RSV-specific antibodies and functional parts thereof

Abstract

To provide means and methods for alleviating and / or preventing a respiratory syncytial virus (RSV) infection.SOLUTION: The present invention provides a synthetic antibody, a recombinant antibody or an isolated antibody that is capable of specifically binding to RSV, serving as an alternative and / or improved antibody against RSV, or functional equivalents of such antibodies, and to provide stable cells capable of producing antibodies or functional equivalents thereof against RSV.SELECTED DRAWING: None

Description

[Technical field]

[0001] Continuing Application Data This application claims the benefit of U.S. Provisional Application No. 61 / 927,819, filed Jan. 15, 2014, the disclosure of which is incorporated herein by reference.

[0002] This application relates to the fields of biotechnology and medicine. [Background technology]

[0003] Respiratory Syncytial Virus (RSV) is a common cold virus that belongs to the Paramyxoviridae family. RSV is highly contagious, easily transmitted, and is the most common cause of lower respiratory tract disease in children under 2 years of age. Up to 98% of children attending day care will become infected during one RSV season. Between 0.5% and 3.2% of children infected with RSV require hospitalization. Approximately 90,000 hospitalizations and 4,500 deaths are reported annually in the United States. The main risk factors for hospitalization due to RSV are premature birth, chronic lung disease, congenital heart disease, compromised immunity, and age less than 6 weeks in otherwise healthy children. RSV-positive bronchiolitis requires additional treatment in addition to supportive care in the form of adequate nutrition and oxygen therapy. Antiviral therapy such as ribavirin has not proven effective in RSV infection. One monoclonal antibody, palivizumab (also called Synagis®), has been registered for the prevention of RSV infection. Palivizumab is a genetically engineered (humanized) monoclonal antibody against the fusion protein of RSV. Although palivizumab is a highly effective prophylactic agent, it would be advantageous for alternative antibodies and therapies to provide further coverage against RSV. Summary of the Invention [Problem to be solved by the invention]

[0004] It is an object to provide means and methods for mitigating and / or preventing RSV infection. It is a further object to provide alternative and / or improved antibodies to RSV or functional equivalents of such antibodies, as well as to provide stable cells capable of producing antibodies to RSV or functional equivalents thereof. [Means for solving the problem]

[0005] In accordance with the present disclosure, a synthetic, recombinant or isolated antibody or functional portion thereof capable of specifically binding to respiratory syncytial virus is a. a heavy chain variable region CDR1 sequence comprising a sequence that is at least 70% identical to the sequence NYIIN (SEQ ID NO:1) or the sequence DYIIN (SEQ ID NO:9); b. a heavy chain variable region CDR2 sequence comprising the sequence GIIPVLGTVHYAPKFQG (SEQ ID NO: 2) or a sequence that is at least 75% identical to the sequence GIIPVLGTVHYGPKFQG (SEQ ID NO: 10); c. a heavy chain variable region CDR3 sequence comprising the sequence ETALVVSTTYLPHYFDN (SEQ ID NO: 3) or a sequence that is at least 70% identical to the sequence ETALVVSTTYRPHYFDN (SEQ ID NO: 11); d. a light chain variable region CDR1 sequence comprising a sequence that is at least 85% identical to the sequence QASQDIVNYLN (SEQ ID NO:4); e. a light chain variable region CDR2 sequence comprising a sequence that is at least 70% identical to the sequence VASNLET (SEQ ID NO:5); f. a light chain variable region CDR3 sequence comprising a sequence that is at least 70% identical to the sequence QQYDNLP (SEQ ID NO:6); Including, At least one amino acid in the heavy chain differs from SEQ ID NO:7, and said at least one amino acid is selected from:

[0006] JPEG2025063143000001.jpg66134

[0007] In another embodiment, the antibody or functional portion comprises: a. the heavy chain variable region CDR1 sequence comprises a sequence that differs by one amino acid from NYIIN (SEQ ID NO:1) or DYIIN (SEQ ID NO:9); b. the heavy chain variable region CDR2 sequence comprises a sequence that differs by one or two amino acids from GIIPVLGTVHYAPKFQG (SEQ ID NO: 2) or GIIPVLGTVHYGPKFQG (SEQ ID NO: 10); c. the heavy chain variable region CDR3 sequence comprises a sequence that differs by one or two amino acids from ETALVVSTTYLPHYFDN (SEQ ID NO: 3) or ETALVVSTTYRPHYFDN (SEQ ID NO: 11); d. the light chain variable region CDR1 sequence comprises a sequence that differs by one amino acid from QASQDIVNYLN (SEQ ID NO:4); e. the light chain variable region CDR2 sequence comprises a sequence that differs by one amino acid from VASNLET (SEQ ID NO:5); and / or f. the light chain variable region CDR3 sequence comprises a sequence that differs by one amino acid from QQYDNLP (SEQ ID NO:6); Includes.

[0008] In another embodiment, the antibody or functional portion comprises: a. the heavy chain variable region CDR1 sequence comprises NYIIN (SEQ ID NO:1) or DYIIN (SEQ ID NO:9); b. the heavy chain variable region CDR2 sequence comprises GIIPVLGTVHYAPKFQG (SEQ ID NO:2) or GIIPVLGTVHYGPKFQG (SEQ ID NO:10); c. the heavy chain variable region CDR3 sequence comprises ETALVVSTTYLPHYFDN (SEQ ID NO:3) or ETALVVSTTYRPHYFDN (SEQ ID NO:11); d. the light chain variable region CDR1 sequence comprises QASQDIVNYLN (SEQ ID NO: 4); e. the light chain variable region CDR2 sequence comprises VASNLET (SEQ ID NO: 5); and f. the light chain variable region CDR3 sequence comprises QQYDNLP (SEQ ID NO:6); Includes.

[0009] In a further embodiment, the antibody or functional portion comprises at least the following amino acids in the heavy chain variable region which differ from SEQ ID NO:7:

[0010] JPEG2025063143000002.jpg45134

[0011] In additional embodiments, at least the following amino acids in the heavy chain variable region differ from SEQ ID NO:7:

[0012] JPEG2025063143000003.jpg29134

[0013] In a further embodiment, the antibody or functional portion comprises at least the following amino acids in the heavy chain variable region which differ from SEQ ID NO:7:

[0014] JPEG2025063143000004.jpg31134

[0015] In further embodiments, the antibody or functional portion comprises at least the following amino acids in the heavy chain variable region that differ from SEQ ID NO:7:

[0016] JPEG2025063143000005.jpg56134

[0017] In a further embodiment, the antibody or functional portion comprises at least the following amino acids in the heavy chain variable region which differ from SEQ ID NO:7:

[0018] JPEG2025063143000006.jpg42134

[0019] In further embodiments, the antibody or functional portion comprises the following amino acids in the heavy chain variable region which differ from SEQ ID NO:7:

[0020] JPEG2025063143000007.jpg48134

[0021] In further embodiments, the antibody or functional portion comprises at least the following amino acids in the heavy chain variable region that differ from SEQ ID NO:7:

[0022] JPEG2025063143000008.jpg61134

[0023] In further embodiments, the antibody or functional portion comprises at least the following amino acids in the heavy chain variable region that differ from SEQ ID NO:7:

[0024] JPEG2025063143000009.jpg51134

[0025] In further embodiments, the antibody or functional portion comprises at least the following amino acids in the heavy chain variable region that differ from SEQ ID NO:7:

[0026] JPEG2025063143000010.jpg59134

[0027] In further embodiments, the antibody or functional portion comprises at least the following amino acids in the heavy chain variable region that differ from SEQ ID NO:7:

[0028] JPEG2025063143000011.jpg56134

[0029] In a further embodiment, the heavy chain variable region CDR1 sequence comprises a sequence that differs by one amino acid from NYIIN (SEQ ID NO: 1) or DYIIN (SEQ ID NO: 9), the heavy chain variable region CDR2 sequence comprises a sequence that differs by one or two amino acids from GIIPVLGTVHYAPKFQG (SEQ ID NO: 2) or GIIPVLGTVHYGPKFQG (SEQ ID NO: 10), and / or the heavy chain variable region CDR3 sequence comprises a sequence that differs by one or two amino acids from ETALVVSTTYLPHYFDN (SEQ ID NO: 3) or ETALVVSTTYRPHYFDN (SEQ ID NO: 11).

[0030] In a further embodiment, the light chain variable region CDR1 sequence comprises a sequence that differs by one amino acid from QASQDIVNYLN (SEQ ID NO: 4), the light chain variable region CDR2 sequence comprises a sequence that differs by one amino acid from VASNLET (SEQ ID NO: 5), and / or the light chain variable region CDR3 sequence comprises a sequence that differs by one amino acid from QQYDNLP (SEQ ID NO: 6).

[0031] In further embodiments, the heavy chain variable region CDR1 sequence comprises NYIIN (SEQ ID NO: 1) or DYIIN (SEQ ID NO: 9), the heavy chain variable region CDR2 sequence comprises GIIPVLGTVHYAPKFQG (SEQ ID NO: 2) or GIIPVLGTVHYGPKFQG (SEQ ID NO: 10), and / or the heavy chain variable region CDR3 sequence comprises ETALVVSTTYLPHYFDN (SEQ ID NO: 3) or ETALVVSTTYRPHYFDN (SEQ ID NO: 11).

[0032] In additional embodiments, the light chain variable region CDR1 sequence comprises QASQDIVNYLN (SEQ ID NO:4), the light chain variable region CDR2 sequence comprises VASNLET (SEQ ID NO:5), and / or the light chain variable region CDR3 comprises QQYDNLP (SEQ ID NO:6).

[0033] In one embodiment, a synthetic, recombinant or isolated antibody or functional portion thereof capable of specifically binding to a RSV F antigen is a. a heavy chain variable region CDR1 sequence comprising the amino acid sequence DYIIN (SEQ ID NO:9); b. a heavy chain variable region CDR2 sequence comprising the amino acid sequence GIIPVLGTVHYGPKFQG (SEQ ID NO: 10); c. a heavy chain variable region CDR3 sequence comprising the amino acid sequence ETALVVSTTYRPHYFDN (SEQ ID NO: 11); d. a light chain variable region CDR1 sequence comprising the amino acid sequence QASQDIVNYLN (SEQ ID NO:4); e. a light chain variable region CDR2 sequence comprising the amino acid sequence VASNLET (SEQ ID NO:5); and f. a light chain variable region CDR3 comprising the amino acid sequence QQYDNLP (SEQ ID NO:6). Includes.

[0034] In another embodiment, a synthetic, recombinant or isolated antibody or functional portion thereof capable of specifically binding to a RSV F antigen is a. a heavy chain variable region CDR1 sequence comprising the amino acid sequence DYIIN (SEQ ID NO:9); b. a heavy chain variable region CDR2 sequence comprising the amino acid sequence GIIPVLGTVHYGPKFQG (SEQ ID NO: 10); c. a heavy chain variable region CDR3 sequence comprising the amino acid sequence ETALVVSTTYLPHYFDN (SEQ ID NO: 3); d. a light chain variable region CDR1 sequence comprising the amino acid sequence QASQDIVNYLN (SEQ ID NO:4); e. a light chain variable region CDR2 sequence comprising the amino acid sequence VASNLET (SEQ ID NO:5); and f. a light chain variable region CDR3 comprising the amino acid sequence QQYDNLP (SEQ ID NO:6). Includes.

[0035] In a further embodiment, the antibody or functional portion has an Fc region having a Y at position 252Y, a T at position 254T and an E at position 256, this numbering corresponding to the EU index in Kabat.

[0036] In a further embodiment, a method of inhibiting RSV infection in a subject, comprising administering to the subject an antibody or functional portion described herein.

[0037] In further embodiments, synthetic, recombinant, or isolated nucleic acid sequences encode the antibodies or functional portions described herein.

[0038] Additional objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice. These objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0039] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the scope of the appended claims.

[0040] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments and, together with the description, serve to explain the principles described herein. [Brief description of the drawings]

[0041] [Figure 1A] FIG. 1 depicts the sequence of the D25 heavy chain and several heavy chain variants (J, L and LA). [Figure 1B] FIG. 1 depicts the sequence of the D25 heavy chain and several heavy chain variants (J, L and LA). [Diagram 2] FIG. 1 shows the heavy chain sequences of several optimized variants (1G7, 1F5, 2D10). [Figure 3A] Figure 1 depicts the heavy chain sequences of four additional variants of optimized variant 1G7. These variants (1G7-GLM, B12-1, E3-5 and E9-2) were created to alter the pI by incorporating germline residues. [Figure 3B] Figure 1 depicts the heavy chain sequences of four additional variants of optimized variant 1G7. These variants (1G7-GLM, B12-1, E3-5 and E9-2) were created to alter the pI by incorporating germline residues. [Figure 4] FIG. 1 is a graph showing the in vitro activity of D25 and several variants in a microneutralization assay. [Diagram 5] Graph showing that D25 and several variants confer protection against RSV A2 and RSV B subtype challenge. [Figure 6]1 is a graph showing the results of cotton rat RSV A2 challenge in animals treated with 1G7, 1F5, D25 (designated D25wt in the figure) or no antibody. [Figure 7] 1 is a graph showing that 1G7 protects against both RSV A2 and RSV B9320. RSV lung titers are plotted as a function of human IgG serum concentration at harvest. [Figure 8] 1 is a graph showing neutralization of both RSV A2 and RSV B9320 by various antibodies. [Figure 9] FIG. 1 shows the binding epitope of 1G7 defined by monoclonal antibody resistant mutants. [Figure 10] 1 is a graph showing the results of a neutralization assay against clinical isolates of RSV. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] Array Description Table 1 below lists some of the sequences referenced in this embodiment.

[0043] [Table 1]

[0044] JPEG2025063143000013.jpg81122

[0045] Description of the embodiments I. Antibodies or Functional Portions Thereof Improved RSV-specific antibodies or functional portions thereof are provided that have improved properties compared to other antibodies. Antibodies or functional portions, such as antibodies or antigen-binding fragments, variants or derivatives thereof, include, but are not limited to, polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies or chimeric antibodies, single chain antibodies, epitope-binding fragments, such as Fab, Fab' and F(ab')2, Fd, Fvs, single chain Fvs (scFv), single chain antibodies, disulfide-linked Fvs (sdFv), fragments containing VL or VH domains, fragments produced by Fab expression libraries. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019. Immunoglobulin or antibody molecules encompassed by this disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), of any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or of any subclass of immunoglobulin molecule.

[0046] For example, the present inventors have succeeded in generating RSV-specific antibodies with improved properties compared to previous RSV-specific antibodies, such as improved protection against RSV A and RSV B subtypes, improved neutralization, and lower IC50 values. Such antibodies have a particularly high or strong affinity for RSV and are therefore particularly suitable for alleviating and / or at least to some extent preventing RSV infection and / or the adverse effects of RSV infection. Antibodies and functional portions thereof are synonymous with RSV-specific binding molecules, and include any full-length antibody or antibody portion that can specifically bind to RSV.

[0047] A. Antibodies or functional moieties in which non-germline residues are changed to germline residues In one embodiment, at least one non-germline residue in the heavy chain variable region of D25 (SEQ ID NO: 7) is changed to a germline residue. In another embodiment, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 non-germline residues in the heavy chain variable region of D25 residues are replaced back to a germline residue. In a further embodiment, at least one non-germline residue can be changed to a germline residue and at least one CDR can be altered relative to SEQ ID NO: 7.

[0048] One embodiment is capable of specifically binding to respiratory syncytial virus, a) a heavy chain CDR1 sequence comprising a sequence that is at least 70%, 75%, 80%, 85%, 90% or 94% identical to the sequence NYIIN (SEQ ID NO: 1) or the sequence DYIIN (SEQ ID NO: 9); b) a heavy chain CDR2 sequence comprising a sequence that is at least 70%, 75%, 80%, 85%, 90% or 94% identical to the sequence GIIPVLGTVHYAPKFQG (SEQ ID NO: 2) or the sequence GIIPVLGTVHYGPKFQG (SEQ ID NO: 10); c) a heavy chain CDR3 sequence comprising the sequence ETALVVSTTYLPHYFDN (SEQ ID NO: 3) or a sequence that is at least 70%, 75%, 80%, 85%, 90% or 94% identical to the sequence ETALVVSTTYRPHYFDN (SEQ ID NO: 11); d) a light chain CDR1 sequence comprising a sequence that is at least 70%, 75%, 80%, 85%, 90% or 94% identical to the sequence QASQDIVNYLN (SEQ ID NO: 4); e) a light chain CDR2 sequence comprising a sequence that is at least 70%, 75%, 80%, 85%, 90% or 94% identical to the sequence VASNLET (SEQ ID NO:5); f) a light chain CDR3 sequence comprising a sequence that is at least 70%, 75%, 80%, 85%, 90% or 94% identical to the sequence QQYDNLP (SEQ ID NO: 6); An isolated, synthetic or recombinant antibody or a functional portion thereof comprising: The present invention includes an isolated, synthetic or recombinant antibody, or a functional portion thereof, wherein at least one amino acid in the heavy chain differs from SEQ ID NO:7, wherein said at least one amino acid is selected from the following:

[0049] [Table 2]

[0050] "At least one amino acid in the heavy chain variable region differs from SEQ ID NO:7" means that the antibody or functional portion must contain at least one amino acid difference from SEQ ID NO:7, but in some embodiments the antibody or functional portion can contain multiple differences from SEQ ID NO:7. In some embodiments, the antibody or functional portion contains substitutions as outlined in Table 2, deletions or replacement amino acids at positions listed in Table 2, or differences (such as deletions, substitutions or additions) at positions not listed in Table 2. The position numbers in Table 2 are listed relative to SEQ ID NO:7, and may not ultimately be the same as the position numbers based on sequential numbering of the amino acids in the antibody or functional portion of interest. Instead, the position numbers are assigned based on sequence comparison to SEQ ID NO:7.

[0051] In one embodiment, the antibody or functional portion comprises a heavy and / or light chain, wherein the heavy chain variable region is at least 90% identical to SEQ ID NO:7 or SEQ ID NO:15 and the light chain variable region is at least 90% identical to SEQ ID NO:8. In another embodiment, the heavy chain variable region is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:7 or SEQ ID NO:15. In another embodiment, the light chain variable region is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:8.

[0052] In one embodiment, the antibody or functional portion comprises a heavy chain variable region CDR1 sequence that comprises a sequence that differs by one amino acid from NYIIN (SEQ ID NO:1) or DYIIN (SEQ ID NO:9).

[0053] In one embodiment, the antibody or functional portion comprises a heavy chain variable region CDR2 sequence that comprises a sequence that differs by one or two amino acids from GIIPVLGTVHYAPKFQG (SEQ ID NO: 2) or GIIPVLGTVHYGPKFQG (SEQ ID NO: 10).

[0054] In one embodiment, the antibody or functional portion comprises a heavy chain variable region CDR3 sequence comprising a sequence which differs by one or two amino acids from ETALVVSTTYLPHYFDN (SEQ ID NO: 3) or ETALVVSTTYRPHYFDN (SEQ ID NO: 11).

[0055] In one embodiment, the antibody or functional portion comprises a light chain variable region CDR1 sequence comprising a sequence differing by one amino acid from QASQDIVNYLN (SEQ ID NO: 4).

[0056] In one embodiment, the antibody or functional portion comprises a light chain variable region CDR2 sequence comprising a sequence that differs by one amino acid from VASNLET (SEQ ID NO:5).

[0057] In one embodiment, the antibody or functional portion comprises a light chain variable region CDR3 comprising a sequence that differs by one amino acid from QQYDNLP (SEQ ID NO: 6).

[0058] In a further embodiment, the antibody or functional portion comprises a heavy chain variable region CDR1 sequence comprising a sequence that differs by one amino acid from NYIIN (SEQ ID NO: 1) or DYIIN (SEQ ID NO: 9), a heavy chain variable region CDR2 sequence comprising a sequence that differs by one or two amino acids from GIIPVLGTVHYAPKFQG (SEQ ID NO: 2) or GIIPVLGTVHYGPKFQG (SEQ ID NO: 10), a heavy chain variable region CDR3 sequence comprising a sequence that differs by one or two amino acids from ETALVVSTTYLPHYFDN (SEQ ID NO: 3) or ETALVVSTTYRPHYFDN (SEQ ID NO: 11), a light chain variable region CDR1 sequence comprising a sequence that differs by one amino acid from QASQDIVNYLN (SEQ ID NO: 4), a light chain variable region CDR2 sequence comprising a sequence that differs by one amino acid from VASNLET (SEQ ID NO: 5), and / or a light chain variable region CDR3 sequence comprising a sequence that differs by one amino acid from QQYDNLP (SEQ ID NO: 6).

[0059] In some embodiments the antibody or functional portion has at least one or up to all CDRs identical to SEQ ID NO: 7 (heavy chain) and / or SEQ ID NO: 8 (light chain).

[0060] In one embodiment, the antibody or functional portion comprises a heavy chain variable region CDR1 sequence comprising NYIIN (SEQ ID NO:1) or DYIIN (SEQ ID NO:9).

[0061] In one embodiment, the antibody or functional portion comprises a heavy chain variable region CDR2 sequence comprising GIIPVLGTVHYAPKFQG (SEQ ID NO: 2) or GIIPVLGTVHYGPKFQG (SEQ ID NO: 10).

[0062] In one embodiment, the antibody or functional portion comprises a heavy chain variable region CDR3 sequence comprising ETALVVSTTYLPHYFDN (SEQ ID NO: 3) or ETALVVSTTYRPHYFDN (SEQ ID NO: 11).

[0063] In one embodiment, the antibody or functional portion comprises a heavy chain variable region CDR1 sequence comprising NYIIN (SEQ ID NO: 1) or DYIIN (SEQ ID NO: 9), a heavy chain variable region CDR2 sequence comprising GIIPVLGTVHYAPKFQG (SEQ ID NO: 2) or GIIPVLGTVHYGPKFQG (SEQ ID NO: 10), and / or a heavy chain variable region CDR3 sequence comprising ETALVVSTTYLPHYFDN (SEQ ID NO: 3) or ETALVVSTTYRPHYFDN (SEQ ID NO: 11).

[0064] In one embodiment, the antibody or functional portion comprises a light chain variable region CDR1 sequence comprising QASQDIVNYLN (SEQ ID NO: 4).

[0065] In one embodiment, the antibody or functional portion comprises that the light chain variable region CDR2 sequence comprises VASNLET (SEQ ID NO:5).

[0066] In one embodiment, the antibody or functional portion comprises that the light chain variable region CDR3 comprises QQYDNLP (SEQ ID NO: 6).

[0067] In one embodiment, the antibody or functional portion comprises a light chain variable region CDR1 sequence comprising QASQDIVNYLN (SEQ ID NO: 4), a light chain variable region CDR2 sequence comprising VASNLET (SEQ ID NO: 5), and / or a light chain variable region CDR3 sequence comprising QQYDNLP (SEQ ID NO: 6).

[0068] In a further embodiment, the antibody or functional portion comprises a heavy chain variable region CDR1 sequence comprising NYIIN (SEQ ID NO: 1) or DYIIN (SEQ ID NO: 9), a heavy chain variable region CDR2 sequence comprising GIIPVLGTVHYAPKFQG (SEQ ID NO: 2) or GIIPVLGTVHYGPKFQG (SEQ ID NO: 10), a heavy chain variable region CDR3 sequence comprising ETALVVSTTYLPHYFDN (SEQ ID NO: 3) or ETALVVSTTYRPHYFDN (SEQ ID NO: 11), a light chain variable region CDR1 sequence comprising QASQDIVNYLN (SEQ ID NO: 4), a light chain variable region CDR2 sequence comprising VASNLET (SEQ ID NO: 5), and / or a light chain variable region CDR3 sequence comprising QQYDNLP (SEQ ID NO: 6).

[0069] In one embodiment, the antibody or functional portion comprises a heavy chain variable region comprising a sequence that is 95% identical in framework regions to a sequence selected from SEQ ID NOs: 12-21.

[0070] In one embodiment, the antibody or functional portion comprises a heavy chain variable region comprising a sequence identical in framework regions to a sequence selected from SEQ ID NOs: 12-21.

[0071] In one embodiment, the antibody or functional portion comprises at least the following amino acids in the heavy chain variable region which differ from SEQ ID NO:7:

[0072] [Table 3]

[0073] In another embodiment, the differences set forth in Table 3 are the only differences from SEQ ID NO: 7. In one embodiment, the differences set forth in Table 3 are not the only differences from SEQ ID NO: 7. In another embodiment, the light chain variable region comprises SEQ ID NO:8.

[0074] In one embodiment, the antibody or functional portion comprises at least the following amino acids in the heavy chain variable region which differ from SEQ ID NO:7:

[0075] [Table 4]

[0076] In another embodiment, the differences set forth in Table 4 are the only differences from SEQ ID NO: 7. In one embodiment, the differences set forth in Table 4 are not the only differences from SEQ ID NO: 7. In another embodiment, the light chain variable region comprises SEQ ID NO:8.

[0077] In one embodiment, the antibody or functional portion comprises at least the following amino acids in the heavy chain variable region which differ from SEQ ID NO:7:

[0078] [Table 5]

[0079] In another embodiment, the differences listed in Table 5 are the only differences from SEQ ID NO: 7. In one embodiment, the differences listed in Table 5 are not the only differences from SEQ ID NO: 7. In another embodiment, the light chain variable region comprises SEQ ID NO:8.

[0080] In one embodiment, the antibody or functional portion comprises at least the following amino acids in the heavy chain variable region which differ from SEQ ID NO:7:

[0081] [Table 6]

[0082] In another embodiment, the differences listed in Table 6 are the only differences from SEQ ID NO: 7. In one embodiment, the differences listed in Table 6 are not the only differences from SEQ ID NO: 7. In another embodiment, the light chain variable region comprises SEQ ID NO:8.

[0083] In one embodiment, the antibody or functional portion comprises at least the following amino acids in the heavy chain variable region which differ from SEQ ID NO:7:

[0084] [Table 7]

[0085] In another embodiment, the differences listed in Table 7 are the only differences from SEQ ID NO: 7. In one embodiment, the differences listed in Table 7 are not the only differences from SEQ ID NO: 7. In another embodiment, the light chain variable region comprises SEQ ID NO:8.

[0086] In one embodiment, the antibody or functional portion comprises at least the following amino acids in the heavy chain variable region which differ from SEQ ID NO:7:

[0087] [Table 8]

[0088] In another embodiment, the differences set forth in Table 8 are the only differences from SEQ ID NO: 7. In one embodiment, the differences set forth in Table 8 are not the only differences from SEQ ID NO: 7. In another embodiment, the light chain variable region comprises SEQ ID NO:8.

[0089] In one embodiment, the antibody or functional portion comprises at least the following amino acids in the heavy chain variable region which differ from SEQ ID NO:7:

[0090] [Table 9]

[0091] In another embodiment, the differences listed in Table 9 are the only differences from SEQ ID NO: 7. In one embodiment, the differences listed in Table 9 are not the only differences from SEQ ID NO: 7. In another embodiment, the light chain variable region comprises SEQ ID NO:8.

[0092] In one embodiment, the antibody or functional portion comprises at least the following amino acids in the heavy chain variable region which differ from SEQ ID NO:7:

[0093] [Table 10]

[0094] In another embodiment, the differences listed in Table 10 are the only differences from SEQ ID NO: 7. In one embodiment, the differences listed in Table 10 are not the only differences from SEQ ID NO: 7. In another embodiment, the light chain variable region comprises SEQ ID NO:8.

[0095] In one embodiment, the antibody or functional portion comprises at least the following amino acids in the heavy chain variable region which differ from SEQ ID NO:7:

[0096] [Table 11]

[0097] In another embodiment, the differences set forth in Table 11 are the only differences from SEQ ID NO: 7. In one embodiment, the differences set forth in Table 11 are not the only differences from SEQ ID NO: 7. In another embodiment, the light chain variable region comprises SEQ ID NO:8.

[0098] In one embodiment, the antibody or functional portion comprises at least the following amino acids in the heavy chain variable region which differ from SEQ ID NO:7:

[0099] [Table 12]

[0100] In another embodiment, the differences set forth in Table 12 are the only differences from SEQ ID NO: 7. In one embodiment, the differences set forth in Table 12 are not the only differences from SEQ ID NO: 7. In another embodiment, the light chain variable region comprises SEQ ID NO:8.

[0101] In one embodiment, the antibody or functional portion comprises a heavy chain variable region sequence comprising a sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21. In another embodiment, the antibody or functional portion comprises a heavy chain variable region sequence comprising a sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the framework (i.e. non-CDR) sequences of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

[0102] In another embodiment, the antibody or functional portion comprises a light chain variable region sequence comprising a sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence DIQMTQSPSSLSAAVGDRVTITCQASQDIVNYLNWYQQKPGKAPKLLIYVASNLETGVPSRFSGSGSGTDFSLTISSLQPEDVATYYCQQYDNLPLTFGGGTKVEIKRTV (SEQ ID NO: 8).

[0103] In another embodiment, conservative amino acid substitutions are made, which include the replacement of one amino acid with another that generally has similar properties (size, hydrophobicity, etc.) such that overall function is unlikely to be seriously affected.

[0104] B. Antibodies or functional portions having modified CDRs In one embodiment, the antibody or functional portion may comprise at least one CDR mutation relative to the CDR present in SEQ ID NO: 7. In one embodiment, the antibody or functional portion may comprise at least one of the alterations in Table 13. In another embodiment, the antibody or functional portion may comprise one, two or all three of the alterations in Table 13.

[0105] [Table 13]

[0106] Another embodiment is an isolated antibody, synthetic antibody or recombinant antibody or functional portion thereof that can specifically bind to the RSV F antigen and includes a heavy chain variable region CDR1 sequence comprising the amino acid sequence DYIIN (SEQ ID NO: 9), a heavy chain variable region CDR2 sequence comprising the amino acid sequence GIIPVLGTVHYAPKFQG (SEQ ID NO: 2), a heavy chain variable region CDR3 sequence comprising the amino acid sequence ETALVVSTTYLPHYFDN (SEQ ID NO: 3), a light chain variable region CDR1 sequence comprising the amino acid sequence QASQDIVNYLN (SEQ ID NO: 4), a light chain variable region CDR2 sequence comprising the amino acid sequence VASNLET (SEQ ID NO: 5), and a light chain variable region CDR3 comprising the amino acid sequence QQYDNLP (SEQ ID NO: 6).

[0107] Another embodiment is an isolated antibody, synthetic antibody or recombinant antibody or functional portion thereof that can specifically bind to the RSV F antigen and includes a heavy chain variable region CDR1 sequence comprising the amino acid sequence NYIIN (SEQ ID NO: 1), a heavy chain variable region CDR2 sequence comprising the amino acid sequence GIIPVLGTVHYGPKFQG (SEQ ID NO: 10), a heavy chain variable region CDR3 sequence comprising the amino acid sequence ETALVVSTTYLPHYFDN (SEQ ID NO: 3), a light chain variable region CDR1 sequence comprising the amino acid sequence QASQDIVNYLN (SEQ ID NO: 4), a light chain variable region CDR2 sequence comprising the amino acid sequence VASNLET (SEQ ID NO: 5), and a light chain variable region CDR3 comprising the amino acid sequence QQYDNLP (SEQ ID NO: 6).

[0108] Another embodiment is an isolated antibody, synthetic antibody or recombinant antibody or functional portion thereof that can specifically bind to the RSV F antigen and includes a heavy chain variable region CDR1 sequence comprising the amino acid sequence NYIIN (SEQ ID NO: 1), a heavy chain variable region CDR2 sequence comprising the amino acid sequence GIIPVLGTVHYAPKFQG (SEQ ID NO: 2), a heavy chain variable region CDR3 sequence comprising the amino acid sequence ETALVVSTTYRPHYFDN (SEQ ID NO: 11), a light chain variable region CDR1 sequence comprising the amino acid sequence QASQDIVNYLN (SEQ ID NO: 4), a light chain variable region CDR2 sequence comprising the amino acid sequence VASNLET (SEQ ID NO: 5), and a light chain variable region CDR3 comprising the amino acid sequence QQYDNLP (SEQ ID NO: 6).

[0109] In another embodiment, the isolated, synthetic or recombinant antibody, or functional portion thereof, comprises a heavy chain variable region comprising a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, and a light chain comprising a light chain CDR1, a light chain CDR2 and a light chain CDR3, wherein at least one, two or all three heavy chain CDRs are selected from column A in Table 14, the remaining heavy chain CDRs (if present) are selected from column B, and the light chain CDRs comprise column C. Thus, in one embodiment, the light chain CDRs are set forth in column C of Table 14, and heavy chain CDRs from columns A and B can be mixed and matched as long as the heavy chain has one CDR1, one CDR2 and one CDR3.

[0110] [Table 14]

[0111] The various alternative CDR embodiments do not necessarily include all of the non-germline to germline mutations discussed above in Section IA, although any or all of these mutations can optionally be present.

[0112] In one embodiment, the antibody or functional portion may comprise at least one of the alternative heavy chain CDRs from Table 14 and may have at least one other CDR modification. Specifically, in yet another embodiment, the antibody or functional portion comprises a heavy chain CDR1 sequence that comprises a sequence that differs by one amino acid from NYIIN (SEQ ID NO: 1), a heavy chain CDR2 sequence that comprises a sequence that differs by one or two amino acids from GIIPVLGTVHYAPKFQG (SEQ ID NO: 2), a heavy chain CDR3 sequence that comprises a sequence that differs by one or two amino acids from ETALVVSTTYLPHYFDN (SEQ ID NO: 3), a light chain CDR1 sequence that comprises a sequence that differs by one or two amino acids from QASQDIVNYLN (SEQ ID NO: 4), and a light chain CDR2 sequence that comprises a sequence that differs by one or two amino acids from GIIPVLGTVHYAPKFQG (SEQ ID NO: 5). the light chain CDR2 sequence comprises a sequence that differs by one amino acid from VASNLET (SEQ ID NO:5), and / or the light chain CDR3 sequence comprises a sequence that differs by one amino acid from QQYDNLP (SEQ ID NO:6), and at least one heavy chain CDR1 is DYIIN (SEQ ID NO:9), the heavy chain CDR2 is GIIPVLGTVHYGPKFQG (SEQ ID NO:10), or the heavy chain CDR3 is ETALVVSTTYRPHYFDN (SEQ ID NO:11).

[0113] C. Antibodies or functional moieties with improved half-life In one embodiment, the antibody or functional portion described herein may be further modified to improve the half-life of the antibody or functional portion. In one embodiment, the antibody or functional portion may be mutated, such as a deletion mutation, an addition mutation, or a substitution mutation, to improve the half-life of the antibody or functional portion. In one embodiment, the Fc region may be mutated to include one, two, or all three of the following substitutions: M252Y, S254T, and T256E, the numbering of which corresponds to the EU index in Kabat. In one embodiment, the Fc region may be mutated to include all of the following substitutions: M252Y, S254T, and T256E, the numbering of which corresponds to the EU index in Kabat. Dall'Acqua et al., Properties of Human IgG1s Engineered for Enhanced Binding to the Neonatal Fc Receptor(FcRn), J Biol Chem 281(33):23514-23524(2006). An embodiment with all three substitutions is designated the YTE variant. Alternatively represented, in one embodiment, the antibody or functional portion has an Fc region with a Y at position 252Y, a T at position 254T, and an E at position 256, this numbering corresponds to the EU index in Kabat.

[0114] D. Other Properties of Antibodies and Functional Portions thereof In one embodiment, the antibody or functional portion has an IC50 value of less than 10 ng / ml in an in vitro neutralization assay in which Hep-2 cells are infected with RSV and the antibody or functional portion. In another embodiment, the IC50 is 9 ng / ml, 8 ng / ml, 7 ng / ml, 6 ng / ml, 5 ng / ml, 4 ng / ml, 3 ng / ml, or 2 ng / ml or less for RSV subtype A and / or RSV subtype B. In one embodiment, the IC50 is measured in an in vitro neutralization assay described in the Examples, and optionally in an in vitro neutralization assay described in the Examples for RSV A2 and / or RSV B9320.

[0115] In one embodiment, the antibodies and functional portions thereof are effective in neutralizing RSV subtype A strains. In one embodiment, the antibodies and functional portions thereof are effective in neutralizing RSV subtype B strains. In another embodiment, the antibodies and functional portions thereof are effective in neutralizing both RSV subtype A strains and RSV subtype B strains.

[0116] As used herein, the term antibody or functional portion thereof is used in the broadest sense. The term antibody or functional portion thereof can be an artificial product such as a monoclonal antibody (mAb) produced by conventional hybridoma technology, recombinant technology, and / or a functional fragment thereof. The term antibody or functional portion thereof can include both intact immunoglobulin molecules, such as polyclonal antibodies, monoclonal antibodies (mAb), monospecific antibodies, bispecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, animal antibodies (e.g. camelid antibodies), chimeric antibodies, and parts, fragments, regions, peptides and derivatives thereof (produced by any known technique, such as, but not limited to, enzymatic cleavage, peptide synthesis or recombinant technology), such as immunoglobulins lacking light chains, Fab, Fab', F(ab')2, Fv, scFv, antibody fragments, diabodies, Fd, CDR regions, or any portion or peptide sequence of an antibody capable of binding to an antigen or epitope. In one embodiment, the functional portion is a single chain antibody, a single chain variable fragment (scFv), a Fab fragment or a F(ab')2 fragment.

[0117] An antibody or functional portion is said to be "capable of binding" to a molecule if the antibody or functional portion is capable of specifically reacting with the molecule and thereby binding the molecule to the antibody. An antibody fragment or portion can lack the Fc fragment of an intact antibody, and can be cleared from the circulation more rapidly and have less non-specific tissue binding compared to an intact antibody. Examples of antibodies can be generated from intact antibodies using methods known in the art, for example, by proteolytic cleavage with enzymes such as papain (to generate Fab fragments) or pepsin (to generate F(ab')2 fragments). Antibody portions can be produced by any of the above methods or by expressing portions of recombinant molecules. For example, the CDR regions of recombinant antibodies can be isolated and subcloned into a suitable expression vector.

[0118] In one embodiment, the antibody or functional portion is a human antibody. The use of human antibodies for human therapeutic purposes can reduce the possibility of side effects due to immunological reactions in human individuals against non-human sequences. In another embodiment, the antibody or functional portion is humanized. In another embodiment, the antibody or functional portion is a chimeric antibody. In this way, a sequence of interest, such as a binding site of interest, can be included in the antibody or functional portion.

[0119] In one embodiment, the antibody can have an IgG, IgA, IgM or IgE isotype, hi one embodiment, the antibody is an IgG.

[0120] II. Nucleic Acids Encoding Antibodies and Functional Portions Thereof The present embodiment further provides isolated, synthetic or recombinant nucleic acid sequences encoding any of the antibodies or functional portions described in Sections IA or IB above. Such nucleic acids are, for example, isolated from B cells capable of producing the antibodies or functional portions. Such nucleic acids encode the heavy and light chain sequences described herein. Alternatively, such nucleic acids encode the heavy and light chain sequences comprising the heavy and light chain CDRs, respectively, described herein. In some embodiments, the nucleic acids can encode the functional portions of the antibodies described herein. Due to the degeneracy of the nucleic acid code, multiple nucleic acids can encode the same amino acid, and all are encompassed by the present application.

[0121] III.How to use A. Methods of Use of Antibodies or Functional Moieties In one embodiment, the antibody or functional portion can be used in a treatment method or as a medicament. The method can be used to alleviate or at least partially prevent RSV infection, or to alleviate or at least partially prevent the adverse effects of RSV infection. The method also includes administering a therapeutically effective amount of the antibody or functional portion described herein to an individual in need. In one embodiment, the individual in need is a human patient.

[0122] In one embodiment, the antibody or functional portion can be administered to an individual before RSV infection occurs, in other words, as a prophylactic, to mitigate RSV. Alternatively, the antibody or functional portion can be administered when the individual is already infected with RSV. The antibody or functional portion can be administered to individuals at increased risk of RSV infection, such as premature children, individuals with chronic lung disease, congenital heart disease, and / or immunocompromised, children under 6 weeks of age. Premature children include both 1-year-old infants and 2-year-old and older children who remain at risk for RSV infection. Elderly people are also at increased risk of RSV infection and therefore may be candidates for administration according to their risk. The antibody or functional portion can also be administered to individuals who have previously been infected with RSV.

[0123] For therapeutic use, the antibody or functional portion is generally combined with a pharma- ceutically acceptable carrier, adjuvant, diluent and / or excipient. In one embodiment, the antibody or functional portion is combined with water for injection. In another embodiment, the antibody or functional portion is prepared in a sterile, preservative-free liquid solution with histidine, glycine and chloride. In another embodiment, suitable carriers include, for example, keyhole limpet hemocyanin (KLH), serum albumin (e.g., BSA or RSA) and ovalbumin. In another embodiment, the suitable carriers include solutions such as, for example, saline. In other embodiments, the antibody or functional portion is provided in lyophilized form and mixed with water for injection prior to administration.

[0124] B. Methods of Using Nucleic Acids Encoding Antibodies or Functional Portions In yet another embodiment, a nucleic acid encoding an antibody or functional portion can be administered. When such a nucleic acid is administered, the host's machinery produces the antibody or functional portion. The produced antibody or functional portion can prevent and / or alleviate RSV infection and / or the adverse effects of RSV infection.

[0125] A nucleic acid encoding a functional portion of an antibody means a nucleic acid at least 30 base pairs in length, at least 50 base pairs in length, or at least 100 base pairs in length that contains at least one expression characteristic (type, not necessarily amount) of a nucleic acid encoding an antibody. In one embodiment, a nucleic acid encoding a functional portion of an antibody encodes at least an amino acid sequence comprising two or optionally three CDRs of an antibody described herein.

[0126] IV. Methods of Producing Antibodies and Functional Portions Also provided are isolated antibody-producing cells capable of producing the antibodies or functional portions. The antibodies or functional portions described herein can be produced from hybridomas that secrete the antibodies, or can be produced from recombinantly produced cells that have been transformed or transfected with a gene encoding the antibody or functional portion.

[0127] One embodiment includes a method of producing the antibody or functional portion by culturing a host cell under conditions in which the nucleic acid is expressed to produce the antibody, and then recovering the antibody. A variety of cell lines can be used to express the antibody or functional portion, including but not limited to mammalian cell lines. In one embodiment, the cell line can be human. In another embodiment, bacterial or insect cell lines can be used. In one embodiment, the cell line includes Chinese Hamster Ovary (CHO) cells, variants of CHO cells (e.g., DG44), 293 cells, and NSO cells. In another embodiment, the cell line includes VERY cells, BHK cells, Hela cells, COS cells, MDCK cells, 293F cells, 293T cells, 3T3 cells, W138 cells, BT483 cells, Hs578T cells, HTB2 cells, BT2O cells, and T47D cells, CRL7O3O cells, and HsS78Bst cells.

[0128] Recombinant expression utilizes the construction of an expression vector that contains a polynucleotide encoding the antibody or functional portion. Once the polynucleotide is obtained, a vector for the production of the antibody can be generated by recombinant DNA techniques known in the art. The expression vector can include appropriate transcriptional and translational regulatory signals. This can be accomplished using in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. In one embodiment, a replicable vector contains a nucleic acid sequence encoding the antibody or functional portion that is operably linked to a heterologous promoter.

[0129] As described in U.S. Pat. No. 5,807,715, various host-expression vector systems can be utilized to express antibodies or functional portions. For example, mammalian cells such as Chinese hamster ovary cells (CHO) are effective expression systems for antibodies with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus (Foecking et al., Gene, 45:101 (1986) and Cockett et al., Bio / Technology, 8:2 (1990)). In addition, a host cell line can be selected that regulates the expression of the sequence of interest or modifies and processes the gene product in the specific manner desired. Such protein product modifications (e.g., glycosylation) and processing (e.g., cleavage) can be important for the function of the protein. Various host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure the correct modification and processing of the proteins of the invention. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.

[0130] In bacterial systems, a number of expression vectors can be selected depending on the intended use of the expressed antibody or functional portion. For example, when large quantities of an antibody or functional portion are to be produced to generate pharmaceutical compositions containing such an antibody or functional portion, a vector that directs high-level expression of a fusion protein product that is easily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO, 12:1791 (1983)), pIN vector (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, 1989, J. Biol. Chem., 24:5503-5509 (1989)), and the like, in which coding sequences can be individually ligated into the vector in frame with the lacZ coding region to produce a fusion protein. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione-S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to a glutathione-agarose affinity matrix followed by elution in the presence of free glutathione. pGEX vectors are designed to introduce thrombin and / or factor Xa protease cleavage sites into the expressed polypeptide so that the cloned target gene product can be released from the GST moiety.

[0131] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. Protein coding sequences can be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0132] In mammalian host cells, a number of virus-based expression systems are available. When adenovirus is used as an expression vector, the coding sequence of interest can be ligated to the adenovirus transcription / translation control complex, for example, to the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into non-essential regions of the viral genome, such as region E1 or region E3, can result in recombinant viruses that are viable and capable of expressing the antibody or the functional portion in an infected host (see, for example, Logan & Shenk, Proc. Natl. Acad. Sci. USA, 81:355-359 (1984)). A specific initiation signal is also required for efficient translation of the inserted antibody or functional portion coding sequence. The signal includes the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon should usually be in frame with the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translational control signals and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, and the like (see, e.g., Bittner et al., Methods in Enzymol., 153:51-544 (1987)).

[0133] Stable expression can be used to produce recombinant proteins in high yields over long periods of time. For example, cell lines can be generated that stably express the protein molecule. Host cells can be transformed with appropriately engineered vectors that contain expression control elements (e.g., promoters, enhancers, transcription terminators, polyadenylation sites, etc.) and selectable marker genes. After introduction of the foreign DNA, cells can be grown in enriched medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, and cells with the plasmid stably integrated into their chromosomes can be grown to form foci and then cloned and expanded into cell lines. Using plasmids encoding antibodies or functional portions, genes / cDNAs can be introduced into any cell line suitable for production in culture.

[0134] A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell, 11:223 (1977)), hypoxanthine guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell, 22:8-17 (1980)) genes, which may be utilized in tk cells, hgprt cells, or aprT cells, respectively. Antimetabolite resistance can also be used as the basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA, 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA, 78:1527 (1981)), gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072 (1981)), neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993) and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIB TECH 11(5):155-215 (1993)), and hygro, which confers resistance to hygromycin (Santerre et al., Gene, 30:147 (1984)).Methods commonly known in the field of recombinant DNA technology can be routinely applied for the selection of the desired recombinant clones, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993), Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990) and Chapters 12 and 13, Dracopoli et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994), Colberre-Garapin et al., 1981, J. Mol. Biol., 150:1.

[0135] Once an antibody or functional portion is produced by recombinant expression, it can be purified by any method known in the art for the purification of immunoglobulin molecules, such as by chromatography (e.g., ion exchange, affinity, particularly affinity for the specific antigens Protein A or Protein G, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for the purification of proteins. Additionally, the proteins or fragments of the invention can be fused to heterologous polypeptide sequences described herein or known in the art to facilitate purification.

[0136] In some embodiments, RSV-specific antibody-producing cells are generated that are stable for at least 6 months. In another embodiment, RSV-specific antibody-producing cells are stable for at least 9 weeks, at least 3 months, or at least 6 months. In another embodiment, alternative methods for producing antibodies and functional moieties are known in the art and are described at least in U.S. Patent No. 8,562,996.

[0137] Reference will now be made in more detail to the exemplary embodiments, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or similar parts. Other embodiments will be apparent to those skilled in the art from consideration of the specification and practice disclosed herein. The embodiments are further described in the examples below. The examples do not limit the scope of the claims, but merely serve to clarify some embodiments. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit being indicated by the following claims. EXAMPLES

[0138] Example 1. Preparation and expression of monoclonal antibodies The DNA fragments of the variable light (VL) and variable heavy (VH) chains of the 1G7 immunoglobulin (each containing the desired mutations that enhance the potency of the antibody) were inserted into a human IgG1 expression vector containing a kappa light constant region and a CH1-hinge-CH2-CH3 IgG1 heavy constant region. To express the 1G7 antibody, human embryonic kidney 293-F cells were transiently transformed with the 1G7-containing vector using 293Fectin™ reagent (Invitrogen, Carlsbad, CA). The cells were grown at 37°C, 120 rpm, 5% CO2 and 80% humidity. Culture medium was fed on day 2 by adding an equal volume of medium and harvested on day 10 after transfection. The supernatant was sterile filtered to remove cells and debris. IgG was purified using a protein A column (Hitrap protein A column, Sigma) and the eluted protein was dialyzed against PBS at 4°C overnight. IgG concentrations were measured by protein quantification in a NanoDrop (Thermo Scientific).

[0139] The same approach was taken to generate other antibodies.

[0140] Example 2. In vitro microneutralization assay Microneutralization assays were performed as follows: Briefly, two-fold serial dilutions of MAbs were introduced into 384-well microtiter plates in HEp-2 cell culture medium at a volume of 15 μL / well. Then, 15 μL of RSV A2 or RSV B9320 virus was diluted in Hep-2 cell culture medium to concentrations ranging from 80 to 150 pfu / well and added to each well, including control wells containing HEp-2 cell culture medium alone, and the plates were incubated at 37° C., 5% CO2 for 1.5 hours. HEp-2 cells were cultured at 2.5×10 in 30 μL. 5 cells / ml were added to each well and plates were incubated at 37° C., 5% CO2. After 3 days for RSV A2 or 4 days for RSV B9320, media was removed and cells were fixed by adding 30 μL of ice-cold 80% acetone / 20% PBS.

[0141] Viral replication was measured by enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase-conjugated anti-RSV F MAb targeting the C site of RSV F (1331H) (Beeler and van Wyke Coelingh, J Virol. 63(7):2941-2950 (1989). 1331H MAb was diluted 1:6,000 in PBS and 30 μL was added to each well. After 2 h incubation at 37°C, plates were washed three times with PBS-T. 30 μL of TMB peroxidase was added to each well and plates were incubated for 15 min at room temperature in the dark. The reaction was stopped by adding 15 μL of 2N H2SO4 to each well. Substrate turnover was measured by monitoring absorbance at 450 nm using a microplate reader. Graphpad software was used to calculate the 1331H MAb using log(inhibitor) vs. response with a variable slope curve fit. IC50 values ​​were calculated using a non-linear fit algorithm in Prism and represent the concentration of MAb required for a 50% decrease in absorbance measured at 450 nm.

[0142] The results are shown in Figure 4, which shows that 1G7, 1F5, 2D10 and D25 each inhibited the replication of RSV A2 and RSV B9320 in microneutralization. 1G7 was the most effective, followed by 1F5, 2D10 and then D25.

[0143] Example 3. Cotton Rat RSV Model A) Comparison of the protective ability of D25, J, L, and LA variants against RSV challenge. Animals were administered 0.1 ml of antibody by intramuscular injection at various dose levels as indicated. After 24 hours, animals were anesthetized using an isoflurane chamber and 1×10 6 pfu / animal was infected with RSV strain A2 by intranasal instillation. Four days later, animals were sacrificed by carbon dioxide asphyxiation and their lungs were surgically removed, bisected, and frozen in liquid nitrogen or immediately processed. Blood samples were obtained by cardiac puncture at the time of sacrifice.

[0144] To evaluate the effect of MAb administration on RSV replication in the lungs of cotton rats, RSV virus titers were measured in lung homogenates of cotton rats for each administration group. For this purpose, excised lungs were individually homogenized in 10 (wt / vol) parts of Hank's balanced salt solution and sucrose phosphate using a Fast Prep 24 tissue homogenizer for 20 seconds in TeenALysing Matrix tubes at room temperature. The resulting suspension was centrifuged at 930 × g for 10 minutes at 4 ° C, and the supernatant was collected and stored at −80 ° C until analysis. Lung homogenate samples were diluted 1:10 and 1:100 in culture medium, and 24 hours before inoculation, 50 μL of undiluted or diluted lung homogenate samples were transferred to 2.5 × 10 24-well plates. 5The inoculum was added to duplicate wells of HEp-2 cells seeded at a cell density of 100 cells / well. After 1 h of incubation at 37°C and 5% CO2, the inoculum was replaced with culture medium containing 0.8% methylcellulose, and the cells were incubated at 37°C and 5% CO2. After 5 days, the overlay was removed, and the cells were fixed and immunostained with a goat anti-RSV polyclonal antibody, followed by a secondary anti-goat HRP antibody. Plaques were visualized by reaction with AEC reagent. Plaques were quantified under a microscope using a 10x objective. The detection limit of this assay is 200 pfu per gram of tissue. For statistical analysis, samples with virus titers below the detection limit (<200 pfu / g) were assigned a value of 100 pfu / g (half the lower limit of detection). The results are shown in Figure 5 and show that the J, L and LA variants were all more effective than D25 in protecting against RSV A challenge, but the L and LA variants were less effective in protecting against RSV B subtypes (L worked better against one B strain than another). Based on this data, J was selected as the starting point for further optimization.

[0145] B) Comparison of the protective ability of the 1G7, 1F5, and D25 variants against RSV challenge. Using the model discussed above in part (A) of this example, 1G7, 1F5 and D25 were compared for their protective ability against RSV challenge. Animals were dosed at 2mg / kg, 1mg / kg, 0.5mg / kg and 0.25mg / kg. Results are shown in Figure 6. The data show that 1G7 performed better than 1F5 in protecting against RSV challenge, but both were able to reduce virus titers to the limit of detection. Both 1G7 and 1F5 performed better than D25.

[0146] C) Detailed evaluation of the 1G7 variant The same cotton rat model described in section (A) above was used. Each animal received 0.1 ml of antibody at various antibody concentrations as reported in Figure 7. 1G7 clearly demonstrates a dose-response relationship with RSV lung titers for both RSV A2 and RSV B9320.

[0147] D) Comparison of the ability of variants to neutralize RSV A2 and RSV B9320 The same Cotton Rad model was used as described in section (A) above.

[0148] The concentration of human IgG in cotton rat serum samples on the day of lung removal was measured using an ELISA method. In this assay, human antibodies were captured with goat anti-human antibodies bound to a microtiter plate. Goat anti-human IgG (H+L) antibodies (0.5 μg / ml in 1×PBS) were coated on Nunc Maxisorp 384-well microtiter plates in a volume of 30 μL overnight at 4° C. The plates were washed and then blocked with a solution of 60 μL of PBS+3% heat-inactivated goat serum for 1 hour at room temperature. The blocking buffer was removed and the samples were fitted as follows: 2-fold serial dilutions of standard human antibodies diluted in assay buffer were used for the standard curve, ranging from 500 ng / ml to 0.488 ng / ml. The standard curve was fitted using a 4 parameter curve fit.

[0149] The results are shown in Figure 8 and show that the variants described herein have lower IC50s than D25 in neutralizing both RSV A2 and RSV B9320. This also shows that there was no loss of activity against A2 or B9320 viruses, E9-2 and B12-1 showed increased activity, and only 1G7 GLM and E3-5 showed a slight loss of activity against B9320 virus.

[0150] Example 4. Epitopes defined by monoclonal antibody resistant mutants RSV A and B virus variants were isolated by three passages in the presence of 250 ng / ml 1G7-YTE. 1G7-YTE is the 1G7 antibody with the YTE mutations described above in Section IC. After the final passage, the sequence of the RSV F protein was confirmed. The mutations correspond to regions previously defined in the co-crystal structure of RSV F with the parent D25 antibody. All resistance mutants contained changes in the region of the RSV F protein in the F1 region between amino acids 200-213. A secondary mutation at position 294 did not show improved resistance, and was as resistant as a single mutation in the region 200-213. A secondary mutation in the F2 region in the N208S mutation background improved resistance. The results are shown in FIG. 9.

[0151] Example 5. Neutralization of clinical isolates of RSV Neutralization assays were performed by preincubating propagated clinically isolated viruses with serial dilutions of antibodies and then infecting HEp2 cells. Cell infection was measured as a function of F protein expression on the cell surface. IC50 values ​​were calculated by nonlinear fitting of neutralization curves. Viral replication was measured as in Example 2 (in vitro microneutralization assay).

[0152] The results are shown in Figure 10. The IC50s of 1G7 and 1F5 are both lower than D25 for neutralization of A or B isolates of RSV in HEp2 cells.

[0153] Incorporation by Reference All references cited herein, e.g., patents, patent applications, articles, texts, etc., and the references cited therein, unless already incorporated by reference herein, are hereby incorporated by reference in their entirety for all purposes.

[0154] Equivalent The above specification is believed to be sufficient to enable one skilled in the art to practice the present embodiments. The above description and examples detail several embodiments and explain the best mode contemplated by the inventors. However, it will be recognized that no matter how detailed the above description and examples are in the text, the embodiments can be practiced in many ways, and the claims include all equivalents thereof.

[0155] [Sequence table] SEQUENCE LISTING <110> MedImmune Limited <120> RSV-SPECIFIC ANTIBODIES AND FUNCTIONAL PARTS THEREOF <130> PA24-719 <150> US 61 / 927,819 <151> 2014-01-15 <160> twenty one <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> artificial <220> <223> D25 Heavy Chain CDR1 <400> 1 Asn Tyr Ile Ile Asn 1 5 <210> 2 <211> 17 <212> PRT <213> artificial <220> <223> D25 Heavy Chain CDR2 <400> 2 Gly Ile Ile Pro Val Leu Gly Thr Val His Tyr Ala Pro Lys Phe Gln 1 5 10 15 Gly <210> 3 <211> 17 <212> PRT <213> artificial <220> <223> D25 Heavy Chain CDR3 <400> 3 Glu Thr Ala Leu Val Val Ser Thr Thr Tyr Leu Pro His Tyr Phe Asp 1 5 10 15 Asn <210> 4 <211> 11 <212> PRT <213> artificial <220> <223> D25 Light Chain CDR1 <400> 4 Gln Ala Ser Gln Asp Ile Val Asn Tyr Leu Asn 1 5 10 <210> 5 <211> 7 <212> PRT <213> artificial <220> <223> D25 Light Chain CDR2 <400> 5 Val Ala Ser Asn Leu Glu Thr 1 5 <210> 6 <211> 7 <212> PRT <213> artificial <220> <223> D25 Light Chain CDR3 <400> 6 Gln Gln Tyr Asp Asn Leu Pro 1 5 <210> 7 <211> 126 <212> PRT <213> artificial <220> <223> D25 heavy chain variable region <220> <221> MISC_FEATURE <222> (19)..(19) <223> M can also be K <220> <221> MISC_FEATURE <222> (23)..(23) <223> Q can also be K <220> <221> MISC_FEATURE <222> (28)..(28) <223> P can also be T or L <220> <221> MISC_FEATURE <222> (29)..(29) <223> L can also be F <220> <221> MISC_FEATURE <222> (30)..(30) <223> R can also be S or E <220> <221> MISC_FEATURE <222> (31)..(31) <223> N can also be D <220> <221> MISC_FEATURE <222> (37)..(37) <223> L can also be V <220> <221> MISC_FEATURE <222> (45)..(45) <223> P can also be L <220> <221> MISC_FEATURE <222> (61)..(61) <223> A can also be G <220> <221> MISC_FEATURE <222> (77)..(77) <223> D can also be S <220> <221> MISC_FEATURE <222> (81)..(81) <223> I can also be M <220> <221> MISC_FEATURE <222> (82)..(82) <223> H can also be E or H <220> <221> MISC_FEATURE <222> (84)..(84) <223> I can also be S <220> <221> MISC_FEATURE <222> (98)..(98) <223> T can also be R <220> <221> MISC_FEATURE <222> (109)..(109) <223> L can also be R <400> 7 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Met Val Ser Cys Gln Ala Ser Gly Gly Pro Leu Arg Asn Tyr 20 25 30 Ile Ile Asn Trp Leu Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Val Leu Gly Thr Val His Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asp Thr Ala Tyr 65 70 75 80 Ile His Leu Ile Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Thr Glu Thr Ala Leu Val Val Ser Thr Thr Tyr Leu Pro His Tyr 100 105 110 Phe Asp Asn Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 8 <211> 110 <212> PRT <213> artificial <220> <223> D25 light chain variable region <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ala Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Val Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Val Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 <210> 9 <211> 5 <212> PRT <213> artificial <220> <223> Alternative Heavy Chain CDR1 <400> 9 Asp Tyr Ile Ile Asn 1 5 <210> 10 <211> 17 <212> PRT <213> artificial <220> <223> Alternative Heavy Chain CDR2 <400> 10 Gly Ile Ile Pro Val Leu Gly Thr Val His Tyr Gly Pro Lys Phe Gln 1 5 10 15 Gly <210> 11 <211> 17 <212> PRT <213> artificial <220> <223> Alternative Heavy Chain CDR3 <400> 11 Glu Thr Ala Leu Val Val Ser Thr Thr Tyr Arg Pro His Tyr Phe Asp 1 5 10 15 Asn <210> 12 <211> 126 <212> PRT <213> artificial <220> <223> J variant heavy chain variable region <400> 12 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Met Val Ser Cys Gln Ala Ser Gly Gly Pro Leu Arg Asn Tyr 20 25 30 Ile Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Val Leu Gly Thr Val His Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Thr Glu Thr Ala Leu Val Val Ser Thr Thr Tyr Leu Pro His Tyr 100 105 110 Phe Asp Asn Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 13 <211> 126 <212> PRT <213> artificial <220> <223> L variant heavy chain variable region <400> 13 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Met Val Ser Cys Gln Ala Ser Gly Gly Pro Leu Arg Asn Tyr 20 25 30 Ile Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Val Leu Gly Thr Val His Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Thr Glu Thr Ala Leu Val Val Ser Thr Thr Tyr Leu Pro His Tyr 100 105 110 Phe Asp Asn Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 14 <211> 126 <212> PRT <213> artificial <220> <223> LA variant heavy chain variable region <400> 14 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr 20 25 30 Ile Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Val Leu Gly Thr Val His Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Thr Glu Thr Ala Leu Val Val Ser Thr Thr Tyr Leu Pro His Tyr 100 105 110 Phe Asp Asn Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 15 <211> 126 <212> PRT <213> artificial <220> <223> 1G7 heavy chain variable region <400> 15 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Met Val Ser Cys Gln Ala Ser Gly Gly Leu Leu Glu Asp Tyr 20 25 30 Ile Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Val Leu Gly Thr Val His Tyr Gly Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Thr Glu Thr Ala Leu Val Val Ser Thr Thr Tyr Leu Pro His Tyr 100 105 110 Phe Asp Asn Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 16 <211> 126 <212> PRT <213> artificial <220> <223> 1F5 heavy chain variable region <400> 16 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Met Val Ser Cys Gln Ala Ser Gly Gly Pro Leu Glu Asp Tyr 20 25 30 Ile Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Val Leu Gly Thr Val His Tyr Gly Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Thr Glu Thr Ala Leu Val Val Ser Thr Thr Tyr Leu Pro His Tyr 100 105 110 Phe Asp Asn Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 17 <211> 126 <212> PRT <213> artificial <220> <223> 2D10 heavy chain variable region <400> 17 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Met Val Ser Cys Gln Ala Ser Gly Gly Pro Leu Glu Asp Tyr 20 25 30 Ile Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Val Leu Gly Thr Val His Tyr Gly Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Thr Glu Thr Ala Leu Val Val Ser Thr Thr Tyr Arg Pro His Tyr 100 105 110 Phe Asp Asn Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 18 <211> 126 <212> PRT <213> artificial <220> <223> 1G7-GLM heavy chain variable region <400> 18 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Leu Leu Glu Asp Tyr 20 25 30 Ile Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Val Leu Gly Thr Val His Tyr Gly Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met His Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Glu Thr Ala Leu Val Val Ser Thr Thr Tyr Leu Pro His Tyr 100 105 110 Phe Asp Asn Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 19 <211> 126 <212> PRT <213> artificial <220> <223> B12-1 heavy chain variable region <400> 19 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Leu Leu Glu Asp Tyr 20 25 30 Ile Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Val Leu Gly Thr Val His Tyr Gly Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met His Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Thr Glu Thr Ala Leu Val Val Ser Thr Thr Tyr Leu Pro His Tyr 100 105 110 Phe Asp Asn Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 20 <211> 126 <212> PRT <213> artificial <220> <223> E3-5 heavy chain variable region <400> 20 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Leu Leu Glu Asp Tyr 20 25 30 Ile Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Val Leu Gly Thr Val His Tyr Gly Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Thr Glu Thr Ala Leu Val Val Ser Thr Thr Tyr Leu Pro His Tyr 100 105 110 Phe Asp Asn Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 21 <211> 126 <212> PRT <213> artificial <220> <223> E9-2 heavy chain variable region <400> 21 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Leu Leu Glu Asp Tyr 20 25 30 Ile Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Val Leu Gly Thr Val His Tyr Gly Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met His Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Thr Glu Thr Ala Leu Val Val Ser Thr Thr Tyr Leu Pro His Tyr 100 105 110 Phe Asp Asn Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125

Claims

1. capable of specifically binding to respiratory syncytial virus, a. a heavy chain variable region CDR1 sequence comprising a sequence that is at least 70% identical to the sequence NYIIN (SEQ ID NO:1) or the sequence DYIIN (SEQ ID NO:9); b. a heavy chain variable region CDR2 sequence comprising the sequence GIIPVLGTVHYAPKFQG (SEQ ID NO: 2) or a sequence that is at least 75% identical to the sequence GIIPVLGTVHYGPKFQG (SEQ ID NO: 10); c. a heavy chain variable region CDR3 sequence comprising the sequence ETALVVSTTYLPHYFDN (SEQ ID NO: 3) or a sequence that is at least 70% identical to the sequence ETALVVSTTYRPHYFDN (SEQ ID NO: 11); d. a light chain variable region CDR1 sequence comprising a sequence that is at least 85% identical to the sequence QASQDIVNYLN (SEQ ID NO:4); e. a light chain variable region CDR2 sequence comprising a sequence that is at least 70% identical to the sequence VASNLET (SEQ ID NO:5); f. a light chain variable region CDR3 sequence comprising a sequence that is at least 70% identical to the sequence QQYDNLP (SEQ ID NO:6); A synthetic or recombinant antibody or a functional part thereof comprising: at least one amino acid in the heavy chain differs from SEQ ID NO:7, and said at least one amino acid is A synthetic or recombinant antibody or a functional portion thereof selected from the group consisting of:

2. a. the heavy chain variable region CDR1 sequence comprises a sequence that differs by one amino acid from NYIIN (SEQ ID NO: 1) or DYIIN (SEQ ID NO: 9); b. the heavy chain variable region CDR2 sequence comprises a sequence that differs by one or two amino acids from GIIPVLGTVHYAPKFQG (SEQ ID NO: 2) or GIIPVLGTVHYGPKFQG (SEQ ID NO: 10); c. The heavy chain variable region CDR3 sequence comprises a sequence that differs from ETALVVSTTYLPHYFDN (SEQ ID NO: 3) or ETALVVSTTYRPHYFDN (SEQ ID NO: 11) by one or two amino acids; d. the light chain variable region CDR1 sequence comprises a sequence that differs by one amino acid from QASQDIVNYLN (SEQ ID NO:4); e. the light chain variable region CDR2 sequence comprises a sequence that differs by one amino acid from VASNLET (SEQ ID NO:5); and / or f. the light chain variable region CDR3 sequence comprises a sequence that differs by one amino acid from QQYDNLP (SEQ ID NO:6); An antibody or functional portion thereof according to claim 1.

3. a. the heavy chain variable region CDR1 sequence comprises NYIIN (SEQ ID NO:1) or DYIIN (SEQ ID NO:9); b. the heavy chain variable region CDR2 sequence comprises GIIPVLGTVHYAPKFQG (SEQ ID NO: 2) or GIIPVLGTVHYGPKFQG (SEQ ID NO: 10); c. The heavy chain variable region CDR3 sequence comprises ETALVVSTTYLPHYFDN (SEQ ID NO: 3) or ETALVVSTTYRPHYFDN (SEQ ID NO: 11); d. the light chain variable region CDR1 sequence comprises QASQDIVNYLN (SEQ ID NO:4); e. the light chain variable region CDR2 sequence comprises VASNLET (SEQ ID NO:5), and f. the light chain variable region CDR3 sequence comprises QQYDNLP (SEQ ID NO:6); An antibody or functional portion thereof according to claim 1.

4. At least the following amino acids in the heavy chain variable region differ from SEQ ID NO:7: An antibody or functional portion thereof according to any one of claims 1 to 3.

5. At least the following amino acids in the heavy chain variable region differ from SEQ ID NO:7: An antibody or functional portion thereof according to any one of claims 1 to 3.

6. At least the following amino acids in the heavy chain variable region differ from SEQ ID NO:7: An antibody or functional portion thereof according to any one of claims 1 to 3.

7. At least the following amino acids in the heavy chain variable region differ from SEQ ID NO:7: An antibody or functional portion thereof according to any one of claims 1 to 3.

8. At least the following amino acids in the heavy chain variable region differ from SEQ ID NO:7: An antibody or functional portion thereof according to any one of claims 1 to 3.

9. At least the following amino acids in the heavy chain variable region differ from SEQ ID NO:7: An antibody or functional portion thereof according to any one of claims 1 to 3.

10. At least the following amino acids in the heavy chain variable region differ from SEQ ID NO:7: An antibody or functional portion thereof according to any one of claims 1 to 3.

11. At least the following amino acids in the heavy chain variable region differ from SEQ ID NO:7: An antibody or functional portion thereof according to any one of claims 1 to 3.

12. At least the following amino acids in the heavy chain variable region differ from SEQ ID NO:7: An antibody or functional portion thereof according to any one of claims 1 to 3.

13. At least the following amino acids in the heavy chain variable region differ from SEQ ID NO:7: An antibody or functional portion thereof according to any one of claims 1 to 3.

14. The antibody or functional portion according to any one of claims 1 to 13, wherein the heavy chain variable region is at least 90% identical to SEQ ID NO: 7 or SEQ ID NO: 13 and the light chain variable region is at least 90% identical to SEQ ID NO:

8.

15. The antibody or functional portion according to any one of claims 1, 2 or 4 to 14, wherein the heavy chain variable region CDR1 sequence comprises a sequence that differs by one amino acid from NYIIN (SEQ ID NO: 1) or DYIIN (SEQ ID NO: 9).

16. The antibody or functional portion according to any one of claims 1, 2 or 4 to 15, wherein the heavy chain variable region CDR2 sequence comprises a sequence that differs by one or two amino acids from GIIPVLGTVHYAPKFQG (SEQ ID NO: 2) or GIIPVLGTVHYGPKFQG (SEQ ID NO: 10).

17. The antibody or functional portion according to any one of claims 1, 2 or 4 to 16, wherein the heavy chain variable region CDR3 sequence comprises a sequence that differs by one or two amino acids from ETALVVSTTYLPHYFDN (SEQ ID NO: 3) or ETALVVSTTYRPHYFDN (SEQ ID NO: 11).

18. The antibody or functional portion according to any one of claims 1, 2 or 4 to 17, wherein the light chain variable region CDR1 sequence comprises a sequence differing by one amino acid from QASQDIVNYLN (SEQ ID NO: 4).

19. The antibody or functional portion according to any one of claims 1, 2 or 4 to 18, wherein the light chain variable region CDR2 sequence comprises a sequence that differs by one amino acid from VASNLET (SEQ ID NO: 5).

20. The antibody or functional portion according to any one of claims 1, 2 or 4 to 19, wherein the light chain variable region CDR3 comprises a sequence that differs by one amino acid from QQYDNLP (SEQ ID NO: 6).

21. The antibody or functional portion according to any one of claims 1, 2 or 4 to 14 or 16 to 20, wherein the heavy chain variable region CDR1 sequence comprises NYIIN (SEQ ID NO: 1) or DYIIN (SEQ ID NO: 9).

22. The antibody or functional portion according to any one of claims 1, 2, 4 to 15 or 17 to 21, wherein the heavy chain variable region CDR2 sequence comprises GIIPVLGTVHYAPKFQG (SEQ ID NO: 2) or GIIPVLGTVHYGPKFQG (SEQ ID NO: 10).

23. The antibody or functional portion according to any one of claims 1, 2, 4 to 16 or 18 to 22, wherein the heavy chain variable region CDR3 sequence comprises ETALVVSTTYLPHYFDN (SEQ ID NO: 3) or ETALVVSTTYRPHYFDN (SEQ ID NO: 11).

24. The antibody or functional portion according to any one of claims 1, 2, 4 to 17 or 19 to 23, wherein the light chain variable region CDR1 sequence comprises QASQDIVNYLN (SEQ ID NO: 4).

25. The antibody or functional portion according to any one of claims 1, 2, 4 to 18 or 20 to 24, wherein the light chain variable region CDR2 sequence comprises VASNLET (SEQ ID NO:5).

26. The antibody or functional portion according to any one of claims 1, 2, 4 to 19 or 21 to 25, wherein the light chain variable region CDR3 comprises QQYDNLP (SEQ ID NO: 6).

27. The antibody or functional portion according to any one of claims 1 to 26, wherein the heavy chain variable region comprises a sequence which is 95% identical in the framework regions to a sequence selected from SEQ ID NO: 7 and SEQ ID NO: 12 to 21.

28. The antibody or functional portion according to any one of claims 1 to 26, wherein the heavy chain variable region comprises a sequence which is identical in the framework regions to a sequence selected from SEQ ID NOs: 12 to 21.

29. The antibody or functional portion according to any one of claims 1 to 26, wherein the heavy chain variable region comprises a sequence that is at least 70% identical to a sequence selected from SEQ ID NOs: 12 to 21.

30. The antibody or functional portion according to any one of claims 1 to 29, having a light chain variable region sequence comprising a sequence that is at least 70% identical to SEQ ID NO:

8.

31. The antibody or functional part according to any one of claims 1 to 29, having a light chain sequence variable region comprising a sequence identical to SEQ ID NO:

8.

32. capable of specifically binding to the RSV F antigen; a. a heavy chain variable region CDR1 sequence comprising the amino acid sequence DYIIN (SEQ ID NO:9); b. a heavy chain variable region CDR2 sequence comprising the amino acid sequence GIIPVLGTVHYGPKFQG (SEQ ID NO: 10); c. a heavy chain variable region CDR3 sequence comprising the amino acid sequence ETALVVSTTYRPHYFDN (SEQ ID NO: 11); d. a light chain variable region CDR1 sequence comprising the amino acid sequence QASQDIVNYLN (SEQ ID NO:4); e. a light chain variable region CDR2 sequence comprising the amino acid sequence VASNLET (SEQ ID NO:5); and f. a light chain variable region CDR3 sequence comprising the amino acid sequence QQYDNLP (SEQ ID NO:6). A synthetic or recombinant antibody or a functional portion thereof comprising:

33. capable of specifically binding to the RSV F antigen; a. a heavy chain variable region CDR1 sequence comprising the amino acid sequence DYIIN (SEQ ID NO:9); b. a heavy chain variable region CDR2 sequence comprising the amino acid sequence GIIPVLGTVHYGPKFQG (SEQ ID NO: 10); c. a heavy chain variable region CDR3 sequence comprising the amino acid sequence ETALVVSTTYLPHYFDN (SEQ ID NO: 3); d. a light chain variable region CDR1 sequence comprising the amino acid sequence QASQDIVNYLN (SEQ ID NO:4); e. a light chain variable region CDR2 sequence comprising the amino acid sequence VASNLET (SEQ ID NO:5); and f. a light chain variable region CDR3 sequence comprising the amino acid sequence QQYDNLP (SEQ ID NO:6). A synthetic or recombinant antibody or a functional portion thereof comprising:

34. The antibody or functional portion according to any one of claims 1 to 33, wherein the antibody or functional portion is fully human, a humanized antibody or a chimeric antibody.

35. The functional portion is a single chain antibody, a single chain variable fragment (scFv), a Fab fragment or an F(ab') 2 A functional part according to any one of claims 1 to 34 which is a fragment.

36. The antibody or functional portion according to any one of claims 1 to 35, wherein the antibody or functional portion has an IC50 of 5.0 ng / ml or less in an RSV A2 neutralization assay.

37. The antibody or functional portion according to any one of claims 1 to 36, wherein the antibody or functional portion has an IC50 of 3.0 ng / ml or less in a neutralization assay for RSV B9320.

38. The antibody or functional portion according to any one of claims 1 to 37, wherein the antibody or functional portion has an Fc region with Y at position 252Y, T at position 254T and E at position 256, said numbering corresponding to the EU index in Kabat.

39. A method for inhibiting RSV infection in a subject, comprising administering to the subject an antibody or functional part according to any one of claims 1 to 38.

40. 40. The method of claim 39, wherein the subject is a human patient.

41. 41. The method of claim 40, wherein the subject is a human patient who has had a preterm birth.

42. A synthetic, recombinant or isolated nucleic acid sequence encoding the antibody or functional part according to any one of claims 1 to 38.

43. 43. A method for inhibiting RSV infection in a subject, comprising administering to the subject a nucleic acid according to claim 42.

44. An isolated antibody-producing cell capable of producing the antibody or functional portion according to any one of claims 1 to 38.

45. 45. The antibody-producing cell of claim 44, which is stable for at least 9 weeks.

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