Antibodies that specifically bind to RSV

Abstract

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

Description

[Technical Field]

[0001] Related Applications Incorporated by Reference This application claims priority to Chinese Patent Application No. 202211571449.4, filed December 8, 2022, and Chinese Patent Application No. 202310007283.1, filed January 4, 2023, which are incorporated herein by reference in their entireties.

[0002] Technical Field The present invention provides a set 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 producing the antibodies, and compositions comprising the antibodies. [Background technology]

[0003] Respiratory syncytial virus (RSV) causes approximately 33 million acute lower respiratory tract infections and over 100,000 deaths annually in children under 5 years of age, and approximately 7.2 deaths per 100,000 adults over 65 years of age annually. Furthermore, RSV infection does not confer permanent immunity, so children cannot be protected from reinfection. RSV is one of the most important causes of acute lower respiratory tract infections worldwide in children under 5 years of age and in people over 65 years of age with impaired memory immunity, and is recognized as a global health problem.

[0004] Viral diseases are generally prevented by vaccines. However, since RSV was isolated in 1957, no vaccines have been approved for prophylactic use in the past several decades. Previous vaccine research and development efforts have not only failed to provide a preventative effect, but have also exacerbated RSV-related pneumonia and even led to death in some children. The druggability risk in vaccine research and development remains extremely high. Therefore, increasing attention is being paid to the development of RSV antibody drugs. Currently, the only preventative drug for this disease is the monoclonal antibody palivizumab, first approved by the FDA in 1998. Therefore, there is an urgent need to develop new anti-RSV drugs, especially those capable of treating RSV infection.

[0005] RSV is a non-segmented, single-stranded, negative-strand RNA virus belonging to the genus Pneumovirus in the family Paramyxoviridae. RSV particles contain an envelope. RSV particles are composed of an envelope, a nucleocapsid, and a core. The RSV genome is 15.2 kb long and contains 10 transcribed genes (NS1, NS2, N, P, M, SH, G, F, M2, and L, from the 3' to 5' end) that encode 11 proteins. Of these genes, M2 contains two open reading frames that encode two proteins, M2-1 and M2-2, and the remaining genes each encode a single protein. NS1 and NS2 are nonstructural proteins; N (nucleocapsid protein), P (phosphoprotein), and L (polymerase subunit protein) are nucleocapsid proteins; M, M2-1, and M2-2 are matrix proteins; and F (fusion protein), G (adhesion protein), and SH (small hydrophobin) are transmembrane glycoproteins. The G and F proteins on the RSV particle surface mediate attachment and fusion between the virus and host cells, and between virus-infected and uninfected cells. Furthermore, these proteins are the major protective antigens of RSV and can induce the production of protective neutralizing antibodies. Research has shown that the G protein is highly variable between the two subtypes, and although it induces the production of type-specific neutralizing antibodies in the body, it does not provide broad protection. The F protein is highly conserved, and the protective antibodies it induces are broadly neutralizing and can inhibit both RSV A and B infection. The F protein is an effective target protein for research into RSV prophylactics, therapeutics, and vaccines. The F protein is only active when cleaved into two fragments, F1 (48 kDa) and F2 (26 kDa), by host proteases. The F protein normally forms a trimeric structure of F1-F2 heterodimers, which plays a role in fusion. The F protein currently contains six epitopes, namely epitopes O, I, II, III, IV, and V, which are thought to be capable of generating neutralizing antibodies. Summary of the Invention

[0006] In one aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis. (1) CDR-H1 shown in SEQ ID NO: 1, CDR-H2 shown in SEQ ID NO: 2, CDR-H3 shown in SEQ ID NO: 3, CDR-L1 shown in SEQ ID NO: 4, CDR-L2 shown in SEQ ID NO: 5, and CDR-L3 shown 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 set forth in SEQ ID NO: 17, CDR-H2 set forth in SEQ ID NO: 18, CDR-H3 set forth in SEQ ID NO: 19, CDR-L1 set forth in SEQ ID NO: 20, CDR-L2 set forth in SEQ ID NO: 21, and CDR-L3 set forth in SEQ ID NO: 22; or (4) CDR-H1 shown in SEQ ID NO: 25, CDR-H2 shown in SEQ ID NO: 26, CDR-H3 shown in SEQ ID NO: 27, CDR-L1 shown in SEQ ID NO: 28, CDR-L2 shown in SEQ ID NO: 29, and CDR-L3 shown in SEQ ID NO: 30 The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to RSV, comprising:

[0007] In one embodiment, the antibody or antigen-binding fragment thereof of the invention comprises: (1) VH set forth in SEQ ID NO: 7 and VL set forth in SEQ ID NO: 8; (2) VH set forth in SEQ ID NO: 15 and VL set forth in SEQ ID NO: 16; (3) VH set forth in SEQ ID NO: 23 and VL set forth in SEQ ID NO: 24; or (4) VH shown in SEQ ID NO: 31 and VL shown in SEQ ID NO: 32 The compound comprises:

[0008] In one embodiment, the antibody or antigen-binding fragment thereof of the invention comprises: (1) a heavy chain constant region set forth in SEQ ID NO: 35, and (2) the light chain constant region shown in SEQ ID NO: 33 or 34 The compound comprises:

[0009] In one embodiment, an 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, an antibody or antigen-binding fragment thereof of the present invention belongs to the IgG1, IgG2, IgG3, or IgG4 subclass. In one embodiment, an antibody or antigen-binding fragment thereof of the present invention comprises a λ light chain or a κ light chain. In one embodiment, an antibody or antigen-binding fragment thereof of the present invention is a full-length antibody. In one embodiment, an 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, an antibody or antigen-binding fragment thereof of the present invention is a chimeric antibody or a human antibody, or an antigen-binding fragment thereof.

[0010] In one embodiment, the antibody or antigen-binding fragment thereof of the invention comprises: (1) capable of specifically binding to the RSV A2 pre-F protein; (2) specifically binds to RSV A2 pre-F protein with an EC50 value of 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 an EC50 value that is between 1% and 99%, e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% lower than the EC50 value of MEDI8897; (3) 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, 0.002 or can specifically bind to RSV A2 pre-F protein with a KD value of less than 0.001 nM, or a KD value that is lower (e.g., 1% to 99%, e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) than the KD value of MEDI8897; (4) capable of inhibiting RSV (e.g., subgroup A and / or subgroup B) from infecting host cells; (5) inhibiting an RSV subgroup A strain (e.g., A2) from infecting a host cell with an IC50 value of 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 that is 1% to 99%, e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% lower than the IC50 value of MEDI8897; (6) inhibiting an RSV subgroup B strain (e.g., B9320) from infecting a host cell with an IC50 value of 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 an IC50 value that is 1% to 99%, e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% lower than the IC50 value of MEDI8897; (7) inhibiting an RSV subgroup B strain (e.g., B18537) from infecting a host cell 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 an IC50 value that is between 1% and 99%, e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% lower than the IC50 value of MEDI8897; (8) capable of competing with or inhibiting the binding of MEDI8897 to the RSV A2 pre-F protein; (9) inhibiting the binding of MEDI8897 to RSV A2 pre-F protein by 1% to 100%, for example, by 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) capable of competing with or inhibiting the binding of MK-1654 to the RSV A2 pre-F protein; (11) inhibiting the binding of MK-1654 to RSV A2 pre-F protein by 1% to 100%, for example, by 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) capable of competing with or inhibiting the binding of motavizumab to the RSV A2 pre-F protein; (13) inhibiting the binding of motavizumab to RSV A2 pre-F protein by 1% to 100%, for example, by 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) capable of competing with or inhibiting the binding of MPE8 to the RSV A2 pre-F protein; (15) can inhibit the binding of MPE8 to RSV A2 pre-F protein by 1% to 100%, for example, by 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 epitope O of the RSV A2 pre-F protein; (17) capable of binding to epitope IV of the RSV A2 pre-F protein; (18) capable of binding to epitopes O and IV of the RSV A2 pre-F protein; (19) capable of binding to epitope II of the RSV A2 pre-F protein; (20) capable of binding to epitope III of the RSV A2 pre-F protein; and / or (21) It can bind to epitopes II and III of the RSV A2 pre-F protein. It has one or more of the following characteristics:

[0011] In one aspect, the invention provides a polynucleotide encoding an antibody or antigen-binding fragment thereof of the invention.

[0012] In one embodiment, the polynucleotide of the invention comprises: (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 The compound comprises:

[0013] In one aspect, the invention provides a vector comprising a polynucleotide of the invention.

[0014] In one aspect, the invention provides a host cell comprising a polynucleotide or vector of the invention. In one embodiment, the host cell is a eukaryotic cell. In one embodiment, the host cell is a CHO cell.

[0015] In one aspect, the invention provides a method for producing an antibody or antigen-binding fragment thereof, comprising: (a) culturing a host cell of the invention under conditions suitable for expression of the antibody or antigen-binding fragment thereof; and (b) optionally recovering said antibody or antigen-binding fragment thereof. The present invention provides a method comprising:

[0016] In one aspect, the invention provides a composition comprising an antibody or antigen-binding fragment thereof of the invention.

[0017] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof or composition of the present invention for use as a medicament.

[0018] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof 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.

[0019] In one aspect, the invention provides use of an antibody or antigen-binding fragment thereof or composition of the invention for the manufacture of a medicament for treating a disease. In one embodiment, the disease is a lower respiratory tract infection.

[0020] In one aspect, the invention provides a method for treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of an antibody or antigen-binding fragment thereof, or a composition of the invention. In one embodiment, the disease is a lower respiratory tract infection. [Brief explanation of the drawings]

[0021] [Figure 1-1] 1 shows the antigen binding curve of the antibody of the present invention (ELISA). [Figure 1-2] 1 shows the antigen binding curve of the antibody of the present invention (ELISA). [Figure 2-1] 1 shows the antigen binding curve of the antibody of the present invention (BA). [Figure 2-2] 1 shows the antigen binding curve of the antibody of the present invention (BA). [Figure 3-1] 1 shows the virus neutralization curve of the antibody of the present invention. [Figure 3-2] 1 shows the virus neutralization curve of the antibody of the present invention. [Figure 4] FIG. 1 shows a schematic diagram of a competitive binding epitope assay for the antibodies of the present invention. Specific Description of the Invention

[0022] Detailed Description of the Embodiments As used 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) derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereto, or may contain amino acid sequence mutations. In some embodiments, the number of amino acid mutations 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.

[0023] "Affinity" refers to the strength of the sum total of 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 specified, 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 as a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific exemplary embodiments for measuring binding affinity are described below.

[0024] An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody that does not possess such alterations, which alterations result in an improvement in the affinity of the antibody for antigen.

[0025] The terms "anti-RSV antibody" and "antibody that binds to RSV" refer to an antibody that can bind to RSV with sufficient affinity so that the antibody can be used as a diagnostic, prophylactic, and / or therapeutic agent in targeting RSV. In one embodiment, the extent of binding of the anti-RSV antibody to unrelated non-RSV proteins is less than about 10% of the antibody's binding to RSV, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to RSV has 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 M or less, e.g., 10 M to 10 M, e.g., 10 M to 10 M). In 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, the anti-RSV antibody binds to a pre-F epitope that is conserved in different RSV strains. In preferred embodiments, "anti-RSV antibodies," "antibodies that specifically bind to RSV," and "antibodies that bind to RSV" refer to antibodies that specifically bind to RSV or an antigen or epitope thereof with a K value of 1.0 x 10 mol / L or less, in one embodiment, a K value of 1.0 x 10 mol / L or less, and in one embodiment, a K value of 1.0 x 10 mol / L to 1.0 x 10 mol / L. In this context, binding affinity is determined using standard binding assays such as surface plasmon resonance (BIAcore®, GE-Healthcare Uppsala, Sweden; SARTORIUS, Octet® R8) using, for example, RSV A2 pre-F protein.

[0026] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0027] "Antibody fragment" refers to a molecule that differs from an intact antibody and comprises a portion of the 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.

[0028] The term "epitope" refers to the site on an antigen (proteinaceous or non-proteinaceous) to which an anti-RSV antibody binds. An epitope may be formed from a continuous string of amino acids (linear epitope) or may comprise non-contiguous amino acids that are spatially adjacent, for example, due to antigen folding (i.e., tertiary folding of a proteinaceous antigen) (conformational epitope). Linear epitopes typically still bind to anti-RSV antibodies after exposing a proteinaceous antigen to a denaturing agent, whereas conformational epitopes typically are destroyed after treatment with a denaturing agent. An 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.

[0029] Screening for antibodies that bind to a specific epitope (i.e., bind to the same epitope) can be performed using methods routine in the art, including, but not limited to, alanine scanning, peptide imprinting (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 Harbor, NY)).

[0030] Antigen structure-based antibody profiling (ASAP), also known as modification-assisted profiling (MAP), allows a population of monoclonal antibodies that specifically bind to RSV to be binned based on the binding profile of each antibody from the population to chemically or enzymatically modified antigen surfaces (see, e.g., U.S. Patent Application Publication No. 2004 / 0101920). Antibodies in each bin bind to the same epitope, which may be unique and different from or overlap with the epitopes represented by other bins.

[0031] Competitive binding can also be used to easily identify whether an antibody binds to the same RSV epitope as a reference anti-RSV antibody or competes for binding. 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 competitive assay; conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competitive assay. As another example, to determine whether an antibody binds to the same epitope as a reference anti-RSV antibody, the reference antibody is bound to RSV under saturating conditions. After removing excess reference anti-RSV antibody, the ability of the target anti-RSV antibody to bind to RSV is evaluated. If the anti-RSV antibody can bind to RSV after saturation binding of the reference anti-RSV antibody, it can be concluded that the target anti-RSV antibody binds to a different epitope than the reference anti-RSV antibody. However, if the target anti-RSV antibody cannot bind to RSV after saturation binding of the reference anti-RSV antibody, the target anti-RSV antibody may bind to the same epitope as the reference anti-RSV antibody. To determine whether the target antibodies bind to the same epitope or are simply inhibited for steric reasons, routine experiments (e.g., peptide mutagenesis and binding analysis using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art) can be performed. This assay should be performed in two settings (i.e., using both antibodies as saturating antibodies). If only the first (saturating) antibody can bind to RSV in both settings, it can be concluded that the target anti-RSV antibody competes with the reference anti-RSV antibody for binding to RSV.

[0032] 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 antibody 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).

[0033] In some embodiments, two antibodies are considered to bind to the same epitope if amino acid mutations in the antigen that reduce or eliminate binding of one antibody also substantially reduce or eliminate binding of the other antibody. Two antibodies are considered to have "overlapping epitopes" if only a subset of the amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.

[0034] 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, and the remainder of the heavy and / or light chain is derived from a different source or species.

[0035] The "class" of an antibody refers to the type of constant domain or constant region contained in its heavy chain. The five major classes of antibodies are IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is an IgG4 isotype with 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.

[0036] 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, radioisotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, 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 inhibitors; enzymes and fragments thereof, such as nucleases; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including fragments and / or mutants thereof); and various antitumor or anticancer drugs as disclosed below.

[0037] "Effector function" refers to the biological activity attributable to the Fc region of an antibody and varies depending on 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 receptors); and B cell activation.

[0038] 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.

[0039] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a 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) of the Fc region may or may not be present. In one embodiment, an anti-RSV antibody as described herein is an IgG1 isotype and comprises a constant heavy chain domain of SEQ ID NO: 35. In one embodiment, it further comprises a C-terminal lysine (Lys447). In one embodiment, an anti-RSV antibody as described herein is an IgG4 isotype. In one embodiment, it further comprises a C-terminal lysine (Lys447). Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to 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.

[0040] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the arrangement of HVRs and FRs is generally found in VH (or VL) as follows: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0041] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to antibodies having a structure substantially similar to a native antibody or having heavy chains including an Fc region as defined herein.

[0042] 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," and include the primary transformed cell and its progeny, regardless of the number of transfers. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the initially transformed cell are also included herein.

[0043] "Human antibody" refers to an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell using the human antibody repertoire or other human antibody coding sequence, or an antibody derived from a non-human source. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues. In certain embodiments, a human antibody is derived from a non-human transgenic mammal, such as a mouse, rat, or rabbit. In certain embodiments, a human antibody is derived from a hybridoma cell line.

[0044] A "human consensus framework" refers to a framework that represents the most frequently occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is made from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup such as those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication 91-3242, Bethesda, MD (1991), Vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI, as described in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III, as described in Kabat et al., supra.

[0045] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody may comprise at least one, and usually two, substantially entire variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the 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.

[0046] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence ("complementarity determining region" or "CDR") and / or forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contacts"). Generally, antibodies comprise six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Illustratively, herein: (a) hypervariable loops located 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)); (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)); (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 (d) combinations of (a), (b) and / or (c) containing 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); Examples include:

[0047] In one embodiment, the HVR residues comprise those found in the amino acid sequence description below.

[0048] Unless otherwise specified, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0049] "Immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules, including, but not limited to, cytotoxic agents.

[0050] "Individual" or "subject" refers to a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, 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.

[0051] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr., B 848: 79-87 (2007).

[0052] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained within a cell that ordinarily contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0053] "Isolated nucleic acid encoding an anti-RSV antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or in different vectors, and including such nucleic acid molecules present in one or more locations within a host cell.

[0054] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies; i.e., except for variant antibodies containing, for example, naturally occurring mutations or that may arise during the generation of a monoclonal antibody preparation (such variants are generally present in minor amounts), the individual antibodies comprising the population are identical and / or bind to the same epitope. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of the human immunoglobulin loci; such methods, as well as other exemplary methods for producing monoclonal antibodies, are described herein.

[0055] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. The naked antibody may be present in a pharmaceutical formulation.

[0056] "Native antibodies" refer to naturally occurring immunoglobulin molecules of different structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains disulfide-linked. From the N-terminus to the C-terminus, each heavy chain contains a variable region (VH) (also called a variable heavy domain or heavy chain variable domain), followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain contains a variable region (VL) (also called a variable light domain or light chain variable domain), followed by a constant light (CL) domain. Antibody light chains can be classified into two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of the constant domains.

[0057] The term "package insert" refers to the instructions typically included in the packaging of a commercially available therapeutic drug product that contain information regarding the indications, usage, dosage, administration, concomitant therapy, contraindications and / or warnings regarding the use of such therapeutic drug product.

[0058] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary. Alignment to determine percent amino acid sequence identity can be performed in a variety of ways within the skill of the art 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 algorithms necessary to achieve maximum alignment across the entire length of the sequences being compared. However, for purposes of this specification, percent amino acid sequence identity values ​​are generated using the ggsearch program and BLOSUM50 comparison matrix of the FASTA package, version 36.3.8c or later. The FASTA package is described in W. R. Pearson and D. J. 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 with default options (BLOSUM50; gap open: -10; gap extension: -2; Ktup=2) to ensure a global alignment rather than a local alignment. The percent amino acid identity is given in the output alignment header.

[0059] The term "pharmaceutical formulation" refers to a preparation in a form such that the biological activity of the active ingredient contained therein is effective and which does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0060] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0061] As used herein, "treatment" (and grammatical variations thereof) refers to clinical intervention that may be performed prophylactically or during the course of clinical pathology in an attempt to alter the natural course of the individual being treated. Desired therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, resolution / reduction of direct or indirect pathological consequences of disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of pathology, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay disease onset or slow disease progression.

[0062] The term "prevention," as used herein, includes slowing the onset of a disease, reducing the risk of developing a disease, inhibiting or delaying the onset or onset of symptoms associated with a disease, reducing the severity of subsequent onset or onset of the disease, ameliorating associated symptoms, and inducing immunity to prevent a disease.

[0063] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR) (see, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated using the VH or VL domain from an antibody that binds the antigen, and then a library of complementary VL or VH domains, respectively, can be screened. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991)).

[0064] 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 as self-replicating nucleic acid structures and vectors that integrate 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 operably linked. Such vectors are referred to herein as "expression vectors."

[0065] I. Compositions and Methods In one aspect, the invention provides an isolated antibody that binds to RSV.

[0066] In certain embodiments, antibodies that bind to RSV are provided. The antibodies of the present invention are useful for diagnosing or treating RSV infections, such as lower respiratory tract infections.

[0067] A. Exemplary Anti-RSV Antibodies In certain embodiments, i) binds to RSV, particularly the RSV pre-F protein, more particularly the RSV A2 pre-F protein; and / or ii) inhibiting RSV, particularly RSV A1, RSV B9320 and / or RSV B18537, from infecting host cells, such as Hep2 cells; Anti-RSV antibodies are provided.

[0068] 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) 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; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0069] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences 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; 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) 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.

[0070] In another aspect, the invention provides an antibody comprising at least one, at least two, or all three VL CDR sequences selected from: (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.

[0071] 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 (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 (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.

[0072] 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.

[0073] 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.

[0074] 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, a 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 that sequence retains its ability to bind to RSV. In certain embodiments, a total of 1 to 10 amino acids of SEQ ID NO: 7 are substituted, inserted, and / or deleted. In certain embodiments, these substitutions, insertions, or deletions occur in regions other than the CDRs (i.e., FRs). Optionally, the anti-RSV antibody comprises the VH sequence of SEQ ID NO: 7, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three 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, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3.

[0075] In another aspect, an anti-RSV antibody is provided comprising 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, a 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 retains the ability of the anti-RSV antibody comprising that sequence to bind to RSV. In certain embodiments, a total of 1 to 10 amino acids of SEQ ID NO: 8 are substituted, inserted, and / or deleted. In certain embodiments, these substitutions, insertions, or deletions occur in regions other than the CDRs (i.e., FRs). Optionally, the anti-RSV antibody comprises the VL sequence of SEQ ID NO: 8, including post-translational modifications of that sequence. In certain embodiments, 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.

[0076] In another aspect, an anti-RSV antibody is provided comprising 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 sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 8, respectively, including post-translational modifications of those sequences.

[0077] In yet 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, an antibody is provided that binds 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.

[0078] In one aspect, the invention provides an anti-RSV antibody comprising at least one, two, three, four, five, or six 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; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:11; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:12; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:13; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:14.

[0079] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences 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; 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) 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.

[0080] In another aspect, the invention provides an antibody comprising at least one, at least two, or all three VL CDR sequences selected from (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.

[0081] 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 (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:9, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:10, and (iii) 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 (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO:12, (ii) 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.

[0082] 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.

[0083] 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.

[0084] 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, a 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 retains the ability of the anti-RSV antibody comprising that sequence to bind to RSV. In certain embodiments, a total of 1 to 10 amino acids of SEQ ID NO: 15 are substituted, inserted, and / or deleted. In certain embodiments, these substitutions, insertions, or deletions occur in regions other than the CDRs (i.e., FRs). Optionally, the anti-RSV antibody comprises the VH sequence of SEQ ID NO: 15, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three 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, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11.

[0085] In another aspect, an anti-RSV antibody is provided comprising 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, a 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 retains the binding ability of the anti-RSV antibody comprising that sequence to RSV. In certain embodiments, a total of 1 to 10 amino acids of SEQ ID NO: 16 are substituted, inserted, and / or deleted. In certain embodiments, these substitutions, insertions, or deletions occur in regions other than the CDRs (i.e., FRs). Optionally, the anti-RSV antibody comprises the VL sequence of SEQ ID NO: 16, including post-translational modifications of that sequence. In certain embodiments, 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.

[0086] In another aspect, an anti-RSV antibody is provided comprising 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 sequence of SEQ ID NO: 15 and the VL sequence of SEQ ID NO: 16, respectively, including post-translational modifications of those sequences.

[0087] In yet 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, an antibody is provided that binds 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.

[0088] 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) 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; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22.

[0089] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences 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; 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) 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.

[0090] In another aspect, the invention provides an antibody comprising at least one, at least two, or all three VL CDR sequences selected from: (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.

[0091] 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 (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and (iii) 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 (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20, (ii) 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.

[0092] 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.

[0093] 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.

[0094] 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, a 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 retains the binding ability of the anti-RSV antibody comprising that sequence to RSV. In certain embodiments, a total of 1 to 10 amino acids of SEQ ID NO: 23 are substituted, inserted, and / or deleted. In certain embodiments, these substitutions, insertions, or deletions occur in regions other than the CDRs (i.e., FRs). Optionally, the anti-RSV antibody comprises the VH sequence of SEQ ID NO: 23, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three 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, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19.

[0095] In another aspect, an anti-RSV antibody is provided comprising 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, a 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 retains the ability of the anti-RSV antibody comprising that sequence to bind to RSV. In certain embodiments, a total of 1 to 10 amino acids of SEQ ID NO: 24 are substituted, inserted, and / or deleted. In certain embodiments, these substitutions, insertions, or deletions occur in regions other than the CDRs (i.e., FRs). Optionally, the anti-RSV antibody comprises the VL sequence of SEQ ID NO: 24, including post-translational modifications of that sequence. In certain embodiments, 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.

[0096] In another aspect, an anti-RSV antibody is provided comprising 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 sequence of SEQ ID NO: 23 and the VL sequence of SEQ ID NO: 24, respectively, including post-translational modifications of these sequences.

[0097] In yet 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, an antibody is provided that binds 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.

[0098] In one aspect, the invention provides an anti-RSV antibody comprising at least one, two, three, four, five, or six 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; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

[0099] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences 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; 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) 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.

[0100] In another aspect, the invention provides an antibody comprising at least one, at least two, or all three VL CDR sequences selected from: (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.

[0101] 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 (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and (iii) 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 (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28, (ii) 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.

[0102] 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: 62; (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.

[0103] 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.

[0104] 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, a 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 retains the binding ability of the anti-RSV antibody comprising that sequence to RSV. In certain embodiments, a total of 1 to 10 amino acids of SEQ ID NO: 31 are substituted, inserted, and / or deleted. In certain embodiments, these substitutions, insertions, or deletions occur in regions other than the CDRs (i.e., FRs). Optionally, the anti-RSV antibody comprises the VH sequence of SEQ ID NO: 31, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three 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, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27.

[0105] In another aspect, an anti-RSV antibody is provided comprising 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, a 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 retains the binding ability of the anti-RSV antibody comprising that sequence to RSV. In certain embodiments, a total of 1 to 10 amino acids of SEQ ID NO: 32 are substituted, inserted, and / or deleted. In certain embodiments, these substitutions, insertions, or deletions occur in regions other than the CDRs (i.e., FRs). Optionally, the anti-RSV antibody comprises the VL sequence of SEQ ID NO: 32, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three CDRs selected from (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.

[0106] In another aspect, an anti-RSV antibody is provided comprising 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 sequence of SEQ ID NO: 31 and the VL sequence of SEQ ID NO: 32, respectively, including post-translational modifications of those sequences.

[0107] In yet 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, an antibody is provided that binds 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.

[0108] In yet another aspect of the invention, the anti-RSV antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized, or human antibody. In one embodiment, the anti-RSV antibody is an antibody fragment, such as an Fv, Fab, Fab', xFab, scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody having an Fc region derived from a human IgG1 Fc region (see, e.g., WO 2012 / 130831), e.g., substitutions L234A, L235A, and P329G (LALA-PG).

[0109] 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, either alone or in combination.

[0110] 1. Antibody affinity 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 M or less, e.g., 10 M to 10 M, e.g., 10 M to 10 M).

[0111] In one embodiment, Kd is measured by radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using a Fab form of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is measured by equilibrating the Fab with a minimal concentration of (I)-labeled antigen in the presence of unlabeled antigen in a titration system, followed by capturing the bound antigen using a plate coated with an anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293: 865-881 (1999)). To establish assay conditions, MICROTITER® multiwell plates (Thermo Scientific) were coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by deblocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [I] antigen is mixed with serially diluted Fabs 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 Fabs of interest are then incubated overnight, although this incubation can be continued for a longer period (e.g., approximately 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate and incubated at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. After drying, 150 μl / well of scintillation fluid (MICROSCINT-20™; Packard) is added, and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). Concentrations of each Fab that give 20% or less of maximal binding are chosen for use in competitive binding assays.

[0112] In another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, this assay is performed using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) with an antigen CM5 chip immobilized at 25°C and approximately 10 response units (RU). 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. The antigen was 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 binding protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were injected at a flow rate of approximately 25 μl / min in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25°C. Association rates (k) and dissociation rates (k) were calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (K) was calculated as the ratio k / k. See, e.g., Chen et al., J. Mol. Biol. 293: 865-881 (1999). According to the surface plasmon resonance assay described above, if the association rate exceeds 106 M-1 S-1, the association rate can be determined using the fluorescence quenching method, i.e., by measuring the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandpass 16 nm) of 20 nM anti-antigen antibody (Fab type) in PBS pH 7.2 at 25°C in the presence of increasing concentrations of antigen, and measured with a spectrometer such as a Stop-Flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

[0113] 2. Antibody fragments In certain embodiments, the antibodies provided herein are antibody fragments. The term "antibody fragment" refers to a molecule other than an intact antibody that comprises 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 fragments (scFvs), and single-domain antibodies (dAbs). For a review of specific antibody fragments, see Holliger and Hudson, Nature Biotechnology 23: 1126-1136 (2005).

[0114] 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 called "Fab" fragments, each containing the variable domain of the heavy chain and the variable domain of the light chain, as well as 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 the VL domain and constant domain (CL) of the light chain and the VH domain and the first constant domain (CH1) of the heavy chain. An "xFab fragment" refers to a Fab fragment in which the VH domain has been swapped for a VL domain or the CH1 domain has been swapped for a CL domain. Fab' fragments differ from Fab fragments by additional residues at the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteines from the hinge region of the antibody. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domains have free sulfhydryl groups. Pepsin treatment yields an F(ab')2 fragment that contains two antigen-binding sites (two Fab fragments) and a portion of the Fc region. See U.S. Patent No. 5,869,046 for a discussion of Fab and F(ab')2 fragments that contain salvage receptor-binding epitope residues and have extended in vivo half-lives.

[0115] In another embodiment, the antibody fragment is a diabody, triabody, or tetrabody. Diabodies are antibody fragments with two antigen-binding sites and 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). Tribodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).

[0116] In yet another embodiment, the antibody fragment is a single-chain Fab fragment. A "single-chain Fab fragment" or "scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), antibody constant domain 1 (CH1), antibody light chain variable domain (VL), antibody light chain constant domain (CL), and a linker, where these antibody domains and linker are arranged in one of the following orders from N- to C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. In particular, the linker is a polypeptide having at least 30 amino acids, preferably 32-50 amino acids. Single-chain Fab fragments are stabilized via a natural disulfide bond between the CL and CH1 domains. Furthermore, these single-chain Fab molecules can be further stabilized through interchain disulfide bonds created by the insertion of cysteine ​​residues (e.g., number 44 in the heavy variable chain and number 100 in the light variable chain according to the Kabat numbering).

[0117] In another embodiment, the antibody fragment is a single-chain variable fragment (scFv). A "single-chain variable fragment (scFv)" is a fusion protein of an antibody's heavy chain variable region (VH) and light chain variable region (VL) linked via a linker. In particular, the linker is a short polypeptide of 10 to 25 amino acids, typically glycine-rich for flexibility and serine or threonine-rich for solubility, that can link the N-terminus of VH to the C-terminus of VL, or vice versa. This protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of the linker. For a review of scFv fragments, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, edited by Rosenburg and Moore, (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.

[0118] In another embodiment, the antibody fragment is a single-domain antibody. A single-domain antibody is an antibody fragment comprising 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, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516).

[0119] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells such as E. coli or phages, as described herein.

[0120] 3. Chimeric and humanized antibodies In certain embodiments, the antibody provided herein is a chimeric antibody. 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 non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In yet another example, a chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. A chimeric antibody includes an antigen-binding fragment thereof.

[0121] In certain embodiments, a chimeric antibody is a humanized antibody. Generally, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. Optionally, a humanized antibody will also comprise at least a portion of a human constant region. In some embodiments, some FR residues of a humanized antibody are substituted with the corresponding residues of a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0122] Humanized antibodies and methods for making them are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), Riechmann et al., Nature 332: 323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86: 10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36: 25-34 (2005) (describing grafting of specificity-determining regions (SDRs)); Padlan, Mol. Immunol. 28: 489-498 (1991) (describing "resurfacing"); Dall'Acqua et 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 "guide selection" for FR shuffling).

[0123] 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 consensus sequences of human antibodies of specific subgroups 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 obtained by screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[0124] 4. Human antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

[0125] Human antibodies can be prepared by administering immunogens to transgenic animals that have been engineered to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci, either replacing the endogenous immunoglobulin loci or present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HUMAB® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining them with different human constant regions.

[0126] Human antibodies can also be produced by hybridoma-based methods.Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (see, for example, 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 by human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Further methods include those described, for example, in U.S. Patent 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).

[0127] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. These variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0128] 5. Library-derived antibodies The antibodies of the present invention can be isolated by screening combinatorial libraries for antibodies with one or more desired activities. Methods for screening combinatorial libraries are reviewed, for example, in Lerner et al., Nature Reviews 16: 498-508 (2016). For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with desired binding properties. 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., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001) and Marks and Bradbury, Methods in Molecular Biology 248: 161-175 (Lo, eds., Human Press, Totowa, NJ, 2003).

[0129] In a specific phage display method, VH and VL gene repertoires are separately cloned by polymerase chain reaction (PCR) and randomly recombined into 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). Phages generally display antibody fragments as either single-chain Fv (scFv) fragments or Fab fragments. Libraries derived from immune sources provide high-affinity antibodies to immunogens without the need for hybridoma construction. Alternatively, naive repertoires can be cloned (e.g., from humans), providing a single source of antibodies against a wide range of non-self and self antigens without immune induction, as described in Griffiths et al., EMBO Journal 12: 725-734 (1993). Finally, naive libraries can be synthesized by cloning unrearranged V gene segments from stem cells and achieving in vitro rearrangement using PCR primers containing random sequences encoding the highly variable CDR3 regions, as described in 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.

[0130] Further 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 the review of the methods used 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).Methods for ribosome display are described, for example, in He et al., Nucleic Acids Research 25: 5132-5134 (1997) and Hanes et al., PNAS 94: 4937-4942 (1997).

[0131] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0132] 6. Multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites (i.e., different epitopes on different antigens or different epitopes on the same antigen). In certain embodiments, multispecific antibodies have three or more binding specificities. In certain embodiments, one of the binding specificities is for RSV, and the other (two or more) specificities are for any other antigen. In certain embodiments, bispecific antibodies can bind to two (or more) different antigens or epitopes of RSV. Multispecific (e.g., bispecific) antibodies can also be used to localize cytotoxic agents or cells to cells infected with RSV. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0133] 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 "knobs-in-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168 and Atwell et al., J. Mol. Biol. 270: 26 (1997)). Multispecific antibodies can also be engineered by engineering electrostatic steering effects to produce antibody-Fc heterodimeric molecules (see, e.g., WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992) and WO 2011 / 034605); circumventing the light chain mispairing problem by using common light chain technology (see, e.g., WO 98 / 50431); generating bispecific antibody fragments by using "diabody" technology (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448). (1993)); and by the use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152: 5368 (1994)); and by the preparation of trispecific antibodies as described, for example, in Tutt et al., J. Immunol. 147: 60 (1991).

[0134] Also included herein are engineered antibodies with three or more antigen-binding sites, including, for example, "octopus antibodies" or DVD-Igs (see, e.g., WO 2001 / 77342 and WO 2008 / 024715). Further examples of multispecific antibodies with three or more antigen-binding sites can be found in WO 2010 / 115589, WO 2010 / 112193, WO 2010 / 136172, WO 2010 / 145792, and WO 2013 / 026831. Bispecific antibodies or antigen-binding fragments thereof also include "dual-acting FAbs" or "DABs" that contain antigen-binding sites that bind to RSV and another different antigen, or two different epitopes of RSV (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820 and WO 2015 / 095539).

[0135] Multispecific antibodies can also be provided in an asymmetric form by domain swapping in one or more binding arms with the same antigen specificity, i.e., swapping VH / VL domains (see, e.g., WO 2009 / 080252 and WO 2015 / 150447), swapping CH1 / CL domains (see, e.g., WO 2009 / 080253), or swapping 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 cross-Fab fragment. The terms "cross-Fab fragment" or "xFab fragment" or "domain-swapped Fab fragment" refer to a Fab fragment in which the variable or constant regions of the heavy and light chains have been swapped. A cross-Fab fragment comprises 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 at the domain interface to induce proper Fab pairing. See, for example, WO 2016 / 172485.

[0136] Various additional molecular versions of multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al., Mol Immunol 67 (2015) 95-106).

[0137] 7. Antibody Variants 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. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion and / or insertion and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be used to arrive at the final construct, as long as the final construct possesses the desired characteristics, e.g., antigen binding.

[0138] a) Substitution, insertion, and deletion mutants In certain embodiments, antibody variants are provided that have one or more amino acid substitutions. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are as set forth in Table A under the heading "Preferred Substitutions." More substantial changes are set forth in Table A under the heading "Exemplary Substitutions," and are further described below with respect to amino acid side chain classes. Amino acid substitutions can be introduced into an antibody of interest, and the products screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0139] [Table 1]

[0140] Amino acids can be classified according to general side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral, hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0141] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0142] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody, such as a humanized or human antibody. Generally, the resulting variants selected for further study have modified (e.g., improved) specific biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or substantially retain specific biological properties of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage-display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for a specific biological activity (e.g., binding affinity).

[0143] For example, modifications (e.g., substitutions) can be made to HVRs to improve antibody affinity. Such modifications can be made at "hot spots" in HVRs, i.e., residues encoded by codons frequently mutated during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and / or at residues that contact antigen, and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by construction and reselection of secondary libraries is described, for example, in Hoogenboom et al., 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 one of a variety of methods, such as error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis. A secondary library is then generated. This library is then screened to identify antibody variants with the desired affinity. An alternative approach to introducing diversity is the HVR-directed 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 using, for example, alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are often targeted in particular.

[0144] In certain embodiments, substitutions, insertions, or deletions may be made within one or more HVRs, so long as such mutations do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative mutations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in HVRs. Such mutations may, for example, be outside the antigen-contacting residues of the HVRs. In certain embodiments of the variant VH and VL sequences set forth above, each HVR is unchanged or contains no more than one, two, or three amino acid substitutions.

[0145] As described in Cunningham and Wells (1989) Science, 244: 1081-1085, a useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis." In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and it is determined whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively / in addition, a crystal structure of an antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact residues and adjacent residues can be targeted or removed as substitution candidates. Screening can then be performed to determine whether the mutants have the desired properties.

[0146] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions, ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody of an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0147] b) Glycosylation variants In certain embodiments, the antibodies provided herein are mutated to increase or decrease the extent of glycosylation of the antibody. Addition or deletion of glycosylation sites in an antibody can be conveniently accomplished by mutating the amino acid sequence to create or remove one or more glycosylation sites.

[0148] If an antibody comprises an Fc region, the oligosaccharides attached thereto may vary. Natural antibodies produced by mammalian cells typically comprise branched, biantennary oligosaccharides, which are typically attached via an N-linkage to Asn297 in the CH2 domain of the Fc region. See Wright et al., TIBTECH 15: 26-32 (1997). Oligosaccharides include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the present invention can be performed to generate antibody variants with specific improved properties.

[0149] In one embodiment, antibody variants are provided having nonfucosylated oligosaccharides, i.e., oligosaccharide structures lacking fucose, attached (directly or indirectly) to the Fc region. Such nonfucosylated oligosaccharides (also referred to as "afucosylated" oligosaccharides) are, in particular, N-linked oligosaccharides lacking a fucose residue attached to the first GlcNAc in the stem of the biantennary oligosaccharide structure. In one embodiment, antibody variants are provided having an increased proportion of nonfucosylated oligosaccharides in the Fc region compared to the native or parent antibody. For example, the proportion of nonfucosylated 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., no fucosylated oligosaccharides are present). The percentage of nonfucosylated oligosaccharides is the (average) amount of oligosaccharides lacking a fucose residue relative to the sum of all oligosaccharides (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, e.g., as described in WO 2006 / 082515. Asn297 refers to the asparagine residue located near position 297 in the Fc region (EU numbering of Fc region residues), although Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, depending on slight antibody sequence variations. Such antibodies with an increased percentage of nonfucosylated oligosaccharides in the Fc region may exhibit improved FcRγIIIa receptor binding and / or improved effector function, particularly improved ADCC function. See, e.g., U.S. Patent Application Publication Nos. 2003 / 0157108; 2004 / 0093621.

[0150] 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); U.S. Patent Application Publication No. 2003 / 0157108; and WO 2004 / 056312, particularly Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (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 2003 / 085107), or cells with reduced or eliminated GDP-fucose synthesis or transporter activity (see, e.g., U.S. Patent Application Publication Nos. 2004 / 259150, 2005 / 031613, 2004 / 132140, and 2004 / 110282).

[0151] In yet another embodiment, the antibody variant provides a bisected oligosaccharide, such as a biantennary oligosaccharide, bisected by GlcNAc, attached to the Fc region of the antibody. Such antibody variants may exhibit reduced fucosylation and / or improved ADCC function, as described above. Examples of such antibody variants are described, for example, in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99 / 54342; WO 2004 / 065540; and WO 2003 / 011878.

[0152] Also provided is an antibody variant that has at least one galactose residue in the oligosaccharide attached to Fc region.Such antibody variants can show improved CDC function.Such antibody variants are described in, for example, WO1997 / 30087; WO1998 / 58964; and WO1999 / 22764.

[0153] c) Fc region mutants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0154] In certain embodiments, the present invention encompasses antibody variants that retain 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 reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas 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 to assess 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); U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assays can be employed (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc., Mountain View, CA); and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.Alternatively / additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an 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 is unable to bind C1q and thus lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may 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).

[0155] Antibodies with reduced effector function include those with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Pat. No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

[0156] Certain antibody variants have been described with improved or reduced binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0157] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0158] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that increase FcRn binding, e.g., substitutions at positions 252, and / or 254, and / or 256 (EU numbering of residues) of the Fc region. In certain embodiments, the antibody variant comprises an Fc region with amino acid substitutions at positions 252, 254, and 256. In one embodiment, the substitutions are M252Y, S254T, and T256E in the Fc region derived from a human IgG1 Fc region.

[0159] In certain embodiments, the antibody variant comprises an Fc region with amino acid substitutions that reduce FcγR binding, e.g., substitutions at positions 234 and 235 (EU numbering of residues) in the Fc region. In one embodiment, the substitutions are L234A and L235A (LALA). In certain embodiments, the antibody variant further comprises D265A and / or P329G in the Fc region derived from a human IgG1 Fc region. In one embodiment, the substitutions are L234A, L235A, and P329G in the Fc region derived from a human IgG1 Fc region (LALA-PG) (see, e.g., WO 2012 / 130831). In another embodiment, the substitutions are L234A, L235A, and D265A in the Fc region derived from a human IgG1 Fc region (LALA-DA).

[0160] In some embodiments, mutations are made to the Fc region resulting in altered (i.e., improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

[0161] Antibodies that exhibit increased half-life and improved binding to the neonatal Fc receptor (FcRn) are documented in U.S. Patent Application Publication No. 2005 / 0014934 (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 binding of the Fc region to FcRn. Such Fc variants include those with a substitution 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, e.g., a substitution at 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).

[0162] The Fc region residues important 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 (according to Kabat's EU numbering system) are involved in this 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 have been found to be important for the interaction between human Fc and 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 important for this interaction (Firan, M. et al., Int. Immunol. 13 (2001) 993; Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604). Various mutants of residues 248-259, 301-317, 376-382, and 424-437 have been described and investigated (Yeung, YA et al., J. Immunol. 182 (2009) 7667-7671).

[0163] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that reduce FcRn binding, e.g., substitutions at positions 253 and / or 310 and / or 435 (EU numbering of residues) of the Fc region. In certain embodiments, the antibody variant comprises an Fc region with amino acid substitutions at positions 253, 310, and 435. In one embodiment, the substitutions are I253A, H310A, and H435A in the Fc region derived from a human IgG1 Fc region. See, e.g., Grevys, A. et al., J. Immunol. 194 (2015) 5497-5508.

[0164] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that reduce FcRn binding, e.g., substitutions at positions 310 and / or 433 and / or 436 (EU numbering of residues) of the Fc region. In certain embodiments, the antibody variant comprises an Fc region with amino acid substitutions at positions 310, 433, and 436. In one embodiment, the substitutions are H310A, H433A, and Y436A in the Fc region derived from a human IgG1 Fc region (see, e.g., WO 2014 / 177460).

[0165] 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 for other examples of Fc region variants.

[0166] B. Recombinant Methods and Compositions Antibodies can be made using recombinant methods and compositions such as those described in U.S. Patent No. 4,816,567, which provide one or more isolated nucleic acids encoding the antibody.

[0167] In the case of a natural antibody or natural antibody fragment, two nucleic acids are required: one for the light chain or fragment thereof and one for the heavy chain or fragment thereof. Such nucleic acids encode an amino acid sequence comprising the VL and / or the VH of the antibody (e.g., the light and / or heavy chains of the antibody). These nucleic acids may be in the same expression vector or in different expression vectors.

[0168] In the case of a bispecific antibody having a heterodimeric heavy chain, four nucleic acids are required: one for the first light chain, one for the second light chain comprising a first heteromonomeric Fc-region polypeptide, one for the second light chain, and one for the second heavy chain comprising a second heteromonomeric Fc-region polypeptide. These four nucleic acids can be configured in one or more nucleic acid molecules or expression vectors. Such nucleic acids encode an amino acid sequence comprising a first VL and / or an amino acid sequence comprising a first VH comprising a first heteromonomeric Fc region and / or an amino acid sequence comprising a second VL and / or an amino acid sequence comprising a second VH comprising a second heteromonomeric Fc region of the antibody (e.g., the first and / or second light chains and / or the first and / or second heavy chains of the antibody). These nucleic acids can be present in the same expression vector or in different expression vectors. Typically, these nucleic acids are present on two or three expression vectors; i.e., one vector can comprise two or more of these nucleic acids. Examples of such bispecific antibodies include CrossMabs and T cell bispecific agents (see, e.g., Schaefer, W. et al., PNAS, 108 (2011) 11187-1191). For example, one of the heteromonomeric 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).

[0169] In one embodiment, an isolated nucleic acid encoding an antibody for use in the methods described herein is provided.

[0170] In yet another embodiment, one or more vectors (eg, expression vectors) comprising such nucleic acids are provided.

[0171] In yet another embodiment, a host cell comprising such nucleic acid is provided.

[0172] In one such embodiment, the host cell comprises (e.g., has been transformed with): In the case of antibodies composed of two identical disulfide-bonded light chains and two identical heavy chains comprising VH and VL fragments, (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and an amino acid sequence comprising the VH of an antibody; or (2) A first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. For bispecific antibodies with heterodimeric heavy chains, (1) a first vector comprising a first nucleic acid portion encoding amino acid sequences, one of which comprises a first VL and the other of which comprises a first VH of an antibody, and a second vector comprising a second nucleic acid portion encoding amino acid sequences, one of which comprises a second VL and the other of which comprises a second VH of an antibody; or (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 nucleic acid portion encoding an amino acid sequence, one of which comprises a light chain variable domain and the other of which comprises a first heavy chain variable domain, and a third vector comprising a nucleic acid portion encoding an amino acid sequence, one of which comprises another light chain variable domain corresponding to the second vector and the other of which comprises a second heavy chain variable domain; or (3) A third vector comprising a first vector comprising a nucleic acid encoding an amino acid sequence comprising a first VL of the antibody, a second vector comprising a nucleic acid encoding an amino acid sequence comprising a first VH of the antibody, a nucleic acid encoding an amino acid sequence comprising a second VL of the antibody, and a fourth vector comprising a nucleic acid encoding an amino acid sequence comprising a second VH of the antibody.

[0173] In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method for producing an anti-RSV antibody is provided, comprising culturing a host cell comprising a nucleic acid encoding the antibody as described above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0174] For recombinant production of anti-RSV antibodies, nucleic acids encoding the antibodies (e.g., as described above) are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody), or can be produced by recombinant methods or obtained by chemical synthesis.

[0175] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly if glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523 (also see Charlton, KA, in Methods in Molecular Biology, Vol. 248, ed. Lo, BKC, Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the antibody can be isolated from the bacterial cell paste as a soluble fraction and further purified.

[0176] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of 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.

[0177] Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Several baculovirus strains have been identified that can be used with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0178] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0179] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells as described in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney (BHK) cells; mouse Sertoli cells (TM4 cells) as described, for example, in Mather, JP, Biol. Reprod. 23 (1980) 243-252; monkey kidney cells (CV1); African 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); e.g., Mather, JP 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 specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, A. M., Methods in Molecular Biology, Vol. 248, Lo, BKC, ed., Humana Press, Totowa, NJ (2004), pp. 255-268.

[0180] C. Assay The anti-RSV antibodies provided herein can be identified, screened, or characterized for physical / chemical properties and / or biological activity by a variety of assays known in the art.

[0181] 1. Binding and Other Assays In one aspect, the antibodies of the invention can be tested for their antigen binding activity by known methods such as, for example, ELISA and Western blotting.

[0182] In another aspect, a competitive assay can be used to identify antibodies that compete with a reference anti-RSV antibody for binding to RSV or its antigen. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear epitope or a conformational epitope) as the reference anti-RSV antibody binds. Detailed exemplary methods for mapping antibody-binding epitopes are described in Morris (1996) "Epitope Mapping Protocols", Methods in Molecular Biology, vol. 66 (Humana Press, Totowa, NJ).

[0183] In an exemplary competitive assay, immobilized RSV or its antigen is incubated in a solution containing a first labeled antibody (which binds to RSV or its antigen) (e.g., a reference anti-RSV antibody) and a second unlabeled antibody (which is being tested for its ability to compete with the first antibody for binding to RSV or its antigen). The second antibody may be present in hybridoma supernatant. As a control, immobilized RSV or its antigen is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to RSV or its antigen, excess unbound antibody is removed and the amount of label associated with the immobilized RSV or its antigen is measured. If the amount of label associated with the immobilized RSV or its antigen is substantially less in the test sample than in the control sample, this indicates that the second antibody competes with the first antibody for binding to RSV or its antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0184] 2. Activity Assay In one aspect, an assay is provided for identifying anti-RSV antibodies with biological activity. Biological activity can include, for example, the inhibition of RSV infection of host cells by anti-RSV antibodies. Antibodies with such biological activity in vivo and / or in vitro are also provided.

[0185] D. Methods and Compositions for Diagnostics and Detection In certain embodiments, any of the anti-RSV antibodies provided herein can be used to detect the presence of RSV in a biological sample. As used herein, the term "detection" includes quantitative detection or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues, such as tumor tissues.

[0186] In one embodiment, an anti-RSV antibody is provided for use in a diagnostic or detection method. In yet another aspect, a method for detecting the presence of RSV in a biological sample is provided. In a specific embodiment, the method comprises contacting a biological sample with an anti-RSV antibody as described herein under conditions that allow binding of the anti-RSV antibody to RSV or its antigen, and detecting whether a complex is formed between the anti-RSV antibody and RSV or its antigen. Such a method may be an in vitro method or an in vivo method. In one embodiment, the anti-RSV antibody is used to select subjects suitable for treatment with the anti-RSV antibody, for example, when a patient is infected with RSV, suspected of being infected with RSV, or at risk of exposure to RSV. In one embodiment, the anti-RSV antibody is used to select subjects suitable for treatment with the anti-RSV antibody, for example, when a patient is infected with RSV, suspected of being infected with RSV, or at risk of exposure to RSV.

[0187] In certain embodiments, labeled anti-RSV antibodies are provided. Labels include, but are not limited to, directly detectable labels or moieties (such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels) and indirectly detectable moieties, such as enzymes or ligands, via enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes 32P, 14C, 125I, 3H, and 131I, 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-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, and the like. Examples of oxidases include β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase (which uses hydrogen peroxide to catalyze dye precursors, such as HRP), lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, phage labels, and stable free radicals.

[0188] E. Pharmaceutical Preparations Pharmaceutical formulations of anti-RSV antibodies as described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing such antibodies of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used and include, but are not limited to, buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride); hexanediamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; low molecular weight (approximately 1000 residues) soluble ... Examples of suitable pharmaceutically acceptable carriers include: polypeptides (<1000 bases); 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 such as Zn-protein complexes; and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersion agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, e.g., rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods, including rHuPH20, are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0189] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulations comprising a histidine-acetate buffer.

[0190] The formulations herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are preferably present in combination in amounts that are effective for the purpose intended.

[0191] The active ingredient can be encapsulated in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared by coacervation techniques or interfacial polymerization, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th ed., Osol, A. ed. (1980).

[0192] Sustained-release preparations may also 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.

[0193] Formulations to be used for in vivo administration are generally sterile. Sterilization can be readily accomplished, for example, by filtration through sterile filtration membranes.

[0194] F. Prophylactic and Therapeutic Methods and Compositions Any of the anti-RSV antibodies provided herein can be used in prophylactic or therapeutic methods.

[0195] In one aspect, anti-RSV antibodies are provided for use as pharmaceuticals. In yet another aspect, anti-RSV antibodies are provided for use in the prevention or treatment of RSV infection. In certain embodiments, anti-RSV antibodies are provided for use in prophylaxis or therapy. In certain embodiments, the invention provides anti-RSV antibodies for use in a method of preventing an individual at risk of RSV infection, wherein the prevention comprises administering to the individual an effective amount of an anti-RSV antibody. In certain embodiments, the invention provides anti-RSV antibodies for use in a method of treating an individual with a RSV infection, wherein the treatment comprises administering to the individual an effective amount of an anti-RSV antibody. In one embodiment, the antibody is for use in treating or delaying the onset of a RSV infection.

[0196] An "individual" according to any of the above embodiments is preferably a human. In yet another aspect, the 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 the treatment of a RSV infection. In yet another embodiment, the medicament is for use in a method of treating a RSV infection, the method comprising administering an effective amount of the medicament to an individual having a RSV infection. An "individual" according to any of the above embodiments can be a human.

[0197] As used herein, the term "RSV infection" can be, for example, a lower respiratory tract infection.

[0198] In yet another aspect, the invention provides a method for treating a RSV infection. In one embodiment, the method comprises administering an effective amount of an anti-RSV agent to an individual having cancer. The "individual" according to any of the above embodiments can be a human.

[0199] In yet another aspect, the invention provides pharmaceutical formulations comprising any of the anti-RSV antibodies provided herein, e.g., 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.

[0200] In yet another aspect, the present invention provides pharmaceutical formulations comprising any of the anti-RSV antibodies provided herein, for example, 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. In another embodiment, the pharmaceutical formulation comprises any of the anti-RSV antibodies provided herein and at least one additional therapeutic agent. In one embodiment, the at least one additional therapeutic agent is, for example, another anti-RSV antibody that binds to a different RSV epitope than the anti-RSV antibody of the present invention.

[0201] The antibodies of the invention may be used alone or in combination with other agents in therapy, for example, the antibodies of the invention may be administered in combination with at least one additional therapeutic agent.

[0202] Such combination therapy includes combined administration (two or more therapeutic agents in the same or different formulations) and separate administration, in which the administration of an antibody of the invention can occur before, simultaneously with, and / or after the administration of one or more additional therapeutic agents. In one embodiment, the administration of the anti-RSV antibody and the administration of the additional therapeutic agent occur within about 1 month, or within about 1, 2, or 3 weeks, or within about 1, 2, 3, 4, 5, or 6 days of each other.

[0203] The antibodies of the invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for localized treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be by any suitable route, e.g., injection, such as intravenous or subcutaneous injection, depending, in part, on whether administration is brief or chronic. A variety of administration schedules are encompassed herein, including, but not limited to, single administration or multiple administrations over various time periods, bolus administration, and pulse infusion.

[0204] The antibodies of the present invention are formulated, dosed, and administered in a manner consistent with good clinical practice. Factors to consider in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to medical professionals. The antibodies need not, but may, be combined with one or more agents currently used to prevent or treat the disorder. The effective amount of such other agents will vary depending on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These agents are generally used in the same dosages and by the same routes of administration as described herein, or at about 1% to 99% of the dosages described herein, or at any dosage and by any route, with the dosage and route being empirically / clinically determined to be appropriate.

[0205] The appropriate dose of the antibody of the present invention (used alone or in combination with one or more other additional therapeutic agents) for the prevention or treatment of disease will vary depending on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is administered to the patient at one time or over a series of treatments, as appropriate. Depending on the type and severity of the disease, approximately 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of antibody can be the initial suggested amount to administer to the patient, whether administered as one or more individual doses or by continuous infusion. Typical daily doses can range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. In the case of repeated administration over several days or longer, depending on the condition, treatment is generally sustained until the desired suppression of disease symptoms occurs. One exemplary dose of the antibody would 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, for example, once per week or once every three weeks (e.g., such that the patient receives from about 2 doses to about 20 doses, or, for example, about 6 doses of antibody). An initial higher loading dose may be administered, followed by one or more lower doses. Exemplary dosing regimens include administration. However, other dosing regimens may also be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0206] It is understood that any of the above formulations or treatment methods can be carried out using the immunoconjugates of the present invention in place of, or in addition to, anti-RSV antibodies.

[0207] G. Preparation In another aspect of the present invention, a formulation comprising a substance useful for the treatment, prevention, and / or diagnosis of the above-mentioned disorders is provided. The formulation comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV infusion bags, etc. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition, either by itself or in combination with another composition effective for the treatment, prevention, and / or diagnosis of a condition, and can have a sterile access port (e.g., the container can be a vial or an infusion 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 used to treat a selected condition. Furthermore, the preparation can comprise (a) a first container containing a composition comprising an antibody of the present invention; and (b) a second container containing a composition comprising an additional therapeutic agent. The formulation in this embodiment of the present invention can further include a package insert indicating that the composition can be used to treat a specific condition. Alternatively / in addition, the formulation 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. The formulation may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0208] [Table 2] JPEG2025528316000004.jpg189170

[0209] The present invention provides a set of antibodies having an affinity for the RSV A2 pre-F protein of 0.001 nM to 3.25 nM, an IC50 of neutralizing activity against the RSV A2 strain of 0.34 ng / mL to 80.04 ng / mL, an IC50 of neutralizing activity against the RSV B9320 strain of 3.65 ng / mL to 153.90 ng / mL, and an IC50 of neutralizing activity against the RSV B18537 strain of 1.87 ng / mL to 67.03 ng / mL. [Example]

[0210] Example 1: Preparation of anti-RSV antibodies Peripheral blood was collected from healthy adult volunteers, and upper plasma and middle PBMCs were isolated by density gradient centrifugation. Memory B cells specifically binding to pre-F protein were isolated from PBMCs by flow sorting using fluorescently labeled pre-F protein. Transfectable PCR fragments with expression activity were obtained by nested PCR. These fragments were transfected into CHO cells for expression, and cell supernatants containing secreted antibodies were obtained. ELISA screening for binding activity yielded several hundred pre-F-binding positive clones. Multiple recombinant antibodies were tested for neutralizing activity against RSV A2 strains. Based on their neutralizing activity against RSV A2, RSV 9320, and RSV 18537, and their affinity for RSV A2 pre-F protein, four antibodies, F6-10, F6-18, F6-113, and F6-125, were ultimately selected.

[0211] The amino acid sequences of the CDRs of these antibodies are shown in Table 1.

[0212] [Table 3]

[0213] The amino acid sequences of VH, VL and CL of these antibodies are shown in Table 2.

[0214] [Table 4] JPEG2025528316000007.jpg61170

[0215] The coding sequences for the antibody light and heavy chain variable regions were cloned into a eukaryotic expression vector containing the coding sequence for the human IgG1 constant region. The resulting vector was transiently transfected into CHO cells for secretory expression. Antibody proteins with over 90% purity, i.e., F6-10, F6-18, F6-113, and F6-125, were obtained by Protein A affinity purification.

[0216] Amino acid sequence of human IgG1 constant region: [ka]

[0217] Amino acid sequence of RSV A2 pre-F: [ka]

[0218] Example 2: Characterization of anti-RSV antibodies - Characterization of antigen binding activity by ELISA RSV A2 pre-F protein (homemade by Vazyme) diluted to 2 μg / mL in carbonate buffer pH 9.6 was added to a 96-well microplate (NEST, 504201) at 100 μL / well and coated overnight at 4°C. The solution was removed, the plate was washed twice with PBST, and blocked with blocking solution (PBS + 5% BSA) at 37°C for 2 hours. The solution was removed, and 3-fold serial dilutions of antibody (starting concentration: 5 μg / mL, total 10 gradient) in diluent (PBS + 5% BSA) were added to the microplate at 100 μL / well and incubated at 37°C for 1 hour. The solution was removed, and the plate was washed three times with PBST. 100 μL of mouse anti-human IgG Fc-HRP (homemade by Vazyme) was added to each well at a 1:10,000 dilution and incubated at 37°C for 1 hour. The solution was removed, and the plate was washed three times with PBST. 100 μL of the chromogenic substrate TMB was added to each well and incubated at 37°C in the dark for 10 minutes. The solution was removed, and the plate was washed three times with PBST. 50 μL of 2 M sulfuric acid was added to each well. The OD values ​​at 450 nm were measured using a multifunction microplate reader (Tecan, Spark). A four-parameter fitting 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 from different experiments; the only difference between Table 3.2 and the experiment was the two-fold serial dilution of the antibody in diluent (PBS + 5% BSA) (starting concentration: 1 μg / mL, total 14 gradients).) Antibody concentrations and OD values ​​were plotted using GraphPad Prism 8.0. The results are shown in Figures 1-1 and 1-2.

[0219] [Table 5] [Table 6]

[0220] Example 3: Characterization of anti-RSV antibodies—affinity characterization by BLI The antibody affinity fitting curves were obtained by biolayer interferometry (BLI) using an Octet protein analysis system (SARTORIUS, Octet R8). The sensor used was HIS1K (SARTORIUS, 18-5120), the capture agent was 47.6 nM RSV A2 pre-F protein, the immobilization time was 120 s, 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 s, the dissociation time was 120 s, the regeneration solution was 10 mM glycine-HCl pH 1.5, and the regeneration time was 180 s. The KD values ​​(M) of the antibody antigen binding affinities were calculated using Octet Analysis Studio 12.2 software. The results are shown in Tables 4.1 and 4.2. The data were recorded using Octet BLI Discovery 12.2 software. The results are shown in Figures 2-1 and 2-2.

[0221] [Table 7] [Table 8]

[0222] Example 4: Characterization of anti-RSV antibodies - Characterization of neutralizing activity by in vitro microneutralization assay Three-fold serial dilutions of antibodies (starting concentration: 0.5 μg / mL, total 7 gradients) in PBS + 5% HIFBS were added at 60 μL / well to a 96-well plate (NEST, 701001) containing virus (RSV A2, ATCC VR-1540; RSV B9320, ATCC VR-955; RSV B18537, ATCC VR-1580) (60 μL / well, 500 pfu) and incubated for 1 hour in a cell culture incubator (37°C, 5% CO). 96-well plates (NEST, 701001) containing 90% confluent Hep2 cells (coated the day before at 3 × 10 cells / mL) were coated with 50 μL / well of the above mixture and incubated for 2 hours in a cell culture incubator (37°C, 5% CO). The supernatant was removed. 100 μL of DMEM medium (Solaibio, P1400) supplemented with 2% FBS and 1% penicillin-streptomycin was added to each well, and incubation continued for 21–22 hours. The supernatant was removed. 100 μL of 4% paraformaldehyde was added to each well and cells were fixed for 10 minutes. The paraformaldehyde was removed. The cells were washed once by adding 250 μL of PBS to each 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. 50 μL of 3D3 fluorescent antibody (homemade by Vazyme) diluted to 5 μg / mL was added to each well and incubated for 1 hour in a cell culture incubator (37°C, 5% CO2). The antibody was then removed. The plates were washed three times with PBST and spun dry. The number of spots per well was read using a fluorescent (enzyme-linked) immunospot analyzer (CTL, S6 Ultra M2). Wells containing only cells (cell wells) were used as negative wells, and wells containing only virus (virus wells) were used as positive wells.

[0223]

number

[0224] Antibody concentrations and virus neutralization were plotted using GraphPad Prism 8.0. The results are shown in Figures 3-1 and 3-2. A four-parameter fitting was performed to calculate the IC50 values ​​(ng / mL) of the antibody virus neutralization activity. The results are shown in Tables 5.1 and 5.2.

[0225] [Table 9] [Table 10]

[0226] Example 5: Characterization of anti-RSV antibodies - Epitope characterization by competitive binding RSV A2 pre-F protein was diluted to 10 μg / mL in buffer (PBS + 0.02% Tween 20 + 0.1% BSA). Antibodies were diluted to 200 nM in the same buffer and added at 200 μL per well. As shown in Figure 4, the above buffer was run through an Octet Protein Analysis System (SARTORIUS, Octet R8) for 120 seconds to reach baseline levels, followed by antigen until the signal reached 0.3 nM. The above buffer was run for 60 seconds until baseline levels were reached, followed by the first antibody for 180 seconds, and then the second antibody for 180 seconds. (If the first antibody had a fast dissociation rate, the same concentration of the first antibody was added to the second antibody to prevent false positive results due to dissociation of the first antibody upon binding to the second antibody.) Regeneration solution (10 mM glycine-HCl, pH 1.5) and buffer were alternately run for a total of 20 seconds, repeated three times. The above cycle was repeated.

[0227] Data Analysis: If the first antibody is Ab1 and the second antibody is Ab2+Ab1, the second signal is H1′2; If the first antibody is Ab1 and the second antibody is Ab1, the second signal is H1'1; then If the first antibody is Ab1 and the second antibody is Ab2, the second signal is H1, = H1'2 - H1'1; If the first antibody is 0 and the second antibody is Ab2, the second signal is H2; then The inhibition of the second antibody by the first antibody is: 1-(H1 / H2)×100% (The two antibodies were swapped to calculate the inhibition of the first antibody by the second antibody).

[0228] Experimental Validation: Autoreactive signal <20%, otherwise, data is invalid.

[0229] Decision criteria >70%: perfect conflict, one direction is sufficient to meet this criterion; 30%~70%: partial conflict; <30%: No conflicts at all, one direction is sufficient to meet this criterion.

[0230] If the test antibody perfectly competes with the reference antibody for the known epitope, then the test antibody is considered to bind to the same epitope as the reference antibody.

[0231] The inhibition rate was calculated as described above. The results are shown in Tables 6.1 and 6.2. Reference antibody MEDI8897 binds to epitope O of the RSV 2A pre-F protein, motavizumab binds to epitope II of the RSV 2A F protein, MPE8 binds to epitope III of the RSV 2A F protein, and MK-1654 binds to epitope IV of the RSV 2A F protein. The results show that F6-10 and F6-113 compete with MEDI8897, i.e., these antibodies also recognize epitope O of the F protein; F6-125 competes with MK-1654, i.e., this antibody also recognizes epitope IV of the pre-F protein; and F6-18 competes with MK-1654, i.e., this antibody also recognizes epitope IV of the pre-F protein.

[0232]

Table 11

[0233]

Table 12

[0234]

Table 13

Claims

1. (1) CDR-H1 shown in SEQ ID NO: 1, CDR-H2 shown in SEQ ID NO: 2, CDR-H3 shown in SEQ ID NO: 3, CDR-L1 shown in SEQ ID NO: 4, CDR-L2 shown in SEQ ID NO: 5, and CDR-L3 shown in SEQ ID NO: 6; (2) CDR-H1 shown in SEQ ID NO: 9, CDR-H2 shown in SEQ ID NO: 10, CDR-H3 shown in SEQ ID NO: 11, CDR-L1 shown in SEQ ID NO: 12, CDR-L2 shown in SEQ ID NO: 13, and CDR-L3 shown in SEQ ID NO: 14; (3) CDR-H1 shown in SEQ ID NO: 17, CDR-H2 shown in SEQ ID NO: 18, CDR-H3 shown in SEQ ID NO: 19, CDR-L1 shown in SEQ ID NO: 20, CDR-L2 shown in SEQ ID NO: 21, and CDR-L3 shown in SEQ ID NO: 22; or (4) CDR-H1 shown in SEQ ID NO: 25, CDR-H2 shown in SEQ ID NO: 26, CDR-H3 shown in SEQ ID NO: 27, CDR-L1 shown in SEQ ID NO: 28, CDR-L2 shown in SEQ ID NO: 29, and CDR-L3 shown in SEQ ID NO: 30 1. An antibody or antigen-binding fragment thereof that specifically binds to RSV, comprising:

2. (1) VH shown in SEQ ID NO: 7 and VL shown in SEQ ID NO: 8; (2) VH represented by SEQ ID NO: 15 and VL represented by SEQ ID NO: 16; (3) VH set forth in SEQ ID NO: 23 and VL set forth in SEQ ID NO: 24; or (4) VH shown in SEQ ID NO: 31 and VL shown in SEQ ID NO: 32 The antibody or antigen-binding fragment thereof of claim 1, comprising:

3. (1) a heavy chain constant region represented by SEQ ID NO: 35, and (2) a light chain constant region represented by SEQ ID NO: 33 or 34 The antibody or antigen-binding fragment thereof of claim 1, comprising:

4. 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.

5. The antibody or antigen-binding fragment thereof according to claim 1, which belongs to the IgG1, IgG2, IgG3 or IgG4 subclass.

6. The antibody or antigen-binding fragment thereof of claim 1, comprising a λ light chain or a κ light chain.

7. The antibody or antigen-binding fragment thereof of claim 1, which is a full-length antibody.

8. Fv, scFv, Fab, Fab', F(ab') ２ 2. The antibody or antigen-binding fragment thereof of claim 1, which is an antibody fragment selected from xFab and xFab.

9. (1) capable of specifically binding to the RSV A2 pre-F protein; (2) 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 an EC50 value that is between 1% and 99%, e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% lower than the EC50 value of MEDI8897; (3) 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, 0.002 or capable of specifically binding to RSV A2 pre-F protein with a K value of less than 0.001 nM, or a K value that is between 1% and 99%, e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% lower than the K value of MEDI8897; (4) capable of inhibiting RSV (e.g., subgroup A and / or subgroup B) from infecting host cells; (5) capable of inhibiting an RSV subgroup A strain (e.g., A2) from infecting 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 an IC50 value that is between 1% and 99%, e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) lower than the IC50 value of MEDI8897; (6) capable of inhibiting an RSV subgroup B strain (e.g., B9320) from infecting a host cell 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 an IC50 value that is between 1% and 99%, e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% lower than the IC50 value of MEDI8897; (7) capable of inhibiting an RSV subgroup B strain (e.g., B18537) from infecting a host cell 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 an IC50 value that is between 1% and 99%, e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% lower than the IC50 value of MEDI8897; (8) capable of competing with or inhibiting the binding of MEDI8897 to RSV A2 pre-F protein; (9) inhibiting binding of MEDI8897 to RSV A2 pre-F protein by 1% to 100%, e.g., 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) capable of competing with or inhibiting the binding of MK-1654 to RSV A2 pre-F protein; (11) It can inhibit binding of MK-1654 to RSV A2 pre-F protein by 1% to 100%, for example, by 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) capable of competing with or inhibiting the binding of motavizumab to RSV A2 pre-F protein; (13) It can inhibit the binding of motavizumab to RSV A2 pre-F protein by 1% to 100%, for example, by 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) capable of competing with or inhibiting the binding of MPE8 to RSV A2 pre-F protein; (15) capable of inhibiting binding of MPE8 to RSV A2 pre-F protein by 1% to 100%, e.g., 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 epitope O of RSV A2 pre-F protein; (17) capable of binding to epitope IV of RSV A2 pre-F protein; (18) capable of binding to epitopes O and IV of the RSV A2 pre-F protein; (19) capable of binding to epitope II of RSV A2 pre-F protein; (20) capable of binding to epitope III of the RSV A2 pre-F protein; and / or (21) Capable of binding to epitopes II and III of RSV A2 pre-F protein The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, having one or more of the following properties:

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

11. (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 The polynucleotide of claim 10, comprising:

12. A vector comprising the polynucleotide of claim 10 or 11.

13. A host cell comprising a polynucleotide according to claim 10 or 11 or a vector according to claim 12.

14. The host cell of claim 13, which is a eukaryotic cell.

15. The host cell of claim 14, which is a CHO cell.

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

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

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

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

20. 20. The antibody or antigen-binding fragment thereof or composition of 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 for the manufacture of a medicament for treating a disease.

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

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

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

25. The invention as described in the specification.

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