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0134] In another aspect, the anti-RSV antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-RSV antibody comprising the sequence retains the ability to bind to RSV. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 15. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the CDRs (i.e., in the FRs). Optionally, the anti-RSV antibody comprises the VH sequence in SEQ ID NO: 15, including post-translational modifications of the sequence. In a specific embodiment, the VH comprises one, two or three CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11.

[0135] In another aspect, an anti-RSV antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-RSV antibody comprising the sequence retains the ability to bind to RSV. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 16. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the CDR (i.e., in the FR). Optionally, the anti-RSV antibody comprises the VL sequence in SEQ ID NO: 16, including post-translational modifications of the sequence. In a specific embodiment, the VL comprises one, two or three CDRs selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14.

[0136] In another aspect, an anti-RSV antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 15 and SEQ ID NO: 16, respectively, including post-translational modifications of those sequences.

[0137] In another aspect, the present invention provides antibodies that bind to the same epitope as the anti-RSV antibodies provided herein. For example, in certain embodiments, antibodies that bind to the same epitope as an anti-RSV antibody comprising the VH sequence of SEQ ID NO: 15 and the VL sequence of SEQ ID NO: 16 are provided.

[0138] In one aspect, the present invention provides an anti-RSV antibody comprising at least one, two, three, four, five, or six CDRs selected from: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22.

[0139] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19. In one embodiment, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19. In another embodiment, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22. In yet another embodiment, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22, and a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18. In yet another embodiment, the antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19.

[0140] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL CDR sequences selected from the group consisting of: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22. In one embodiment, the antibody comprises (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22.

[0141] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from the group consisting of: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19; and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from the group consisting of: (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22.

[0142] In another aspect, the present invention provides an antibody comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (f) a CDR-L3 comprising an amino acid sequence selected from SEQ ID NO: 22.

[0143] In any of the above embodiments, the anti-RSV antibody is human or humanized.In one embodiment, the anti-RSV antibody comprises the CDRs as in any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0144] In another aspect, the anti-RSV antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 23. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-RSV antibody comprising the sequence retains the ability to bind to RSV. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 23. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the CDRs (i.e., in the FRs). Optionally, the anti-RSV antibody comprises the VH sequence in SEQ ID NO: 23, including post-translational modifications of the sequence. In a specific embodiment, the VH comprises one, two or three CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19.

[0145] In another aspect, an anti-RSV antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 24. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-RSV antibody comprising the sequence retains the ability to bind to RSV. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 24. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the CDR (i.e., in the FR). Optionally, the anti-RSV antibody comprises the VL sequence in SEQ ID NO: 24, including post-translational modifications of the sequence. In a specific embodiment, the VL comprises one, two or three CDRs selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22.

[0146] In another aspect, an anti-RSV antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 23 and SEQ ID NO: 24, respectively, including post-translational modifications of those sequences.

[0147] In another aspect, the present invention provides antibodies that bind to the same epitope as the anti-RSV antibodies provided herein. For example, in certain embodiments, antibodies that bind to the same epitope as an anti-RSV antibody comprising the VH sequence of SEQ ID NO: 23 and the VL sequence of SEQ ID NO: 24 are provided.

[0148] In one aspect, the present invention provides an anti-RSV antibody comprising at least one, two, three, four, five, or six CDRs selected from: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

[0149] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from the group consisting of: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27. In one embodiment, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27. In another embodiment, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30. In yet another embodiment, the antibody comprises a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30, and a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26. In yet another embodiment, the antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:25; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:26; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:27.

[0150] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL CDR sequences selected from the group consisting of: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30. In one embodiment, the antibody comprises (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

[0151] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from the group consisting of: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from the group consisting of: (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3Q.

[0152] In another aspect, the present invention provides an antibody comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (f) a CDR-L3 comprising an amino acid sequence selected from SEQ ID NO: 30.

[0153] In any of the above embodiments, the anti-RSV antibody is human or humanized.In one embodiment, the anti-RSV antibody comprises the CDRs as in any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0154] In another aspect, the anti-RSV antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 31. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-RSV antibody comprising the sequence retains the ability to bind RSV. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 31. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the CDRs (i.e., in the FRs). Optionally, the anti-RSV antibody comprises the VH sequence in SEQ ID NO: 31, including post-translational modifications of that sequence. In a specific embodiment, the VH comprises one, two or three CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27.

[0155] In another aspect, an anti-RSV antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 32. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-RSV antibody comprising the sequence retains the ability to bind to RSV. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 32. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the CDR (i.e., in the FR). Optionally, the anti-RSV antibody comprises the VL sequence in SEQ ID NO: 32, including post-translational modifications of the sequence. In a specific embodiment, the VL comprises one, two or three CDRs selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

[0156] In another aspect, an anti-RSV antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 31 and SEQ ID NO: 32, respectively, including post-translational modifications of those sequences.

[0157] In another aspect, the present invention provides antibodies that bind to the same epitope as the anti-RSV antibodies provided herein. For example, in certain embodiments, antibodies that bind to the same epitope as an anti-RSV antibody comprising the VH sequence of SEQ ID NO: 31 and the VL sequence of SEQ ID NO: 32 are provided.

[0158] In another aspect of the invention, the anti-RSV antibody according to any of the above embodiments is a monoclonal antibody, including chimeric, humanized or human antibodies. In one embodiment, the anti-RSV antibody is an antibody fragment, such as Fv, Fab, Fab', xFab, scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as one having substitutions L234A, L235A and P329G (LALA-PG) in the Fc region derived from a human IgG1 Fc region (see, e.g., WO2012 / 130831 A1).

[0159] In yet another aspect, an anti-RSV antibody according to any of the above embodiments may incorporate any of the features described in Sections 1-7 below, singly or in combination:

[0160] 1. Antibody affinity

[0161] In certain embodiments, the antibodies provided herein have a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, such as 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 M).

[0162] In one embodiment, Kd is measured by radiolabeled antigen binding assay (RIA). In one embodiment, RIA is performed using a Fab version of the antibody of interest and its antigen. For example, by titrating the antibody with a minimal concentration of ( 125I) Label the antigen to equilibrate the Fab, then use an anti-Fab antibody-coated plate to capture the bound antigen to measure the solution binding affinity of the Fab for the antigen (see, e.g., Chen et al., J. Mol. Biol. 293: 865-881 (1999)). To establish the conditions for the assay, Multiwell plates (Thermo Scientific) were coated overnight with 5 μg / ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and then blocked with 2% (w / v) bovine serum albumin in PBS at room temperature (approximately 23°C) for 2-5 hours. In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 The antigen is mixed with serially diluted Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation can be continued for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate and incubated at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed with 0.1% polysorbate 20 in PBS. Wash the plate 8 times. After the plate is dry, add 150 μl / well scintillation fluid (MICROSCINT-20 TM ; Packard), then in TOPCOUNT TM Plates were counted for 10 minutes on a gamma counter (Packard).Concentrations of each Fab that gave less than or equal to 20% of maximal binding were selected for competitive binding assays.

[0163] According to another embodiment, Kd is obtained using For example, at 25°C using an immobilized antigen CM5 chip at approximately 10 response units (RU) or The assay is performed using a biosensor chip (BIAcore, Inc., Piscataway, NJ). In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μl / min to obtain approximately 10 response units (RU) of coupled protein. Following antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, a 5% ethanolamine solution containing 0.05% polysorbate 20 (TWEEN-20) is injected at 25°C at a flow rate of approximately 25 μl / min. TM ) surfactant in PBS (PBST) two-fold serial dilution of Fab (0.78nM to 500nM). Using a simple one-to-one Langmuir binding model ( Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams to calculate the association rate (k on ) and dissociation rate (k off The equilibrium dissociation constant (Kd) is expressed as the ratio k off / k on Calculation. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10 according to the surface plasmon resonance assay above, 6 M -1 S -1 , the binding rate can be measured using the fluorescence quenching technique, i.e., on a spectrometer such as a spectrophotometer equipped with a cut-off flow device (Aviv Instruments) or a 8000 series SLM-AMINCO TM The increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS pH 7.2 at 25°C was measured in a spectrophotometer (ThermoSpectronic) using a stirred cuvette in the presence of increasing concentrations of antigen.

[0164] 2. Antibody fragments

[0165] In certain embodiments, the antibodies provided herein are antibody fragments. The term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that retains the ability to specifically bind to an antigen. Antibody fragments include, but are not limited to, Fab, Fab', Fab'SH, F(ab')2, xFab, Fv, single-chain Fab (scFab), single-chain variable fragment (scFv), and single-domain antibodies (dAb). For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23: 1126-1136 (2005).

[0166] In one embodiment, the antibody fragment is a Fab, Fab', Fab'SH, xFab, or F(ab')2 fragment, particularly a Fab fragment. Papain digestion of an intact antibody produces two identical antigen-binding fragments, termed "Fab" fragments, each containing the variable domains of the heavy and light chains, the constant domain of the light chain, and the first constant domain (CH1) of the heavy chain. Thus, the term "Fab fragment" refers to an antibody fragment comprising a light chain fragment including the VL domain and constant domain (CL) of the light chain, and the VH domain and first constant domain (CH1) of the heavy chain. The term "xFab fragment" refers to a Fab fragment in which the VH domain is exchanged with the VL domain, or the CH1 domain is exchanged with the CL domain. Fab' fragments differ from Fab fragments by having additional residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domains have free sulfhydryl groups. Pepsin treatment produces a F(ab')2 fragment with two antigen-binding sites (two Fab fragments) and a portion of the Fc region. For a discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-lives, see US Pat. No. 5,869,046.

[0167] In another embodiment, the antibody fragment is a diabody, a triabody, or a tetrabody. Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0168] In another embodiment, the antibody fragment is a single-chain Fab fragment." Single-chain Fab fragment" or "scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a joint, wherein the antibody domain and the joint have one of the following orders in the N-terminal to C-terminal direction: a) VH-CH1-joint-VL-CL, b) VL-CL-joint-VH-CH1, c) VH-CL-joint-VL-CH1 or d) VL-CH1-joint-VH-CL. In particular, the joint is a polypeptide having at least 30 amino acids, preferably between 32 and 50 amino acids. The single-chain Fab fragment is stabilized via the natural disulfide bond between the CL domain and the CH1 domain. In addition, interchain disulfide bonds are generated by inserting cysteine ​​residues (e.g., according to Kabat numbering at position 44 in the variable heavy chain and position 100 in the variable light chain), and these single-chain Fab molecules can be further stabilized.

[0169] In another embodiment, the antibody fragment is a single chain variable fragment (scFv). A "single chain variable fragment (scFv)" is a fragment of the heavy chain variable region (V) of an antibody connected by a linker. H ) and light chain variable region (V L In particular, the linker is a short polypeptide of 10 to 25 amino acids and is usually rich in glycine for flexibility, and serine or threonine for solubility, and can be V H The N-terminal and V L The C-terminus of the antibody is linked to the C-terminus of the antibody, or vice versa. Despite the removal of the constant region and the introduction of a linker, this protein retains the specificity of the original antibody. For a review of scFv fragments, see, for example, Pluckthün, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds. (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458.

[0170] In another embodiment, the antibody fragment is a single-domain antibody. A single-domain antibody is an antibody fragment that contains all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0171] Antibody fragments can be produced by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production in recombinant host cells (eg, E. coli or phage), as described herein.

[0172] 3. Chimeric and humanized antibodies

[0173] In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody, in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0174] In certain embodiments, chimeric antibodies are humanized antibodies. Generally, non-human antibodies are humanized to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibody. Generally, humanized antibodies include one or more variable domains, wherein HVR, such as CDR (or part thereof) are derived from non-human antibodies, and FR (or part thereof) are derived from human antibody sequences. Optionally, humanized antibodies also include at least a portion of human constant region. In some embodiments, some FR residues in humanized antibodies are replaced with corresponding residues from non-human antibodies (such as antibodies derived from HVR residues), for example, to restore or improve antibody specificity or affinity.

[0175] Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer 83:252-260 (2000) (describing the "guided selection" method of FR shuffling).

[0176] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al., J. Immunol. 151: 2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); and Presta et al., J. Immunol., 151: 2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and framework regions derived by screening FR libraries (see, e.g., Baca et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); and Presta et al., J. Immunol., 151: 2623 (1993)). al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[0177] 4. Human Antibodies

[0178] In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be generated using a variety of techniques known in the art. Generally, human antibodies are described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

[0179] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to an antigenic challenge. Such animals typically contain all or part of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or randomly integrated into the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). Also see, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE TM technology; U.S. Patent No. 5,770,429, which describes technology; U.S. Patent No. 7,041,870, which describes KM technology, and U.S. Patent Application Publication No. US 2007 / 0061900, which describes Technology). The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with a different human constant region.

[0180] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described (see, e.g., Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991)). Human antibodies produced via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Other methods include those described, for example, in U.S. Pat. No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which describes human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0181] It is also possible to generate human antibodies by separating the Fv clone variable domain sequence selected from the phage display library derived from humans.Then, such variable domain sequence can be combined with the desired human constant domain.The technology of selecting human antibodies from the antibody library is described below.

[0182] 5. Library-derived Antibodies

[0183] The antibodies of the present invention can be isolated by screening combinatorial libraries for antibodies with the desired one or more activities. Methods for screening combinatorial libraries are reviewed in, for example, Lerner et al., Nature Reviews 16: 498-508 (2016). For example, a variety of methods for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics are known in the art. Such methods are reviewed, for example, in Frenzel et al., mAbs 8: 1177-1194 (2016); Bazan et al., Human Vaccines and Immunotherapeutics 8: 1817-1828 (2012) and Zhao et al., Critical Reviews in Biotechnology 36: 276-289 (2016) and Hoogenboom et al., in Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001) and Marks and Bradbury, in Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003).

[0184] In certain phage display methods, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be screened for antigen-binding phage, as described in Winter et al., Annual Review of Immunology 12: 433-455 (1994). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, non-immune repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a large number of non-self and also self-antigens without any immunization, as described by Griffiths et al., EMBO Journal 12: 725-734 (1993). Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences encoding the highly variable CDR3 regions and achieving rearrangement in vitro, as described by Hoogenboom and Winter, Journal of Molecular Biology 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent Nos. 5,750,373; 7,985,840; 7,785,903 and 8,679,490 and U.S. Patent Publication Nos. 2005 / 0079574, 2007 / 0117126, 2007 / 0237764 and 2007 / 0292936.

[0185] Other examples of methods known in the art for screening combinatorial libraries for antibodies with one or more desired activities include ribosome and mRNA display, and methods for displaying and selecting antibodies on bacteria, mammalian cells, insect cells, or yeast cells. For a review of methods for yeast surface display, see, for example, Scholler et al., Methods in Molecular Biology 503: 135-56 (2012) and Cherf et al., Methods in Molecular biology 1319: 155-175 (2015) and Zhao et al., Methods in Molecular Biology 889: 73-84 (2012). For a description of methods for ribosome display, see, for example, He et al., Nucleic Acids Research 25: 5132-5134 (1997) and Hanes et al., PNAS 94: 4937-4942 (1997).

[0186] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0187] 6. Multispecific Antibodies

[0188] In certain embodiments, the antibody provided herein is a multispecific antibody, such as a bispecific antibody. A multispecific antibody is a monoclonal antibody that has binding specificity to at least two different sites (i.e., different epitopes on different antigens or different epitopes on the same antigen). In certain embodiments, a multispecific antibody has three or more binding specificities. In certain embodiments, one of the binding specificities is for RSV, and other (two or more) specificities are for any other antigen. In certain embodiments, a bispecific antibody can be in conjunction with two (or more) different antigens or epitopes of RSV. It is also possible to use multispecific (e.g., bispecific) antibodies to localize cytotoxic agents or cells to cells infected with RSV. Multispecific antibodies can be prepared with full-length antibodies or antibody fragments.

[0189] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature, 305:537 (1983)), and "knob-into-hole" engineering (see, e.g., U.S. Patent No. 5,731,168 and Atwell et al., J. Mol. Biol. 270:26 (1997)). It is also possible to engineer electrostatic manipulation effects for generating antibody Fc-heterodimer molecules (see, e.g., WO 2009 / 089004); cross-link two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); use of leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605); use of common light chain technology to circumvent the light chain mispairing problem (see, e.g., WO 98 / 50431); use of "diabody" technology for generating bispecific antibody fragments (see, e.g., Hollinger et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605). al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147:60 (1991). Multispecific antibodies are generated.

[0190] Also included herein are engineered antibodies with three or more antigen binding sites, including, for example, "octopus antibodies" or DVD-Ig (see, for example, WO 2001 / 77342 and WO 2008 / 024715). Other examples of multispecific antibodies with three or more antigen binding sites can be found in WO 2010 / 115589, WO 2010 / 112193, WO 2010 / 136172, WO2010 / 145792, and WO 2013 / 026831. Bispecific antibodies or their antigen-binding fragments also include "dual action FAb" or "DAF" comprising an antigen binding site in conjunction with RSV and another different antigen, or two different epitopes of RSV (see, for example, US 2008 / 0069820 and WO 2015 / 095539).

[0191] Multispecific antibodies can also be provided in an asymmetric format having one or more domains in the binding arms with the same antigen specificity exchanged, i.e., by exchanging VH / VL domains (see, e.g., WO 2009 / 080252 and WO 2015 / 150447), CH1 / CL domains (see, e.g., WO 2009 / 080253), or entire Fab arms (see, e.g., WO 2009 / 080251, WO 2016 / 016299, see also Schaefer et al., PNAS, 108 (2011) 1187-1191, and Klein et al., MAbs 8 (2016) 1010-20). In one embodiment, the multispecific antibody comprises a crossover Fab fragment. The term "crossover Fab fragment" or "xFab fragment" or "crossover Fab fragment" refers to a Fab fragment in which the variable regions or constant regions of the heavy and light chains are exchanged. The crossover Fab fragment contains a polypeptide chain consisting of a light chain variable region (VL) and a heavy chain constant region (CH1), and a polypeptide chain consisting of a heavy chain variable region (VH) and a light chain constant region (CL). Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations into the domain interfaces to guide correct Fab pairing. See, for example, WO 2016 / 172485.

[0192] Various additional molecular formats of multispecific antibodies are known in the art and are encompassed herein (see, e.g., Spiess et al., Mol Immunol 67 (2015) 95-106).

[0193] 7. Antibody variants

[0194] In certain embodiments, amino acid sequence variants of the antibodies provided herein are encompassed. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Suitable modifications may be introduced into the nucleotide sequence encoding the antibody, or the amino acid sequence variants of the antibody may be prepared by peptide synthesis. Such modifications include, for example, deletion of the residues in the amino acid sequence of the antibody, and / or insertion and / or substitution. Any combination of deletion, insertion, and substitution may be performed to obtain the final construct, as long as the final construct possesses desired characteristics, such as antigen binding.

[0195] a) Substitution, insertion, and deletion variants

[0196] In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table A under the heading "Preferred Substitutions." More substantial changes are provided in Table A under the heading "Exemplary Substitutions," and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the product screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0197] Table A

[0198] According to common side chain properties, amino acids can be grouped as follows:

[0199] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0200] (2) Neutral, hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0201] (3) Acidic: Asp, Glu;

[0202] (4) Basic: His, Lys, Arg;

[0203] (5) Residues that affect chain orientation: Gly, Pro;

[0204] (6) Aromatic: Trp, Tyr, Phe.

[0205] Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.

[0206] A class of substitution variants involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further study will have changes (e.g., improvements) in certain biological properties relative to the parent antibody (e.g., increased affinity, reduced immunogenicity) and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which can be conveniently generated, for example, using affinity maturation techniques based on phage display such as those described herein. In short, one or more HVR residues are mutated, and the variant antibody is displayed on phage and screened for specific biological activity (e.g., binding affinity).

[0207] Changes (e.g., substitutions) can be made to the HVRs, for example, to improve antibody affinity. Such changes can be made to HVR "hot spots," residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and / or residues that contact the antigen, with the resulting variant VH or VL tested for binding affinity. Affinity maturation by construction and reselection of secondary libraries has been described, for example, by Hoogenboom et al., in Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). Then, a secondary library is created. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves an HVR-guided approach in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0208] In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind to antigen. For example, conservative changes (e.g., conservative substitutions, as provided herein) may be made to HVRs that do not substantially reduce binding affinity. Such changes may be, for example, outside the antigen contact residues in the HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unchanged or contains no more than 1, 2, or 3 amino acid substitutions.

[0209] A method that can be used to identify residues or regions in an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or a group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitutions. Alternatively or additionally, the crystal structure of the antigen-antibody complex can be used to identify contact points between the antibody and the antigen. Such contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. The variants can be screened to determine whether they contain the desired properties.

[0210] Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from 1 residue to polypeptides containing 100 or more residues, and intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N or C terminus of an antibody with an enzyme (e.g., for ADEPT) or a polypeptide that extends the serum half-life of the antibody.

[0211] b) Glycosylation variants

[0212] In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of antibody glycosylation. Addition or deletion of glycosylation sites of an antibody can be conveniently achieved by altering the amino acid sequence such that one or more glycosylation sites are created or eliminated.

[0213] In the case where the antibody comprises an Fc region, the oligosaccharides attached thereto can be altered. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally attached to Asn297 of the CH2 domain of the Fc region via an N-linkage. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "backbone" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the invention can be modified to create antibody variants with certain improved properties.

[0214] In one embodiment, antibody variants are provided that have non-fucosylated oligosaccharides, i.e., oligosaccharide structures lacking fucose, attached (directly or indirectly) to the Fc region. Such non-fucosylated oligosaccharides (also referred to as "afucosylated" oligosaccharides) are particularly N-linked oligosaccharides that lack the fucose residue attached to the first GlcNAc in the backbone of the biantennary oligosaccharide structure. In one embodiment, antibody variants are provided that have an increased proportion of non-fucosylated oligosaccharides in the Fc region compared to a native or parent antibody. For example, the proportion of non-fucosylated oligosaccharides can be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., the absence of fucosylated oligosaccharides). The percentage of non-fucosylated oligosaccharides is the (average) amount of oligosaccharides lacking fucose residues relative to the sum of all oligosaccharides attached to Asn297 (e.g., complex, hybrid, and high-mannose structures), as measured by MALDI-TOF mass spectrometry, as described, for example, in WO 2006 / 082515. Asn297 refers to the asparagine residue located at approximately position 297 (EU numbering of Fc region residues) in the Fc region; however, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such antibodies with an increased proportion of non-fucosylated oligosaccharides in the Fc region may have improved FcRγIIIa receptor binding and / or improved effector function, particularly improved ADCC function. See, for example, US 2003 / 0157108; US 2004 / 0093621.

[0215] Examples of cell lines capable of producing antibodies with reduced fucosylation include protein fucosylation-deficient Lec13 CHO cells (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US 2003 / 0157108; and WO 2004 / 056312, particularly in Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2004 / 056312). 2003 / 085107), or cells in which GDP-fucose synthesis or transporter activity is reduced or abolished (see, e.g., US2004259150, US2005031613, US2004132140, US2004110282).

[0216] In another embodiment, antibody variants having bisected oligosaccharides are provided, for example, biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function as described above. Examples of such antibody variants are described in, for example, Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99 / 54342; WO 2004 / 065540, WO 2003 / 011878.

[0217] Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0218] c) Fc region variants

[0219] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0220] In certain embodiments, the present invention encompasses antibody variants that possess some, but not all, effector functions, making them desirable candidates for applications where the in vivo half-life of the antibody is important, while certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduction / ablation of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore likely lacks ADCC activity), but retains FcRn binding ability. NK cells, the primary cells mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991). Non-limiting examples of in vitro assays for evaluating ADCC activity of a molecule of interest are described in U.S. Pat. Nos. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, e.g., the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA; and CytoTox Non-radioactive cytotoxicity assays (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example, in a animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind to C1q and, therefore, lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006); WO 2013 / 120929 A1).

[0221] Antibodies with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 ( U.S. Patent No. 6,737,056 ). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine ( U.S. Patent No. 7,332,581 ).

[0222] Certain antibody variants with improved or decreased binding to FcRs have been described (see, eg, US Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0223] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues).

[0224] In certain embodiments, the antibody variants comprise an Fc region having one or more amino acid substitutions that improve FcRn binding, such as substitutions at positions 252, and / or 254, and / or 256 (EU residue numbering) of the Fc region. In certain embodiments, the antibody variants comprise an Fc region having amino acid substitutions at positions 252, 254, and 256. In one embodiment, the substitutions are M252Y, S254T, and T256E in an Fc region derived from a human IgG1 Fc region.

[0225] In certain embodiments, the antibody variants include an Fc region with an amino acid substitution that reduces FcγR binding, such as substitutions at positions 234 and 235 (EU residue numbering) of the Fc region. In one embodiment, the substitutions are L234A and L235A (LALA). In certain embodiments, the antibody variants further include D265A and / or P329G in an Fc region derived from a human IgG1 Fc region. In one embodiment, the substitutions are L234A, L235A, and P329G (LALA-PG) in an Fc region derived from a human IgG1 Fc region (see, e.g., WO 2012 / 130831 A1). In another embodiment, the substitutions are L234A, L235A, and D265A (LALA-DA) in an Fc region derived from a human IgG1 Fc region.

[0226] In some embodiments, alterations are made to the Fc region that result in altered (i.e., improved or decreased) C1q binding and / or complement-dependent cytotoxicity (CDC), such as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0227] Antibodies with extended half-life and improved binding to the neonatal Fc receptor (FcRn) are described in US 2005 / 0014934A1 (Hinton et al.), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). These antibodies comprise an Fc region with one or more substitutions that improve Fc region binding to FcRn. Such Fc variants include those having substitutions at one or more of Fc region residues 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434, for example, substitution of Fc region residue 434 (see, e.g., U.S. Patent No. 7,371,826; Dall'Acqua, WF, et al. J. Biol. Chem. 281 (2006) 23514-23524).

[0228] Fc region residues critical for mouse Fc-mouse FcRn interaction have been identified by site-directed mutagenesis (see, e.g., Dall'Acqua, WF, et al. J. Immunol 169 (2002) 5171-5180). Residues I253, H310, H433, N434, and H435 (EU numbering according to Kabat) are involved in the interaction (Medesan, C. et al., Eur. J. Immunol. 26 (1996) 2533; Firan, M. et al., Int. Immunol. 13 (2001) 993; Kim, JK et al., Eur. J. Immunol. 24 (1994) 542). Residues I253, H310, and H435 were found to be crucial for the interaction of human Fc with mouse FcRn (Kim, JK et al., Eur. J. Immunol. 29 (1999) 2819). Studies on the human Fc-human FcRn complex have shown that residues I253, S254, H435, and Y436 are crucial for the interaction (Firan, M. et al., Int. Immunol. 13 (2001) 993; Shields, R. Let al., J. Biol. Chem. 276 (2001) 6591-6604). Various mutants of residues 248 to 259 and 301 to 317 and 376 to 382 and 424 to 437 have been reported and examined (Yeung, YA et al., J. Immunol. 182 (2009) 7667-7671).

[0229] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, such as substitutions at positions 253, and / or 310, and / or 435 (EU residue numbering) of the Fc region. In certain embodiments, the antibody variant comprises an Fc region having amino acid substitutions at positions 253, 310, and 435. In one embodiment, the substitutions are I253A, H310A, and H435A in an Fc region derived from a human IgG1 Fc region. See, e.g., Grevys, A. et al., J. Immunol. 194 (2015) 5497-5508.

[0230] In certain embodiments, the antibody variants comprise an Fc region having one or more amino acid substitutions that reduce FcRn binding, such as substitutions at positions 310, and / or 433, and / or 436 (EU residue numbering) of the Fc region. In certain embodiments, the antibody variants comprise an Fc region having amino acid substitutions at positions 310, 433, and 436. In one embodiment, the substitutions are H310A, H433A, and Y436A in an Fc region derived from a human IgG1 Fc region (see, e.g., WO 2014 / 177460 A1).

[0231] See also Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351, which concern other examples of Fc region variants.

[0232] B. Recombinant Methods and Compositions

[0233] Antibodies can be produced using recombinant methods and compositions, such as those described in US 4,816, 567. For these methods, one or more isolated nucleic acids encoding the antibodies are provided.

[0234] In the case of natural antibodies or natural antibody fragments, two nucleic acids are required, one for the light chain or its fragment and one for the heavy chain or its fragment. Such nucleic acid encoding comprises the amino acid sequence of the antibody VL and / or comprises the amino acid sequence of VH (e.g., the light and / or heavy chain of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors.

[0235] In the case of a bispecific antibody with a heterodimeric heavy chain, four nucleic acids are required, one for the first light chain, one for the second light chain comprising the first heteromonomer Fc region polypeptide, one for the second light chain, and one for the second heavy chain comprising the second heteromonomer Fc region polypeptide. The four nucleic acids can be contained in one or more nucleic acid molecules or expression vectors. Such nucleic acids encode the amino acid sequence of the first VL of the antibody and / or the amino acid sequence of the first VH comprising the first heteromonomer Fc region and / or the amino acid sequence of the second VL and / or the amino acid sequence of the second VH comprising the second heteromonomer Fc region (e.g., the first and / or second light and / or first and / or second heavy chain of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors. Normally, these nucleic acids are located on two or three expression vectors, i.e., one vector can contain more than one of these nucleic acids. Examples of these bispecific antibodies are CrossMab and T cell bispecific agents (see, for example, Schaefer, W. et al, PNAS, 108 (2011) 11187-1191). For example, one of the heteromonomer heavy chains comprises a so-called "knob mutation" (T366W and optionally one of S354C or Y349C) and the other comprises a so-called "hole mutation" (T366S, L368A and Y407V and optionally Y349C or S354C) (see e.g. Carter, P. et al., Immunotechnol. 2 (1996) 73).

[0236] In one embodiment an isolated nucleic acid encoding the antibody used in the methods reported herein is provided.

[0237] In yet another embodiment, one or more vectors (eg, expression vectors) comprising such nucleic acids are provided.

[0238] In yet another embodiment, a host cell comprising such nucleic acid is provided.

[0239] In one such embodiment, the host cell comprises (e.g., has been transformed with):

[0240] In the case of antibodies composed of two identical light chains and two identical heavy chains forming disulfide bonds, their VH and VL containing fragments:

[0241] (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL and an amino acid sequence comprising an antibody VH, or

[0242] (2) A first vector and a second vector, wherein the first vector comprises a nucleic acid encoding an amino acid sequence comprising antibody VL, and the second vector comprises a nucleic acid encoding an amino acid sequence comprising antibody VH.

[0243] - In the case of bispecific antibodies with heterodimeric heavy chains:

[0244] (1) a first vector comprising a first pair of nucleic acids encoding the following amino acid sequences, one of which comprises a first VL and the other comprises a first VH of an antibody, and a second vector comprising a second pair of nucleic acids encoding the following amino acid sequences, one of which comprises a second VL and the other comprises a second VH of an antibody, or

[0245] (2) a first vector comprising a first nucleic acid encoding an amino acid sequence comprising one of the variable domains (preferably a light chain variable domain), a second vector comprising a pair of nucleic acids encoding amino acid sequences, one of which comprises a light chain variable domain and the other comprises a first heavy chain variable domain, and a third vector comprising a pair of nucleic acids encoding amino acid sequences, one of which comprises the other light chain variable domain corresponding to the second vector and the other comprises the second heavy chain variable domain, or

[0246] (3) A first vector comprising a nucleic acid encoding an amino acid sequence comprising a first VL of an antibody, a second vector comprising a nucleic acid encoding an amino acid sequence comprising a first VH of an antibody, a third vector comprising a nucleic acid encoding an amino acid sequence comprising a second VL of an antibody, and a fourth vector comprising a nucleic acid encoding an amino acid sequence comprising a second VH of an antibody.

[0247] In one embodiment, the host cell is eukaryotic, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method for generating an anti-RSV antibody is provided, wherein the method includes culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expressing the antibody, as provided above, and optionally, recovering the antibody from the host cell (or host cell culture fluid).

[0248] For the recombinant generation of anti-RSV antibodies, the nucleic acid (such as described above) of the encoding antibody is separated, and inserted into one or more vectors, to further clone and / or express in a host cell. Such nucleic acid can be easily separated and sequenced using conventional procedures (such as by using oligonucleotide probes that can specifically bind the gene encoding antibody heavy chain and light chain), or generated by recombinant method or obtained by chemical synthesis.

[0249] Host cells suitable for cloning or expressing antibody encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies can be generated in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, US 5,648,237, US 5,789,199 and US 5,840,523 (also see Charlton, KA, in Methods in Molecular Biology, Vol. 248, Lo, BKC ed., Humana Press, Totowa, NJ (2003), pp. 245-254, which describes expression of antibody fragments in Escherichia coli (E. coli.). After expression, the antibody can be separated from the bacterial cell mass in a soluble fraction and can be further purified.

[0250] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partially or fully human glycosylation patterns. See Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414; Li et al., Nat. Biotech. 24 (2006) 210-215.

[0251] Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used with insect cells, particularly for transfecting Spodoptera frugiperda cells.

[0252] Plant cell cultures can also be used as hosts. See, for example, US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978 and US 6,417,429 (which describe plant cell cultures for producing antibodies in transgenic plants). TM technology).

[0253] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney line (293 or 293 cells, as described, for example, in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, in Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); non-derived green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, in Mather, JP et al., Biol. Reprod. 23 (1980) 243-252. et al., Annals NY Acad. Sci. 383 (1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC ed., Humana Press, Totowa, NJ (2004), pp. 255-268.

[0254] C. Assay

[0255] The anti-RSV antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art.

[0256] 1. Binding assays and other assays

[0257] In one aspect, the antibodies of the invention are tested for their antigen binding activity, for example, by known methods such as ELISA, Western blotting, and the like.

[0258] On the other hand, competition assays can be used to identify antibodies that compete for the combination of RSV or its antigens with reference to anti-RSV antibodies. In certain embodiments, such competitive antibodies bind to the same epitope (e.g., linear or conformational epitope) as that bound by an anti-RSV antibody. Detailed exemplary methods for locating antibody-bound epitopes are described in Morris (1996) "Epitope Mapping Protocols," Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0259] In an exemplary competitive assay, immobilized RSV or its antigen is incubated in a solution comprising a first labeled antibody (which binds RSV or its antigen) (e.g., with reference to an anti-RSV antibody) and a second unlabeled antibody (which is to test the ability of the first antibody to compete with the first antibody for the binding of RSV or its antigen). The second antibody may be present in a hybridoma supernatant. As a control, immobilized RSV or its antigen is incubated in a solution comprising the first labeled antibody but not comprising the second unlabeled antibody. After incubation under conditions allowing the first antibody to bind RSV or its antigen, excess unbound antibody is removed, and the amount of the label associated with immobilized RSV or its antigen is measured. If the amount of the label associated with immobilized RSV or its antigen in the test sample is substantially reduced compared to the control sample, this indicates that the second antibody competes with the first antibody for the binding of RSV or its antigen. Referring to Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0260] 2. Activity Assay

[0261] On the one hand, an assay method for identifying anti-RSV antibodies with biological activity is provided. Biological activity can include, for example, that anti-RSV antibodies suppress RSV infection of host cells. Antibodies with this type of biological activity in vivo and / or in vitro are also provided.

[0262] D. Methods and compositions for diagnosis and detection

[0263] In certain embodiments, any anti-RSV antibody provided herein can be used to detect the presence of RSV in a biological sample. When used herein, the term "detection" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues, such as tumor tissue.

[0264] In one embodiment, the anti-RSV antibody used in diagnosis or detection method is provided.On the other hand, the method for detecting the existence of RSV in biological sample is provided.In certain embodiments, the method is included in allowing the anti-RSV antibody to bind RSV or its antigen and making the biological sample contact with the anti-RSV antibody, as described herein, and detecting whether to form a complex between the anti-RSV antibody and RSV or its antigen.Such method can be an in vitro or in vivo method.In one embodiment, anti-RSV antibody is used to select a subject suitable for treating with anti-RSV antibodies, for example, wherein the patient is infected with RSV, or suspects infection with RSV, or has the risk of contacting RSV.

[0265] In certain embodiments, labeled anti-RSV antibodies are provided. Labels include, but are not limited to, directly detected labels or modules (such as fluorescence, chromogenic, electron-dense, chemiluminescent, and radioactive labels), and modules such as enzymes or ligands that are indirectly detected, for example, via enzyme reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes. 32 P, 14 C, 125 I, 3 H, and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase (which are coupled to enzymes such as HRP that use hydrogen peroxide to oxidize dye precursors), lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0266] E. Pharmaceutical Formulations

[0267] Pharmaceutical formulations of anti-RSV antibodies as described herein are prepared by mixing such antibodies having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)) in the form of lyophilized formulations or aqueous solutions. Generally, pharmaceutically acceptable carriers are non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) Polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further comprise interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinase.

[0268] Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO 2006 / 044908, the latter formulation comprising a histidine-acetate buffer.

[0269] The formulations herein may also contain more than one active ingredient necessary for the particular indication being treated, preferably those whose activities complement each other and do not adversely affect each other. Such active ingredients are suitably present in combination in amounts effective for the desired purpose.

[0270] The active ingredient can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A., ed. (1980).

[0271] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg films, or microcapsules.

[0272] Formulations for in vivo administration are generally sterile. Sterility can be readily accomplished, for example, by filtration through sterile filtration membranes.

[0273] F. Prophylactic, Therapeutic Methods and Compositions

[0274] Any of the anti-RSV antibodies provided herein can be used in prophylactic or therapeutic methods.

[0275] In one aspect, the anti-RSV antibody used as a medicine is provided. In other aspects, the anti-RSV antibody used in preventing or treating RSV infection is provided. In certain embodiments, the anti-RSV antibody used in a prevention or treatment method is provided. In certain embodiments, the invention provides the anti-RSV antibody used in the method for preventing an individual with RSV infection risk, the prevention comprising administering an effective amount of the anti-RSV antibody to the individual. In certain embodiments, the invention provides the anti-RSV antibody used in the method for treating an individual with RSV infection, the treatment comprising administering an effective amount of the anti-RSV antibody to the individual. In one embodiment, the antibody is used to treat RSV infection or delay its progression.

[0276] According to any of the above embodiments, the "individual" is preferably a human. In another aspect, the present invention provides the use of an anti-RSV antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is for use in treating RSV infection. In another embodiment, the medicament is for use in a method of treating RSV infection, comprising administering an effective amount of the medicament to an individual with RSV infection. According to any of the above embodiments, the "individual" can be a human.

[0277] As used herein, the term "RSV infection" may be, for example, a lower respiratory tract infection.

[0278] In yet another aspect, the present invention provides a method for treating RSV infection. In one embodiment, the method comprises administering an effective amount of an anti-RSV to an individual with cancer. According to any of the above embodiments, the "individual" can be a human.

[0279] In yet another aspect, the invention provides a pharmaceutical formulation comprising any of the anti-RSV antibodies provided herein, eg, for use in any of the above-described therapeutic methods. In one embodiment, the pharmaceutical formulation comprises any of the anti-RSV antibodies provided herein and a pharmaceutically acceptable carrier.

[0280] In yet another aspect, the invention provides pharmaceutical formulations comprising any anti-RSV antibody provided herein, for example, used in any of the above-mentioned therapeutic methods. In one embodiment, pharmaceutical formulations comprise any anti-RSV antibody provided herein and a pharmaceutically acceptable carrier. In another embodiment, pharmaceutical formulations comprise any anti-RSV antibody provided herein and at least one other therapeutic agent. In one embodiment, the at least one other therapeutic agent is, for example, another anti-RSV antibody that binds to a different epitope of RSV with the anti-RSV antibody herein.

[0281] The antibodies of the present invention can be used alone or in combination with other agents in therapy. For example, the antibodies of the present invention can be co-administered with at least one other therapeutic agent.

[0282] Such combination therapies noted above encompass both co-administration (wherein two or more therapeutic agents are contained in the same or different formulations), and separate administration, in which case administration of the antibodies of the invention can occur before, simultaneously with, and / or after administration of one or more other therapeutic agents. In one embodiment, administration of the anti-RSV antibody and administration of the other therapeutic agent occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other.

[0283] The antibodies of the invention (and any other therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and if desired for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules are contemplated herein, including but not limited to single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

[0284] The antibodies of the present invention should be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this regard include the specific condition being treated, the specific mammal being treated, the clinical status of the individual patient, the cause of disease, the site of drug delivery, the method of administration, the dosing schedule, and other factors known to medical practitioners. The antibodies need not be, but may optionally be, formulated with one or more agents currently used to prevent or treat the condition. The effective amount of the other agents described above depends on the amount of the antibody present in the formulation, the type of condition or treatment, and other factors discussed above. These agents are generally used in the same dosages and routes of administration as described herein, or in about 1-99% of the dosages described herein, or in any dosage and by any route, which dosage and route are empirically / clinically determined to be appropriate.

[0285] For the prevention or treatment of disease, the appropriate dosage of the antibodies of the present invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, the preventive or therapeutic purpose of the antibody administered, previous treatment, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitable for administration to the patient in one or a series of treatments. Depending on the type and severity of the disease, about 1 μg / kg-15 mg / kg (e.g., 0.1 mg / kg-10 mg / kg) of the antibody can be administered to the patient as a first candidate dose, whether, for example, by one or more separate administrations or by continuous infusion. Depending on the factors mentioned above, a typical daily dose may be in the range of about 1 μg / kg-100 mg / kg or more. For repeated administration over several days or longer, depending on the condition, treatment will generally continue until the desired suppression of disease symptoms occurs. An exemplary dosage of the antibody will be in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) can be administered to the patient. The above doses can be administered intermittently, such as once a week or every three weeks (such as so that the patient receives about 2 to about 20 doses, or, for example, about 6 doses of the antibody). An initial higher loading dose can be administered, followed by one or more lower doses. An exemplary dosing regimen includes administration. However, other dosing regimens may be useful. The progress of the treatment is easily monitored by conventional techniques and assays.

[0286] It will be understood that any of the above-described formulations or therapeutic methods can be practiced using the immunoconjugates of the invention in place of or in addition to anti-RSV antibodies.

[0287] G. Products

[0288] In another aspect of the present invention, an article of manufacture is provided that contains materials useful for treating, preventing, and / or diagnosing the conditions described above. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition that is effective for treating, preventing, and / or diagnosing the condition, alone or in combination with another composition, and can have a sterile access port (e.g., the container can be a vial or intravenous solution bag with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody of the present invention. The label or package insert indicates that the composition is to be used to treat a selected condition. Additionally, the article of manufacture may comprise: (a) a first container containing a composition comprising an antibody of the present invention; and (b) a second container containing a composition comprising another therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the composition can be used to treat a specific condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0289] Description of amino acid and nucleic acid sequences

[0290] The present invention provides a group of antibodies having an affinity for RSV A2 pre-F protein ranging from 0.001 nM to 3.25 nM, a neutralization activity IC50 against RSV A2 strain ranging from 0.34 ng / mL to 80.04 ng / mL, a neutralization activity IC50 against RSV B9320 strain ranging from 3.65 ng / mL to 153.90 ng / mL, and a neutralization activity IC50 against RSV B18537 strain ranging from 1.87 ng / mL to 67.03 ng / mL.

[0291] Example 1: Preparation of anti-RSV antibodies

[0292] Peripheral blood was collected from healthy adult volunteers, and density gradient centrifugation was used to obtain the upper plasma layer and the middle PBMC fraction. Memory B cells that specifically bind to the pre-F protein were isolated from the PBMCs by flow cytometry using a fluorescently labeled pre-F protein. Nested PCR was used to obtain a transfectable PCR fragment with expression activity. This fragment was then transfected into CHO cells for expression, yielding cell supernatants containing secreted antibodies. Binding activity was screened by ELISA, resulting in the identification of hundreds of pre-F binding-positive clones. Several recombinant antibodies were tested for neutralizing activity against RSV A2. Based on the neutralizing activity of each recombinant antibody against RSV A2, RSV 9320, and RSV 18537, as well as their affinity for the RSV A2 pre-F protein, four antibodies were ultimately selected: F6-10, F6-18, F6-113, and F6-125.

[0293] The amino acid sequences of the antibody CDRs are shown in Table 1.

[0294] Table 1: Amino acid sequences of CDRs of anti-RSV antibodies

[0295] The amino acid sequences of the antibodies VH, VL, and CL are shown in Table 2.

[0296] Table 2: Amino acid sequences of VH, VL and CL of anti-RSV antibodies

[0297] The coding sequences of the light and heavy chain variable regions of the antibodies were cloned into a eukaryotic expression vector carrying the coding sequence of the human IgG1 constant region, transiently transfected into CHO cells for secretory expression, and antibody proteins with a purity of >90% were obtained by protein A affinity purification, namely F6-10, F6-18, F6-113 and F6-125.

[0298] Amino acid sequence of human IgG1 constant region:

[0299] RSV A2 pre-F amino acid sequence:

[0300] Example 2: Characterization of anti-RSV antibodies - Characterization of antigen binding activity by ELISA

[0301] RSV A2 pre-F protein (manufactured by Vazyme), diluted to 2 μg / mL in carbonate buffer (pH 9.6), was added to a 96-well microtiter plate (NEST, 504201) at 100 μL / well and coated overnight at 4°C. The plate was then removed, washed twice with PBST, and blocked with blocking buffer (PBS + 5% BSA) at 37°C for 2 hours. The plate was then removed, and the antibody, serially diluted three-fold in diluent (PBS + 5% BSA) (starting at 5 μg / mL, 10 steps total), was added at 100 μL / well and incubated at 37°C for 1 hour. The plate was then removed, washed three times with PBST, and 100 μL of a 1:10,000 diluted mouse anti-human IgG Fc-HRP (manufactured by Vazyme) was added to each well and incubated at 37°C for 1 hour. The plate was then removed, washed three times with PBST, and 100 μL of the chromogenic substrate TMB was added to each well and incubated at 37°C for 10 minutes in the dark. The solution was removed, the cells were washed three times with PBST, and 50 μL of 2M sulfuric acid was added to each well. The OD value at 450 nm was measured on a multifunctional microplate reader (Tecan, Spark). A four-parameter fit was performed to calculate the EC50 value (ng / mL) of the antibody's antigen-binding activity. The results are shown in Tables 3.1 and 3.2 (Tables 3.2 and 3.1 are the results of different experiments, and the only difference in the experimental procedures in Table 3.2 is the "antibody was serially diluted 2-fold with diluent (PBS + 5% BSA) (starting concentration 1 μg / mL, a total of 14 gradients)"). Antibody concentration and OD value were plotted using GraphPad Prism 8.0, and the results are shown in Figures 1-1 and 1-2.

[0302] Table 3.1: EC50 values ​​(ng / mL) of RSV A2 pre-F binding activity of anti-RSV antibodies

[0303] Table 3.2: EC50 values ​​(ng / mL) of RSV A2 pre-F binding activity of antibody F6-18

[0304] Example 3: Characterization of anti-RSV antibodies - affinity characterization by BLI

[0305] The affinity curves for the antibodies were obtained using bio-layer interferometry (BLI) using the Octet Protein Analysis System (SARTORIUS, Octet R8). The sensor used HIS1K (SARTORIUS, 18-5120), the capture agent was 47.6 nM RSV A2 pre-F protein, the immobilization time was 120 seconds, the antibody concentrations were 500 nM, 167 nM, 56 nM, 19 nM, 6 nM, 2 nM, and 0.69 nM, the association time was 60 seconds, the dissociation time was 120 seconds, and the regeneration solution was 10 mM glycine-HCl pH 1.5 with a regeneration time of 180 seconds. The KD values ​​(M) for the antigen-binding affinities of the antibodies were calculated using Octet Analvsis Studio 12.2. The results are shown in Tables 4.1 and 4.2. The software Octet BLI Discovery 12.2 was used for recording, and the results are shown in Figures 2-1 and 2-2.

[0306] Table 4.1: KD values ​​(M) for RSV A2 pre-F binding affinities of anti-RSV antibodies

[0307] Table 4.2: KD values ​​(M) for RSV A2 pre-F binding affinity of antibody F6-18

[0308] Example 4: Characterization of anti-RSV antibodies - Characterization of neutralization activity by in vitro microneutralization assay

[0309] Antibodies serially diluted 3-fold with PBS + 5% HIFBS (starting concentration 0.5 μg / mL, 7 steps in total) were added at 60 μL / well to a 96-well plate (NEST, 701001) containing viruses (RSV A2, ATCC VR-1540; RSV B9320, ATCC VR-955; RSV B18537, ATCC VR-1580) (60 μL / well, 500 pfu) and incubated in a cell culture incubator (37°C, 5% CO2) for 1 hour. 50 μL / well of the above mixture was plated onto a 96-well plate (NEST, 701001) containing Hep2 cells at a confluence of 90% (3 × 10 cells were plated the day before). 5Cells were plated at 400 cells / mL and incubated in a cell culture incubator (37°C, 5% CO2) for 2 hours. The supernatant was removed, and 100 μL of DMEM medium supplemented with 2% FBS and 1% penicillin-streptomycin (Solaibio, P1400) was added to each well and incubated for another 21-22 hours. The supernatant was removed, and 100 μL of 4% paraformaldehyde was added to each well to fix the cells for 10 minutes. The paraformaldehyde was removed, and the cells were washed once with 250 μL of PBS per well. 100 μL of PBS + 4% BSA was added to each well and blocked for 30 minutes in a cell culture incubator (37°C, 5% CO2). The blocking solution was removed, and 50 μL of 3D3 fluorescent antibody diluted to 5 μg / mL (manufactured by Vazyme) was added to each well and incubated in a cell culture incubator (37°C, 5% CO2) for 1 hour. The antibodies were removed, and the cells were washed three times with PBST and spun dry. The number of spots in each well was read using a fluorescent (enzyme-linked) immunospot analyzer (CTL, S6 Ultra M2). Wells with cells added alone (cell wells) were designated as negative wells, and wells with virus added alone (virus wells) were designated as positive wells.

[0310] GraphPad Prism 8.0 was used to plot antibody concentration against neutralization efficiency, as shown in Figures 3-1 and 3-2. Four-parameter fitting was performed to calculate the IC50 values ​​(ng / mL) of the virus-neutralizing activity of the antibodies, as shown in Tables 5.1 and 5.2.

[0311] Table 5.1: IC50 values ​​of virus neutralization activity of anti-RSV antibodies (ng / mL)

[0312] Table 5.2: IC50 values ​​of virus neutralization activity of antibody F6-18 (ng / mL)

[0313] Example 5: Characterization of anti-RSV antibodies - Characterization of epitopes by competition binding

[0314] RSV A2 pre-F protein was diluted to 10 μg / mL with a buffer solution (PBS + 0.02% Tween 20 + 0.1% BSA), and the antibody was diluted to 200 nM with the above buffer solution, both added at 200 μL / well. As shown in Figure 4, the above buffer solution was run on the Octet Protein Analysis System (SARTORIUS, OctetR8) for 120 seconds to reach the baseline level, the antigen was run until the signal reached 0.3 nm, the above buffer solution was run for 60 seconds to reach the baseline level, the first antibody was run for 180 seconds, and the second antibody was run for 180 seconds (the same concentration of the first antibody was added to the second antibody to prevent false positive results caused by the dissociation of the first antibody when the second antibody binds in the case where the first antibody is a fast-dissociating antibody). Repeat three times, alternating between running the regeneration solution (10 mM glycine-HCl, pH 1.5) and the buffer solution for a total of 20 seconds. Repeat the above cycle.

[0315] Data Analysis:

[0316] The second signal when the first antibody is Ab1 and the second antibody is Ab2+Ab1 is set as H1'2;

[0317] The second signal when the first antibody is Ab1 and the second antibody is Ab1 is set to H1'1;

[0318] but

[0319] The second signal is H1, = H1'2 - H1'1, when the first antibody is Ab1 and the second antibody is Ab2;

[0320] The second signal when the first antibody is 0 and the second antibody is Ab2 is set to H2;

[0321] but

[0322] The inhibition rate of the first antibody on the second antibody is: 1-(H1 / H2)×100%

[0323] (The two antibodies were swapped to calculate the inhibition rate of the second antibody on the first antibody).

[0324] Experimental validity judgment:

[0325] The self-reaction signal is less than 20%; otherwise, the data is invalid.

[0326] Judgment criteria:

[0327] >70%: Perfect competition, only one direction needs to meet this standard;

[0328] 30%-70%: partial competition;

[0329] <30%: No competition at all, only one direction needs to reach this standard.

[0330] When a test antibody completely competes with a reference antibody of known epitope, the test antibody is considered to bind to the same epitope as the reference antibody.

[0331] The inhibition rate was calculated as described above, and the results are shown in Table 6.1 and Table 6.2. Epitope, Motavizumab binds to epitope II of RSV 2A F protein, MPE8 binds to epitope III of RSV 2A F protein, and MK-1654 binds to epitope IV of RSV 2A F protein. The results showed that F6-10 and F6-113 compete with MEDI8897, that is, these antibodies also recognize epitope IV of RSV 2A F protein. epitope; F6-125 competes with MK-1654, ie, this antibody also recognizes epitope IV of the pre-F protein; F6-18 competes with MK-1654, ie, this antibody also recognizes epitope IV of the pre-F protein.

[0332] Table 6.1: Binding competition of anti-RSV antibodies

[0333] Table 6.2: Binding competition of antibody F6-18

[0334] Antibody variable region coding sequence

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to RSV, comprising: (1) CDR-H1 set forth in SEQ ID NO: 1, CDR-H2 set forth in SEQ ID NO: 2, CDR-H3 set forth in SEQ ID NO: 3, CDR-L1 set forth in SEQ ID NO: 4, CDR-L2 set forth in SEQ ID NO: 5, and CDR-L3 set forth in SEQ ID NO: 6; (2) CDR-H1 set forth in SEQ ID NO: 9, CDR-H2 set forth in SEQ ID NO: 10, CDR-H3 set forth in SEQ ID NO: 11, CDR-L1 set forth in SEQ ID NO: 12, CDR-L2 set forth in SEQ ID NO: 13, and CDR-L3 set forth in SEQ ID NO: 14; (3) CDR-H1 as set forth in SEQ ID NO: 17, CDR-H2 as set forth in SEQ ID NO: 18, CDR-H3 as set forth in SEQ ID NO: 19, CDR-L1 as set forth in SEQ ID NO: 20, CDR-L2 as set forth in SEQ ID NO: 21, and CDR-L3 as set forth in SEQ ID NO: 22; or (4) CDR-H1 as shown in SEQ ID NO: 25, CDR-H2 as shown in SEQ ID NO: 26, CDR-H3 as shown in SEQ ID NO: 27, CDR-L1 as shown in SEQ ID NO: 28, CDR-L2 as shown in SEQ ID NO: 29, and CDR-L3 as shown in SEQ ID NO:

30.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising: (1) VH as shown in SEQ ID NO: 7 and VL as shown in SEQ ID NO: 8; (2) VH as shown in SEQ ID NO: 15 and VL as shown in SEQ ID NO: 16; (3) VH as shown in SEQ ID NO: 23 and VL as shown in SEQ ID NO: 24; or (4) VH shown in SEQ ID NO: 31 and VL shown in SEQ ID NO:

32.

3. The antibody or antigen-binding fragment thereof according to claim 1, comprising: (1) the heavy chain constant region as shown in SEQ ID NO: 35, and (2) The light chain constant region shown in SEQ ID NO: 33 or 34. The antibody or antigen-binding fragment thereof according to claim 1 , comprising an α heavy chain, a δ heavy chain, an ε heavy chain, a γ heavy chain or a μ heavy chain. The antibody or antigen-binding fragment thereof according to claim 1 , which belongs to the IgG1, IgG2, IgG3 or IgG4 subclass. The antibody or antigen-binding fragment thereof according to claim 1 , comprising a λ light chain or a κ light chain. The antibody or antigen-binding fragment thereof according to claim 1 , which is a full-length antibody. The antibody or antigen-binding fragment thereof according to claim 1 , which is an antibody fragment selected from the group consisting of Fv, scFv, Fab, Fab′, F(ab′) 2 and xFab.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, which has one or more of the following characteristics: (1) Ability to specifically bind to RSV A2 pre-F protein; (2) is capable of specifically binding to RSV A2 pre-F protein with an EC50 value of less than 60, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 ng / mL, or with an EC50 value that is less than (e.g., 1% to 99%, such as at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) the EC50 value of MEDI8897; (3) It can be less than 5.0, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.009, 0.008, 0.007, 0.006, 0.005, 0.004, 0.003, that specifically binds to RSV A2 pre-F protein with a KD value of 0.002 or 0.001 nM, or with a KD value that is less than (e.g., 1% to 99%, such as at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) the KD value of MEDI8897; (4) being able to inhibit RSV (e.g., type A and / or type B) from infecting host cells; (5) capable of producing an IC50 value of less than 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 ng / mL, or an IC50 value less than that of MEDI8897 (e.g., 1% to 99%, such as at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%), inhibiting infection of host cells by RSV type A strain (e.g., A2); (6) capable of inhibiting RSV B strain (e.g., B9320) infection of host cells with an IC50 value of less than 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, or 3 ng / mL, or with an IC50 value that is less than (e.g., 1% to 99%, such as at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) the IC50 value of MEDI8897; (7) capable of inhibiting RSV B strain (e.g., B18537) infection of host cells with an IC50 value of less than 50, 45, 40, 35, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 ng / mL, or with an IC50 value that is less than the IC50 value of MEDI8897 (e.g., 1% to 99%, such as at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%); (8) can compete with or inhibit the binding of MEDI8897 to RSV A2 pre-F protein; (9) capable of inhibiting the binding of MEDI8897 to RSV A2 pre-F protein by 1% to 100%, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%; (10) can compete with or inhibit the binding of MK-1654 to RSV A2 pre-F protein; (11) capable of inhibiting the binding of MK-1654 to RSV A2 pre-F protein by 1% to 100%, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%; (12) can compete with or inhibit the binding of Motavizumab to RSV A2 pre-F protein; (13) capable of inhibiting the binding of Motavizumab to RSV A2 pre-F protein by 1% to 100%, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%; (14) can compete with or inhibit the binding of MPE8 to RSV A2 pre-F protein; (15) capable of inhibiting the binding of MPE8 to RSV A2 pre-F protein by 1% to 100%, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%; (16) Capable of binding to RSV A2 pre-F protein epitope; (17) can bind to the IV epitope of RSV A2 pre-F protein; (18) Capable of binding to RSV A2 pre-F protein epitope and IV epitope; (19) can bind to epitope II of RSV A2 pre-F protein; (20) capable of binding to epitope III of RSV A2 pre-F protein; and / or (21) It is able to bind to epitopes II and III of RSV A2 pre-F protein.

10. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

11. The polynucleotide according to claim 10, comprising: (1) SEQ ID NOs: 36 and 37; (2) SEQ ID NOs: 38 and 39; (3) SEQ ID NOs: 40 and 41; or (4) SEQ ID NOs: 42 and 43.

12. A vector comprising the polynucleotide according to claim 10 or 11.

13. A host cell comprising the polynucleotide according to claim 10 or 11 or the vector according to claim 12. The host cell according to claim 13 , which is a eukaryotic cell. The host cell according to claim 14 , which is a CHO cell.

16. A method for producing an antibody or an antigen-binding fragment thereof, comprising: (a) culturing the host cell according to any one of claims 13 to 15 under conditions suitable for expressing the antibody or antigen-binding fragment thereof, and (b) optionally, recovering the antibody or antigen-binding fragment thereof.

17. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

18. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 or the composition according to claim 17 for use as a medicament.

19. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 or the composition according to claim 17, for use in treating a disease.

20. The antibody or antigen-binding fragment thereof or composition according to claim 19, wherein the disease is a lower respiratory tract infection.

21. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 or the composition according to claim 17 in the manufacture of a medicament for treating a disease.

22. The use according to claim 21, wherein the disease is a lower respiratory tract infection.

23. A method of treating a disease in an individual comprising administering to the individual a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 or the composition according to claim 17.

24. The method of claim 23, wherein the disease is a lower respiratory tract infection.

25. The invention as herein described.

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