HUMAN NEUTRALIZING ANTIBODIES AGAINST RESPIRATORY SYNCYTIAL VIRUS (RSV) AND HUMAN METAPNEUMOVIRUS (hMPV)

Abstract

The invention provides novel neutralizing antibodies and related antibody compositions against respiratory syncytial virus (RSV) and human metapneumovirus (hMPV). Also provided in the invention are polynucleotides and vectors encoding such antibodies, as well as pharmaceutical compositions containing the antibodies or polynucleotides. Therapeutic uses of the antibodies or pharmaceutical compositions in preventing or treating RAV and hMPV infections are also encompassed by the invention.

Description

TSRI 2230.1PC HUMAN NEUTRALIZING ANTIBODIES AGAINST RESPIRATORY SYNCYTIAL VIRUS (RSV) AND HUMAN METAPNEUMOVIRUS (hMPV) CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The subject patent application claims the benefit of priority to U.S. Provisional Patent Application Numbers 63 / 548,874 (filed February 2, 2024; now pending). The full disclosure of the priority application is incorporated herein by reference in its entirety and for all purposes. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing, which is submitted in .xml format and is hereby incorporated by reference in its entirety. Said .xml file is named “2231_1PC_Sequence Listing”, was created on January 29, 2025, and is 50 KB bytes in size. BACKGROUND OF THE INVENTION

[0003] Respiratory syncytial virus (RSV), also called human respiratory syncytial virus, (hRSV), and human metapneumovirus (hMPV) are enveloped, non-segmented, negative- sense, single-stranded RNA viruses in the Paramyxoviridae family, which also includes parainfluenza virus (PIV). RSV has been extensively studied since its discovery over 60 years ago, and hMPV was identified ~20 years ago with active ongoing research. RSV and hMPV pose a major burden on human health. RSV is a prevalent cause of acute lower respiratory infection (ALRI) in neonates and infants. RSV causes acute respiratory infection, accounting for ~66,000-200,000 deaths and 3.5 million hospitalizations worldwide in children under 5 years of age. Children infected with hMPV usually exhibit symptoms similar to those of RSV infection. Since the discovery of hMPV, this virus has been detected in 4–16% of patients with ALRIs. Like RSV, hMPV causes disease primarily in children but can also infect adults and immunocompromised individuals. RSV and hMPV share similar genomic compositions with several key differences. The RSV genome encodes three envelope glycoproteins, the attachment (G) protein, fusion (F) protein, and small hydrophobic (SH) protein, and eight non-structural proteins (NS1, NS2, N, P, M, M2-1, M2- 2, and L). The hMPV genome contains eight open reading frames (ORFs) encodingnucleoprotein (N), phosphoprotein (P), matrix protein (M), transcription enhancer protein (M2), and three envelope glycoproteins (G, F, and SH). For both RSV and hMPV, F and G are crucial for infectivity and pathogenesis and can be recognized by host neutralizing antibodies (NAbs).

[0004] Over the last decade, a series of advancements have been made in RSV and hMPV research. First, multiple antigenic sites (AS) on the RSV F protein have been identified that can be recognized by NAbs and non-NAbs. Co-crystallization of a therapeutic antibody, Motavizumab, with the F peptide epitope was the first of a series of RSV antigen / NAb complex structures that defined the atomic interactions between NAbs and RSV F. Importantly, co-crystallization of a potent human NAb D25 with RSV F resulted in the first atomic structure of prefusion F trimer and revealed a novel antigenic site (Ø) near the apex. Second, a structural understanding has been achieved for the F protein at both prefusion and postfusion states and for RSV and hMPV. Their prefusion trimers share a high similarity with a closed, “football” shaped trimer conformation. A number of anti-RSV antibodies have undergone clinical development, with palivizumab (brand name Synagis) currently licensed for human use. In two phase III clinical trials in the pediatric population, palivizumab reduced the risk of hospitalization due to RSV infection by 55% and 45%. Palivizumab is dosed once a month via intramuscular (IM) injection, to be administered throughout the duration of the RSV season. Of note, palivizumab is indicated for the prevention of serious ALRI in children at high risk for RSV disease. Motavizumab, as an improved version of palivizumab with 20-fold higher potency, was tested in phase III trials but failed to get the FDA approval. MEDI8897, an optimized version of the human NAb D25, was granted the Breakthrough Therapy Designation by the US FDA in 2019.

[0005] Nonetheless, there is still a need for NAbs with superior potency and cross- reactivity, which will provide new treatment for RSV and hMPV infections in the high-risk human population. The present invention is directed to addressing such unmet needs in the art. SUMMARY OF THE INVENTION

[0006] In one aspect, the invention antibodies or antigen-binding fragments thereof that neutralize human respiratory syncytial virus (RSV). These antibodies contain heavy chain variable region and light chain variable region sequences that are at least 95% identical to,respectively, (1) SEQ ID NOs:1 and 2 (A4), (2) SEQ ID NOs:3 and 4 (D1), (3) SEQ ID NOs:5 and 6 (F3), (4) SEQ ID NOs:7 and 8 (E3), (5) SEQ ID NOs:9 and 10 (D12), (6) SEQ ID NOs:11 and 12 (E5), (7) SEQ ID NOs:13 and 14 (H9), or (8) SEQ ID NOs:15 and 16 (F1). In some of these embodiments, the antibody or antigen-binding contains heavy chain CDR sequences (HCDR1-3) and light chain CDR sequences (LCDR1-3) as set forth respectively in (1) SEQ ID NOs:17-20, DVS, and SEQ ID NO:21 (A4), (2) SEQ ID NOs:22- 25, GNT, and SEQ ID NO:26 (D1), (3) SEQ ID NOs:27-30, GNT, and SEQ ID NO:31 (F3), (4) SEQ ID NOs:32-35, GNT, and SEQ ID NO:36 (E3), (5) SEQ ID NOs:37-40, GNT, and SEQ ID NO:41 (D12), (6) SEQ ID NOs:42-45, DNN, and SEQ ID NO:46 (E5), (7) SEQ ID NOs:47-50, AAS, and SEQ ID NO:51 (H9), or (8) SEQ ID NOs:52-55, EDK, and SEQ ID NO:56 (F1). In some of these embodiments, the antibody or antigen-binding fragment contains heavy chain variable region and light chain variable region sequences that are at least 98% or 99% identical to, respectively, (1) SEQ ID NOs:1 and 2, (2) SEQ ID NOs:3 and 4, (3) SEQ ID NOs:5 and 6, (4) SEQ ID NOs:7 and 8, (5) SEQ ID NOs:9 and 10, (6) SEQ ID NOs:11 and 12, (7) SEQ ID NOs:13 and 14, or (8) SEQ ID NOs:15 and 16. In some of these embodiments, the antibody or antigen-binding fragment contains heavy chain variable region and light chain variable region sequences that are respectively identical to (1) SEQ ID NOs:1 and 2, (2) SEQ ID NOs:3 and 4, (3) SEQ ID NOs:5 and 6, (4) SEQ ID NOs:7 and 8, (5) SEQ ID NOs:9 and 10, (6) SEQ ID NOs:11 and 12, (7) SEQ ID NOs:13 and 14, or (8) SEQ ID NOs:15 and 16.

[0007] In a related aspect, the invention provides antibodies or antigen-binding fragments thereof that neutralize human respiratory syncytial virus (RSV). These antibody molecules contain heavy chain CDR sequences (HCDR1-3) and light chain CDR sequences (LCDR1-3) as set forth respectively in (1) SEQ ID NOs:17-20, DVS, and SEQ ID NO:21 (A4), (2) SEQ ID NOs:22-25, GNT, and SEQ ID NO:26 (D1), (3) SEQ ID NOs:27-30, GNT, and SEQ ID NO:31 (F3), (4) SEQ ID NOs:32-35, GNT, and SEQ ID NO:36 (E3), (5) SEQ ID NOs:37-40, GNT, and SEQ ID NO:41 (D12), (6) SEQ ID NOs:42-45, DNN, and SEQ ID NO:46 (E5), (7) SEQ ID NOs:47-50, AAS, and SEQ ID NO:51 (H9), or (8) SEQ ID NOs:52-55, EDK, and SEQ ID NO:56 (F1). In some of these embodiments, in addition to the identical CDR sequences, the antibody also contains heavy chain variable region and light chain variable region sequences that are substantially identical (e.g., at least 95%, 98% or 99% identical) to, respectively, (1) SEQ ID NOs:1 and 2, (2) SEQ ID NOs:3 and 4, (3)SEQ ID NOs:5 and 6, (4) SEQ ID NOs:7 and 8, (5) SEQ ID NOs:9 and 10, (6) SEQ ID NOs:11 and 12, (7) SEQ ID NOs:13 and 14, or (8) SEQ ID NOs:15 and 16.

[0008] Some RSV-neutralizing antibodies or antigen-binding fragments of the invention also cross-neutralize human metapneumovirus (hMPV). In some of these embodiments, the antibody molecule contains heavy chain CDR sequences (HCDR1-3) and light chain CDR sequences (LCDR1-3) as set forth respectively in (1) SEQ ID NOs:22-25, GNT, and SEQ ID NO:26, or (2) SEQ ID NOs:27-30, GNT, and SEQ ID NO:31. In some embodiments, the antibody molecule contains heavy chain variable region and the light chain variable region sequences that are substantially identical (e.g., at least 95%, 98%, 99% or 100% identical) to (1) SEQ ID NOs:3 and 4, or (2) SEQ ID NOs:5 and 6.

[0009] In some embodiments, the RSV-neutralizing antibodies of the invention contain heavy chain and light chain variable region sequences that, except for substitutions of one or more amino acid residues in the heavy chain framework region and the light chain framework region, are respectively identical to (1) SEQ ID NOs:1 and 2, (2) SEQ ID NOs:3 and 4, (3) SEQ ID NOs:5 and 6, (4) SEQ ID NOs:7 and 8, (5) SEQ ID NOs:9 and 10, (6) SEQ ID NOs:11 and 12, (7) SEQ ID NOs:13 and 14, or (8) SEQ ID NOs:15 and 16. In some embodiments, the RSV-neutralizing antibodies of the invention contain (a) one or more non- natural amino acid residues in the Fc domain, and (b) heavy chain CDR sequences (HCDR1- 3) and light chain CDR sequences (LCDR1-3) as set forth respectively in (1) SEQ ID NOs:17-20, DVS, and SEQ ID NO:21, (2) SEQ ID NOs:22-25, GNT, and SEQ ID NO:26, (3) SEQ ID NOs:27-30, GNT, and SEQ ID NO:31, (4) SEQ ID NOs:32-35, GNT, and SEQ ID NO:36, (5) SEQ ID NOs:37-40, GNT, and SEQ ID NO:41, (6) SEQ ID NOs:42-45, DNN, and SEQ ID NO:46, (7) SEQ ID NOs:47-50, AAS, and SEQ ID NO:51, or (8) SEQ ID NOs:52-55, EDK, and SEQ ID NO:56. In various embodiments, the RSV-neutralizing antibodies of the invention can be IgG, scFv-Fc, scFv, Fab, or F(ab’)2.

[0010] In another aspect, the invention provides pharmaceutical compositions or kits that contain a therapeutically effective amount of an RSV-neutralizing antibody molecule disclosed herein, and a pharmaceutically acceptable carrier. In another aspect, the invention provides conjugate molecules that contain (i) an RSV-neutralizing antibody molecule disclosed herein, and (ii) a second moiety that is fused or conjugated to the antibody molecule. In some of these embodiments, the second moiety is a polypeptide or a small organic molecule. In some of these embodiments, the second moiety is a drug.

[0011] In another aspect, the invention provides novel polynucleotide molecules. These molecules each encode an antibody heavy chain variable region sequence and an antibody light chain variable region sequence that are substantially identical (e.g., at least 95%, 98%, 99% or 100% identical) to, respectively, (1) SEQ ID NOs:1 and 2, (2) SEQ ID NOs:3 and 4, (3) SEQ ID NOs:5 and 6, (4) SEQ ID NOs:7 and 8, (5) SEQ ID NOs:9 and 10, (6) SEQ ID NOs:11 and 12, (7) SEQ ID NOs:13 and 14, or (8) SEQ ID NOs:15 and 16. In some embodiments, CDR1-3 sequences (HCDR1-3) of the encoded heavy chain variable region and CDR1-3 sequences (LCDR1-3) of the encoded light chain variable region are respectively identical to (1) SEQ ID NOs:17-20, DVS, and SEQ ID NO:21, (2) SEQ ID NOs:22-25, GNT, and SEQ ID NO:26, (3) SEQ ID NOs:27-30, GNT, and SEQ ID NO:31, (4) SEQ ID NOs:32-35, GNT, and SEQ ID NO:36, (5) SEQ ID NOs:37-40, GNT, and SEQ ID NO:41, (6) SEQ ID NOs:42-45, DNN, and SEQ ID NO:46, (7) SEQ ID NOs:47-50, AAS, and SEQ ID NO:51, or (8) SEQ ID NOs:52-55, EDK, and SEQ ID NO:56. In some embodiments, the encoded heavy chain variable region and light chain variable region sequences are respectively identical to (1) SEQ ID NOs:1 and 2, (2) SEQ ID NOs:3 and 4, (3) SEQ ID NOs:5 and 6, (4) SEQ ID NOs:7 and 8, (5) SEQ ID NOs:9 and 10, (6) SEQ ID NOs:11 and 12, (7) SEQ ID NOs:13 and 14, or (8) SEQ ID NOs:15 and 16. In some embodiments, the novel polynucleotides of the invention are cDNA molecules or mRNA molecules. Some related embodiments of the invention are directed to vectors that contain one or more polynucleotide molecules of the invention. Some other embodiments of the invention are directed to host cells that harbor such a vector. In some other related embodiments, the invention provides methods of producing an RSV-neutralizing antibody or antigen-binding fragment disclosed herein. These methods entail expressing in a suitable host cell a vector that contains operably a novel polynucleotide molecule of the invention.

[0012] In still another aspect, the invention provides methods or therapeutic uses of the novel antibody molecules disclosed herein to treat or ameliorate symptoms associated with a respiratory syncytial virus (RSV) infection in a subject (e.g., a human patient). These methods involve administering to the subject a pharmaceutical composition containing one RSV-neutralizing antibody or antibody-binding fragment disclosed herein, thereby treating or ameliorating symptoms associated with RSV infection in the subject. In some embodiments, the administered antibody or antigen-binding fragment contains a heavy chain variable region and a light chain variable region sequences that are at least 95% identical to,respectively, (1) SEQ ID NOs:1 and 2, (2) SEQ ID NOs:3 and 4, (3) SEQ ID NOs:5 and 6, (4) SEQ ID NOs:7 and 8, (5) SEQ ID NOs:9 and 10, (6) SEQ ID NOs:11 and 12, (7) SEQ ID NOs:13 and 14, or (8) SEQ ID NOs:15 and 16. In some embodiments, administered antibody or antigen-binding fragment contains heavy chain CDR sequences (HCDR1-3) and light chain CDR sequences (LCDR1-3) as set forth respectively in (1) SEQ ID NOs:17-20, DVS, and SEQ ID NO:21, (2) SEQ ID NOs:22-25, GNT, and SEQ ID NO:26, (3) SEQ ID NOs:27-30, GNT, and SEQ ID NO:31, (4) SEQ ID NOs:32-35, GNT, and SEQ ID NO:36, (5) SEQ ID NOs:37-40, GNT, and SEQ ID NO:41, (6) SEQ ID NOs:42-45, DNN, and SEQ ID NO:46, (7) SEQ ID NOs:47-50, AAS, and SEQ ID NO:51, or (8) SEQ ID NOs:52-55, EDK, and SEQ ID NO:56. In some embodiments, the administered antibody or antigen- binding fragment contains heavy chain and a light chain variable region sequences that are respectively identical to (1) SEQ ID NOs:1 and 2, (2) SEQ ID NOs:3 and 4, (3) SEQ ID NOs:5 and 6, (4) SEQ ID NOs:7 and 8, (5) SEQ ID NOs:9 and 10, (6) SEQ ID NOs:11 and 12, (7) SEQ ID NOs:13 and 14, or (8) SEQ ID NOs:15 and 16. In some embodiments, the subject to be treated is also afflicted a human metapneumovirus infection in addition to RSV infection.

[0013] In another aspect, the invention provides methods of diagnosing an RSV infection in a subject. The methods entail (a) obtaining a biological sample from the subject, and (b) contacting the sample with an RSV neutralizing antibody or antigen-binding fragment to detect a specific binding between an antigen in the sample and the antibody or antigen- binding fragment, thereby diagnosing an RSV infection in the subject. In some of these methods, the RSV-neutralizing antibody or antigen-binding fragment contains heavy chain CDR sequences (HCDR1-3) and light chain CDR sequences (LCDR1-3) as set forth respectively in (1) SEQ ID NOs:17-20, DVS, and SEQ ID NO:21, (2) SEQ ID NOs:22-25, GNT, and SEQ ID NO:26, (3) SEQ ID NOs:27-30, GNT, and SEQ ID NO:31, (4) SEQ ID NOs:32-35, GNT, and SEQ ID NO:36, (5) SEQ ID NOs:37-40, GNT, and SEQ ID NO:41, (6) SEQ ID NOs:42-45, DNN, and SEQ ID NO:46, (7) SEQ ID NOs:47-50, AAS, and SEQ ID NO:51, or (8) SEQ ID NOs:52-55, EDK, and SEQ ID NO:56. In some of these methods, the biological sample is a blood sample or a saliva sample.

[0014] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims.DESCRIPTION OF THE DRAWINGS

[0015] Figure 1. Samples for phage library construction. (A) Healthy human donor information. (B) ELISA of normal donor plasma against an RSV antigen using a commercial kit. All work was performed by iXCells Biotechnologies USA, LLC.

[0016] Figure 2. Identification RSV-reactive antibodies. (A) Phage ELISA was performed to assess randomly picked phage clones for their ability to bind an RSV prefusion F trimer, sc9-10 DS-Cav1, which has a disulfide bond-locked “closed” conformation. The 36 scFv clones that were sequenced are shown in bars. Of these, 16 scFv clones with complete variable sequences are labeled with solid circles and 8 representative scFv clones with diamond symbols. (B) Gene family analysis of the phage library-derived antibodies and their yield in transient expression of HEK293 F cells. Key antibody features are summarized in the table. Notably, the scFv gene was cloned into an Fc vector for expression, and as such, the antibodies tested in the initial characterization were in the scFv-Fc form. CDR sequences of the 8 listed scFv antibodies are shown in the figure, including HCDR1 sequences (SEQ ID NOs:22, 32, 27, 37, 42, 17, 52 and 47), HCDR2 sequences (SEQ ID NOs:23, 33, 28, 38, 43, 18, 53 and 48), HCDR3 sequences (SEQ ID NOs:24, 34, 29, 39, 44, 19, 54 and 49), LCDR1 sequences (SEQ ID NOs:25, 35, 30, 40, 45, 20, 55 and 50), LCDR2 sequences, and LCDR3 sequences (SEQ ID NOs:26, 36, 31, 41, 46, 21, 56 and 51).

[0017] Figure 3. Functional and structural analyses of phage library-derived antibodies (A) ELISA binding of eight antibodies, in scFv-Fc and some in IgG forms, tested against RSV-F (sc9-10 DS-Cav1) and hMPV-F (UFCM1-P2-iSS). The EC50values are plotted to facilitate comparison between different clones, while the binding curves are shown below the EC50 plot. (B) Neutralization of eight antibodies, in scFv-Fc and some in IgG forms, tested against live RSV-A2-GFP and hMPV-GFP viruses. The ID50values are plotted to facilitate comparison between different clones, while the neutralization curves are shown below. (C) The nsEM analysis of sc9-10 DS-Cav1 in complex with A4. The representative 2D classes are shown on the left, while the 3D reconstruction of the complex is shown on the left. A 3.50 Å-resolution crystal structure of RSD5 / DS-Cav1 complex (PDB ID: 6DC3) is used for density fitting. (D) The nsEM analysis of sc9-10 DS-Cav1 in complex with D1 and F3. The representative 2D classes are shown on the left, while the 3D reconstruction of the complex is shown on the right. A 3.08 Å-resolution crystal structure of MPE8-bound DS- Cav1 (PDB ID: 5U68) is used for density fitting.

[0018] Figure 4. Identity-divergence 2D analysis of three selected scFv clones (A4, D1 and F3) in the human antibody library during the biopanning process. For the heatmaps, after data processing using an Antibodyomics pipeline, each sequence is plotted as a function of sequence identity from a reference antibody chain and sequence divergence from the assigned germline gene. Color indicates sequence density at a particular point on the 2D plot. Sequences with the same germline gene, a CDR3 identity of 95% or greater, and a 1- residue error margin of CDR length calculation with respect to the reference antibody chain are plotted as orange dots with the number of sequences and gene family percentage labeled. DETAILED DESCRIPTION I. Overview

[0019] The present invention resides in part on the construction and identification of a panel of novel RSV-neutralizing antibodies. The antibodies were identified from a human single-chain antibody (scFv) library containing about 108- 1011scFv antibodies. The scFv library was constructed by the inventors through coupling of antibody heavy chain and light fragments that were obtained by PCR amplification of total RNAs from human donor cells. The phage-displayed scFv library was subjected to four rounds of biopanning against a prefusion RSV-F trimer conjugated to epoxy beads. Phagemid clones were then randomly selected from the output titer plate from the last round of biopanning and these clones were subjected to phage enzyme-linked immunosorbent assay (ELISA). RSV-F reactive clones were selected for Sanger sequencing to determine scFv sequences. A total of eight functional clones were identified. All scFv clones were transiently expressed in the scFv-Fc form in HEK293 F cells and purified by affinity for further characterization, such as ELISA and live virus neutralization. IgGs and Fabs were also produced for selected functional scFv clones to facilitate functional and structural analyses. Next-generation sequencing (NGS), or deep sequencing, was performed on the scFv library after each biopanning step to capture the somatic variants of functional scFv clones. In brief, one antibody (A4) potently neutralizes RSV by binding to sites Ø and V, and another antibody (F3) cross-neutralizes RSV and hMPV by recognizing site III. These novel antibodies and their somatic variants identified from the NGS analysis can be developed towards effective RSV and hMPV therapeutics.

[0020] In accordance with these studies, the invention provides neutralizing antibodies against RAV and hMPV. The antibodies and their variants described herein can be used asRSV and hMPV therapeutics to reduce viral loads and ameliorate disease symptoms in vulnerable populations such as infants and elderly. They can also be used as antibody reagents to evaluate rationally designed RSV-F and hMPV-F antigens and to facilitate RSV and hMPV vaccine development. The antibodies of the invention have demonstrated significant technological advantages over RSV binding antibodies known in the art. In particular, antibody A4 recognizes a unique prefusion-specific epitope that overlaps sites Ø and V on the F trimer. It potently neutralizes RSV by binding to both sites Ø and V. In contrast, the best known RSV therapeutic antibody, MEDI8897 (Nirsevimab), MEDI8897 only recognizes antigenic site Ø. In the scFv-Fc form, A4 has a half maximal inhibitory concentration (IC50) of 0.0021 µg / ml, 1.8-fold higher than MEDI8897. Antibody F3 is a potent cross-pneumovirus neutralizer that recognizes site III on the trimer. In RSV neutralization assays, F3 achieves a comparable IC50 value to the well-known site Ø antibody, D25 (0.013 µg / ml vs.0.011 µg / ml, respectively). In hMPV neutralization assays, F3 shows a ~11-fold-higher potency, estimated by IC50, than a widely studied cross- pneumovirus neutralizing antibody, MPE8. Similarly, antibody D1 has a similar potency to a site II directed NAb, Motavizumab, and can neutralize hMPV as well.

[0021] The invention can employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. (See, for example, Sambrook et al, ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al, ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No.4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al, eds., Methods In Enzymology, Vols.154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV;Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); and in Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0022] General principles of antibody engineering are set forth in Borrebaeck, ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). General principles of protein engineering are set forth in Rickwood et al, eds. (1995) Protein Engineering, A Practical Approach (IRL Press at Oxford Univ. Press, Oxford, Eng.). General principles of antibodies and antibody- hapten binding are set forth in: Nisonoff (1984) Molecular Immunology (2nd ed.; Sinauer Associates, Sunderland, Mass.); and Steward (1984) Antibodies, Their Structure and Function (Chapman and Hall, New York, N.Y.). Additionally, standard methods in immunology known in the art and not specifically described can be followed as in Current Protocols in Immunology, John Wiley & Sons, New York; Stites et al, eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange, Norwalk, Conn.) and Mishell and Shiigi (eds) (1980) Selected Methods in Cellular Immunology (W.H. Freeman and Co., NY).

[0023] Standard reference works setting forth general principles of immunology include Current Protocols in Immunology, John Wiley & Sons, New York; Klein (1982) J., Immunology: The Science of Self-Nonself Discrimination (John Wiley & Sons, NY); Kennett et al, eds. (1980) Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses (Plenum Press, NY); Campbell (1984) "Monoclonal Antibody Technology" in Laboratory Techniques in Biochemistry and Molecular Biology, ed. Burden et al, (Elsevier, Amsterdam); Goldsby et al, eds. (2000) Kuby Immunology (4th ed.; W.H. Freeman & Co.); Roitt et al. (2001) Immunology (6th ed.; London: Mosby); Abbas et al. (2005) Cellular and Molecular Immunology (5th ed.; Elsevier Health Sciences Division); Kontermann and Dubel (2001) Antibody Engineering (Springer Verlag); Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press); Lewin (2003) Genes VIII (Prentice Hall, 2003); Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press); Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press). II. Definitions

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which thisinvention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Academic Press Dictionary of Science and Technology, Morris (Ed.), Academic Press (1sted., 1992); Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic Dictionary of Chemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3rded., 2002); Dictionary of Chemistry, Hunt (Ed.), Routledge (1sted., 1999); Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer-Verlag Telos (1994); Dictionary of Organic Chemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), Oxford University Press (4thed., 2000). In addition, the following definitions are provided to assist the reader in the practice of the invention.

[0025] The term "antibody" also synonymously called "immunoglobulin" (Ig), or "antigen-binding fragment" refers to polypeptide chain(s) which exhibit a strong monovalent, bivalent or polyvalent binding to a given antigen, epitope or epitopes. Unless otherwise noted, antibodies or antigen-binding fragments used in the invention can have sequences derived from any vertebrate species. They can be generated using any suitable technology, e.g., single B cell cloning, hybridoma technology, ribosome display, phage display, gene shuffling libraries, semi-synthetic or fully synthetic libraries or combinations thereof. Unless otherwise noted, the term “antibody” as used in the present invention includes intact antibodies, antigen-binding polypeptide fragments and other designer antibodies that are described below or well known in the art (see, e.g., Serafini, J Nucl. Med. 34:533-6, 1993).

[0026] An “intact antibody” (or “full length antibody”) typically comprises at least two heavy (H) chains (about 50-70 kD) and two light (L) chains (about 25 kD) inter-connected by disulfide bonds. The recognized immunoglobulin genes encoding antibody chains include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0027] Each heavy chain of an antibody is comprised of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region of most IgGisotypes (subclasses) is comprised of three domains, CH1, CH2and CH3, some IgG isotypes, like IgM or IgE comprise a fourth constant region domain, CH4. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system and the first component (Clq) of the classical complement system.

[0028] The VH and VL regions of an antibody can be further subdivided into regions of hypervariability, also termed complementarity determining regions (CDRs), which are interspersed with the more conserved framework regions (FRs). Each VHand VLis composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The locations of CDR and FR regions and a numbering system have been defined by, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, U.S. Government Printing Office (1987 and 1991).

[0029] An antibody-based binding protein, as used herein, may represent any protein that contains at least one antibody-derived VH, VL, or CHimmunoglobulin domain in the context of other non-immunoglobulin, or non-antibody derived components. The antibody-based binding proteins of the invention include, but are not limited to (i) Fc-fusion proteins of binding proteins, including receptors or receptor components with all or parts of the immunoglobulin CH domains, (ii) binding proteins, in which VH and or VL domains are coupled to alternative molecular scaffolds, or (iii) molecules, in which immunoglobulin VH, and / or VL, and / or CHdomains are combined and / or assembled in a fashion not normally found in naturally occurring antibodies or antibody fragments.

[0030] “Binding affinity” is generally expressed in terms of equilibrium association or dissociation constants (KAor KD, respectively), which are in turn reciprocal ratios of dissociation and association rate constants (koffand kon, respectively). Thus, equivalent affinities may correspond to different rate constants, so long as the ratio of the rate constants remains the same. The binding affinity of an antibody is usually be expressed as the KD of a monovalent fragment (e.g. a Fabfragment) of the antibody, with KDvalues in the single-digitnanomolar range or below (subnanomolar or picomolar) being considered as very high and of therapeutic and diagnostic relevance.

[0031] As used herein, the term "binding specificity" refers to the selective affinity of one molecule for another such as the binding of antibodies to antigens (or an epitope or antigenic determinant thereof), receptors to ligands, and enzymes to substrates. Thus, all monoclonal antibodies that bind to a particular antigenic determinant of an entity (e.g., a specific epitope of RSV F protein) are deemed to have the same binding specificity for that entity.

[0032] A "conservative substitution" with respect to proteins or polypeptides refers to replacement of one amino acid with another amino acid having a side chain with similar chemical properties. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate protein activity are well-known in the art (see, e.g., Brummell et ah, Biochem.32: 1180-1187 (1993); Kobayashi et ah, Protein Eng.12(10):879-884 (1999); and Burks et al, Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)).

[0033] The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule.Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0034] For polypeptide sequences, “conservatively modified variants” refer to a variant which has conservative amino acid substitutions, amino acid residues replaced with other amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0035] The term “contacting” has its normal meaning and refers to combining two or more agents (e.g., polypeptides or chemical compounds), combining agents and cells or biological samples, or combining two populations of different cells. Contacting can occur in vitro, e.g., mixing an antibody and a biological sample, or mixing a population of antibodies with a population of cells in a test tube or growth medium. Contacting can also occur in a cell or in situ, e.g., contacting two polypeptides in a cell by co-expression in the cell of recombinant polynucleotides encoding the two polypeptides, or in a cell lysate. Contacting can also occur in vivo inside a subject, e.g., by administering an agent to a subject for delivery the agent to a target cell.

[0036] A “humanized antibody” is an antibody or antibody fragment, antigen-binding fragment, or antibody-based binding protein comprising antibody VH or VL domains with a homology to human VHor VLantibody framework sequences having a T20 score of greater than 80, as defined by defined by Gao et al. (2013) BMC Biotechnol.13, pp.55.

[0037] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one ofthe following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0038] Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math.2:482c, 1970; by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol.48:443, 1970; by the search for similarity method of Pearson and Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444, 1988; by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI); or by manual alignment and visual inspection (see, e.g., Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003)). Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively.

[0039] “Pharmaceutically acceptable”, “physiologically tolerable” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.

[0040] The term "subject" refers to human and non-human animals (especially non- human mammals). The term "subject" is used herein, for example, in connection with therapeutic and diagnostic methods, to refer to human or animal subjects. Animal subjects include, but are not limited to, animal models, such as, mammalian models of conditions or disorders associated with RSV infections. Other specific examples of non-human subjects include, e.g., cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys.

[0041] The terms "treat," "treating," "treatment," and "therapeutically effective" used herein do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment recognized by one of ordinary skill in the art as having a potential benefit or therapeutic effect. In this respect, the inventive method can provide any amount ofany level of treatment. Furthermore, the treatment provided by the inventive method can include the treatment of one or more conditions or symptoms of the disease being treated.

[0042] A "vector" is a replicon, such as plasmid, phage or cosmid, to which another polynucleotide segment may be attached so as to bring about the replication of the attached segment. Vectors capable of directing the expression of genes encoding for one or more polypeptides are referred to as "expression vectors". III. Antibodies and related compositions for neutralizing RSV and hMPV

[0043] The invention provides novel neutralizing antibodies against RSV and hMPV, modified antibodies derived from the antibodies exemplified herein, as well as related fusion or conjugate molecules (e.g., antibody-drug conjugates), pharmaceutical compositions, other related compositions, and methods for producing the antibody compositions. As detailed herein, the antibody compositions are capable of neutralizing RSV by specifically binding to RSV prefusion F trimer. In some embodiments, the antibodies or antigen-binding fragments thereof of the invention are capable of neutralizing both RSV and hMPV. In general, the RSV-neutralizing antibodies or antigen-binding fragments of the invention are derived from the scFv molecules described in the Examples below (e.g., Figure 2). They exhibit antigen- binding activities that are substantially identical to one of the scFv molecules exemplified herein, e.g., antibody “A4” which has heavy and light chain variable region sequences (VH and VL) set forth in SEQ ID Nos:1 and 2, respectively. Typically, the RSV-neutralizing antibodies of the invention contain heavy chain and light chain VHand VLsequences that are substantially identical to the VH and VL sequences, respectively, of one of the exemplified scFv antibodies. In some embodiments, the antibodies of the invention have identical or substantially identical heavy chain and light chain CDR sequences as that of one of the exemplified antibodies. Defined alternatively, they have the same binding specificity as that of one of the exemplified antibodies. In some embodiments, the antibodies or antigen- binding fragments have heavy chain and light CDR sequences that are respectively identical to the heavy chain and light chain CDR sequences listed in Figure 2. In some embodiments, the antibodies or antigen-binding fragments thereof contain one or more amino acid substitutions relative to the heavy chain sequence and the light chain sequence of one of the exemplified antibodies (e.g., antibody “A4”). The substitutions can be located either in the framework region, in the Fc domain, or in the CDRs of the antibodies exemplified herein.

[0044] In some embodiments, the antibodies or antigen-binding fragments of the invention contain VH and VL sequences that are substantially identical (e.g., at least 90% or 95%) to, respectively, (1) SEQ ID NOs:1 and 2, (2) SEQ ID NOs:3 and 4, (3) SEQ ID NOs:5 and 6, (4) SEQ ID NOs:7 and 8, (5) SEQ ID NOs:9 and 10, (6) SEQ ID NOs:11 and 12, (7) SEQ ID NOs:13 and 14, or (8) SEQ ID NOs:15 and 16. In some of these embodiments, the VH and VL sequences are at least 98% or 99% identical to, respectively, that of one the exemplified antibodies (e.g., SEQ ID NOs:1 and 2). In some of these embodiments, the VHand VLsequences are 100% identical to, respectively, that of one the exemplified antibodies (e.g., SEQ ID NOs:1 and 2). In some of these embodiments, in addition to the substantial identity of the VH and VL sequences, the antibodies or antigen- binding fragments of the invention contain heavy chain CDR sequences (HCDR1-3) and light chain CDR sequences (LCDR1-3) that are respectively identical to that of one of the exemplified scFv molecules. In various embodiments, the antibodies or antigen-binding fragments have HCDR1-3 and LCDR1-3 sequences set forth respectively in (1) SEQ ID NOs:17-20, DVS, and SEQ ID NO:21 (antibody A4), (2) SEQ ID NOs:22-25, GNT, and SEQ ID NO:26 (antibody D1), (3) SEQ ID NOs:27-30, GNT, and SEQ ID NO:31 (antibody F3), (4) SEQ ID NOs:32-35, GNT, and SEQ ID NO:36 (antibody E3), (5) SEQ ID NOs:37- 40, GNT, and SEQ ID NO:41 (antibody D12), (6) SEQ ID NOs:42-45, DNN, and SEQ ID NO:46 (antibody E5), (7) SEQ ID NOs:47-50, AAS, and SEQ ID NO:51 (antibody H9), or (8) SEQ ID NOs:52-55, EDK, and SEQ ID NO:56 (antibody F1).

[0045] In some embodiments, the antibodies or antigen-binding fragments of the invention contain of the invention contain HCDR1-3 and LCDR1-3 that are respectively identical to one of the exemplified scFv molecules. In some of these embodiments, in addition to the identical CDR sequences, the antibody or antigen-binding fragment also contains VHand VLsequences that are substantially identical (e.g., at least 90%, at least 95%, at least 99%, or 100% identical) to, respectively, the VH and VL sequences of one of the exemplified scFv molecules. In various embodiments, the VHand VLsequences can be substantially identical to, respectively, (1) SEQ ID NOs:1 and 2 (antibody A4), (2) SEQ ID NOs:3 and 4 (antibody D1), (3) SEQ ID NOs:5 and 6 (antibody F3), (4) SEQ ID NOs:7 and 8 (antibody E3), (5) SEQ ID NOs:9 and 10 (antibody D12), (6) SEQ ID NOs:11 and 12 (antibody E5), (7) SEQ ID NOs:13 and 14 (antibody H9), or (8) SEQ ID NOs:15 and 16 (antibody F1).

[0046] Antibodies of the invention include intact antibodies (e.g., IgG1 antibodies), antibody fragments or antigen-binding fragments, antibody-based binding proteins, which contain the antigen-binding portions of an intact antibody that retain capacity to bind to RSV F trimer. Examples of such antibody fragments include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CLand CH1domains; (ii) a F(ab’)2fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VHand CH1domains; (iv) a Fv fragment consisting of the VLand VHdomains of a single arm of an intact antibody; (v) disulfide stabilized Fvs (dsFvs) which have an interchain disulfide bond engineered between structurally conserved framework regions; (vi) a single domain antibody (dAb) which consists of a VH or VL domain (see, e.g., Ward et al., Nature 341:544-546, 1989); and (vii) an isolated complementarity determining region (CDR) as a linear or cyclic peptide. Examples of antibody-based binding proteins are polypeptides in which the binding domains of the antibodies are combined with other polypeptides or polypeptide domains, e.g. alternative molecular scaffolds, Fc regions, other functional or binding domains of other polypeptides or antibodies resulting in molecules with additional binding properties, e.g. bi- or multispecific proteins or antibodies. Such polypeptides can create an arrangement of binding or functional domains normally not found in naturally occurring antibodies or antibody fragments.

[0047] Antibodies of the invention also encompass “antibody fragments” (also termed “antigen-binding fragments” herein) that contain portions of an intact IgG antibody (e.g., the variant regions) responsible for target antigen recognition and binding. One example of such antibody fragments is single chain antibodies. The term "single chain antibody" refers to a polypeptide comprising a VH domain and a VL domain in polypeptide linkage, generally linked via a spacer peptide, and which may comprise additional domains or amino acid sequences at the amino- and / or carboxyl-termini. For example, a single-chain antibody may comprise a tether segment for linking to the encoding polynucleotide. As an example, a single chain variable region fragment (scFv) is a single-chain antibody. Compared to the VLand VHdomains of the Fv fragment which are coded for by separate genes, a scFv has the two domains joined (e.g., via recombinant methods) by a synthetic linker. This enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules. In some embodiments, the RSV neutralizing antigen-binding fragments of the invention are in the scFv-Fc format, as exemplified herein. This antibody format consists ofa scFv fused in one polypeptide chain to the hinge region, CH2 and CH3 domains of human IgG1. The scFv-Fc format allows for rapid characterization of candidate scFvs isolated from phage display libraries before conversion into a full-length IgG. This format offers several advantages over the phage display-derived scFv, including bivalent binding, longer half-life, and Fc-mediated effector functions.

[0048] In some embodiments, the invention provides modified antibodies or antigen- binding fragments that are derived from one of the RSV neutralizing antibodies exemplified herein. In some of these embodiments, the modified antibodies contain heavy chain CDRs and light chain CDRs that are identical to that of the exemplified antibody, and substitution of one or more amino acid residues in the framework regions (e.g., conservative substitutions). In some of these embodiments, the modified antibodies contain heavy chain CDRs and light chain CDRs that are identical to that of the exemplified antibody, and one or more substitutions with non-natural amino acid residues (e.g., substitutions in the Fc domain). In some other embodiments, the modified antibodies contain heavy chain CDRs and light chain CDRs that are embedded in a heterologous antibody scaffold.

[0049] In general, the antibodies or antigen-binding fragments of the invention can be generated in accordance with routinely practiced immunology methods. Some of such methods are exemplified herein in the Examples. General methods for preparation of monoclonal or polyclonal antibodies are well known in the art. See, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1998; Kohler & Milstein, Nature 256:495-497, 1975; Kozbor et al., Immunology Today 4:72, 1983; and Cole et al., pp.77-96 in Monoclonal Antibodies and Cancer Therapy, 1985. In some embodiments, antigen-binding fragments of the RSV- neutralizing antibodies of the invention can be produced by enzymatic or chemical modification of the intact antibodies, or synthesized de novo using recombinant DNA methodologies, or identified using phage display libraries. Methods for generating these antibody fragments are all well known in the art. For example, single chain antibodies can be identified using phage display libraries or ribosome display libraries, gene shuffled libraries (see, e.g., McCafferty et al., Nature 348:552-554, 1990; and U.S. Pat. No.4,946,778). In particular, scFv antibodies can be obtained using methods described in, e.g., Bird et al., Science 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988. Fv antibody fragments can be generated as described in Skerra and Plückthun, Science240:1038-41, 1988. Disulfide-stabilized Fv fragments (dsFvs) can be made using methods described in, e.g., Reiter et al., Int. J. Cancer 67:113-23, 1996. Similarly, single domain antibodies (dAbs) can be produced by a variety of methods described in, e.g., Ward et al., Nature 341:544-546, 1989; and Cai and Garen, Proc. Natl. Acad. Sci. USA 93:6280-85, 1996. Camelid single domain antibodies can be produced using methods well known in the art, e.g., Dumoulin et al., Nat. Struct. Biol.11:500–515, 2002; Ghahroudi et al., FEBS Letters 414:521–526, 1997; and Bond et al., J. Mol. Biol.332:643-55, 2003. Other types of antigen-binding fragments (e.g., Fab, F(ab’)2or Fd fragments) can also be readily produced with routinely practiced immunology methods.

[0050] The invention also provides conjugate molecules containing an RSV-neutralizing antibody that is linked to a second or heterologous entity such as a carrier protein (e.g., BSA or KLH), an antibody moiety, a polypeptide, or a small molecule moiety. In some embodiments, the second moiety to be conjugated to the antibody or antigen-binding fragment of the invention is a small organic molecule or a synthetic moiety. In some of these embodiments, the synthetic molecule can be a marker or detectable moiety (or label). In some other embodiments, the second moiety is a drug or toxin moiety. In some of these embodiments, the conjugated drug moiety is a compound known to be effective or useful for countering RSV or hMPV infections. Examples include ribavirin for treating RSV, antiviral agents such as Laninamivir, Interferon-β-1a, Cidofovir, and Rupintrivir. Conjugation of a second moiety to the antibodies of the invention can be readily carried out via routinely practiced methods that are well known in the art. In some embodiments, conjugation of the second moiety to the antibody can utilize covalent coupling to native or engineered lysine side-chain amines or cysteine side-chain thiols on the antibody. See, e.g., Wu et al., Nat. Biotechnol, 23: 1137-1146 (2005). These non-site-specific conjugation of the antibody or antigen-binding fragment via lysine or cysteine amino acid side chains can employ classical chemical linkers with maleimide functionality, or other chemical known in the art that can mediate conjugation to lysine or cysteine amino acid side chains. Alternatively, the second moiety can be conjugated site-specifically either by chemical, chemo-enzymatic, or enzymatic conjugations known in the art, like e.g. with bifunctional linkers, linkers allowing Pictet-Spengler chemistry on formyl-glycine forming enzyme modified antibodies, by glycan-remodeled antibodies, or by bacterial transglutaminase or sortase enzymes. In some embodiments, recombinant engineering and incorporated selenocysteine (e.g., as describedin U.S. Patent 8,916,159) can be used to site-specifically conjugate the second moiety. In some other embodiments, the antibody can be conjugated to the second moiety by site- specific sortase-enzyme mediated antibody conjugation as described in, e.g., WO2014140317. IV. Polynucleotides, vectors and host cells for producing RSV-neutralizing Abs

[0051] The invention provides substantially purified polynucleotides (DNA or RNA) that are identical or complementary to sequences encoding polypeptides containing the heavy chain and light chain variable region sequences of the RSV-neutralizing antibodies or antigen-binding fragments described herein, including segments or domains of the antibodies. The polynucleotides of the invention can encode only the variable region sequences of the exemplified antibodies. They can also encode both VH and VL of one of the exemplified antibodies, plus one or more constant regions. In some embodiments, the polynucleotides of the invention encode a mature heavy chain variable region and a mature light chain variable region that are substantially identical (e.g., at least 90%, 95% or 99%) to, respectively, SEQ ID NOs:1 and 2, SEQ ID NOs:3 and 4, SEQ ID NOs:5 and 6, SEQ ID NOs:7 and 8, SEQ ID NOs:9 and 10, SEQ ID NOs:11 and 12, SEQ ID NOs:13 and 14, or SEQ ID NOs:15 and 16. In some embodiments, the polynucleotides of the invention are cDNAs. In some embodiments, the polynucleotides of the invention are mRNAs.

[0052] The polynucleotide sequences can be produced by de novo solid-phase DNA synthesis or by PCR of a sequence encoding the heavy chain and light chain of an exemplified RSV-neutralizing antibody. Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al., Meth. Enzymol.68:90, 1979; the phosphodiester method of Brown et al., Meth. Enzymol.68:109, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859, 1981; and the solid support method of U.S. Patent No.4,458,066. As noted above, the antibodies of the invention can contain one or more amino acid substitutions in the framework region or in the Fc domain, relative to one of the exemplified RSV-neutralizing antibodies. Introducing mutations to a polynucleotide sequence by PCR can be performed as described in, e.g., PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods andApplications, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res.19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

[0053] Also provided in the invention are expression vectors and host cells for producing the RSV-neutralizing antibodies described herein. Specific examples of plasmid and transposon based vectors for expressing the antibodies are described in the Examples below. Various other expression vectors can also be employed to express the polynucleotides encoding the RSV-neutralizing antibody chains or binding fragments. Both viral-based and nonviral expression vectors can be used to produce the antibodies in a mammalian host cell. Nonviral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g., Harrington et al., Nat. Genet.15:345, 1997). For example, nonviral vectors useful for expression of the antibody polynucleotides and polypeptides in mammalian (e.g., human) cells include pCEP4, pREP4, pThioHis A, B & C, pcDNA3.1 / His, pEBVHis A, B & C (Invitrogen, San Diego, CA), MPSV vectors, and numerous other vectors known in the art for expressing other proteins. Other useful nonviral vectors include vectors that comprise expression cassettes that can be mobilized with Sleeping Beauty, PiggyBack and other transposon systems. Useful viral vectors include vectors based on lentiviruses or other retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brent et al., supra; Smith, Annu. Rev. Microbiol.49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992.

[0054] The choice of expression vector depends on the intended host cells in which the vector is to be expressed. Typically, the expression vectors contain a promoter and other regulatory sequences (e.g., enhancers) that are operably linked to the polynucleotides encoding an RSV-neutralizing antibody chain or fragment. In some embodiments, an inducible promoter is employed to prevent expression of inserted sequences except under inducing conditions. Inducible promoters include, e.g., arabinose, lacZ, metallothionein promoter or a heat shock promoter. Cultures of transformed organisms can be expanded under non-inducing conditions without biasing the population for coding sequences whose expression products are better tolerated by the host cells. In addition to promoters, other regulatory elements may also be required or desired for efficient expression of an RSV- neutralizing antibody chain or fragment. These elements typically include an ATG initiationcodon and adjacent ribosome binding site (Kozak consensus sequence) or other sequences. In addition, the efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (see, e.g., Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or CMV enhancer may be used to increase expression in mammalian host cells.

[0055] The expression vectors may also provide a secretion signal sequence position to form a fusion protein with polypeptides encoded by inserted RSV-neutralizing antibody sequences. More often, the inserted RSV-neutralizing antibody sequences are linked to a signal sequences before inclusion in the vector. Vectors to be used to receive sequences encoding the RSV-neutralizing antibody light and heavy chain variable domains sometimes also encode constant regions or parts thereof. Such vectors allow expression of the variable regions as fusion proteins with the constant regions thereby leading to production of intact antibodies or fragments thereof. Typically, such constant regions are human, and preferably of human IgG1 antibodies.

[0056] The host cells for harboring and expressing the RSV-neutralizing antibody chains can be either prokaryotic or eukaryotic. In some preferred embodiments, mammalian host cells are used to express and to produce the antibody polypeptides of the present invention. For example, they can be either a hybridoma cell line expressing endogenous immunoglobulin genes or a mammalian cell line harboring an exogenous expression vector. These include any normal mortal or normal or abnormal immortal animal or human cell. In addition to the cell lines exemplified herein, a number of other suitable host cell lines capable of secreting intact immunoglobulins are also known in the art. These include, e.g., the CHO cell lines, various HEK 293 cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B-cells and hybridomas. The use of mammalian tissue cell culture to express polypeptides is discussed generally in, e.g., Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y., 1987. Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer, and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell type-specific, stage-specific, and / or modulatable or regulatable. Useful promoters include, but are not limited to, EF1α and human UbCpromoters exemplified herein, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP pol III promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[0057] Methods for introducing expression vectors containing the polynucleotide sequences of interest vary depending on the type of cellular host. For example, calcium chloride transformation is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts (see generally Sambrook et al., supra). Other methods include, e.g., electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic methods, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion to the herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), agent-enhanced uptake of DNA, and ex vivo transduction. For long-term, high-yield production of recombinant proteins, stable expression will often be desired. For example, cell lines which stably express the antibody chains or binding fragments can be prepared using expression vectors of the invention which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth of cells which successfully express the introduced sequences in selective media. Resistant, stably transfected cells can be proliferated using tissue culture techniques appropriate for the cell type.

[0058] The invention further provides eukaryotic or non-eukaryotic cells (e.g., T lymphocytes) that have been recombinantly engineered to produce the antibodies, antibody- based binding proteins or antibody fragments thereof of the invention. The eukaryotic or non-eukaryotic cells can be used as an expression system to produce the antibody of the invention. In some embodiments, the invention provides RSV targeting immune cells that are engineered to recombinantly express an RSV-neutralizing antibody of the invention. For example, the invention provides a T cell engineered to express an antibody of the invention (e.g., an scFv, scFv-Fc, or (scFv)2), which is linked to a synthetic molecule containing one or more of the following domains: a spacer or hinge region (e.g., a CD28 sequence or a IgG4hinge-Fc sequence), a transmembrane region (e.g., a transmembrane canonical domain), and an intracellular T-cell receptor (TCR) signaling domain, thereby forming a chimeric antigen receptor (CAR) or T-body. Intracellular TCR signaling domains that can be included in a CAR (or T-body) include, but are not limited to, CD3ζ, FcR-γ, and Syk-PT signaling domains as well as the CD28, 4-1BB, and CD134 co-signaling domains. Methods for constructing T-cells expressing a CAR (or T-body) are known in the art. See, e.g., Marcu- Malina et al., Expert Opinion on Biological Therapy, Vol.9, No.5 (posted online on April 16, 2009). V. Therapeutic and diagnostic applications

[0059] The RSV-neutralizing antibodies or antigen-binding fragments thereof disclosed herein can be used in various therapeutic and diagnostic applications. For example, they can be used alone or in a combination therapy in the prophylactic or therapeutic treatment of RSV and hMPV infections. In some embodiments, the invention provides methods of using the RSV-neutralizing antibodies or fragments thereof to treat patients having infection by RSV and hMPV or patients having other diseases or conditions associated with RSV infections. In some embodiments, the antibodies or antigen-binding fragments of the invention can be used to prevent infections by RSV and / or hMPV, or to reduce or manage virus-induced symptoms in a subject infected with RSV and / or hMPV. In some other embodiments, the invention provides diagnostic methods for detecting RSV and / or hMPV infections or the presence of the viruses in biological samples obtained from human subjects.

[0060] Pharmaceutical compositions containing one or more of the RSV-neutralizing antibodies or antigen-binding fragments described herein are encompassed by the invention. In some embodiments, the pharmaceutical compositions are employed in therapeutic methods for treating RSV and / or hMPV infections. Typically, the subject or patient suitable for treatment is one who has been or is suspected of having been exposed to RSV and hMPV, is infected or suspected of being infected by RSV and hMPV, or has a symptom of an RSV- or hMPV-related infection. For example, the subject to be treated can be one who has been diagnosed of RSV and hMPV infection and / or possess symptoms associated with infections by RSV and hMPV. The RSV-neutralizing antibody or antigen-binding fragment thereof for use in the methods of the invention can a human or humanized antibody containing the same CDR sequences as that of one of the antibodies exemplified herein. In some embodiments,the neutralizing antibody or antigen-binding fragment thereof contains a binding domain that binds to the same epitope as, or competitively inhibits binding of, one or more of the antibodies exemplified herein.

[0061] In addition to the antibodies, pharmaceutical compositions of the invention typically also contain a pharmaceutically acceptable carrier, which is a molecule or substance that is normally not co-present naturally in a subject (e.g., a human patient) with an RSV neutralizing antibody described herein. Pharmaceutically carriers enhance or stabilize the composition, or to facilitate preparation of the composition. They should also be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the subject. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The pharmaceutically acceptable carrier employed should be suitable for various routes of administration. Additional guidance for selecting appropriate pharmaceutically acceptable carriers is provided in the art, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20thed., 2000. Some of the pharmaceutical compositions of the invention are vaccines. For vaccine compositions, appropriate adjuvants can be additionally included. Examples of suitable adjuvants include, e.g., aluminum hydroxide, lecithin, Freund's adjuvant, MPLTMand IL-12.

[0062] Therapeutic methods of the invention typically involve administering to a subject in need of treatment a pharmaceutical composition that contains a therapeutically amount of a neutralizing antibody or antigen-binding fragment described herein (e.g., an antibody shown in Figure 2). A therapeutically effective amount refers to an amount sufficient to achieve a therapeutic benefit, e.g., to ameliorate symptoms associated with RSV or hMPV infections. Suitable amount to be administered can be readily determined by one of ordinary skill in the art without undue experimentation given the invention. Factors influencing the mode of administration and the respective amount of an RSV neutralizing antibody or antigen-binding fragment thereof include, but are not limited to, the severity of the disease, the history of the disease, and the age, height, weight, health, and physical condition of the individual undergoing therapy. Similarly, the amount of an RSV-neutralizing immunotherapeutic to be administered will be dependent upon the mode of administration and whether the subject will undergo a single dose or multiple doses of this agent. In someembodiments, the therapeutic methods of the invention can be employed in combination with other regimens for treating or controlling RSV or hMPV infections. These include, e.g., ribavirin and interferon β-1a, or intravenous fluids and balancing electrolytes.

[0063] Methods of preparing and administering an RSV-neutralizing antibody or antigen-binding fragment thereof provided herein, to a subject in need thereof are well known to or can be readily determined by those skilled in the art. The route of administration of can be, for example, oral, parenteral, by inhalation or topical. The term parenteral as used herein includes, e.g., intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. While all these forms of administration are clearly contemplated as suitable forms, another example of a form for administration would be a solution for injection, in particular for intravenous or intraarterial injection or drip. In some cases, a suitable pharmaceutical composition can comprise a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. human albumin), etc. In other methods compatible with the teachings herein, an RSV-neutralizing antibody or antigen-binding fragment thereof as provided herein can be delivered directly to a site where the binding molecule can be effective in virus neutralization, e.g., the endosomal region of an RSV-infected cell. Preparation of pharmaceutical compositions of the invention and their various routes of administration can be carried out in accordance with methods well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20thed., 2000; and Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0064] The invention also provided methods for using the antibodies or related antigen- binding fragments described herein in diagnostic methods for detecting RSV infections or the presence of RSV. Various assays routinely practiced in the art can be employed for performing the diagnostic methods. These include, e.g., competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, (1994) Current Protocols in Molecular Biology (John Wiley & Sons, Inc., NY) Vol.1, which is incorporated by reference herein in its entirety). Methodsand reagents suitable for determination of binding characteristics of an antibody or antigen- binding fragment thereof are known in the art and / or are commercially available. Equipment and software designed for such kinetic analyses are commercially available (e.g., BIAcore®, BIAevaluation® software, GE Healthcare; KINEXA® Software, Sapidyne Instruments).

[0065] The diagnostic methods of the invention typically involve obtaining a biological sample from a subject that has or is suspected of having been infected with RSV and / or hMPV. Preferably, the subject is a human. In various embodiments, the biological sample suitable for the assays can be blood or any fraction thereof (e.g., serum, plasma, or whole blood), urine, feces, saliva, vomitus, or any combination thereof. Utilizing the novel antibodies disclosed herein, presence of an RSV antigen in the biological sample can be readily determined with any of the various immunoassays described herein, e.g., ELISA.

[0066] The invention further provides kits that contain an RSV-neutralizing immunotherapeutic of the invention for performing the therapeutic or diagnostic applications described herein. Typically, the kits contain two or more components required for performing the therapeutic or diagnostic methods of the invention. Kit components include, but are not limited to, one or more the disclosed antibodies or antibody fragments thereof, appropriate reagents, and / or equipment. In some embodiments, the kits can contain an antibody or antibody fragment thereof of the invention and an immunoassay buffer suitable for detecting RSV antigens (e.g. by ELISA, flow cytometry, magnetic sorting, or FACS). The kit may also contain one or more microtiter plates, standards, assay diluents, wash buffers, adhesive plate covers, magnetic beads, magnets, and / or instructions for carrying out a method of the invention using the kit. The kits can include an antibody or antigen-binding fragment thereof of the invention bound to a substrate (e.g., a multi-well plate or a chip), which is suitably packaged and useful to detect RSV antigens. In some embodiments, the kits include an antibody or antibody fragment thereof of the invention that is conjugated to a label, such as, a fluorescent label, a biologically active enzyme label, a luminescent label, or a chromophore label. The kits can further include reagents for visualizing the conjugated antibody or antibody fragment thereof, e.g., a substrate for the enzyme. In some embodiments, the kits include an antibody or antibody fragment thereof of the invention that is conjugated to a contrast agent and, optionally, one or more reagents or pieces of equipment useful for imaging the antibody in a subject.

[0067] Generally, the RSV neutralizing antibodies or antibody fragments thereof of the invention in a kit are suitably packaged, e.g., in a vial, pouch, ampoule, and / or any container appropriate for a therapeutic or detection method. Kit components can be provided as concentrates (including lyophilized compositions), which may be further diluted prior to use, or they can be provided at the concentration of use. For use of the antibody of the invention in vivo, single dosages may be provided in sterilized containers having the desired amount and concentration of components.

[0068] In various applications, the RSV neutralizing antibodies of the invention can be employed to produce antibody derivatives such as immunoconjugates. In some embodiments, the antibodies of the invention can be linked to a therapeutic moiety, such as a cytotoxin, a drug or a radioisotope. When conjugated to a cytotoxin, these antibody conjugates are referred to as "immunotoxins." A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Techniques for conjugating such therapeutic moiety to antibodies are well known in the art. In some embodiments, antibodies of the invention can be conjugated to an appropriate detectable agent to form immunoconjugates for use in diagnostic applications and in vivo imaging. The detectable agents can be any chemical moieties that contain a detectable label, e.g., radioisotopes, enzymes, fluorescent labels and various other antibody tags. In some other embodiments, the RSV neutralizing antibodies of the invention can be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers, e.g., polyethylene glycol (PEG). EXAMPLES

[0069] The following examples are provided to further illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. Example 1. Construction of normal human scFv antibody library

[0070] We received a notice of waiver from the Scripps IRB for using human blood mononuclear cell (PBMC) samples that were purchased from a commercial entity (iXCells Biotechnologies USA, LLC.). These PBMCs are collected from healthy donors by thecompany with an established IRB protocol, which made sure that researchers would have no access to identifiable private information and no direct interaction with the individual donors. A total of 11 human whole blood samples (10 mL) were collected during October 2015 to January 2016 (Figure 1, Panel A). The PBMC (5 million cells each) and plasma samples were prepared and cryopreserved on the day of collection. The plasma samples were used with dilution of 1:2 for the ELISA assay using a commercial detection kit (Respiratory syncytial virus IgG ELISA kit, DIAGNOSTIC AUTOMATION, Cat # 5114-8). The kit included a negative control (no IgG antibody against RSV), cut-off standard (a low concentration of IgG antibody against RSV), weak positive control (a medium concentration of IgG antibody against RSV), and positive control (a high concentration of IgG antibody against RSV). All 11 samples showed high titers of RSV-specific IgG antibodies (Figure 1, Panel B). Example 2. Identification of RSV reactive scFv antibodies by phage display and biopanning

[0071] The normal donor scFv library was constructed using a standard protocol reported in our previous publication, in which a phage library was constructed from PBMCs of an HIV-1 donor (He et al., Front Immunol 2017, 8:1025). In this study, PBMCs from 10 healthy donors excluding sample 4 were used to construct the scFv library. Four rounds of biopanning were performed against our lead design of prefusion RSV-F trimer, UFCR1-D- NQ, to enrich RSV-specific antibodies in the phage library. In our previous study, we found that four biopanning steps were sufficient to reach convergence in antibody enrichment, as demonstrated by multiple biopanning experiments using the HIV-1 donor-17 scFv library (He et al., Front Immunol 2017, 8:1025). In this study, to select RSV reactive clones, phage ELISA was performed by random selection of 96 scFv clones after the last biopanning step, using a previously reported prefusion RSV-F trimer (Joyce et al., Nat Struct Mol Biol 2016, 23: 811-820), sc9-10 DS-Cav1(1TD0), as the coating antigen. A total of 60 scFv antibodies showed detectable binding as indicated by the ultraviolet (UV) absorbance value and were identified as “RSV-reactive” clones for further characterization (Figure 2, Panel A). Of these, 36 scFv clones were selected and subjected to Sanger sequencing to determine their nucleotide sequences. High-quality Sanger sequencing data were obtained for 16 clones. Of these, 8 scFv clones with complete heavy chain (HC) and light chain (LC) variable regionsequences, as well as unique HCDR3 loops, were selected for further characterization. IMGT analysis revealed that these eight clones are derived from five HC variable (VH) genes and five λ / κ-LC variable (VL / VK) genes (Figure 2, Panel B). These clones showed a high yield of 2.2 mg to 4.3 mg when expressed as scFv-Fc forms except for F1 (17.5µg) when their His-tagged version was transiently expressed in 50 ml HEK293 F cells (Figure 2, Panel B). The amino acid sequences of heavy chains (HCs) and light chains (LCs) are shown below for the eight scFv clones. CDR motifs are indicated in bold and italicized font.

[0072] >IXL-A4 HC (SEQ ID NO:1) with CDRs1-3 (SEQ ID NOs:17-19, respectively) noted. EVQLVESGGGLVQPGGSLRLSCAASGFTFTTYAMHWVRQSPGKGLEWLASISYDGT SQYYAESVKGRFTVSRDNSKKTLLLQVNSLRPEDTAVYYCAGSPSGHYSWLGYFYA MDVWGQGTLVTVSS LC (SEQ ID NO:2) with CDRs1-3 (SEQ ID NO:20, DVS , SEQ ID NO:21) noted. QSALTQPASVSGSPGQSITISCTGASSDVGGYDYVSWYQQHPGKAPKLLIYDVSDRPS GVSNRFSGSKSGNTASLTISGLQTEDEADYYCSSYTSKNTLIFGGGTKLTVL

[0073] >IXL-D1 HC (SEQ ID NO:3) with CDRs1-3 (SEQ ID NOs:22-24, respectively) noted. EVQLVESGGGLVKPGGSLKLSCAASGFTLTSYTMNWVRQAPGKGLQWVSSITSGG NFLNYAYSMKGLFTISKDYARNSLSLQMNSLTVDDTAVYFCVKYGPPGIYGVASGL DVWGQGTRVTVSS LC (SEQ ID NO:4) with CDR1-3 (SEQ ID NO:25, GNT, SEQ ID NO:26) noted. HAVLTQPSSVSGAPGQTVTISCTGTSSNLGAGYDVHWYQQFPGTAPKLLIYGNTNRP SGSLTDSLAPSLAPPLLAITGLQAEDESDYYCHSYDNSLSGWVFGGGTQLTVL

[0074] >IXL-F3 HC (SEQ ID NO:5) with CDRs1-3 (SEQ ID NOs:27-29, respectively) noted. EVQLVESGGGLVKPGGSLRLSCAASGFSLTSYAMNWVRQAPGKGLEWVASISSGSN YIHYGDSVMGRFTISKDNARNSLDLQMKSLRVEDTAMYYCVRDGPPSIYGVPNGFD VWGQGTLVTVSS LC (SEQ ID NO:6) with CDRs1-3 (SEQ ID NO:30, GNT, SEQ ID NO:31) noted. QAVLTQPSSVSGAPGQRVTISCTGTSSNLGAGYDVHWYQQFPGTAPKLLIYGNTNRP SGVPDRFSGSKSGSSASLAITGLQAEDESDYYCQSYDNSLSGWVFGGGTKLTVL

[0075] >IXL-E3 HC (SEQ ID NO:7) with CDRs1-3 (SEQ ID NOs:32-34, respectively) noted. EVQLVESGGGLVKPGGSLKLSCAASGFSLTGYTMEWVRQAPGKGLQWVSSITSGSNFINYADSVKGRFTISRDNAGNSVSLQMNSLRVDDTAVYFCVRDGPPGIYGVASGLD VWGQGTTVTVSS LC (SEQ ID NO:8) with CDRs1-3 (SEQ ID NO:35, GNT, SEQ ID NO:36) noted. QAVLTQPSSVSGAPGQRVTISCTGTSSNLGAGYDVHWYQQFPGTAPKLLIYGNTNRP SGVPDRFSGSKSGSSASLAITGLQAEDESDYYCQSYDNSLSGWVFGGGTQLTVL

[0076] >IXL-D12 HC (SEQ ID NO:9) with CDRs1-3 (SEQ ID NOs:37-39, respectively) noted. EVQLVQSGGGLVQPGGSLRLSCAASGFTVSSNYMSWVRQAPGKGLEWVSVIYSGG STYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDYGDAIDYWGQGT MVTVSS LC (SEQ ID NO:10) with CDRs1-3 (SEQ ID NO:40, GNT, SEQ ID NO:41) noted. QAVLTQPSSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGNTNRP SGVPDRFSGSNSGTSASLAITGLQAGDEADYYCQSYDTSLSGWVFGGGTKVTVL

[0077] >IXL-E5 HC (SEQ ID NO:11) with CDRs1-3 (SEQ ID NOs:42-44, respectively) noted. EVQLVETGGGVVQPGRSLRLSCAASGFIFSDYGMHWVRQTPGKALEWVAVMSND GTNIYYADSVKGRFTISRDNSKNTLDLQMNSLRPEDTAVYYCAKDNYEWFEYYHGI DVWGQGTTVTVSS LC (SEQ ID NO:12) with CDRs1-3 (SEQ ID NO:45, DNN, SEQ ID NO:46) noted. QAVLTHPSSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPS GIPDRFSGSQSGTSATLGITGLQTGDEADYYCGTWDSSLSAVVFGGGTQLTVL

[0078] >IXL-H9 HC HC (SEQ ID NO:13) with CDRs1-3 (SEQ ID NOs:47-49, respectively) noted. EVQLVQSGGGVVQPGGSLRLSCKASGFNFDEYAMHWVRQRPGKGLEWVSLISGD GDNTYYPDSLKGRFTISRENNKYTLYLQMKSLTTDDTGFYFCAKDLDYDFWGASYT NSIDYSGMDVWGQGTTVTVSS LC (SEQ ID NO:14) with CDRs1-3 (SEQ ID NO:50, AAS, SEQ ID NO:51) noted. SDIQLTQSPPSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGV PSRFRGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKLEIK

[0079] >IXL-F1 HC (SEQ ID NO:15) with CDRs1-3 (SEQ ID NOs:52-54, respectively) noted. EVQLQQSGAEVKKPGSSVNVSCKASGGTFSSHAISWVRQAPGQGLEWMGGIIPIFG TPHYAQNFQGRVTISADESTSTAYMEPNSLRSEDTAVYYCARSYGNYDFWSGYYNW FDPWGQGTLVTVSS LC (SEQ ID NO:16) with CDRs1-3 (SEQ ID NO:55, EDK, SEQ ID NO:56) noted. QAVLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSAPTTVIYEDKQRPSG VPDRFSGTVDSSSNSASLTISGLKTEDEADYYCQSYDSSNHAVFGGGTKLTVLExample 3. Functional and structural characterization of phage library-derived antibodies

[0080] The eight identified RSV antibody clones noted above were evaluated in ELISA against a disulfide-locked prefusion-closed RSV-F trimer “sc9-10 DS-Cav1” (Joyce et al., Nat Struct Mol Biol 2016, 23: 811-820) and a disulfide-locked prefusion-closed hMPV-F trimer “UFCM1-P2-iSS” (Figure 3, Panel B). In the scFv-Fc form, A4, D1, and F3 showed higher affinities for sc9-10 DS-Cav1 than other RSV-F binding clones, such as E3, E5, and H9, with a ~4.8-18.9-fold difference in EC50. The affinities of A4, D1, and F3 scFv-Fc antibodies were largely comparable to that of D25 IgG. In the IgG form, A4, D1 and F3 still displayed similar affinities for sc9-10 DS-Cav1, but slightly lower than D25, with a 2.9-3.9- fold difference in EC50. When tested against UFCM1-P2-iSS, only D1 and F3 exhibited any measurable binding to this prefusion hMPV-F trimer. Specifically, D1 and F3 had similar affinities for hMPV-F in the scFv-Fc form, which were reduced by 25- and 3.8-fold when changed to the IgG form, respectively. These library-derived antibodies showed different potencies in live RSV and hMPV neutralization assays (Figure 3, Panel B). In the scFv-Fc form, A4 appeared to be the best RSV neutralizer with a half maximal inhibitory concentration (IC50) of 0.0021 µg / ml, 1.8-fold more potent than a highly optimized therapeutic antibody, MEDI8897 (Nirsevimab). In the IgG form, A4 showed a nearly identical IC50 value to MEDI8897, F3 yielded a comparable IC50 value to D25 (0.013 µg / ml vs.0.011 µg / ml, respectively), and D1 exhibited similar potency to a site II directed NAb, Motavizumab. Both D1 and F3 IgGs neutralized live hMPV, with F3 showing a ~11-fold- higher potency, estimated by IC50, than a widely studied cross-NAb, MPE8. Of the eight scFv clones, A4, D1, and F3 displayed distinct binding and neutralization profiles. To investigate how they recognize prefusion RSV-F, we generated A4, D1, and F3 Fabs and formed complexes with sc9-10 DS-Cav1. We first used nsEM to identify antibody epitopes on the prefusion trimer. For A4, the 2D classification revealed sc9-10 DS-Cav1 trimers with Fabs bound to an epitope near the trimer apex (Figure 3, Panel C, left). In comparison with the D25-bound DS-Cav1 trimer (McLellan et al., Science 2013, 340:6136), A4 shifted sidewards and created a larger angle with respect to the trimer axis in the side views (Figure 3, Panel C, left). Visual inspection of previously reported RSV-F NAbs revealed that the RSD5 / DS-Cav1 complex (PDB ID: 6DC3) could be fitted into the A4 / sc9-10 DS-Cav1density with a nearly perfect match (Figure 3, Panel C, right). RSD5 was reported to target an alternative conformation of site Ø. Indeed, crystal structure revealed a distinctive RSD5 epitope that mainly overlaps with site Ø, as defined by D25, but also extends to site V, as defined by hRSV90, 01.4B and ADI-14442. For D1 and F3, nsEM revealed an angle of approach resembling that of the site III-specific NAb, MPE8 (Figure 3, Panel D, left), which was confirmed by fitting the MPE8 / DS-Cav1 complex into the D1- and F3-bound sc9-10 DS-Cav1 densities (Figure 3, Panel D, right). The MPE8-like epitope specificity explained the cross-reactivity of D1 and F3 with both RSV-F and hMPV-F. Example 4. Deep sequencing analysis of phage libraries to identify the variants of functional scFv clones

[0081] To understand how these functional antibodies were selected during the biopanning process, we pooled the pre-panning and four post-panning scFv libraries for NGS on the Ion GeneStudio S5 platform using an Ion 530 chip. NGS yielded over ~18.8 million raw reads, which were processed using our Antibodyomics 2.0 pipeline to generate full-length VH and VL / K sequences for bioinformatics analyses. A two-dimensional (2D) identity-divergence analysis was used to visualize the scFv-derived HC and LC populations during the biopanning process (Figure 4). Given a template HC or LC, a somatic variant can be identified as the sequence that shares the same VH and VL / VK germline genes and a CDR3 identity of 95% or greater with a CDR3 length error of ≤ 1. The 2D plots revealed rapid enrichment of D1, E3, and D12 clones and modest expansion of A4, F3 and E5 clones after two panning steps, but little selection pressure was noted for F1 and H9. Between the two most potent neutralizers, F3 exhibited more pronounced expansion than A4, which accounted for 1.0% and 0.03% of their germline gene families, respectively. D1 showed the most dramatic expansion, accounting for 28.0% of its germline gene family. The HC and LC somatic variants of these functional scFv clones identified by 2D plots can be synthesized, and the reconstituted antibodies can be assessed for RSV-F / hMPV-F binding and live virus neutralization. ***

[0082] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certainchanges and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0083] All publications, databases, GenBank sequences, patents, and patent applications cited in this specification are herein incorporated by reference as if each was specifically and individually indicated to be incorporated by reference.

Claims

WE CLAIM:

1. An antibody or antigen-binding fragment thereof that neutralizes human respiratory syncytial virus (RSV), comprising heavy chain variable region (VH) and light chain variable region (VL) sequences that are at least 95% identical to, respectively, (1) SEQ ID NOs:1 and 2, (2) SEQ ID NOs:3 and 4, (3) SEQ ID NOs:5 and 6, (4) SEQ ID NOs:7 and 8, (5) SEQ ID NOs:9 and 10, (6) SEQ ID NOs:11 and 12, (7) SEQ ID NOs:13 and 14, or (8) SEQ ID NOs:15 and 16.

2. The antibody or antigen-binding fragment of claim 1, comprising heavy chain CDR sequences (HCDR1-3) and light chain CDR sequences (LCDR1-3) as set forth respectively in (1) SEQ ID NOs:17-20, DVS, and SEQ ID NO:21, (2) SEQ ID NOs:22-25, GNT, and SEQ ID NO:26, (3) SEQ ID NOs:27-30, GNT, and SEQ ID NO:31, (4) SEQ ID NOs:32-35, GNT, and SEQ ID NO:36, (5) SEQ ID NOs:37-40, GNT, and SEQ ID NO:41, (6) SEQ ID NOs:42-45, DNN, and SEQ ID NO:46, (7) SEQ ID NOs:47-50, AAS, and SEQ ID NO:51, or (8) SEQ ID NOs:52-55, EDK, and SEQ ID NO:

56.

3. The antibody or antigen-binding fragment of claim 1, comprising VHand VL sequences that are at least 98% or 99% identical to, respectively, (1) SEQ ID NOs:1 and 2, (2) SEQ ID NOs:3 and 4, (3) SEQ ID NOs:5 and 6, (4) SEQ ID NOs:7 and 8, (5) SEQ ID NOs:9 and 10, (6) SEQ ID NOs:11 and 12, (7) SEQ ID NOs:13 and 14, or (8) SEQ ID NOs:15 and 16.

4. The antibody or antigen-binding fragment claim 1, comprising VHand VLsequences that are respectively identical to (1) SEQ ID NOs:1 and 2, (2) SEQ ID NOs:3 and 4, (3) SEQ ID NOs:5 and 6, (4) SEQ ID NOs:7 and 8, (5) SEQ ID NOs:9 and 10, (6) SEQ ID NOs:11 and 12, (7) SEQ ID NOs:13 and 14, or (8) SEQ ID NOs:15 and 16.

5. An antibody or antigen-binding fragment thereof that neutralizes human respiratory syncytial virus (RSV), comprising heavy chain CDR sequences (HCDR1-3) and light chain CDR sequences (LCDR1-3) as set forth respectively in (1) SEQ ID NOs:17-20, DVS, and SEQ ID NO:21, (2) SEQ ID NOs:22-25, GNT, and SEQ ID NO:26, (3) SEQ ID NOs:27-30, GNT, and SEQ ID NO:31, (4) SEQ ID NOs:32-35, GNT, and SEQ ID NO:36, (5) SEQ ID NOs:37-40, GNT, and SEQ ID NO:41, (6) SEQ ID NOs:42-45, DNN, and SEQ IDNO:46, (7) SEQ ID NOs:47-50, AAS, and SEQ ID NO:51, or (8) SEQ ID NOs:52-55, EDK, and SEQ ID NO:

56.

6. The antibody or antigen-binding fragment of claim 5, comprising VH and VLsequences that are at least 95% identical to, respectively, (1) SEQ ID NOs:1 and 2, (2) SEQ ID NOs:3 and 4, (3) SEQ ID NOs:5 and 6, (4) SEQ ID NOs:7 and 8, (5) SEQ ID NOs:9 and 10, (6) SEQ ID NOs:11 and 12, (7) SEQ ID NOs:13 and 14, or (8) SEQ ID NOs:15 and 16.

7. The antibody or antigen-binding fragment of claim 1 or claim 5, which cross-neutralizes human metapneumovirus (hMPV).

8. The antibody or antigen-binding fragment of claim 7, comprising HCDR1-3 and LCDR1-3 as set forth respectively in (1) SEQ ID NOs:22-25, GNT, and SEQ ID NO:26, or (2) SEQ ID NOs:27-30, GNT, and SEQ ID NO:

31.

9. The antibody or antigen-binding fragment of claim 7, comprising VH and VLsequences that are respectively identical to (1) SEQ ID NOs:3 and 4, or (2) SEQ ID NOs:5 and 6.

10. The antibody or antigen-binding fragment of claim 1 or claim 5, comprising VHand VLsequences that, except for substitutions of one or more amino acid residues in the heavy chain framework region and the light chain framework region, are respectively identical to (1) SEQ ID NOs:1 and 2, (2) SEQ ID NOs:3 and 4, (3) SEQ ID NOs:5 and 6, (4) SEQ ID NOs:7 and 8, (5) SEQ ID NOs:9 and 10, (6) SEQ ID NOs:11 and 12, (7) SEQ ID NOs:13 and 14, or (8) SEQ ID NOs:15 and 16.

11. The antibody or antigen-binding fragment of claim 1 or claim 5, comprising (a) one or more non-natural amino acid residues in the Fc domain, and (b) HCDR1-3 and LCDR1-3 sequences as set forth respectively in (1) SEQ ID NOs:17-20, DVS, and SEQ ID NO:21, (2) SEQ ID NOs:22-25, GNT, and SEQ ID NO:26, (3) SEQ ID NOs:27-30, GNT, and SEQ ID NO:31, (4) SEQ ID NOs:32-35, GNT, and SEQ ID NO:36, (5) SEQ ID NOs:37-40, GNT, and SEQ ID NO:41, (6) SEQ ID NOs:42-45, DNN, and SEQ ID NO:46, (7) SEQ ID NOs:47-50, AAS, and SEQ ID NO:51, or (8) SEQ ID NOs:52-55, EDK, and SEQ ID NO:

56.

12. The antibody or antigen-binding fragment of claim 1 or claim 5, which is an IgG, a scFv-Fc, a scFv, a Fab, or a F(ab’)2fragment.

13. A pharmaceutical composition or kit, comprising a therapeutically effective amount of the antibody or antigen-binding fragment of claim 1 or claim 5, and a pharmaceutically acceptable carrier.

14. A molecule comprising (i) an antibody or antigen-binding fragment thereof that neutralizes human respiratory syncytial virus (RSV) as set forth in claim 1 or claim 5, and (ii) a second moiety that is fused or conjugated to the antibody or antigen-binding fragment thereof.

15. The molecule of claim 14, wherein the antibody or antigen-binding fragment comprises HCDR1-3 and LCDR1-3 sequences as set forth respectively in (1) SEQ ID NOs:17-20, DVS, and SEQ ID NO:21, (2) SEQ ID NOs:22-25, GNT, and SEQ ID NO:26, (3) SEQ ID NOs:27-30, GNT, and SEQ ID NO:31, (4) SEQ ID NOs:32-35, GNT, and SEQ ID NO:36, (5) SEQ ID NOs:37-40, GNT, and SEQ ID NO:41, (6) SEQ ID NOs:42-45, DNN, and SEQ ID NO:46, (7) SEQ ID NOs:47-50, AAS, and SEQ ID NO:51, or (8) SEQ ID NOs:52-55, EDK, and SEQ ID NO:

56.

16. The molecule of claim 14, wherein the second moiety is a polypeptide or a small organic molecule.

17. The molecule of claim 14, wherein the second moiety is a drug.

18. A polynucleotide, encoding an antibody heavy chain variable region (VH) sequence and an antibody light chain variable region (VL) sequence that are at least 95% identical to, respectively, (1) SEQ ID NOs:1 and 2, (2) SEQ ID NOs:3 and 4, (3) SEQ ID NOs:5 and 6, (4) SEQ ID NOs:7 and 8, (5) SEQ ID NOs:9 and 10, (6) SEQ ID NOs:11 and 12, (7) SEQ ID NOs:13 and 14, or (8) SEQ ID NOs:15 and 16.

19. The polynucleotide of claim 18, wherein CDR1-3 of the heavy chain (HCDR1-3) and CDR1-3 of the light chain (LCDR1-3) are respectively identical to (1) SEQ ID NOs:17-20, DVS, and SEQ ID NO:21, (2) SEQ ID NOs:22-25, GNT, and SEQ ID NO:26, (3) SEQ ID NOs:27-30, GNT, and SEQ ID NO:31, (4) SEQ ID NOs:32-35, GNT, and SEQ ID NO:36, (5) SEQ ID NOs:37-40, GNT, and SEQ ID NO:41, (6) SEQ ID NOs:42-45, DNN, and SEQ ID NO:46, (7) SEQ ID NOs:47-50, AAS, and SEQ ID NO:51, or (8) SEQ ID NOs:52-55, EDK, and SEQ ID NO:56.

20. The polynucleotide of claim 18, encoding VHand VLsequences that are respectively identical to (1) SEQ ID NOs:1 and 2, (2) SEQ ID NOs:3 and 4, (3) SEQ ID NOs:5 and 6, (4) SEQ ID NOs:7 and 8, (5) SEQ ID NOs:9 and 10, (6) SEQ ID NOs:11 and 12, (7) SEQ ID NOs:13 and 14, or (8) SEQ ID NOs:15 and 16.

21. The polynucleotide of claim 18, which is a cDNA.

22. A vector comprising the polynucleotide of claim 18.

23. A host cell harboring the vector of claim 22.

24. A method of producing an antibody or antigen-binding fragment thereof that specifically neutralizes respiratory syncytial virus, comprising expressing the vector of claim 22 in a suitable host cell.

25. A method for treating or ameliorating symptoms associated with a respiratory syncytial virus (RSV) infection in a subject, comprising administering to the subject a pharmaceutical composition comprising one antibody or antibody-binding fragment that neutralizes RSV, thereby treating or ameliorating symptoms associated with RSV infection in the subject; wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL) sequences that are at least 95% identical to, respectively, (1) SEQ ID NOs:1 and 2, (2) SEQ ID NOs:3 and 4, (3) SEQ ID NOs:5 and 6, (4) SEQ ID NOs:7 and 8, (5) SEQ ID NOs:9 and 10, (6) SEQ ID NOs:11 and 12, (7) SEQ ID NOs:13 and 14, or (8) SEQ ID NOs:15 and 16.

26. The method of claim 25, wherein the antibody or antigen-binding fragment comprises heavy chain CDR sequences (HCDR1-3) and light chain CDR sequences (LCDR1- 3) as set forth respectively in (1) SEQ ID NOs:17-20, DVS, and SEQ ID NO:21, (2) SEQ ID NOs:22-25, GNT, and SEQ ID NO:26, (3) SEQ ID NOs:27-30, GNT, and SEQ ID NO:31, (4) SEQ ID NOs:32-35, GNT, and SEQ ID NO:36, (5) SEQ ID NOs:37-40, GNT, and SEQ ID NO:41, (6) SEQ ID NOs:42-45, DNN, and SEQ ID NO:46, (7) SEQ ID NOs:47-50, AAS, and SEQ ID NO:51, or (8) SEQ ID NOs:52-55, EDK, and SEQ ID NO:

56.

27. The method of claim 25, wherein the antibody or antigen-binding fragment comprises VHand VLsequences that are respectively identical to (1) SEQ ID NOs:1 and 2, (2) SEQ ID NOs:3 and 4, (3) SEQ ID NOs:5 and 6, (4) SEQ ID NOs:7 and 8, (5) SEQ ID NOs:9and 10, (6) SEQ ID NOs:11 and 12, (7) SEQ ID NOs:13 and 14, or (8) SEQ ID NOs:15 and 16.

28. The method of claim 25, wherein the subject is also afflicted a human metapneumovirus infection.