WO2025106427A1

WO2025106427A1 - Neutralizing and protective monoclonal antibodies against respiratory syncytial virus (rsv)

Info

Publication number: WO2025106427A1
Application number: PCT/US2024/055505
Authority: WO
Inventors: Surender Khurana
Original assignee: US Department of Health and Human Services
Current assignee: US Department of Health and Human Services
Priority date: 2023-11-14
Filing date: 2024-11-12
Publication date: 2025-05-22
Anticipated expiration: 2026-05-14

Abstract

Antibodies and antigen binding fragments that specifically bind to an attachment (G) protein of RSV are disclosed. Nucleic acids encoding these antibodies, vectors and host cells are also provided. The disclosed antibodies, antigen binding fragments, nucleic acids and vectors can be used, for example, to reduce disease in a subject with an RSV infection, or to detect G protein in a sample.

Description

NEUTRALIZING AND PROTECTIVE MONOCLONAL ANTIBODIES AGAINST RESPIRATORY SYNCYTIAL VIRUS (RSV)

CROSS REFERENCE TO RELATED APPLICATIONS

This claims the benefit of U.S. Provisional Application No. 63/598,628, filed November 14, 2023, which is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

This relates to monoclonal antibodies and antigen binding fragments that specifically bind to an attachment (G) protein of respiratory syncytial virus (RSV) and their use, for example, in methods of reducing RSV disease in a subject.

INCORPORATION OF AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (“Sequence.xml”; Size: 171,857 bytes; and Date of Creation: October 21, 2024) is herein incorporated by reference in its entirety.

BACKGROUND

RSV is the major cause of lower respiratory tract disease in infants and young children, resulting in approximately 3.2 million hospitalizations and 118,200 deaths per year worldwide in children under the age of 5 years. It also causes severe disease in elderly. RSV has been classified into two antigenically distinct subtypes RSV A and RSV B, with these strains having antigenic differences and unique clinical characteristics. RSV subtypes often co-circulate during the same season and have equivalent severity.

RSV contains two major surface glycoproteins, the attachment (G) and fusion (F) glycosylated proteins, which are both targets of neutralizing and/or protective antibodies. RSV F is highly conserved between RSV A and B subtypes, and a recently approved vaccine against RSV in older adults demonstrated cross-subtype protection after vaccination with adjuvanted pre-fusion stabilized F protein from RSV A2. However, a need remains for other therapeutic for RSV.

SUMMARY OF THE DISCLOSURE

Disclosed are isolated monoclonal antibodies and antigen binding fragments thereof, wherein the monoclonal antibody or antigen binding fragment specifically binds to an attachment (G) protein of RSV. In some aspects, the antibody or antigen binding fragment includes a heavy chain variable (VH) region and a light chain variable region (VL) comprising a heavy chain complementarity determining region (HCDR)l, a HCDR2, and a HCDR3, and a light chain complementarity determining region (LCDR)l, a LCDR2, and a LCDR3 of the VH and Vi. set forth as any one of: a) SEQ ID NOs: 41 and 45, respectively (68C7); b) SEQ ID NOs: 73 and 77, respectively (73C1); c) SEQ ID NOs: 81 and 85, respectively (77D2); d) SEQ ID NOs: 49 and 53, respectively (40D8); e) SEQ ID NOs: 9 and 13, respectively (1D9); f) SEQ ID NOs: 33 and 37, respectively (12G11); g) SEQ ID NOs: 1 and 5, respectively (36E10); h) SEQ ID NOs: 25 and 29, respectively (7H11); i) SEQ ID NOs: 57 and 61, respectively (43A11); j) SEQ ID NOs: 17 and 21, respectively (48E2); k) SEQ ID NOs: 65 and 69, respectively (7H9); l) SEQ ID NOs: 89 and 93, respectively (22B11); m) SEQ ID NOs: 97 and 101, respectively (75F10); n) SEQ ID NOs: 105 and 109, respectively (7G6); o) SEQ ID NOs: 113 and 117, respectively (72E6); or p) SEQ ID NOs: 121 and 125, respectively (23B4).

Also disclosed are compositions including the antibodies and antigen binding fragments, nucleic acids encoding the antibodies and antigen binding fragments, expression vectors comprising the nucleic acids, and isolated host cells that comprise the nucleic acids. Such compositions can include pharmaceutically acceptable carriers. In several aspects, the nucleic acid molecule encoding a disclosed antibody or antigen binding fragment can be a cDNA or RNA molecule that encodes the antibody or antigen binding fragment. In additional aspects, the nucleic acid molecule can be a bicistronic expression construct encoding the CDR, VH and VL of the antibody, or antigen binding fragment.

Methods are disclosed for inhibiting (including preventing) disease caused by RSV in a subject. The method can include administering an effective of one or more of the disclosed antibodies, antigen binding fragments, nucleic acid molecules, vectors, mimics, or compositions, to the subject.

The antibodies, antigen binding fragments, nucleic acid molecules, vectors, and compositions disclosed herein can be used for a variety of additional purposes, such as for diagnosing RSV in a subject, or detecting RSV in a sample.

The foregoing and other features of the disclosure will become more apparent from the following detailed description of several aspects which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE FIGURES

FIGs. 1A-1E. RSV G Amino acid sequence alignment of the phylogeny, subtype sequence alignment. Sequence alignment of RSV G ectodomain from diverse RSV strains. Strains include A2, A-TX- Tracey, A2001-2-20, A/Riyadh/2009, A1998-12-21, Long, RSV-12, ABernett-61, Memphis-37, ON67-1210A, Bl, B-CH18537, and BA/3833/99. RSV G peptides that were used to immunize rabbits and mice are displayed. Plotting identities and similarities in alignment sequences shown with boxes. Shown are A2 (SEQ ID NO: 164), Tracey (SEQ ID NO: 165), 2-20 (SEQ ID NO: 166), Riyadh91/2009 (SEQ ID NO: 167), A1998-12-21 (SEQ ID NO: 168), Long (SEQ ID NO: 169), RSV-12 (SEQ ID NO: 170), Bernett (SEQ ID NO: 171), Memphis-37 (SEQ ID NO: 172), ON67-1210A (SEQ ID NO: 173), Bl (SEQ ID NO: 174), B-CH18537 (SEQ ID NO: 175), and BA/3833/99 (SEQ ID NO: 176).

FIGs. 2A-2B. Anti-G Monoclonal Antibodies (Mabs) and RSV challenge studies in BALB/c mice. FIG. 2A: Schematic representation of MAb injection and RSV challenge schedule in mice. FIG. 2B: Female BALB/c mice (N = 5 per group; 4-6 weeks old) were prophylactically treated intraperitoneally (IP) with the indicated MAbs at the 20 mcg/mouse dose or with phosphate buffered saline (PBS) as a control. MAbs labelled in bold were cross-reactive antibodies that were evaluated in challenge studies against both RSV-A2 and RSV- Bl. Twenty-four hours after MAb injection, mice were challenged intranasally with 10⁶ PFU of either RSV rA2- Line-19F-FFL or firefly luciferase expressing RSV Bl virus. In vivo imaging of lungs and the nasal cavity was performed daily for 5 days following RSV infection. Mice were sacrificed on day 5 post-challenge, when lungs, and blood were collected.

FIGs. 3A-3D. RSV titers and histopathology in the lungs of BALB/c mice at day 5 following RSV challenge. BALB/c mice were primed intraperitoneally (i.p.) with RSV G-specific MAbs (RSV A2: 36E10, 1D9, 7H9, 22B11, 7G6, 12G11, and 23B4; RSV Bl: 68C7, 75F10 and 72E6; or both RSV A2 and RSV Bl: 48E2, 7H11, 40D8, 77D2 and 131-2G). Mice were challenged 24 hours after antibody treatment with RSV-A2- L19-FFL or RSV-B1-FFL i.n., and lung viral titer of RSV-A2-L19-FFL (FIG. 3A) and RSV-B1-FFL (FIG. 3B) on day 5 post-RSV challenge were determined. (FIGs. 3C-3D) Lung histopathology on day 5 following RSV- A2 or RSV-B1 virus challenge. Lung tissue of the mice were collected at 5 days after RSV-A2-FFL (FIG. 3C) or RSV-B1-FFL (FIG. 3D) challenge and were stained with hematoxylin and eosin. Individual lungs were scored blindly using a 0-3 severity scale for pulmonary inflammation: bronchiolitis (mucous metaplasia of bronchioles), perivasculitis (inflammatory cell infiltration around the small blood vessels), interstitial pneumonia (inflammatory cell infiltration and thickening of alveolar walls), and alveolitis (cells within the alveolar spaces). The scores were subsequently converted to a combined histopathology scale of 0-12. Results are presented as mean ± SEM. One-way ANOVA test -multiple comparisons was performed in GraphPad Prism; *** p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

FIGs. 4A-4D. Live imaging of RSV infection in nasal cavity and lung in BALB/c mice at day 5 post-RSV challenge. MAb treated mice were challenged 24 hours afterwards with firefly expressing RSV-A2- L19-FFL or RSV-B1-FFL virus. Live whole-body imaging was performed to detect firefly luciferase activity in the lungs (FIGs. 4A-4B) and nasal cavity (FIGs. 4C-4D) of either RSV-A2-FFL (FIGs. 4A & 4C) or RSV-B1- FFL (FIGs. 4B & 4D) virus expressing firefly luciferase. Graphs represent the quantification of total flux (photons/sec) in these organs on day 5 post challenge. Results are presented as mean ± SEM. One-way ANOVA test -multiple comparisons was performed in GraphPad Prism. The asterisks show significance compared to negative control (SFM). ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

FIGs. 5A-5D. Relationship of lung pathology and lung fluxes or viral load titer on day 5 postchallenge with either RSV-A2 or RSV-B1. Correlation of lung pathology scores versus bioluminescence flux (FIGs. 5C-5D) signal in the infected lungs on day 5 post-challenge or viral load measured by plaque assay in lungs on day 5 post-challenge (FIGs. 5A-5B) with either RSV-A2-FFL (FIGs. 5A & 5C) or RSV-B1-FFL (FIGs. 5B & 5D) for all MAb treated BALB/c mice. Correlations show Pearson correlation coefficient (r) and two-tailed p values for all samples. The black line in the scatter plots depict the linear fit with shaded area showing 95% confidence interval.

FIG 6. Cross-reactivity and protective efficacy of different classes of anti-G MAbs against RSV-A and RSV-B. Schematic summarizing the classification of RSV-G binding MAbs by mapping studies and protective efficacy based on lung viral loads, lung flux and lung pathology in BALB/c mice challenge studies against RSV-A2 and RSV-B 1. High protective MAbs were defined as the MAbs that significantly reduced lung infectious viral titers, lung flux and lung pathology; moderately protective MAbs were defined as those who reduced lung infectious viral titers and lung flux and/or lung pathology; while low protective MAbs only reduced lung infectious viral titers but neither lung flux nor lung pathology.

FIGs. 7A-7B. Classification of Mab reactivity against G protein of RSV-A2 and RSV-B 1. *ELISA endpoint titers of the Mabs were determined as the reciprocal of the highest dilution proving an optical density (OD) twice that of the negative control. **SPR binding to protein/peptides are shown as maximum resonance units (RU).

FIG. 8. Neutralization titers measured by PRNT assay (in the presence of GPC).

FIGs. 9A-9C. RSV G protein sequences. Shown are A2 (SEQ ID NO: 129), Tracey (SEQ ID NO: 130), 2-20 (SEQ ID NO: 131), Riyadh91/2009 (SEQ ID NO: 132), A1998-12-21 (SEQ ID NO: 133), Long (SEQ ID NO: 134), RSV-12 (SEQ ID NO: 135), Bernett (SEQ ID NO: 136), Memphis-37 (SEQ ID NO: 137), ON67-1210A (SEQ ID NO: 138), Bl (SEQ ID NO: 139), B-CH18537 (SEQ ID NO: 140), and BA/3833/99 (SEQ ID NO: 141).

SEQUENCES

SEQ ID NOs: 1-128 arc exemplary heavy chain variable domain (VH), heavy chain complementarity determining region (HCDR)-l, HCDR-2, HCDR-3, light chain variable domain (VL), and heavy chain complementarity determining region (LCDR)-l, LCDR-2, and LCDR3 sequences of the disclosed antibodies, see Table 1.

SEQ ID NOs: 129-141 and 164-176 are amino acid sequences of attachment (G) proteins of RSV.

SEQ ID NOs: 142-163 are exemplary nucleic acid sequences encoding VH and VL domains of the disclosed antibodies, see Table 2.

DETAILED DESCRIPTION

RSV is a negative sense single stranded RNA virus that causes infections of the upper respiratory tract. RSV is the most common cause of respiratory hospitalization in infants, and reinfection remains common in later life: it is a notable pathogen in all age groups. Infection rates are typically higher during the cold winter months, causing bronchiolitis in infants, common colds in adults, and more serious respiratory illnesses such as pneumonia in the elderly and immunocompromised subjects. RSV can cause outbreaks both in the community and in hospital settings. Initial infection usually occurs via the eyes or nose, and the virus infects the epithelial cells of the upper and lower airway, causing inflammation, cell damage, and airway obstruction.

RSV is a member of the Pneumoviridae family and, as such, is an enveloped virus that replicates in the cytoplasm and matures by budding at the host cell plasma membrane. The genome of RSV is 15.2 kilobases that is transcribed by the viral polymerase into 10 mRNAs by a sequential stop-start mechanism that initiates at a single viral promoter at the 3' end of the genome. Each mRNA encodes a single major protein, with the exception of the M2 mRNA that has two overlapping open reading frames (ORFs) encoding two separate proteins M2-1 and M2-2. The 11 RSV proteins are: the RNA-binding nucleoprotein (N), the phosphoprotein (P), the large polymerase protein (L), the attachment glycoprotein (G), the fusion protein (F), the small hydrophobic (SH) surface glycoprotein, the internal matrix protein (M), the two nonstructural proteins NS1 and NS2, and the M2-1 and M2-2 proteins. The RSV gene order is: 3’-NSl-NS2-N-P-M-SH-G-F-M2-L. Each gene is flanked by short, conserved transcription signals called the gene-start (GS) signal, present on the upstream end of each gene and involved in initiating transcription of the respective gene, and the gcnc-cnd (GE) signal, present at the downstream end of each gene and involved in directing synthesis of a polyA tail followed by release of the mRNA. Transcription initiates at a single promoter at the 3' end and proceeds sequentially.

The anti-G MAb 131-2G targeting CCD motif was shown to block the interaction of RSV G protein with surface CX3CR1 and to block RSV G protein induced chemotaxis (Tripp et al., Nat Immunol 2, 732-738 (2001)), and protects animals from disease (see, for example Choi et al., Viral Immunol 25, 193-203 (2012)), but this antibody does not neutralize RSV in neutralization assays (Fuentes et al., Vaccine 31, 3987-3994 (2013)). This disclosure provides monoclonal antibodies and antigen binding fragments that specifically bind an G protein of RSV. The RSV-specific antibodies and antigen binding fragments provided herein can reduce (such as inhibit) disease caused by RSV, and are effective for protecting and treating infants, elderly and immunocompromised subjects. The antibodies and antigen binding fragments are also of use in assays to detect

RSV.

I. Summary of Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin ’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar' or equivalent to those described herein can be used, particular' suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:

Administration: The introduction of a composition into a subject by a chosen route. Administration can be local or systemic. For example, if the chosen route is intravenous, the composition is administered by introducing the composition into a vein of the subject. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation routes.

Antibody and Antigen Binding Fragment: An immunoglobulin, antigen-binding fragment, or derivative thereof, that specifically binds and recognizes an analyte (antigen) such as RSV protein, such as G protein. The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen binding fragments, so long as they exhibit the desired antigen-binding activity.

Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof that retain binding affinity for the antigen. Examples of antigen binding fragments include but are not limited to Fv, Fab, Fab’, Fab’-SH, Flab’)?; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen binding fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Diibel (Eds.), Antibody Engineering, Vols. 1-2, 2^nd ed., Springer-Verlag, 2010). Antibodies also include genetically engineered forms such as chimeric antibodies (such as humanized murine antibodies) and heteroconjugate antibodies (such as multispecific antibodies, including bispecific antibodies).

An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For instance, a naturally-occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment or Fv fragment has one binding site, while a bispecific or bifunctional antibody has two different binding sites.

Typically, a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable domain genes. There are two types of light chain, lambda (X) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region (or constant domain) and a variable region (or variable domain). In combination, the heavy and the light chain variable regions specifically bind the antigen.

References to “VH” or “VH” refer to the variable region of an antibody heavy chain, including that of an antigen binding fragment, such as Fv, scFv, dsFv or Fab. References to “VL” or “VL” refer to the variable domain of an antibody light chain, including that of an Fv, scFv, dsFv or Fab.

The VH and VL contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs” (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5^th ed., NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, U.S. Department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of an antigen. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, 5^th ed., NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, U.S. Department of Health and Human Services, 1991; “Kabat” numbering scheme), Al-Lazikani et al., (“Standard conformations for the canonical structures of immunoglobulins,” J. Mol. Bio., 273(4):927-948, 1997; “Chothia” numbering scheme), and Lefranc et al. (“IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev. Comp. Immunol., 27(l):55-77, 2003; “IMGT” numbering scheme). The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (from the N-terminus to C-terminus), and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is the CDR3 from the VH of the antibody in which it is found, whereas a Vi, CDR1 is the CDR1 from the Vi, of the antibody in which it is found. Light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.

In some aspects, a disclosed antibody includes a heterologous constant domain. For example, the antibody includes a constant domain that is different from a native constant domain, such as a constant domain including one or more modifications (such as the “LS” mutations) to increase half-life.

A “monoclonal antibody” is an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein. In some examples monoclonal antibodies arc isolated from a subject. Monoclonal antibodies can have conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. (See, for example, Greenfield (Ed.), Antibodies: A Laboratory Manual , 2^nd ed. New York: Cold Spring Harbor Laboratory Press, 2014.)

A “humanized” antibody or antigen binding fragment includes a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) antibody or antigen binding fragment. The non-human antibody or antigen binding fragment providing the CDRs is termed a “donor,” and the human antibody or antigen binding fragment providing the framework is termed an “acceptor.” In one aspect, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they can be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized antibody or antigen binding fragment, except possibly the CDRs, are substantially identical to corresponding parts of natural human antibody sequences.

A “chimeric antibody” is an antibody which includes sequences derived from two different antibodies, which typically are of different species. In some examples, a chimeric antibody includes one or more CDRs and/or framework regions from one human antibody and CDRs and/or framework regions from another human antibody.

A “fully human antibody” or “human antibody” is an antibody which includes sequences from (or derived from) the human genome, and does not include sequence from another species. In some aspects, a human antibody includes CDRs, framework regions, and (if present) an Fc region from (or derived from) the human genome. Human antibodies can be identified and isolated using technologies for creating antibodies based on sequences derived from the human genome, for example by phage display or using transgenic animals (see, e.g., Barbas et al. Phage display: A Laboratory Manuel . 1^st Ed. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008).

Antibody or antigen binding fragment that inhibits RSV : An antibody or antigen binding fragment that specifically binds to an RSV antigen, such as the attachment (G) protein, in such a way as to inhibit a biological function associated with RSV. In some aspects, the antibody, or an antigen binding fragment thereof, reduces or inhibits disease resulting from an RSV infection.

In some aspects, an antibody or antigen binding fragment that specifically binds to RSV and can reduce viral load, virus entry, or virus attachment, or cell-to-cell dissemination/infection, for example, by at least 50% (such as at least 60%, at least 70%, at least 80%, at least 90%, or more) compared to a control antibody or antigen binding fragment, or without the antibody present. In some aspects, an antibody or antigen binding fragment that specifically binds to RSV reduces infection or dissemination in a human subject by RSV, for example, by at least 50% compared to a control antibody or antigen binding fragment, or without antibody present, at a specified concentration.

Biological sample: A sample obtained from a subject. Biological samples include all clinical samples useful for detection of disease or infection (for example, an RSV infection) in subjects, including, but not limited to, cells, tissues, and bodily fluids, such as blood, derivatives and fractions of blood (such as serum), sputum, cerebrospinal fluid; as well as biopsied or surgically removed tissue, for example tissues that are unfixed, frozen, or fixed in formalin or paraffin. In a particular example, a biological sample is obtained from a subject having or suspected of having an RSV infection.

Bispecific antibody: A recombinant molecule composed of two different antigen binding domains that consequently binds to two different antigenic epitopes. Bispecific antibodies include chemically or genetically linked molecules of two antigen-binding domains. The antigen binding domains can be linked using a linker. The antigen binding domains can be monoclonal antibodies, antigen-binding fragments (e.g., Fv, Fab, scFv), or combinations thereof. A bispecific antibody can include one or more constant domains, but does not necessarily include a constant domain. A “multi-specific antibody” is a recombinant molecule composed of two or more different antigen binding domains, such as 3, 4, 5 or 6 antigen binding domains, that consequently binds to more than two different antigenic epitopes. Conditions sufficient to form an immune complex: Conditions which allow an antibody or antigen binding fragment to bind to its cognate epitope to a detectably greater degree than, and/or to the substantial exclusion of, binding to substantially all other epitopes. Conditions sufficient to form an immune complex are dependent upon the format of the binding reaction and typically are those utilized in immunoassay protocols or those conditions encountered in vivo. See Greenfield (Ed.), Antibodies: A Laboratory Manual, 2^nd ed. New York: Cold Spring Harbor Laboratory Press, 2014, for a description of immunoassay formats and conditions. The conditions employed in the methods are “physiological conditions” which include reference to conditions (e.g., temperature, osmolarity, pH) that are typical inside a living mammal or a mammalian cell. While it is recognized that some organs are subject to extreme conditions, the intra-organismal and intracellular environment normally lies around pH 7 (e.g., from pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), contains water as the predominant solvent, and exists at a temperature above 0°C and below 50°C. Osmolarity is within the range that is supportive of cell viability and proliferation.

The formation of an immune complex can be detected through conventional methods, for instance immunohistochemistry (IHC), immunoprecipitation (IP), flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (for example, Western blot), magnetic resonance imaging (MRI), computed tomography (CT) scans, radiography, and affinity chromatography. Immunological binding properties of selected antibodies may be quantified using known methods.

Conjugate: A complex of two molecules linked together, for example, linked together by a covalent bond. In one aspect, an antibody is linked to an effector molecule; for example, an antibody that specifically binds to RSV covalently linked to an effector molecule or a detectable label. The linkage can be by chemical or recombinant means. In one aspect, the linkage is chemical, wherein a reaction between the antibody moiety and the effector molecule has produced a covalent bond formed between the two molecules to form one molecule. A peptide linker (short peptide sequence) can optionally be included between the antibody and the effector molecule. Because conjugates can be prepared from two molecules with separate functionalities, such as an antibody and an effector molecule, they are also sometimes referred to as “chimeric molecules.”

Conservative variants: “Conservative” amino acid substitutions are those substitutions that do not substantially affect or decrease a function of a protein, such as the ability of the protein to interact with a target protein. For example, a RSV-specific antibody can include up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 conservative substitutions compared to a reference antibody sequence and retain specific binding activity to RSV, and/or neutralization activity. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid.

Individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (for instance less than 5%, in some aspects less than 1%) in an encoded sequence are conservative variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid.

The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

1 ) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Non-conservative substitutions are those that reduce an activity or function of the RSV-specific antibody, such as the ability to specifically bind to the RSV or neutralize RSV. For instance, if an amino acid residue is essential for a function of the protein, even an otherwise conservative substitution may disrupt that activity. Thus, a conservative substitution does not alter the basic function of a protein of interest.

Contacting: Placement in direct physical association; includes both in solid and liquid form, which can take place either in vivo or in vitro. Contacting includes contact between one molecule and another molecule, for example the amino acid on the surface of one polypeptide, such as an antigen, that contacts another polypeptide, such as an antibody. Contacting can also include contacting a cell for example by placing an antibody in direct physical association with a cell.

Control: A reference standard. In some aspects, the control is a negative control, such as sample obtained from a healthy patient not infected with RSV. In other aspects, the control is a positive control, such as a tissue sample obtained from a patient diagnosed with an RSV infection. In still other aspects, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of patients with known prognosis or outcome, or group of samples that represent baseline or normal values).

A difference between a test sample and a control can be an increase or conversely a decrease. The difference can be a qualitative difference or a quantitative difference, for example a statistically significant difference. In some examples, a difference is an increase or decrease, relative to a control, of at least about 5%, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, or at least about 500%.

Detectable marker: A detectable molecule (also known as a label) that is conjugated directly or indirectly to a second molecule, such as an antibody, to facilitate detection of the second molecule. For example, the detectable marker can be capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as CT scans. MRIs, ultrasound, fiberoptic examination, and laparoscopic examination). Specific, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI). Methods for using detectable markers and guidance in the choice of detectable markers appropriate for various purposes are discussed for example in Green and Sambrook (Molecular Cloning: A Laboratory Manual, 4^th ed., New York: Cold Spring Harbor Laboratory Press, 2012) and Ausubel et al. (Eds.) (Current Protocols in Molecular Biology, New York: John Wiley and Sons, including supplements, 2017).

Detecting: To identify the existence, presence, or fact of something, such as the presence of RSV in a sample.

Effective amount: A quantity of a specific substance sufficient to achieve a desired effect in a subject to whom the substance is administered. For instance, this can be the amount of an antibody, antigen binding fragment, multispecific antibody, or nucleic acid molecule necessary to reduce or inhibit disease in a subject with an RSV infection.

In some aspects, administration of an effective amount of a disclosed antibody or antigen binding fragment that binds to RSV can reduce or inhibit disease in a subject with RSV infection (for example, as measured by number or percentage of subjects infected by the RSV, or by an increase in the survival time of infected subjects, or reduction in symptoms associated with the RSV infection) by, for example at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of disease), as compared to a suitable control.

The effective amount of an antibody or antigen binding fragment that specifically binds RSV that is administered to a subject can vary depending upon a number of factors associated with that subject, for example the overall health and/or weight of the subject. An effective amount can be determined by varying the dosage and measuring the resulting response, such as, for example, a reduction in pathogen titer. Effective amounts also can be determined through various in vitro, in vivo or in situ immunoassays.

An effective amount encompasses a fractional dose that contributes in combination with previous or subsequent administrations to attaining an effective response. For example, an effective amount of an agent can be administered in a single dose, or in several doses, for example daily, during a course of treatment lasting several days or weeks. However, the effective amount can depend on the subject being treated, the severity and type of the condition being treated, and the manner of administration. A unit dosage form of the agent can be packaged in an amount, or in multiples of the effective amount, for example, in a vial (e.g., with a pierceable lid) or syringe having sterile components.

Effector molecule: A molecule intended to have or produce a desired effect; for example, a desired effect on a cell to which the effector molecule is targeted. Effector molecules can include, for example, polypeptides and small molecules. In one non-limiting example, the effector molecule is a toxin. Some effector molecules may have or produce more than one desired effect.

Epitope: An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, i.e., that elicit a specific immune response. An antibody specifically binds a particular antigenic epitope on a polypeptide. In some examples a disclosed antibody specifically binds to an epitope on an RSV antigen.

Expression: Transcription or translation of a nucleic acid sequence. For example, an encoding nucleic acid sequence (such as a gene) can be expressed when its DNA is transcribed into RNA or an RNA fragment, which in some examples is processed to become mRNA. An encoding nucleic acid sequence (such as a gene) may also be expressed when its mRNA is translated into an amino acid sequence, such as a protein or a protein fragment. In a particular example, a heterologous gene is expressed when it is transcribed into an RNA. In another example, a heterologous gene is expressed when its RNA is translated into an amino acid sequence. Regulation of expression can include controls on transcription, translation, RNA transport and processing, degradation of intermediar y molecules such as mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they are produced.

Expression Control Sequences: Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus, expression control sequences can include appropriate promoters, enhancers, transcriptional terminators, a start codon (ATG) in front of a proteinencoding gene, splice signals for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term “control sequences” is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.

Expression vector: A vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Non-limiting examples of expression vectors include cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses) that incorporate the recombinant polynucleotide.

A polynucleotide can be inserted into an expression vector that contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells.

Fc region: The constant region of an antibody excluding the first heavy chain constant domain. Fc region generally refers to the last two heavy chain constant domains of IgA, IgD, and IgG, and the last three heavy chain constant domains of IgE and IgM. An Fc region may also include part or all of the flexible hinge N-terminal to these domains. For IgA and IgM, an Fc region may or may not include the tailpiece, and may or may not be bound by the J chain. For IgG, the Fc region is typically understood to include immunoglobulin domains Cy2 and Cy3 and optionally the lower part of the hinge between Cyl and Cy2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues following C226 or P230 to the Fc carboxyl-terminus, wherein the numbering is according to Kabat. For IgA, the Fc region includes immunoglobulin domains Ca2 and Ca3 and optionally the lower part of the hinge between Cal and Ca2.

IgA: A polypeptide belonging to the class of antibodies that are substantially encoded by a recognized immunoglobulin alpha gene. In humans, this class or isotype comprises IgAi and IgA2. IgA antibodies can exist as monomers, polymers (referred to as plgA) of predominantly dimeric form, and secretory IgA. The constant chain of wild-type IgA contains an 18-amino-acid extension at its C-terminus called the tail piece (tp). Polymeric IgA is secreted by plasma cells with a 15-kDa peptide called the J chain linking two monomers of IgA through the conserved cysteine residue in the tail piece.

IgG: A polypeptide belonging to the class or isotypc of antibodies that arc substantially encoded by a recognized immunoglobulin gamma gene. In humans, this class comprises IgGi, IgG2, IgG;. and IgGr.

Immune complex: The binding of antibody or antigen binding fragment (such as a scFv, Fv or Fab) to a soluble antigen forms an immune complex. The formation of an immune complex can be detected through conventional methods, for instance immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (for example, Western blot), magnetic resonance imaging, CT scans, radiography, and affinity chromatography.

Inhibiting a disease or condition: Reducing the full development of a disease or condition in a subject, for example, reducing the full development of disease caused by RSV in a subject who has, or is at risk of having, disease caused by an RSV infection. This includes neutralizing, antagonizing, prohibiting, preventing, restraining, slowing, disrupting, stopping, or reversing progression or severity of the disease or condition. Complete elimination of disease caused by RSV is not requit ed.

Inhibiting a disease or condition can refer to a prophylactic intervention administered before the disease or condition has begun to develop (for example a treatment initiated in a subject at risk of an RSV infection, but not infected by the RSV ) that reduces subsequent development of the disease or condition and/or ameliorates a sign or symptom of the disease or condition following development. The term “ameliorating,” with reference to inhibiting a disease or condition refers to any observable beneficial effect of the prophylactic intervention intended to inhibit the disease or condition. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease or condition in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease or condition, a slower progression of the disease or condition, an improvement in the overall health or well-being of the subject, a reduction in infection, inhibition of clinical symptoms of disease caused by RSV, or by other parameters known in the art that are specific to the particular disease or condition.

Isolated: A biological component (such as a nucleic acid, peptide, protein or protein complex, for example an antibody) that has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, that is, other chromosomal and extra-chromosomal DNA and RNA, and proteins. Thus, isolated nucleic acids, peptides and proteins include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell, as well as, chemically synthesized nucleic acids. An isolated nucleic acid, peptide or protein, for example an antibody, can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

Kabat position: A position of a residue in an amino acid sequence that follows the numbering convention delineated by Kabat et al. Sequences of Proteins of Immunological Interest, 5^th Edition, Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, NIH Publication No. 91-3242, 1991).

Linker: A bi-functional molecule that can be used to link two molecules into one contiguous molecule, for example, to link an effector molecule to an antibody. Non-limiting examples of peptide linkers include glycine-serine linkers.

The terms “conjugating,” “joining,” “bonding,” or “linking” can refer to making two molecules into one contiguous molecule; for example, linking two polypeptides into one contiguous polypeptide, or covalently attaching an effector molecule or detectable marker radionuclide or other molecule to a polypeptide, such as an scFv. The linkage can be either by chemical or recombinant means. “Chemical means” refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule.

Nucleic acid (molecule or sequence): A deoxyribonucleotide or ribonucleotide polymer or combination thereof including without limitation, cDNA, mRNA, genomic DNA, and synthetic (such as chemically synthesized) DNA or RNA. The nucleic acid can be double stranded (ds) or single stranded (ss). Where single stranded, the nucleic acid can be the sense strand or the antisense strand. Nucleic acids can include natural nucleotides (such as A, T/U, C, and G), and can include analogs of natural nucleotides, such as labeled nucleotides. “cDNA” refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form.

“Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and non-coding strand, used as the template for transcription, of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

Operably linked: A fir st nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter, such as the CMV promoter, is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington: The Science and Practice of Pharmacy, 22^nd ed., London, UK: Pharmaceutical Press, 2013, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed agents.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, added preservatives (such as non-natural preservatives), and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In particular examples, the pharmaceutically acceptable carrier is sterile and suitable for parenteral administration to a subject for example, by injection. In some aspects, the active agent and pharmaceutically acceptable carrier are provided in a unit dosage form such as a pill or in a selected quantity in a vial. Unit dosage forms can include one dosage or multiple dosages (for example, in a vial from which metered dosages of the agents can selectively be dispensed). Polypeptide: A polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred. The terms “polypeptide” or “protein” as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. A polypeptide includes both naturally occurring proteins, as well as those that are recombinantly or synthetically produced. A polypeptide has an amino terminal (N-terminal) end and a carboxy-terminal end. In some aspects, the polypeptide is a disclosed antibody or a fragment thereof. A polypeptide can be glycosylated, but is not necessarily glycosylated.

Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein (such as an antibody) is more enriched than the peptide or protein is in its natural environment within a cell. In one aspect, a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation.

Recombinant: A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. A recombinant protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. In several aspects, a recombinant protein is encoded by a heterologous (for example, recombinant) nucleic acid that has been introduced into a host cell, such as a bacterial or eukaryotic cell. The nucleic acid can be introduced, for example, on an expression vector having signals capable of expressing the protein encoded by the introduced nucleic acid or the nucleic acid can be integrated into the host cell chromosome.

Respiratory Syncytial Virus (RSV): An enveloped non-segmented negative-sense single-stranded RNA virus of the family Pneumoviridae, genus Orthopneumovirus. The RSV genome is -15,000 nucleotides in length (15,223 for KT 992094) and includes 10 genes encoding 11 proteins, including the glycoproteins SH, G and F. The F protein mediates fusion, allowing entry of the virus into the cell cytoplasm and also promoting the formation of syncytia. Two antigenic subgroups of human RSV strains have been described, the A and B subgroups, based primarily on differences in the antigenicity of the G protein, which is glycosylated. RSV strains for other species are also known, including bovine RSV. Several animal models of infection by human RSV and closely-related animal counterparts are available, including model organisms infected with human RSV, as well as model organisms infected with species-specific RSV, such as use of bRS V infection in cattle (see, e.g. , Bern et al. , Am J, Physiol. Lung Cell Mol. Physiol. , 301 : L148-L156, 2011; and Nam and Kun (Eds.). Respiratory Syncytial Virus: Prevention, Diagnosis and Treatment. Nova Biomedical Nova Science Publisher, 2011; and Cane (Ed.) Respiratory Syncytial Virus. Elsevier Science, 2007.) RSV is classified into two subtypes, A and B, based on antigenic differences in the G attachment protein (see Mufson et al. J Gen

Virol. 1985;66:2111-2124, doi: 10.1099/0022-1317-66-10-2111; Garcia et al., J. Virol. 1994;68:5448-59; and Peret et aL, J. Gen. Virol. 1998;79:2221-2229, doi: 10.1099/0022-1317-79-9-2221).

The positioning of RSV nucleic acid residues can be defined according to the reference RSV antigenomic sequence provided herein as SEQ ID NO: 1 of PCT Publication No. WO2021/248086, which is an RSV strain A2 antigenomic sequence, also provided as GENBANK® accession number KT 992094.1, as available on June 12, 2023, also known as “D46.”

RSV attachment glycoprotein (G protein): An RSV envelope glycoprotein that is a type II membrane protein and facilitates attachment of RSV to host cell membranes.

The RSV G protein is expressed during RSV infection in two forms. One is the full-length transmembrane form (mG), which is expressed on the cell surface and is packaged into the virus particle. The other form is an N-terminally-truncated, secreted form, sG. The full-length G protein (mG) is a type II protein that has an N-terminal cytoplasmic tail (CT, predicted to comprise approximately amino acids 1-37), a hydrophobic transmembrane domain (TM, comprising approximately amino acids 38-66), and an ectodomain (comprising approximately amino acids 67-298). The sG form is produced by alternative translation initiation at the second AUG codon (M48) in the ORF, whose corresponding position in the protein lies within the TM domain. The N-terminus is then subjected to intracellular proteolytic trimming that creates a new N-terminus at approximately N66.

Most of the vaccine and therapeutics targeting RSV G are focused on the central conserved domain (CCD) of G (for example, amino acids 172-186), which is bound by some of the disclosed antibodies, for example 40D8, and the fractalkine-like CX3C motif (see Yoder et al., J Med Virol 72, 688-694 (2004)). RSV A2 G protein is provided as gb:AAC55969|gi: 1695262, and RSV_B l_G_protein is provided as gi|2582029|gb|AAB82435.1, both as available on November 6, 2023. Exemplary RSV G protein sequences are provided in FIGS. 9A-9C.

RSV Fusion protein (F): An RSV envelope glycoprotein that facilitates fusion of viral and cellular membranes. In nature, the RSV F protein is initially synthesized as a single polypeptide precursor approximately 574 amino acids in length, designated Fo. Fo includes an N-terminal signal peptide that directs localization to the endoplasmic reticulum, where the signal peptide (approximately the first 25 residues of Fo) is proteolytically cleaved. The remaining Fo residues oligomerize to form a trimer and are proteolytically processed by a cellular protease at two conserved consensus furin cleavage sequences (approximately Fo positions 109 and 136 to generate two disulfide-linked fragments, Fi and F2. The smaller of these fragments, F , originates from the N-terminal portion of the Fo precursor and includes approximately residues 26-109 of Fo. The larger of these fragments, Fi, includes the C-tcrminal portion of the Fo precursor (approximately residues 137-574) including an extracellular/lumenal region (-residues 137-529), a transmembrane domain (-residues 530-550), and a cytoplasmic tail (-residues 551-574) at the C-terminus. The extracellular portion of the RSV F protein is the RSV F ectodomain, which includes the F2 protein and the Fi ectodomain.

Sequence identity: The identity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences. Homologs and variants of a VL or a VH of an antibody that specifically binds a target antigen are typically characterized by possession of at least about 75% sequence identity, for example at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full-length alignment with the amino acid sequence of interest.

Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2(4):482-489, 1981; Needleman and Wunsch, J. Mol. Biol. 48(3):443-453, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85(8) :2444- 2448, 1988; Higgins and Sharp, Gene, 73(l):237-244, 1988; Higgins and Sharp, Bioinformatics, 5(2): 151-3, 1989; Corpet, Nucleic Acids Res. 16(22):10881-10890, 1988; Huang et al. Bioinformatics, 8(2): 155-165, 1992; and Pearson, Methods Mol. Biol. 24:307-331, 1994. Altschul et al., J. Mol. Biol. 215(3):403-410, 1990, presents a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215(3):403-410, 1990) is available from several sources, including the National Center for Biological Information and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Blastn is used to compare nucleic acid sequences, while blastp is used to compare amino acid sequences. Additional information can be found at the NCBI web site.

Generally, once two sequences are aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity between the two sequences is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100.

Specifically bind: When referring to an antibody or antigen binding fragment, refers to a binding reaction which determines the presence of a target protein, such as a G protein of RSV, in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated conditions, an antibody binds preferentially to a particular target protein, peptide or polysaccharide (such as an antigen present on a pathogen, such as RSV ) and does not bind in a significant amount to other proteins present in the sample or subject. Specific binding can be determined by standard methods. See Harlow & Lane, Antibodies, A Laboratory Manual. 2^nd ed.. Cold Spring Harbor Publications, New York (2013), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. With reference to an antibody-antigen complex, specific binding of the antigen and antibody has a KD of less than about 10⁷ Molar, such as less than about 10⁸ Molar, 10⁹, or even less than about 10 ¹⁰ Molar. KD refers to the dissociation constant for a given interaction, such as a polypeptide ligand interaction or an antibody antigen interaction. For example, for the bimolccular interaction of an antibody or antigen binding fragment and an antigen it is the concentration of the individual components of the bimolecular interaction divided by the concentration of the complex.

An antibody that specifically binds to RSV, such as RSV glycoprotein (G), is an antibody that binds substantially to RSV G in a cell transformed to express G, a substrate to which RSV is attached, or RSV in a biological specimen. It is, of course, recognized that a certain degree of non-specific interaction may occur between an antibody or conjugate including an antibody (such as an antibody that specifically binds RSV or conjugate including such antibody) and a non-target (such as a sample of from a subject that is not infected wit RSV). Typically, specific binding results in a much stronger association between the antibody and protein or cells bearing the antigen than between the antibody and protein or cells lacking the antigen. Specific binding typically results in greater than 2-fold, such as greater than 5-fold, greater than 10-fold, or greater than 100-fold increase in amount of bound antibody (per unit time) to a protein including the epitope or cell or tissue expressing the target epitope as compared to a protein or cell or tissue lacking this epitope. Specific binding to a protein under such conditions requires an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats are appropriate for selecting antibodies or other ligands specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein.

Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals. In an example, a subject is a human. In an additional example, a subject is selected that is in need of inhibiting an RSV infection. In examples, the subject is uninfected and at risk of RSV infection. The subject can be immunocompromised. A subject in some aspects is an infant or a child. In some aspects, the subject is an adult, such as 60 years or older.

Transformed: A transformed cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term transformed and the like (e.g., transformation, transfection, transduction, etc.) encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transduction with viral vectors, transformation with plasmid vectors, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.

Vector: An entity containing a nucleic acid molecule (such as a DNA or RNA molecule) bearing a promoter(s) that is operationally linked to the coding sequence of a protein of interest and can express the coding sequence. Non-limiting examples include a naked or packaged (lipid and/or protein) DNA, a naked or packaged RNA, a subcomponent of a virus or bacterium or other microorganism that may be replication- incompetent, or a vims or bacterium or other microorganism that may be replication-competent. A vector is sometimes referred to as a construct. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements. Viral vectors are recombinant nucleic acid vectors having at least some nucleic acid sequences derived from one or more viruses. In some aspects, a viral vector includes a nucleic acid molecule encoding a disclosed antibody or antigen binding fragment that specifically binds RSV. In some aspects, the viral vector can be an adeno- associated virus (AAV) vector.

II. Description of Several Aspects

A. Monoclonal Antibodies to RSV G and Antigen Binding Fragments Thereof

Isolated monoclonal antibodies and antigen binding fragments are provided that specifically bind an attachment (G) protein of RSV. The antibodies and antigen binding fragments can inhibit disease in a subject infected with RSV. Examples include 68C7, 73C1, 77D2, 40D8, 1D9, I2G11, 36E10, 7H11, 43A11, 48E2, 7H9, 22B11, 75F10, 7G6, 72E6, or 23B4. and antigen binding fragments thereof. Also disclosed herein are compositions comprising the antibodies and antigen binding fragments and a pharmaceutically acceptable carrier. Nucleic acids encoding the antibodies or antigen binding fragments, expression vectors (such as DNA and RNA vectors for expression and delivery, as well as adeno-associated virus (AAV) viral vectors) comprising these nucleic acids are also provided.

The antibodies, antigen binding fragments, nucleic acid molecules, host cells, and compositions, disclosed herein, can be used for research, diagnostic, therapeutic and prophylactic purposes. For example, the antibodies and antigen binding fragments can be used to diagnose a subject an RSV infection, or can be administered prophylactically or therapeutically to inhibit disease caused by RSV in a subject. All of the disclosed antibodies, antigen binding fragments, nucleic acid molecules, conjugates, and multispecific antibodies are of use in these methods.

In some aspects, disclosed are the antibodies 68C7, 73C1, 77D2, 40D8, 1D9, 12G11, 36E10, 7H11, 43A11, 48E2, 7H9, 22B11, 75F10, 7G6, 72E6, or 23B4, and an antigen binding fragments thereof. The antibodies 68C7, 73C1, 77D2, 40D8, 1D9, 12G11, 36E10, 7H11, 43A11, 48E2, 7H9, 22B11, 75F10, 7G6, 72E6, or 23B4, antigen binding fragments, nucleic acid molecules, conjugates, and multispecific antibodies, as disclosed herein can be used to inhibit disease caused by RSV, diagnose a subject with disease caused by RSV, or can be administered prophylactically to inhibit disease caused by RSV.

1. Exemplary monoclonal antibodies and antigen binding fragments

The discussion of monoclonal antibodies below refers to isolated monoclonal antibodies that include heavy and/or light chain variable domains (or antigen binding fragments thereof) comprising a CDR1, CDR2, and/or CDR3 with reference to the IMGT numbering scheme (unless the context indicates otherwise). Various CDR numbering schemes (such as the Kabat, Chothia or IMGT numbering schemes) can be used to determine CDR positions. The amino acid sequence and the CDR positions (according to the IMGT numbering scheme, using the reference positions in IMGT) of the heavy and light chains of exemplary monoclonal antibodies that bind to RSV are shown in Table 1 below:

Table 1. IMGT CDRs of antibodies

CDR sequences are identified in the VH and VL sequences using underlining.

a. Monoclonal antibody 68C7

In some aspects, the antibody or antigen binding fragment is based on or derived from the 68C7 antibody, and specifically binds to the attachment (G protein) of RSV.

In some examples, the antibody or antigen binding fragment comprises a VH and a VL comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the 68C7 antibody, and specifically binds to a G protein of RSV. In further aspects, the antibody or antigen binding fragment binds to the intact glycosylated G proteins but not to an individual RSV A2 derived G peptide, sec FIGs. 7A-7B.

In some aspects, the antibody or antigen binding fragment comprises a Vu comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 41 and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 45, and specifically binds to a G protein of RSV. In additional aspects, the antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 41 and 45 respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 42, 43, and 44, respectively, and/or a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 46, 47, and 48, respectively, and specifically binds to a G protein of RSV. In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 42, 43, and 44, respectively, a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 46, 47, and 48, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 41, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 41, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 45, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 45, and the antibody or antigen binding fragment specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 41 and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 45, and specifically binds to a G protein of RSV. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 41 and 45, respectively, and specifically binds to a G protein of RSV.

In some aspects, the disclosed antibodies and antigen binding fragments inhibit disease in a subject with an RSV infection. b. Monoclonal antibody 73 Cl

In some aspects, the antibody or antigen binding fragment is based on or derived from the 73C1 antibody, and specifically binds to the attachment (G protein) of RSV.

In some examples, the antibody or antigen binding fragment comprises a VH and a VL comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the 73C1 antibody, and specifically binds to a G protein of RSV. In further aspects, the antibody or antigen binding fragment binds to the intact glycosylated G proteins but not to an individual RSV A2 derived G peptide.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 73, and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 77, and specifically binds to a G protein of RSV. In additional aspects, the antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 73 and 77, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 74, 75, and 76, respectively, and/or a Vi, comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 78, 79, and 80, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 74, 75, and 76, respectively, a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 78, 79, and 80, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 73 such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 73, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 77, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 77, and the antibody or antigen binding fragment specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 73, and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 77, and specifically binds to a G protein of RSV. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 73 and 77, respectively, and specifically binds to a G protein of RSV.

In some aspects, the disclosed antibodies and antigen binding fragments inhibit disease in a subject with an RSV infection. c. Monoclonal antibody 77D2

In some aspects, the antibody or antigen binding fragment is based on or derived from the 77D2 antibody, and specifically binds to the attachment (G protein) of RSV.

In some examples, the antibody or antigen binding fragment comprises a VH and a VL comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the 77D2 antibody, and specifically binds to a G protein of RSV. In further aspects, the antibody or antigen binding fragment binds to the intact glycosylated G proteins but not to an individual RSV A2 derived G peptide, see FIGs. 7A-7B.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 81, and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 85, and specifically binds to a G protein of RSV. In additional aspects, the antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 81 and 85, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 82, 83, and 84, respectively, and/or a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 86, 87, and 88, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 82, 83, and 84, respectively, a VLComprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 86, 87, and 88, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 81, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 81, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 85, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 85, and the antibody or antigen binding fragment specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 81 and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 85, and specifically binds to a G protein of RSV. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 81 and 85, respectively, and specifically binds to a G protein of RS V.

In some aspects, the disclosed antibodies and antigen binding fragments inhibit disease in a subject with an RSV infection. d. Monoclonal antibody 40D8

In some aspects, the antibody or antigen binding fragment is based on or derived from the 40D8 antibody, and specifically binds to the attachment (G protein) of RSV.

In some examples, the antibody or antigen binding fragment comprises a VH and a VL comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the 40D8 antibody, and specifically binds to a G protein of RSV. In further aspects, the antibody or antigen binding fragment binds to the CCD region (amino acids (aa) 172-186) of a G protein.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 49 and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 53, and specifically binds to a G protein of RSV. In additional aspects, the antibody or antigen binding fragment comprises a VH and a Vi, independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 49 and 53, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 50, 51, and 52, respectively, and/or a VLComprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 54, 55, and 56, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 50, 51, and 52, a VLComprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 54, 55, and 56, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 49, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 49, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 53, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 53, and the antibody or antigen binding fragment specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 49, and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 53, and specifically binds to a G protein of RSV. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 49 and 53, respectively, and specifically binds to a G protein of RSV.

In some aspects, the disclosed antibodies and antigen binding fragments inhibit disease in a subject with an RSV infection. e. Monoclonal antibody 1D9

In some aspects, the antibody or antigen binding fragment is based on or derived from the ID9 antibody, and specifically binds to the attachment (G protein) of RSV.

In some examples, the antibody or antigen binding fragment comprises a VH and a VL comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the 1D9 antibody, and specifically binds to a G protein of RSV. In further aspects, the antibody or antigen binding fragment binds to the CCD region (aa 172-186) of a G protein.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical, to the amino acid sequence set forth as SEQ ID NO: 9 and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 13, and specifically binds to a G protein of RSV. In additional aspects, the antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 9 AND 13, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 10, 11, and 12, respectively, and/or a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 14, 15, and 16, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 10, 11, and 12, respectively, a VLComprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 14, 15, and 16, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 9, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 9, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 13, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13, and the antibody or antigen binding fragment specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 9, and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 13, and specifically binds to a G protein of RSV. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 9 and 13, respectively, and specifically binds to a G protein of RSV.

In some aspects, the disclosed antibodies and antigen binding fragments inhibit disease in a subject with an RSV infection. f Monoclonal antibody 12G11

In some aspects, the antibody or antigen binding fragment is based on or derived from the 12G11 antibody, and specifically binds to the attachment (G protein) of RSV.

In some examples, the antibody or antigen binding fragment comprises a VH and a VL comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the 12G11 antibody, and specifically binds to a G protein of RSV. In further aspects, the antibody or antigen binding fragment binds to the CCD region (aa 172-186) of a G protein.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 33, and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 37, and specifically binds to a G protein of RSV. In additional aspects, the antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 33 and 37, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 34, 35, and 36, respectively, and/or a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 38, 39, and 40, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 34, 35, and 36, respectively, a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 38, 39, and 40, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 11 such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 33, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 37, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 37, and the antibody or antigen binding fragment specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 33, and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 37, and specifically binds to a G protein of RSV. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 33 and 37, respectively, and specifically binds to a G protein of RSV.

In some aspects, the disclosed antibodies and antigen binding fragments inhibit disease in a subject with an RSV infection. g. Monoclonal antibody 36E10

In some aspects, the antibody or antigen binding fragment is based on or derived from the 36E10 antibody, and specifically binds to the attachment (G protein) of RSV.

In some examples, the antibody or antigen binding fragment comprises a VH and a VL comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the 36E10 antibody, and specifically binds to a G protein of RSV. In further aspects, the antibody or antigen binding fragment binds to the CCD region (aa 172-186) of a G protein In some aspects, the antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 1, and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 5, and specifically binds to a G protein of RSV. In additional aspects, the antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 1 and 5, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 2, 3, and 4, and/or a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 6, 7, and 8, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 2, 3, and 4, a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 6, 7, and 8, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 5, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 5, and the antibody or antigen binding fragment specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 1 and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 5, and specifically binds to a G protein of RSV. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 1 and 5, respectively, and specifically binds to a G protein of RSV.

In some aspects, the disclosed antibodies and antigen binding fragments inhibit disease in a subject with an RSV infection. h. Monoclonal antibody 7H11

In some aspects, the antibody or antigen binding fragment is based on or derived from the 7H11 antibody, and specifically binds to the attachment (G protein) of RSV.

In some examples, the antibody or antigen binding fragment comprises a VH and a VL comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the 7H11 antibody, and specifically binds to a G protein of RSV. In further aspects, the antibody or antigen binding fragment specifically binds to a G protein of RSV. In further aspects, the antibody or antigen binding fragment specifically binds RMG-A2, and to a lesser degree with RMG- Bl. In more aspects, the monoclonal antibody or antigen binding fragment specifically binds CCD-deleted REG-A2 protein (REG-A2 delCCD) and to the peptide encompassing residues 169-297 (downstream of CCD).

In some aspects, the antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 25 and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 29 and specifically binds to a G protein of RSV. In additional aspects, the antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 25 and 29, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 26, 27, and 28, respectively, and/or a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 30, 31, and 32, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 26, 27, and 28, respectively, a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 30, 31, and 32, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 25, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 25, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 29, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 29, and the antibody or antigen binding fragment specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 25, and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 29, and specifically binds to a G protein of RSV. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 25 and 29, respectively, and specifically binds to a G protein of RSV.

In some aspects, the disclosed antibodies and antigen binding fragments inhibit disease in a subject with an RSV infection. i. Monoclonal antibody 43A11

In some aspects, the antibody or antigen binding fragment is based on or derived from the 43A11 antibody, and specifically binds to the attachment (G protein) of RSV.

In some examples, the antibody or antigen binding fragment comprises a VH and a VL comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the 43A11 antibody, and specifically binds to a G protein of RSV. In further aspects, the antibody or antigen binding fragment specifically binds RMG-A2, and to a lesser degree with RMG-B1. In more aspects, the monoclonal antibody or antigen binding fragment specifically binds CCD- deleted REG-A2 protein (REG-A2 delCCD) and to the peptide encompassing residues 169-297 (downstream of CCD).

In some aspects, the antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 57 and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 61, and specifically binds to a G protein of RSV. In additional aspects, the antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 57 and 61, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 58, 59, and 60, respectively, and/or a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 62, 63, and 64, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1 , a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 58, 59, and 60, respectively, a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 62, 63, and 64, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 57 such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 57, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 61, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 61, and the antibody or antigen binding fragment specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 57, and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 61, and specifically binds to a G protein of RSV. In some aspects, the antibody or antigen binding fragment comprises a VH and a Vi, comprising the amino acid sequences set forth as SEQ ID NOs: 57 and 61, respectively, and specifically binds to a G protein of RSV.

In some aspects, the disclosed antibodies and antigen binding fragments inhibit disease in a subject with an RSV infection. j. Monoclonal antibody 48E2

In some aspects, the antibody or antigen binding fragment is based on or derived from the 48E2 antibody, and specifically binds to the attachment (G protein) of RSV.

In some examples, the antibody or antigen binding fragment comprises a VH and a VL comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the 48E2 antibody, and specifically binds to a G protein of RSV. In further aspects, the antibody or antigen binding fragment specifically binds a discontinuous epitope consisting of two peptides (residues 129-152 and 169-207) that flank the CCD motif and form the stem of the CCD loop.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 17 and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 21 , and specifically binds to a G protein of RSV. In additional aspects, the antibody or antigen binding fragment comprises a Vn and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID Nos: 17 and 21, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID Nos: 18, 19, and 20, respectively, and/or a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID Nos: 22, 23, and 24, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID Nos: 18, 19, and 20, respectively, a VLComprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID Nos: 22, 23, and 24, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 17, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 17, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 21, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 21, and the antibody or antigen binding fragment specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 17, and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 21, and specifically binds to a G protein of RSV. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 17 and 21, respectively, and specifically binds to a G protein of RSV.

In some aspects, the disclosed antibodies and antigen binding fragments inhibit disease in a subject with an RSV infection. k. Monoclonal antibody 7H9

In some aspects, the antibody or antigen binding fragment is based on or derived from the 7H9 antibody, and specifically binds to the attachment (G protein) of RSV.

In some examples, the antibody or antigen binding fragment comprises a VH and a VL comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the 7H9 antibody, and specifically binds to a G protein of RSV. In further aspects, the antibody or antigen binding fragment specifically binds to the CCD region (aa 172-186) of a G protein.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 65, and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 69, and specifically binds to a G protein of RSV. In additional aspects, the antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID Nos: 65 and 69, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID Nos: 66, 67, and 68, respectively, and/or a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID Nos: 70, 71, and 72, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID Nos: 66, 67, and 68, respectively, a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID Nos: 70, 71, and 72, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 65, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 65, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 69, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 69, and the antibody or antigen binding fragment specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 65, and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 69, and specifically binds to a G protein of RSV. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 65 and 69, 7H9 respectively, and specifically binds to a G protein of RSV.

In some aspects, the disclosed antibodies and antigen binding fragments inhibit disease in a subject with an RSV infection.

I. Monoclonal antibody 22B1I

In some aspects, the antibody or antigen binding fragment is based on or derived from the 22B11 antibody, and specifically binds to the attachment (G protein) of RSV.

In some examples, the antibody or antigen binding fragment comprises a VH and a V comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the 22B 11 antibody, and specifically binds to a G protein of RSV. In further aspects, the antibody or antigen binding fragment specifically binds to the CCD region (aa 172-186) of a G protein

In some aspects, the antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 89, and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 93, and specifically binds to a G protein of RSV. In additional aspects, the antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID Nos: 89 and 93, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID Nos: 90, 91, and 92, respectively, and/or a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID Nos: 94, 95, and 96, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID Nos: 90, 91, and 92, respectively, a V comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID Nos: 94, 95, and 96, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 89, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 89, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 93, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 93, and the antibody or antigen binding fragment specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 89, and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 93, and specifically binds to a G protein of RSV. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 89 and 93, respectively, and specifically binds to a G protein of RSV.

In some aspects, the disclosed antibodies and antigen binding fragments inhibit disease in a subject with an RSV infection. m. Monoclonal antibody 75F10

In some aspects, the antibody or antigen binding fragment is based on or derived from the 75F10 antibody, and specifically binds to the attachment (G protein) of RSV.

In some examples, the antibody or antigen binding fragment comprises a VH and a VL comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the 75F10 antibody, and specifically binds to a G protein of RSV. In further aspects, the antibody or antigen binding fragment specifically binds to the intact glycosylated G proteins but not to an individual RSV A2 derived G peptide, see FIGs. 7A-7B.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 97, and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 101, and specifically binds to a G protein of RSV. In additional aspects, the antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 97 and 101, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 98, 99, and 100, respectively, and/or a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 102, 103, and 104, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 98, 99, and 100, respectively, a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 102, 103, and 104, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 97, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 97, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 101, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 101, and the antibody or antigen binding fragment specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 97, and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 101, and specifically binds to a G protein of RSV. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 97 and 101, respectively, and specifically binds to a G protein of RSV.

In some aspects, the disclosed antibodies and antigen binding fragments inhibit disease in a subject with an RSV infection. n. Monoclonal antibody 7G6

In some aspects, the antibody or antigen binding fragment is based on or derived from the 7G6 antibody, and specifically binds to the attachment (G protein) of RSV.

In some examples, the antibody or antigen binding fragment comprises a VH and a VL comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the 7G6 antibody, and specifically binds to a G protein of RSV. In further aspects, the antibody or antigen binding fragment specifically binds RMG-A2 but not RMG-B1, see FIGs. 7A-7B. In further aspects, the antibody or antigen binding fragment specifically binds to the N-terminal region (aa 61-90) of a G protein.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 105, and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 109, and specifically binds to a G protein of RSV. In additional aspects, the antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 105 and 109, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 106, 107, and 108, respectively, and/or a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 110, 111, and 112, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 106, 107, and 108, respectively, a Vrcomprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 110, 111, and 112, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 105, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 105, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 109, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 109, and the antibody or antigen binding fragment specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 105, and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 109, and specifically binds to a G protein of RSV. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 105 and 109, respectively, and specifically binds to a G protein of RS V.

In some aspects, the disclosed antibodies and antigen binding fragments inhibit disease in a subject with an RSV infection. o. Monoclonal antibody 72E6

In some aspects, the antibody or antigen binding fragment is based on or derived from the 72E6 antibody, and specifically binds to the attachment (G protein) of RSV.

In some examples, the antibody or antigen binding fragment comprises a VH and a VL comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the 72E6 antibody, and specifically binds to a G protein of RSV. In further aspects, the antibody or antigen binding fragment specifically binds more strongly to RMG-B1 than to RMG-A2, and targets a discontinuous epitope flanking the CCD motif consisting of the peptides upstream and downstream of the CCD loop.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 113, and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 117, and specifically binds to a G protein of RSV. In additional aspects, the antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 113 and 117, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 114, 115, and 116 (DEY), respectively, and/or a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 118, 119, and 120, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 114, 115, and 116 (DEY), respectively, a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 118, 119, and 120, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 113, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 113, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 117, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 117, and the antibody or antigen binding fragment specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 113, and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 117, and specifically binds to a G protein of RSV. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 113 and 117, respectively, and specifically binds to a G protein of RSV.

In some aspects, the disclosed antibodies and antigen binding fragments inhibit disease in a subject with an RSV infection. p. Monoclonal antibody 23B4

In some aspects, the antibody or antigen binding fragment is based on or derived from the 23B4 antibody, and specifically binds to the attachment (G protein) of RSV.

In some examples, the antibody or antigen binding fragment comprises a VH and a VL comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the 23B4 antibody, and specifically binds to a G protein of RSV. In further aspects, the antibody or antigen binding fragment specifically binds to a G protein of RSV. In further aspects, the antibody or antigen binding fragment specifically binds RMG-A2, and to a lesser degree with RMG- Bl. In more aspects, the monoclonal antibody or antigen binding fragment specifically binds CCD-deleted REG-A2 protein (REG-A2 delCCD) and to the peptide encompassing residues 169-297 (downstream of CCD). In some aspects, the antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 121 , and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 125, and specifically binds to a G protein of RSV. In additional aspects, the antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 121 and 125, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 122, 123, and 124 (EGY), respectively, and/or a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 126, 127, and 128, respectively, and specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 122, 123, and 124 (EGY), respectively, a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 126, 127, and 128, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 121, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 121, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 125, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 125, and the antibody or antigen binding fragment specifically binds to a G protein of RSV.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 121, and specifically binds to a G protein of RSV. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 125, and specifically binds to a G protein of RSV. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 121 and 125, respectively, and specifically binds to a G protein of RSV.

2. Additional Description of Antibodies and Antigen Binding Fragments

The antibody or antigen binding fragment can be a human antibody or fragment thereof. Chimeric antibodies are also provided. The antibody or antigen binding fragment can include any suitable framework region, such as (but not limited to) a human framework region. Human framework regions, and mutations that can be made in human antibody framework regions, are known (see, for example, in U.S. Patent No. 5,585,089). Alternatively, a heterologous framework region, such as, but not limited to a mouse or monkey framework region, can be included in the heavy or light chain of the antibodies. (See, for example, Jones et al.. Nature, 321(6069):522-525, 1986; Riechmann et al., Nature, 332(6162):323-327, 1988; Verhoeyen et al., Science 239(4847): 1534-1536, 1988; Carter et al., Proc. Natl. Acad. Set. U.S.A. 89(10):4285-4289, 1992; Sandhu, Crit. Rev. Biotechnol. l2(5-6)'A3~l -462, 1992; and Singer et al., J. Immunol. 150(7):2844-2857 , 1993.) In some aspects, antigen binding fragments of 68C7, 73C1, 77D2, 40D8, 1D9, 12G11, 36E10, 7H11, 43A11, 48E2, 7H9, 22B11, 75F10, 7G6, 72E6, or 23B4are provided.

The antibody can be of any isotype. The antibody can be, for example, an IgM or an IgG antibody, such as IgGi, IgG:, IgG:, or IgG4- The class of an antibody that specifically binds RSV can be switched with another. In one aspect, a nucleic acid molecule encoding VL or VH is isolated such that it does not include any nucleic acid sequences encoding the constant region of the light or heavy chain, respectively. A nucleic acid molecule encoding VL or VH is then operatively linked to a nucleic acid sequence encoding a CL or CH from a different class of immunoglobulin molecule. This can be achieved, for example, using a vector or nucleic acid molecule that comprises a CL or CH chain. For example, an antibody that specifically binds RSV, that was originally IgG may be class switched to an IgM or IgA. Class switching can be used to convert one IgG subclass to another, such as from IgGi to IgG:. IgG:, or IgG:. In some aspects, the antibody is IgA or IgM. In other aspects the antibody can be IgG:- Class switching can be used to improve valency or tissue distribution.

In some examples, the disclosed antibodies are oligomers of antibodies, such as dimers, trimers, tetramers, pentamers, hexamers, septamers, octomcrs and so on.

The antibody or antigen binding fragment can be derivatized or linked to another molecule (such as another peptide or protein). In general, the antibody or antigen binding fragment is derivatized such that the binding to RSV is not affected adversely by the derivatization or labeling. For example, the antibody or antigen binding fragment can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (for example, a bi-specific antibody or a diabody), a detectable marker, an effector molecule, or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).

(a) Binding affinity

In several aspects, the antibody or antigen binding fragment specifically binds an G protein with an affinity (e.g., measured by KD) of no more than 1.0 x 10⁸ M, no more than 5.0 x 10⁸ M, no more than 1.0 x 10 M, no more than 5.0 x 10⁹ M, no more than 1.0 x 10 ¹⁰M, no more than 5.0 x 10 ¹⁰ M, or no more than 1.0 x 10 ¹¹ M. KD can be measured, for example, by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen. In one assay, solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of (¹²⁵I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293(4): 865-881 , 1999). To establish conditions for the assay, MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 pg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23° C. ). In a non-adsorbent plate (NUNC™ Catalog #269620), 100 pM or 26 pM [^l25I]-antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57(20):4593-4599, 1997). The Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed, and the plate washed eight times with 0.1% polysorbate 20 (TWEE -20®) in PBS. When the plates have dried, 150 pl Ave 11 of scintillant (MICROSCINT™-20; PerkinElmer) is added, and the plates are counted on a TOPCOUNT™ gamma counter (PerkinElmer) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays.

In another assay, KD can be measured using surface plasmon resonance assays using a BIACORE®- 2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C with immobilized antigen CM5 chips at -10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE®, Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N- hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 pg/ml (~0.2 pM) before injection at a flow rate of 5 1/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C at a flow rate of approximately 25 1/min. Association rates (k_on) and dissociation rates (k_off) are calculated using a simple one- to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (KD) is calculated as the ratio koff/kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10⁶ M^-1 s^_| by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette. (b) Multispecific antibodies

In some aspects, the antibody or antigen binding fragment is included on a multispecific antibody, such as a bi-specific antibody. The multispecific antibody can include more than one of the presently disclosed antibodies or antigen binding fragments thereof. In some aspects, these multispecific antibodies include one or more of the following monoclonal antibodies 68C7, 73C1, 77D2, 40D8, 1D9, 12G11, 36E10, 7H11, 43A11, 48E2, 7H9, 22B11, 75F10, 7G6, 72E6, or 23B4.

The multispecific antibody can include two or more different classes of antibodies (G0-G5), see the Examples below and FIGs. 7A-7B for the list of classes. The multispecific antibody can include three or more different classes of antibodies (G0-G5). In some examples, a multispecific antibody can include an antibody in the GO class with a CCD binding antibody. Examples include the combination of antibody 77D2 (GO) with a CCD binding antibody. Thus, the multispecific antibody can include the 77D2 and 40D8, or 77D2 and G56 Mab 7H11.

Such multispecific antibodies can be produced by known methods, such as crosslinking, cloning or displaying two or more antibodies, antigen binding fragments (such as scFvs) of the same type or of different types. Exemplary methods of making multispecific antibodies include those described in PCT Pub. No. WO2013/163427. Suitable crosslinkers include those that are heterobi functi n al, having two distinctly reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (such as disuccinimidyl subcratc). Such linkers arc available from Pierce Chemical Company, Rockford, Ill. In some aspects, the bispecific antibodies include a monoclonal antibody, or antigen binding fragment thereof, that specifically binds a G protein of RSV.

Various types of multi-specific antibodies are known. Bispecific single chain antibodies can be encoded by a single nucleic acid molecule. Examples of bispecific single chain antibodies, as well as methods of constructing such antibodies are known in the art (see, e.g., U.S. Patent Nos. 8,076,459, 8,017,748, 8,007,796, 7,919,089, 7,820,166, 7,635,472, 7,575,923, 7,435,549, 7,332,168, 7,323,440, 7,235,641, 7,229,760, 7,112,324, 6,723,538). Additional examples of bispecific single chain antibodies can be found in PCT application No. WO 99/54440; Mack et al., J. Immunol., 158(8):3965-3970, 1997; Mack et al., Proc. Natl. Acad. Sci. U.S.A., 92(15):7021-7025, 1995; Kufer et al.. Cancer Immunol. Immunother. , 45(3-4):193-197, 1997; Loffler et al., Blood, 95(6):2098-2103, 2000; and Briihl et al., J. Immunol., 166(4):2420-2426, 2001. Production of bispecific Fab-scFv (“bibody”) molecules are described, for example, in Schoonjans et al. J. Immunol., 165(12):7050-7057, 2000) and Willems et al. (J. Chromatogr. B Analyt. Technol. Biomed Life Sci. 786(1-2): 161-176, 2003). For bibodies, a scFv molecule can be fused to one of the VL-CL (L) or VH-CH1 chains, e.g., to produce a bibody one scFv is fused to the C-temi of a Fab chain.

In some aspects, a multi-specific antibody, or a bi-specific antibody, such as a dual variable domain antibody (DVD-IG™) is provided that comprises an antibody or antigen binding fragment. The bispccific tetravalent immunoglobulin known as the dual variable domain immunoglobulin or DVD-immunoglobulin molecule is disclosed in Wu et al., MAbs. 2009;1:339-47, doi: 10.4161/mabs.l.4.8755, and Nat Biotechnol. 2007 Nov;25(l l): 1290-7. doi: 10.1038/nbtl345. Epub 2007 Oct 14. A DVD-immunoglobulin molecule includes two heavy chains and two light chains. Unlike IgG, however, both heavy and light chains of a DVD- immunoglobulin molecule contain an additional variable domain (VD) connected via a linker sequence at the N- termini of the VH and VL of an existing monoclonal antibody. Thus, when the heavy and the light chains combine, the resulting DVD-immunoglobulin molecule contains four antigen recognition sites, see Jakob et al., Mabs 5: 358-363, 2013, see Fig. 1 of this reference for schematic and space-filling diagrams. A DVD- immunoglobulin molecule functions to bind two different antigens on each DFab simultaneously.

The outermost or N-terminal variable domain is termed VD1 and the innermost variable domain is termed VD2; the VD2 is proximal to the C-terminal CHI or CL. As disclosed in Jakob et al., supra, DVD- immunoglobulin molecules can be manufactured and purified to homogeneity in large quantities, have pharmacological properties similar to those of a conventional IgGi , and show in vivo efficacy. Any of the disclosed monoclonal antibodies can be included in a DVD-immunoglobulin format.

In other aspects, any suitable method can be used to design and produce a bispecific antibody (or a multispecific antibody), such as crosslinking two or more antibodies, antigen binding fragments (such as scFvs) of the same type or of different types. Exemplary methods of making multispecific antibodies, such as bispecific antibodies, include those described in PCT Pub. No. WO2013/163427. Non-limiting examples of suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (such as disuccinimidyl suberate).

Bispecific single chain antibodies can be encoded by a single nucleic acid molecule. Non-limiting examples of bispecific single chain antibodies, as well as methods of constructing such antibodies are provided in U.S. Pat. Nos. 8,076,459, 8,017,748, 8,007,796, 7,919,089, 7,820,166, 7,635,472, 7,575,923, 7,435,549, 7,332,168, 7,323,440, 7,235,641 , 7,229,760, 7,1 12,324, 6,723,538. Additional examples of bispecific single chain antibodies can be found in PCT application No. WO 99/54440; Mack et al., J. Immunol., 158(8):3965- 3970, 1997; Mack et al., Proc. Natl. Acad. Sci. U.S.A., 92(15):7021-7025, 1995; Kufer et al., Cancer Immunol. Immunother., 45(3-4): 193-197, 1997; Loffler et al., Blood, 95(6):2098-2103, 2000; and Briihl et al., J. Immunol., 166(4):2420-2426, 2001. Production of bispecific Fab-scFv (“bibody”) molecules are described, for example, in Schoonjans et al. (J. Immunol., 165(12):7050-7057, 2000) and Willems et al. (J. Chromatogr. B Analyt. Technol. Biomed Life Sci. 786(1 -2): 161 -176, 2003). For bibodies, a scFv molecule can be fused to one of the VL-CL (L) or VH-CH1 chains, e.g., to produce a bibody one scFv is fused to the C-term of a Fab chain.

(c) Fragments

Antigen binding fragments are encompassed by the present disclosure, such as Fab, F(ab')2 , and Fv which include a heavy chain and Vi, and specifically bind a G protein of RSV. These antibody fragments retain the ability to selectively bind with the antigen and are “antigen-binding” fragments. Non-limiting examples of such fragments include:

(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;’(2) Fab’, the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain;

(3) (Fab' , the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; Ffab' is a dimer of ’two Fab' fragments held together by two disulfide bonds;

(4) Fv, a genetically engineered fragment containing the VH and VL expressed as two chains; and

(5) Single chain antibody (such as scFv), defined as a genetically engineered molecule containing the VH and the VL linked by a suitable polypeptide linker as a genetically fused single chain molecule (see, e.g., Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155/2012/980250; Marbry and Snavely, IDnigs, 13(8):543- 549, 2010). The intramolecular orientation of the VH-domain and the V,. -domain in a scFv, is not decisive for the provided antibodies (e.g., for the provided multispccific antibodies). Thus, scFvs with both possible arrangements (VH-domain-linker domain-VL-domain; VL-domain-linker domain- VH-domain) may be used.

(6) A dimer of a single chain antibody (scFVi), defined as a dimer of a scFV. This has also been termed a “miniantibody.”

Methods of making these fragments are known (see for example, Harlow and Lane, Antibodies: A Laboratory Manual, 2^nd, Cold Spring Harbor Laboratory, New York, 2013).

Antigen binding fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in a host cell (such as an E. coli cell) of DNA encoding the fragment. Antigen binding fragments can also be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antigen binding fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce monovalent fragments.

Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent lightheavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

(d) Variants

In some aspects, amino acid sequence variants of the antibodies provided herein are provided. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.

In some aspects, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the CDRs and the framework regions. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

The variants typically retain amino acid residues necessary for correct folding and stabilizing between the VH and the VL regions, and will retain the charge characteristics of the residues in order to preserve the low pl and low toxicity of the molecules. Amino acid substitutions can be made in the VH and the VL regions to increase yield.

In some aspects, the heavy chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of sEQ ID NOs : 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, and 121. In some aspects, the light chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of sEQ ID NOs: 5, 9, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, and 125.

In some aspects, the antibody or antigen binding fragment can include up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) in the framework regions of the heavy chain of the antibody, or the light chain of the antibody, or the heavy and light chains of the antibody, compared to a known framework region, or compared to a known framework region, or compared to the framework regions of the antibody, and maintain the specific binding activity for RSV.

In some aspects, substitutions, insertions, or deletions may occur' within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in CDRs. In some aspects of the variant VH and VL sequences provided above, each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.

To increase binding affinity of the antibody, the VL and VH segments can be randomly mutated, such as within HCDR3 region or the LCDR3 region, in a process analogous to the in vivo somatic mutation process responsible for affinity maturation of antibodies during a natural immune response. Thus in vitro affinity maturation can be accomplished by amplifying VH and VL regions using PCR primers complementary to the HCDR3 or LCDR3, respectively. In this process, the primers have been “spiked” with a random mixture of the four nucleotide bases at certain positions such that the resultant PCR products encode Vnand VL segments into which random mutations have been introduced into the VH and/or VL CDR3 regions. These randomly mutated VH and VL segments can be tested to determine the binding affinity for RSV. In particular examples, the VH amino acid sequence is one of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, and 121. In other examples, the VL amino acid sequence is one of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, and 125. Methods of in vitro affinity maturation are known (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001).)

In some aspects, an antibody or antigen binding fragment, as disclosed herein, is altered to increase or decrease the extent to which the antibody or antigen binding fragment is glycosylated. Addition or deletion of glycosylation sites may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. Trends Biotechnol. 15(l):26-32, 1997. The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some aspects, modifications of the oligosaccharide in an antibody may be made in order to create antibody variants with certain improved properties.

In one aspect, antibody variants are provided having a carbohydrate structure with a modified amount of fucose attached (directly or indirectly) to an Fc region. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region; however, Asn297 may also be located about ±3 amino acids upstream or downstr eam of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. In some aspects, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. Antibody variants also are provided that lack fucose attached to the Fc region. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.J; US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; W02005/053742; WO 2002/031140; Okazaki et al., J. Mol. Biol., 336(5): 1239-1249, 2004; Yamane-Ohnuki et al., Biotechnol. Bioeng. 87(5):614-622, 2004. Examples of cell lines capable of producing defucosylated antibodies include Lee 13 CHO cells deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249(2):533-545, 1986; US Pat. Appl. No. US 2003/0157108 and WO 2004/056312, especially at Example 11), and knockout cell lines, such as alpha- 1 ,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotechnol. Bioeng., 87(5): 614-622, 2004; Kanda et al., Biotechnol. Bioeng., 94(4):680-688, 2006; and W02003/085107).

Antibodies variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al.) U.S. Pat. No. 6,602,684 (Umana et al.) and US 2005/0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087; WO 1998/58964; and WO 1999/22764.

In several aspects, the constant region of the antibody includes one or more amino acid substitutions to optimize in vivo half-life of the antibody. The serum half-life of IgG Abs is regulated by the neonatal Fc receptor (FcRn). Thus, in several aspects, the antibody comprises an amino acid substitution that increases binding to the FcRn. Several such substitutions are known, such as substitutions at IgG constant regions T250Q and M428L (see, e.g., Hinton et al., J Immunol., 176(l):346-356, 2006); M428L and N434S (the “LS” mutation, see, e.g., Zalevsky. et al.. Nature Biotechnol., 28(2): 157-159, 2010); N434A (see, e.g., Petkova et al., Int. Immunol., 18( 12) : 1759- 1769, 2006); T307A, E38OA, and N434A (see, e.g., Petkova et al., Int. Immunol., 18(12): 1759-1769, 2006); and M252Y, S254T, and T256E (see, e.g., Dall’Acqua et al., J. Biol. Chem., 281(33):23514-23524, 2006). The disclosed antibodies and antigen binding fragments can be linked to or comprise a Fc polypeptide including any of the substitutions listed above, for example, the Fc polypeptide can include the M428L and N434S substitutions. In some aspects, the M428L and N434S (“LS”, See Zalevsky, J. et al., Nat Biotechnol. 28, 157-159, doi: 10.1038/nbt,1601 (2010)) or M252Y/S254T/T256E (“YTE,” see Dall’Acqua, W. F. et al., J Immunol 169, 5171-5180) mutations are used, for example, to extend to increase antibody stability and half-life.

In some aspects, the constant region of the antibody comprises one or more amino acid substitutions to optimize ADCC. ADCC is mediated primarily through a set of closely related Fey receptors. In some aspects, the antibody comprises one or more amino acid substitutions that increase binding to FcyRIIIa. Several such substitutions arc known, such as substitutions at IgG constant regions S239D and I332E (sec, e.g., Lazar et al., Proc. Natl., Acad. Sci. U.S.A., 103(11):4005-4010, 2006); and S239D, A330L, and I332E (see, e.g., Lazar et al., Proc. Natl., Acad. Sci. U.S.A., 103(11):4005-4010, 2006).

Combinations of the above substitutions are also included, to generate an IgG constant region with increased binding to FcRn and FcyRIIIa. The combinations increase antibody half-life and ADCC. For example, such combinations include antibodies with the following amino acid substitutions in the Fc region: (1) S239D/I332E and T250Q/M428L; (2) S239D/I332E and M428L/N434S; (3) S239D/I332E and N434A; (4) S239D/I332E and T307A/E380A/N434A; (5) S239D/I332E and M252Y/S254T/T256E; (6) S239D/A330L/I332E and 250Q/M428L; (7) S239D/A330L/I332E and M428L/N434S; (8) S239D/A330L/I332E and N434A; (9) S239D/A330L/I332E and T307A/E380A/N434A; or (10) S239D/A330L/I332E and M252Y/S254T/T256E.

In some aspects, mutations are introduced (e.g., LAL A or GRLR) that reduce antibody binding to FcyRs. A “LALA” mutation substitutes the leucine residues at position 1.3 and 1.2 of the CH2 domain (based on the IMGT numbering for C-domains) with alanine residues (L234A and L235A), which abrogates antibody binding to FcyRI, FcyRII and FcyRIIIa (Hessell, A. J. et al., Nature 449, 101-104, doi:10.1038/nature06106 (2007); Beltramello, M. et al., Cell Host Microbe 8, 271-283, doi:10.1016/j.chom.2010.08.007 (2010)). Similarly, a “GRLR” mutation can be utilized that substitutes the glycine at position 236 and leucine at 328 with arginines (G236R/L328R) to abolish antibody binding to all FcyRs (Horton, H. M. et al., Blood 116, 3004- 3012, doi:10.1182/blood-2010-01-265280 (2010); Horton, H. M. et al., Cancer Res 68, 8049-8057, doi:10.1158/0008-5472.CAN-08-2268 (2008)).

LALA mutations can abrogate some Fc function. These and other modifications can be included to produce an antibody with desired properties or functions for better protection from RSV disease.

In some aspects, an antibody provided herein may be further modified to contain additional nonproteinaceous moieties. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-l,3-dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, poly aminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in an application under defined conditions, etc.

B. Conjugates

The antibodies and antigen binding fragments that specifically bind to a G protein of RSV provided herein can be conjugated to an agent, such as an effector molecule or detectable marker. Both covalent and noncovalent attachment means may be used. Various effector molecules and detectable markers can be used, including (but not limited to) toxins and radioactive agents such as ¹²⁵I, ³²P, ^l4C, ³H and ³⁵S and other labels, target moieties and ligands, etc. The choice of a particular effector molecule or detectable marker depends on the particular target molecule or cell, and the desired biological effect.

The procedure for attaching an effector molecule or detectable marker to an antibody or antigen binding fragment varies according to the chemical structure of the effector. Polypeptides typically contain a variety of functional groups, such as car boxyl (-COOH), free amine (-NH2) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on a polypeptide to result in the binding of the effector molecule or detectable marker. Alternatively, the antibody or antigen binding fragment is derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of known linker molecules, such as those available from Thermo Fisher Scientific, Waltham, MA and MilliporcSigma Corporation, St. Louis, MO. The linker is capable of forming covalent bonds to both the antibody or antigen binding fragment and to the effector molecule or detectable marker. Suitable linkers include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody or antigen binding fragment and the effector molecule or detectable marker are polypeptides, the linkers may be joined to the constituent amino acids through their side chains (such as through a disulfide linkage to cysteine) or the alpha carbon, or through the amino, and/or carboxyl groups of the terminal amino acids.

In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules), toxins, and other agents to antibodies, a suitable method for attaching a given agent to an antibody or antigen binding fragment or other polypeptide can be determined.

In some aspects, the antibody or antigen binding fragment can be conjugated with effector molecules such as small molecular weight drags such as Monomethyl Auristatin E (MMAE), Monomethyl Auristatin F (MMAF), maytansine, maytansine derivatives, including the derivative of maytansine known as DM1 (also known as mertansine), or other agents to make an antibody drag conjugate (ADC). In several aspects, conjugates of an antibody or antigen binding fragment and one or more small molecule toxins, such as a calicheamicin, maytansinoids, dolastatins, auristatins, a trichothecene, and CC1065, and the derivatives of these toxins that have toxin activity, are provided. In some embodiments, the antibody or antigen binding fragment can be conjugated to a toxin.

Toxins can be employed with the disclosed antibodies and antigen binding fragments. Exemplary toxins include Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin and subunits thereof, ribotoxin, ribonuclease, saporin, and calicheaniicin, as well as botulinum toxins A through F. These toxins are readily available from commercial sources (for example, Sigma Chemical Company, St. Louis, MO). Contemplated toxins also include variants of the toxins (see, for example, see, U.S. Patent Nos. 5,079,163 and 4,689,401).

Saporin is a toxin derived from Saponaria officinalis that disrupts protein synthesis by inactivating the 60S portion of the ribosomal complex (Stirpe et al., Bio/Technology, 10:405-412, 1992). However, the toxin has no mechanism for specific entry into cells, and therefore requires conjugation to an antibody or antigen binding fragment that recognizes a cell-surface protein that is internalized in order to be efficiently taken up by cells.

Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxin for use in immunotoxins is mutated to reduce or to eliminate non-specific toxicity. A mutant known as CRM107, which has full enzymatic activity but markedly reduced non-specific toxicity, has been known since the 1970's (Laird and Groman, J. Virol. 19:220, 1976), and has been used in human clinical trials. See, U.S. Patent No. 5,792,458 and U.S. Patent No. 5,208,021.

Ricin is the lectin RCA60 from Ricinus communis (Castor bean). For examples of ricin, see, U.S. Patent No. 5,079,163 and U.S. Patent No. 4,689,401. Ricinus communis agglutinin (RCA) occurs in two forms designated RCAso and RCA120 according to their molecular weights of approximately 65 and 120 kD, respectively (Nicholson & Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). The A chain is responsible for inactivating protein synthesis and killing cells. The B chain binds ricin to cell-surface galactose residues and facilitates transport of the A chain into the cytosol (Olsnes et al., Nature 249:627-631, 1974 and U.S. Patent No. 3,060.165).

Ribonucleases have also been conjugated to targeting molecules for use as immunotoxins (see Suzuki et al., Nat. Biotech. 17:265-70, 1999). Exemplary ribotoxins such as a-sarcin and restrictocin are discussed in, for example Rathore et al., Gene 190:31-5, 1997; and Goyal and Batra, Biochem. 345 Pt 2:247-54, 2000. Calicheamicins were first isolated from Micromonospora echinospora and are members of the enediyne antitumor antibiotic family that cause double strand breaks in DNA that lead to apoptosis (see, for example Lee et al., J. Antibiot. 42:1070-87,1989). The drug is the toxic moiety of an immunotoxin in clinical trials (see, for example, Gillespie et al., Ann. Oncol. 11:735-41, 2000).

Abrin includes toxic lectins from Abrus precatorius. The toxic principles, abrin a, b, c, and d, have a molecular weight of from about 63 and 67 kD and ar e composed of two disulfide-linked polypeptide chains A and B. The A chain inhibits protein synthesis; the B chain (abrin-b) binds to D-galactose residues (see, Funatsu et al., Agr. Biol. Chem. 52:1095, 1988; and Olsnes, Methods Enzymol. 50:330-335, 1978). In one embodiment, the toxin is Pseudomonas exotoxin (PE) (U.S. Patent No. 5,602,095). As used herein, PE includes full-length native (naturally occurring) PE or a PE that has been modified. Such modifications can include, but are not limited to, elimination of domain la, various amino acid deletions in domains lb, II and III, single amino acid substitutions and the addition of one or more sequences at the carboxyl terminus (for example, see Siegall et al., J. Biol. Chem. 264:14256-14261, 1989). PE employed with the provided antibodies can include the native sequence, cytotoxic fragments of the native sequence, and conservatively modified variants of native PE and its cytotoxic fragments. Cytotoxic fragments of PE include those which are cytotoxic with or without subsequent proteolytic or other processing in the target cell. Cytotoxic fragments of PE include PE25, PE40, PE38, and PE35. For additional description of PE and variants thereof, see for example, U.S. Patent Nos. 4,892,827; 5,512,658; 5,602,095; 5,608,039; 5,821,238; and 5,854,044; PCT Publication No. WO 99/51643; Pai et al., Proc. Natl. Acad. Sci. USA, 88:3358-3362, 1991; Kondo et al., J. Biol. Chem., 263:9470-9475, 1988; Pastan et al., Biochim. Biophys. Acta, 1333:C1-C6, 1997.

Also contemplated herein are protease-resistant PE variants and PE variants with reduced immunogenicity, such as, but not limited to PE-LR, PE-6X, PE-8X, PE-LR/6X and PE-LR/8X (see, for example, Weldon et al., Blood 113(16):3792-3800, 2009; Onda et aL, Proc. Natl. Acad. Sci. USA, 105(32):11311-11316, 2008; and PCT Publication Nos. WO 2007/016150, WO 2009/032954 and WO 2011/032022, which are herein incorporated by reference). The PE variant can be PE25, see Weldon et al., Blood 2009; 113:3792-3800, herein incorporated by reference.

In some examples, the PE is a variant that is resistant to lysosomal degradation, such as PE-LR (Weldon et al., Blood 113(16):3792-3800, 2009; PCT Publication No. WO 2009/032954). In other examples, the PE is a variant designated PE-LR/6X (PCT Publication No. WO 2011/032022). In other examples, the PE is a variant designated PE-LR/8M (PCT Publication No. WO 2011/032022).

The antibody or antigen binding fragment can be conjugated with a detectable marker; for example, a detectable marker capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as CT, computed axial tomography (CAT), MRI, magnetic resonance tomography (MTR), ultrasound, fiberoptic examination, and laparoscopic examination). Specific, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like. Bioluminescent markers arc also of use, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP). An antibody or antigen binding fragment can also be conjugated with enzymes that are useful for detection, such as horseradish peroxidase, - galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. When an antibody or antigen binding fragment is conjugated with a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is visually detectable. An antibody or antigen binding fragment may also be conjugated with biotin, and detected through indirect measurement of avidin or streptavidin binding. It should be noted that the avidin itself can be conjugated with an enzyme or a fluorescent label.

The antibody or antigen binding fragment can be conjugated with a paramagnetic agent, such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide are also of use as labels. Antibodies can also be conjugated with lanthanides (such as europium and dysprosium), and manganese. An antibody or antigen binding fragment may also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (such as leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).

The antibody or antigen binding fragment can also be conjugated with a radiolabeled amino acid, for example, for diagnostic purposes. For instance, the radiolabel may be used to detect expressing cells expressing an RSV protein by radiography, emission spectra, or other diagnostic techniques. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes: ’H, ¹⁴C, ³⁵S, ⁹⁰Y, "^mTc, ¹¹ 'In, ¹²⁵I, ¹³¹I. The radiolabels may be detected, for example, using photographic film (or digital equivalent) or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted illumination. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels arc detected by simply visualizing the colored label.

The average number of effector molecule or detectable marker moieties per antibody or antigen binding fragment in a conjugate can range, for example, from 1 to 20 moieties per antibody or antigen binding fragment. In some aspects, the average number of effector molecules or detectable marker moieties per antibody or antigen binding fragment in a conjugate range from about 1 to about 2, from about 1 to about 3, about 1 to about 8; from about 2 to about 6; from about 3 to about 5; or from about 3 to about 4. The loading (for example, effector molecule per antibody ratio) of a conjugate may be controlled in different ways, for example, by: (i) limiting the molar excess of effector molecule-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limiting reducing conditions for cysteine thiol modification, (iv) engineering by recombinant techniques the amino acid sequence of the antibody such that the number and position of cysteine residues is modified for control of the number or position of linker-effector molecule attachments.

C. Polynucleotides and Expression

Nucleic acid molecules (for example, cDNA or RNA molecules) encoding the amino acid sequences of antibodies, antigen binding fragments, and conjugates that specifically bind to a G protein of RSV are provided. In some aspects, the antibody is 68C7, 73C1, 77D2, 40D8, 1D9, 12G11, 36E10, 7H11, 43A11, 48E2, 7H9, 22B11, 75F10, 7G6, 72E6, or 23B4.

Nucleic acids encoding these molecules can readily be produced using the amino acid sequences provided herein (such as the CDR sequences and VH and VL sequences), sequences available in the art (such as framework or constant region sequences), and the genetic code. In several aspects, a nucleic acid molecule can encode the VH, the VL, or both the VH and VL (for example in a bicistronic expression vector) of a disclosed antibody or antigen binding fragment. In several aspects, the nucleic acid molecules can be expressed in a host cell (such as a mammalian cell) to produce a disclosed antibody or antigen binding fragment. The genetic code can be used to construct a variety of functionally equivalent nucleic acid sequences, such as nucleic acids which differ in sequence but which encode the same antibody sequence, or encode a conjugate or fusion protein including the VL and/or Vn nucleic acid sequence. Exemplary nucleic acid molecules are shown in Table 2 below. Table 2: Exemplary Nucleotide Sequences

Nucleic acid molecules encoding the antibodies, antigen binding fragments, and conjugates that specifically bind to RSV can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by standard methods. Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.

Exemplary nucleic acids can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques can be found, for example, in Green and Sambrook Molecular Cloning: A Laboratory Manual, 4^th ed., New York: Cold Spring Harbor Laboratory Press, 2012) and Ausubel et al. (Eds.) (Current Protocols in Molecular Biology, New York: John Wiley and Sons, including supplements, 2017).

Nucleic acids can also be prepared by amplification methods. Amplification methods include the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), and the self-sustained sequence replication system (3SR). The nucleic acid molecules can be expressed in a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells. The antibodies, antigen binding fragments, and conjugates can be expressed as individual proteins including the VH and/or VL (linked to an effector molecule or detectable marker as needed), or can be expressed as a fusion protein. Methods of expressing and purifying antibodies and antigen binding fragments are known and further described herein (see, e.g., Al-Rubeai (Ed.), Antibody Expression and Production, Dordrecht; New York: Springer, 2011). An immunoadhesin can also be expressed. Thus, in some examples, nucleic acids encoding a VH and VL, and immunoadhesin are provided. The nucleic acid sequences can optionally encode a leader sequence.

To create a scFv the VH- and VL-encoding DNA fragments can be operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)a, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH domains joined by the flexible linker (see, e.g., Bird et al., Science. 242(4877):423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85(16):5879-5883, 1988; McCafferty et al., Nature, 348:552-554, 1990; Kontermann and Diibel (Eds.), Antibody Engineering, Vols. 1-2, 2^nd ed., Springer-Verlag, 2010; Greenfield (Ed.), Antibodies: A Laboratory Manual, 2^nd ed. New York: Cold Spring Harbor Laboratory Press, 2014). Optionally, a cleavage site can be included in a linker, such as a furin cleavage site.

The single chain antibody may be monovalent, if only a single VH and VL are used, bivalent, if two VH and VL are used, or polyvalent, if more than two VH and VL are used. Bispecific or polyvalent antibodies may be generated that bind specifically a G protein of RSV and another antigen. The encoded VH and VL optionally can include a furin cleavage site between the VH and VL domains. These single chain antibodies can be encoded by DNA molecules. These single chain antibodies can be encoded by RNA molecules.

One or more DNA sequences encoding the antibodies, antigen binding fragments, or conjugates can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. Numerous expression systems available for expression of proteins including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines, can be used to express the disclosed antibodies and antigen binding fragments. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art. Hybridomas expressing the antibodies of interest are also encompassed by this disclosure.

The expression of nucleic acids encoding the antibodies and antigen binding fragments described herein can be achieved by operably linking the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression cassette. The promoter can be any promoter of interest, including a cytomegalovirus promoter. Optionally, an enhancer, such as a cytomegalovirus enhancer, is included in the construct. The cassettes can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression cassettes contain specific sequences useful for regulation of the expression of the DNA encoding the protein. For example, the expression cassettes can include appropriate promoters, enhancers, transcription and translation terminators, initiation sequences, a start codon (z.e., ATG) in front of a protein-encoding gene, splicing signals for introns, sequences for the maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The vector can encode a selectable marker, such as a marker encoding drug resistance (for example, ampicillin or tetracycline resistance).

To obtain high level expression of a cloned gene, it is desirable to construct expression cassettes which contain, for example, a strong promoter to direct transcription, a ribosome binding site for translational initiation (e.g., internal ribosomal binding sequences), and a transcription/translation terminator. For E. coli, this can include a promoter such as the T7, trp, lac, or lambda promoters, a ribosome binding site, and preferably a tanscription termination signal. For eukaryotic cells, the control sequences can include a promoter and/or an enhancer derived from, for example, an immunoglobulin gene, HTLV, SV40 or cytomegalovirus, and a polyadenylation sequence, and can further include splice donor and/or acceptor sequences (for example, CMV and/or HTLV splice acceptor and donor sequences). The cassettes can be transferred into the chosen host cell by well-known methods such as transformation or electroporation for E. coli and calcium phosphate treatment, electroporation or lipofection for mammalian cells. Cells transformed by the cassettes can be selected by resistance to antibiotics conferred by genes contained in the cassettes, such as the amp, gpt, neo and hyg genes.

Modifications can be made to a nucleic acid encoding a polypeptide described herein without diminishing its biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications include, for example, termination codons, sequences to create conveniently located restriction sites, and sequences to add a methionine at the amino terminus to provide an initiation site, or additional amino acids (such as poly His) to aid in purification steps.

Once expressed, the antibodies, antigen binding fragments, and conjugates can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, Simpson et al. (Eds.), Basic methods in Protein Purification and Analysis: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2009). The antibodies, antigen binding fragment, and conjugates need not be 100% pure. Once purified, partially or to homogeneity as desired, if to be used prophylatically, the polypeptides should be substantially free of endotoxin.

Methods for expression of antibodies, antigen binding fragments, and conjugates, and/or refolding to an appropriate active form, from mammalian cells, and bacteria such as E. coli have been described and are applicable to the antibodies disclosed herein. See, e.g., Greenfield (Ed.), Antibodies: A Laboratory Manual, 2^nd ed. New York: Cold Spring Harbor Laboratory Press, 2014, Simpson et al. (Eds.), Basic methods in Protein Purification and Analysis: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2009, and Ward et al., Nature 341(6242):544-546, 1989. D. Methods and Compositions

1. Inhibiting Disease Caused by RSV

Methods are disclosed herein for the inhibition of disease caused by RSV in a subject. The methods include administering to the subject an effective amount (that is, an amount effective to inhibit a sign or symptom or disease caused by RSV in the subject) of a disclosed antibody, antigen binding fragment, conjugate, or a nucleic acid encoding such an antibody, antigen binding fragment, or conjugate, to a subject that has disease caused by an RSV infection, or is at risk of getting disease caused by an RSV infection. The methods can be used pre-exposure or post-exposure. The method can include administration of one or more of 68C7, 73C1 , 77D2, 40D8, 1D9, 12G1 1. 36E10, 7H1 1 , 43A1 1 , 48E2, 7H9, 22B1 1 , 75F10, 7G6, 72E6, or 23B4, or an antigen binding fragments thereof, as described herein.

The disease does not need to be completely eliminated or inhibited for the method to be effective. For example, the method can decrease a sign or symptom caused by an RSV infection by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable disease) as compared to the absence of the treatment. In some aspects, the subject can also be treated with an effective amount of an additional agent, such as anti-viral agent. In a non-limiting example, the agent is ribavirin. In another example, the agent is a bronchodilator. In a further example, the agent is 3 ’-fluorouridine. In yet other examples, the agent is Palivizumab. In more aspects the agent can be another drug. In further aspects, the agent can be a toxin.

In some aspects, the methods include administering to the subject an effective amount (that is, an amount effective to inhibit disease caused by RSV in the subject) of a disclosed antibody, antigen binding fragment, conjugate, or a nucleic acid encoding such an antibody, antigen binding fragment, or conjugate, to a subject (such as a mammal). The subject can be a human. The subject can be immunocompromised. The subject can be a child or an infant less than one year of age. In some examples the subject is an adult. In some examples the subject is at least 50, at least 55, at least 60 or at least 65 years old. One exemplary route of administration is injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, or intravenous).

In some aspects, administration of an effective amount of a disclosed antibody, antigen binding fragment, conjugate, or nucleic acid molecule, inhibits disease progression in a subject, which can encompass any statistically significant reduction in RSV activity (for example, replication or viral load) or reduction of symptoms of RSV infection in the subject. In some aspects, these methods include the use of one or more of the following monoclonal antibodies 68C7, 73C1, 77D2, 40D8, 1D9, 12G11, 36E10, 7H11, 43A11, 48E2, 7H9, 22B11, 75F10, 7G6, 72E6, or 23B4. An antigen binding fragment of these monoclonal antibodies, conjugate, or nucleic acid molecule encoding the antibody or antigen binding fragment are also of use. The method can include administration of antibodies of different classes of antibodies (G0-G5), see the Examples below and FIGs. 7A-7B for the list of classes. The method can include administration of multispecific antibody, including antibodies of two or more different classes of antibodies (G0-G5), see the Examples below and FIGs. 7A-7B for the list of classes. The method can be protective.

Antibodies and antigen binding fragments thereof are typically administered by intravenous infusion. Doses of the antibody or antigen binding fragment can vary, but generally range between about 0.5 mg/kg to about 50 mg/kg, such as a dose of about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kg. In some aspects, the dose of the antibody or antigen binding fragment can be from about 0.5 mg/kg to about 5 mg/kg, such as a dose of about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg or about 5 mg/kg. The antibody or antigen binding fragment is administered according to a dosing schedule determined by a medical practitioner. In some examples, the antibody or antigen binding fragment is administered weekly, every two weeks, every three weeks, every four weeks, or every 8 weeks. In some examples, the antibody or antigen binding fragment is administered yearly.

In some aspects, the method further comprises administration of one or more additional agents to the subject. Additional agents of interest include, but are not limited to, anti-viral agents.

In some aspects, the method of inhibiting disease caused by RSV in a subject comprises administration of a first antibody that specifically binds to G protein of RSV, as disclosed herein and a second antibody that that specifically binds to G protein of RSV. In some aspects, the method of inhibiting RSV infection in a subject comprises administration of a first antibody that specifically binds to G protein of RSV as disclosed herein and a second antibody that that specifically binds to G protein of RSV. The method can include the administration of 2, 3, 4, 5, 6, 7, 8, 9, or 10 of these antibodies, antigen binding fragments thereof, mutlispecific antibodies (such as bispecific antibodies), conjugates, or nucleic acid molecules encoding these antibodies, antigen binding fragments, or multispecific antibodies (such as bispecific antibodies), or vectors include these nucleic acid molecules. Classes of antibodies are disclosed in the Examples section, labeled G0-G5. In some aspects, the disclosed method includes administration of an antibody from two different classes. In more aspects, the disclosed method includes administration of an antibody from 3, 4 or 5 different classes.

Exemplary combinations include a Go, Gi and/or G5 monoclonal antibody. A combination can include all of a Go, Gi and G5 antibody. In some aspects, the combination includes 40D8. In other embodiments, the combination includes 7H11. In more embodiments, the combination includes one or more of 40D8, 1D9, 12G11, and 36E10. Additional combination s include i) 77D2, ii) one or more of 40D8, 1D9, 12G11, and 36E10, and iii) 7H11.

In some aspects, a subject is administered DNA or RNA encoding a disclosed antibody to provide in vivo antibody production, for example using the cellular machinery of the subject. Administration of nucleic acid constructs is known in the art and taught, for example, in U.S. Patent No. 5,643,578, U.S. Patent No. 5,593,972 and U.S. Patent No. 5,817,637. U.S. Patent No. 5,880,103 describes several methods of delivery of nucleic acids encoding proteins to an organism. One approach to administration of nucleic acids is direct administration with plasmid DNA, such as with a mammalian expression plasmid. The nucleotide sequence encoding the disclosed antibody, or antigen binding fragments thereof, can be placed under the control of a promoter to increase expression. The methods include liposomal delivery of nucleic acids. Such methods can be applied to the production of an antibody, or antigen binding fragments thereof. In some aspects, a disclosed antibody or antigen binding fragment is expressed in a subject using the pVRC8400 vector (described in Barouch et al., J. Virol., 79(14), 8828-8834, 2005).

In several aspects, a subject (such as a human subject at risk of RSV infection) can be administered an effective amount of an AAV viral vector that includes one or more nucleic acid molecules encoding a disclosed antibody or antigen binding fragment. The AAV viral vector is designed for expression of the nucleic acid molecules encoding a disclosed antibody or antigen binding fragment, and administration of the effective amount of the AAV viral vector to the subject leads to expression of an effective amount of the antibody or antigen binding fragment in the subject. Non-limiting examples of AAV viral vectors that can be used to express a disclosed antibody or antigen binding fragment in a subject include those provided in Johnson et al., Nat. Med., 15(8):901 -906, 2009 and Gardner et al., Nature, 519(7541): 87-91 , 2015.

In one aspect, a nucleic acid encoding a disclosed antibody, or antigen binding fragment thereof, is introduced directly into tissue. For example, the nucleic acid can be loaded onto gold microspheres by standard methods and introduced into the skin by a device such as Bio-Rad’s HELIOS™ Gene Gun. The nucleic acids can be “naked,” consisting of plasmids under control of a strong promoter.

Typically, the DNA is injected into muscle, although it can also be injected directly into other sites. Dosages for injection are usually around 0.5 |ig/kg to about 50 mg/kg, and typically are about 0.005 mg/kg to about 5 mg/kg (see, e.g., U.S. Patent No. 5,589,466).

Single or multiple administrations of a composition including a disclosed RSV-specific antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules, can be administered depending on the dosage and frequency as required and tolerated by the patient. The dosage can be administered once, but may be applied periodically until cither a desired result is achieved or until side effects warrant discontinuation of therapy. Generally, the dose is sufficient to inhibit the RSV infection without producing unacceptable toxicity to the patient.

Data obtained from cell culture assays and animal studies can be used to formulate a range of dosage for use in humans. The dosage normally lies within a range of circulating concentrations that include the ED50, with little or minimal toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The effective dose can be determined from cell culture assays and animal studies. The G protein-specific antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules, or a composition including such molecules, can be administered to subjects in various ways, including local and systemic administration, such as, e.g., by injection subcutaneously, intravenously, intra-arterially, intraperitoneally, intramuscularly, intradermally, or intrathecally. In an aspect, the antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules, or a composition including such molecules, is administered by a single subcutaneous, intravenous, intra-arterial, intraperitoneal, intramuscular, intradermal or intrathecal injection once a day. The antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules, or a composition including such molecules, can also be administered by direct injection at or near the site of disease. A further method of administration is by osmotic pump (e.g., an Alzet pump) or mini-pump (e.g., an Alzet mini-osmotic pump), which allows for controlled, continuous and/or slow-release delivery of the antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules, or a composition including such molecules, over a pre -determined period. The osmotic pump or mini-pump can be implanted subcutaneously, or near a target site.

2. Compositions

Compositions are provided that include one or more of the G protein-specific antibody, antigen binding fragment, multispccific antibody conjugate, or nucleic acid molecule encoding such molecules, that arc disclosed herein in a earner. In some aspects, the composition includes one or more of 68C7, 73C1, 77D2, 40D8, 1D9, 12G11, 36E10, 7H11, 43A11, 48E2, 7H9, 22B11, 75F10, 7G6, 72E6, or 23B4. The composition can include at least 2, at least 3, at least 4, at least 5 or at least 10 of these antibodies, such as 2, 3, 4 or 5 of these antibodies, or one or more multispecific antibodies.

Exemplary combinations include a Go, Gi and/or Gs monoclonal antibody. A combination can include all of a Go, Gi and Gs antibody. In some aspects, the combination includes 40D8. In other embodiments, the combination includes 7H11. In more embodiments, the combination includes one or more of 40D8, 1D9, 12G11, and 36E10. Additional combination s include: i) 77D2, ii) one or more of 40D8, 1D9, 12G11, and 36E10, and iii) 7H11.

The compositions are useful, for example, for the inhibition of disease induced by RSV, or detection of an RSV infection. The compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration are at the discretion of the administering physician to achieve the desired purposes. The G protein-specific antibody, antigen binding fragment, conjugate, multispecific antibody, or nucleic acid molecule encoding such molecules can be formulated for systemic or local administration. In one example, the G protein-specific antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules, is formulated for parenteral administration, such as intravenous or intramuscular' administration.

In some aspects, the antibody, antigen binding fragment, multispecific antibody, conjugate thereof, or nucleic acid molecule, in the composition is at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) pure. In some aspects, the composition contains less than 10% (such as less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or even less) of macromolecular contaminants, such as other mammalian (e.g., human) proteins.

The compositions for administration can include a solution of the G protein-specific antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules, dissolved in a pharmaceutically acceptable earner, such as an aqueous carrier. A variety of aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of antibody in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject’s needs.

A typical composition for intravenous administration includes about 0.01 to about 30 mg/kg of antibody or antigen binding fragment or conjugate per subject per day (or the corresponding dose of a conjugate including the antibody or antigen binding fragment). Actual methods for preparing administrable compositions are known and are described in more detail in such publications as Remington: The Science and Practice of Pharmacy, 22^nd ed., London, UK: Pharmaceutical Press, 2013. In some aspects, the composition can be a liquid formulation including one or more antibodies, antigen binding fragments (such as an antibody or antigen binding fragment that specifically binds to RSV), in a concentration range from about 0.1 mg/ml to about 20 mg/ml, or from about 0.5 mg/ml to about 20 mg/ml, or from about 1 mg/ml to about 20 mg/ml, or from about 0.1 mg/ml to about 10 mg/ml, or from about 0.5 mg/ml to about 10 mg/ml, or from about 1 mg/ml to about 10 mg/ml.

Antibodies, or an antigen binding fragment thereof, multispecific antibody, conjugate, or a nucleic acid encoding such molecules, can be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration. The antibody solution, an antigen binding fragment, multispecific antibody or a nucleic acid encoding such antibodies, antigen binding fragments, or multispeciifc antibody, can then be added to an infusion bag containing 0.9% sodium chloride, USP, and typically administered at a dosage of from 0.5 to 15 mg/kg of body weight. Considerable experience is available in the art in the administration of antibody drugs, which have been marketed in the U.S. since the approval of Rituximab in 1997. Antibodies, antigen binding fragments, conjugates, or a nucleic acid encoding such molecules, can be administered by slow infusion, rather than in an intravenous push or bolus. In one example, a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level. For example, an initial loading dose of 4 mg/kg may be infused over a period of some 90 minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kg infused over a 30-minute period if the previous dose was well tolerated.

Controlled-release parenteral formulations can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems see, Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Lancaster, PA: Technomic Publishing Company, Inc., 1995. Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the active protein agent, such as a cytotoxin or a drug, as a central core. In microspheres, the active protein agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 pm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 pm so that only nanoparticles arc administered intravenously. Microparticles are typically around 100 pm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, Colloidal Drug Delivery Systems, J. Kreuter (Ed.), New York, NY: Marcel Dekker, Inc., pp. 219-342, 1994; and Tice and Tabibi, Treatise on Controlled Drug Delivery: Fundamentals, Optimization, Applications, A. Kydonieus (Ed.), New York, NY: Marcel Dekker, Inc., pp. 315-339, 1992.

Polymers can be used for ion-controlled release of the antibody compositions disclosed herein. Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known in the art (Langer, Acc. Chem. Res. 26(10):537-542, 1993). For example, the block copolymer, polaxamer 407, exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has been shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. , 9(3):425-434, 1992; and Pec et al., J. Parent. Sci. Tech. , 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm. 112(3):215-224, 1994). In yet another aspect, liposomes are used for controlled release as well as drag targeting of the lipid-capsulated drag (Betageri et al., Liposome Drug Delivery Systems, Lancaster, PA: Technomic Publishing Co., Inc., 1993). Numerous additional systems for controlled delivery of active protein agent are known (see U.S. Patent No. 5,055,303; U.S. Patent No. 5,188,837; U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; U.S. Patent No. 4,957,735; U.S. Patent No. 5,019,369; U.S.

Patent No. 5,055,303; U.S. Patent No. 5,514,670; U.S. Patent No. 5,413,797; U.S. Patent No. 5,268,164; U.S.

Patent No. 5,004,697; U.S. Patent No. 4,902,505; U.S. Patent No. 5,506,206; U.S. Patent No. 5,271,961; U.S. Patent No. 5,254,342 and U.S. Patent No. 5,534,496).

3. Methods of detection and diagnosis

Methods are also provided for the detection of the presence of RSV in vitro or in vivo. In one example, the presence of RSV is detected in a biological sample from a subject, and can be used to identify a subject with disease caused by RSV. An antigen binding fragment of the disclosed monoclonal antibodies, conjugate of the antibody or antigen binding fragment, and multispecific antibody are also of use.

The sample can be any sample, including, but not limited to, tissue from biopsies, autopsies and pathology specimens. Biological samples also include sections of tissues, for example, frozen sections taken for histological purposes. Biological samples further include body fluids, such as blood, serum, plasma, sputum, spinal fluid or urine. The method of detection can include contacting a cell or sample, with an antibody or antigen binding fragment that specifically binds to RSV, or conjugate thereof (e.g., a conjugate including a detectable marker) under conditions sufficient to form an immune complex, and detecting the immune complex (e.g., by detecting a detectable marker conjugated to the antibody or antigen binding fragment).

In one aspect, the antibody or antigen binding fragment is directly labeled with a detectable marker. In another aspect, the antibody that binds G protein of RSV (the primary antibody) is unlabeled and a secondary antibody or other molecule that can bind the primary antibody is utilized for detection. The secondary antibody is chosen that is able to specifically bind the specific species and class of the first antibody. For example, if the first antibody is a human IgG, then the secondary antibody may be an anti-human-IgG. Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially. Suitable labels for the antibody, antigen binding fragment or secondary antibody are known and described above, and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials.

In some aspects, the disclosed antibodies or antigen binding fragments thereof are used to test vaccines. For example, to test if a vaccine composition including an G protein or fragment thereof assumes a conformation including the epitope of a disclosed antibody. Thus, provided herein is a method for testing a vaccine, wherein the method includes contacting a sample containing the vaccine, such as a G protein immunogen, with a disclosed antibody or antigen binding fragment under conditions sufficient for formation of an immune complex, and detecting the immune complex, to detect the vaccine with a G protein immunogen including the epitope in the sample. In one example, the detection of the immune complex in the sample indicates that vaccine component, such as a G protein immunogen assumes a conformation capable of binding the antibody or antigen binding fragment. The following examples are provided to illustrate particular features of certain aspects, but the scope of the claims should not be limited to those features exemplified.

III. EXAMPLES

RSV is the leading cause of respiratory disease in children worldwide and the primary cause of hospitalization for viral respiratory infections and a major cause of overall mortality in infants and children, especially premature infants (Weinberg et aL, Lancet Glob Health 5:e951-e952, 2017). Therefore, effective RSV prevention strategies are needed to address this major public health issue and burden (Prescott et al., Pharmocoeconomics 28:279-293, 2010). A recent meta-analysis concluded that the licensed monoclonal antibodies, Palivizumab and Nirsevimab, both targeting the F protein, were associated with substantial benefits in the prevention of RSV infection, without a significant increase in adverse events compared with placebo (Sun et al., JAMA Netw Open 6:e230023, 2023).

It was found that young infants prior to RSV exposure have lower titers of anti-G antibodies, compared with anti-F antibodies, that were increased 100-fold after primary RSV infection and were mapped to multiple regions in the RSV G protein, in addition to the CCD region (Fuentes et al., PLoS Pathog 12:el005554, 2016). Subsequently, it has been shown that un-glycosylated G protein (REG), as well as REG with CCD deletion can elicit protective immunity in mice (Fuentes et al., J Virol 89:8193-8205, 2015). Furthermore, several G-derived peptides outside the CCD/CXCR3 induced protective immunity with lower viral loads and pathology scores in RSV challenged animals (Lee et aL, EMBO Mol Med 14:el3847, 2022). These findings show that monoclonal antibodies targeting different sites in G proteins of RSV type A and type B given prophylactically provide protection against RSV disease.

EXAMPLE 1: Materials and Methods

Cell culture and virus production

A549 cells (Cat. No. #CCL-185) were from the American Type Culture Collection (ATCC, Manassas, VA). RSV rA2-Line!9F-Firefly Luciferase virus (rRSV-A2-L19-FFL) or RSV-B1 -FFL virus expressing the firefly luciferase gene upstream of the NS1 gene were prepared by infecting A549 cell at 0.02 multiplicity of infection (MOI). Virus was harvested by freeze/thaw cycles and were then purified via a sucrose gradient prior to being aliquoted and stored at -80°C. To determine the titer of the virus stock, immune plaque assays were performed on A549 cells. The optimal challenge dose (10⁶ PFU intranasally) and peak days of viral infection was determined by BALB/c model in which viral loads were measured by traditional plaque assay in Hep-2 cells, and by live imaging flux (Fuentes et al., Vaccine 35:694-702, 2017). Production of Recombinant E. coli expressed G (REG) proteins

Codon-optimized RSV G coding DNA for E. coli was chemically synthesized. Notl and Pad restriction sites were used for cloning the RSV A2 G ectodomain coding sequence (amino acids 67 to 298) into the TVbased pSK expression vector for bacterial expression. DNA coding REG ACCD with residues 172-186 deleted and replaced with a (G4S)2 linker was prepared by a two-step overlapping PCR (Lee et al., PLoS Pathog 14:el007262, 2018). The deleted sequence contains the cysteine noose in addition to the CX3CR1 binding motif present in all RSV G proteins. All amplified DNA was digested with Notl and Pad and ligated into the TVbased pSK expression vector for bacterial expression.

Recombinant RSV G 67-298 (REG 67-298) and REG ACCD proteins were expressed in E. coli BL21(DE3) cells (Novagen) and were purified as described previously (Fuentes et al., J Virol 89:8193-8205, 2015; Fuentes et al., Sci Rep 7:42428, 2017). Briefly, REG proteins expressed and localized in E. coli inclusion bodies (IB) were isolated by cell lysis, denatured and renatured by slowly diluting in redox folding buffer followed by dialysis. The dialysate was purified through a HisTrap™ Fast Flow chromatography column (GE Healthcare). The protein concentrations were analyzed by bicinchoninic acid (BCA) assay (Pierce), and the purity of the recombinant G proteins from E. coli (REG) determined by SDS-PAGE. Linear peptides were synthesized chemically using Fmoc chemistry, purified by HPLC, conjugated to KLH, and dialyzed, as described before (Lee et al., EMBO Mol Med 14:el3847, 2022).

Production of Recombinant glycosylated G protein using 293 Flp-In cells (RMG)

The 293-Flp-In cell line (Cat. No. #R75007; ThermoFisher Scientific) stably expressing the G protein of either the RSV A2 G or the RSV B 1 with secretory signal peptide from IgG kappa chain was developed as described previously. Briefly, 293-Flp-in cells were co-transfected with the plasmids expressing Flp-in recombinase and the RSV G ectodomain in DMEM media (Invitrogen). Twenty-four hours after transfection, culture medium was replaced with fresh DMEM containing 150 g/mL of hygromycin for selection of stably transfected cells. For protein expression, cells were maintained in 293-Expression media (Invitrogen), and culture supernatant was collected every 3-4 days. Supernatant was cleared by centrifugation and filtered through a 0.45 pm filter before purification through a His-Trap™ fast flow column (GE healthcare).

Gel filtration chromatography

Proteins at a concentration of 2 mg/ml were analyzed on a Superdex™ 200 Increase 10/300 GL column (GE Healthcare) pre-equilibrated with phosphate-buffered saline (PBS), and protein elution was monitored at 280 nm. Protein molecular weight (MW) marker standards (GE Healthcare) were used for column calibration and for the generation of standard curves to identify the molecular' weights of each purified protein. Monoclonal Antibody production

To generate mouse hybridomas against RSV-G protein, two sets of 5 female C57BL/6 mice were immunized and boosted with recombinant non-glycosylated RSV G 67-298 from RSV-A2 strain (REG A2) or from RSV-B1 strains (REG B2) at 28 days apart. After fusion of post-immunization splenocytes with mouse myeloma cell line, an initial screen of mouse hybridoma supernatant was performed against glycosylated RMG A2 and RMG B 1 by ELISA. After screening, the RSV G-binding positive hybridoma were single cell purified, and hybridomas screened again for clonality and screen for glycosylated RMG A2 or RMG Bl positive binders. All positive hybridoma clones were subjected for antibody production in serum free media and MAbs were purified using protein A chromatography (GE Healthcare, Uppsala, Sweden).

RSV G protein ELISA for MAb characterization

1MMUL0N® 2 HB 96-well microtiter plates were coated with 100 pl of purified recombinant G protein expressed in mammalian cells from either RSV-A2 (RMG-A2) or RSV-B1 (RMG-B1) in PBS (50 ng/well) per well at 4 °C overnight. After blocking with PBST containing 2% BSA, 100-fold dilutions of MAbs in blocking solution were added to each well, incubated for Ih at RT, followed by addition of 5,000-fold dilution of HRP- conjugated goat anti-mouse IgG-Fc specific antibody, and developed by 100 pl of OPD substrate solution. Absorbance was measured at 490 nm.

Surface Plasmon Resonance (SPR)

Steady-state equilibrium binding of MAbs was monitored at 25°C using a PROTEON™ surface plasmon resonance (SPR) biosensor (Bio-Rad). The recombinant G protein from 293T cells (REG-A2 dclCCD) was coupled to a GLC sensor chip via amine coupling with 500 resonance units (RU) in the test flow channels. Biotinylated RSV-G peptides were captured using a NLC sensor chip. Samples of 100 pl of freshly prepared dilution of MAbs (1 pg/ml) were injected at a flow rate of 50 pl/min (contact duration, 120 seconds) for association. Disassociation was performed over a 600 second interval. Responses from the protein or peptide surface were corrected for the response from a mock surface and for responses from a buffer-only injection. Anti-CCR5 (2D7) MAb was used as a negative control. Total antibody binding and data analysis results were calculated with BIO-RAD PROTEON™ Manager software (version 3.0.1).

Plaque reduction neutralization test (PRNT)

For the plaque reduction neutralization test (PRNT), heat-inactivated serum was diluted 4-fold and incubated with RSV-A2 virus (diluted to yield 20-50 plaques/well) containing 10% guinea pig complement (Rockland Immunochemical; Philadelphia, PA, USA) and incubated for 1 h at 37 °C. After incubation, 100 pl of the antibody-virus mixtures were inoculated in duplicate onto A549 monolayers in 48-well plates and incubated for 1 h at 37 °C. Inoculum was removed prior to adding infection medium containing 0.8% methylcellulose. Plates were incubated for 5 to 7 days at which time the overlay medium was removed and cell monolayers fixed with 100% methanol; plaques were detected by immunostaining with rabbit RSV polyclonal anti-F sera, followed by addition of alkaline phosphatase goat anti-rabbit IgG (H+L) (Jackson, 111-055-144) antibody. The reactions were developed by using Vector® Black Alkaline Phosphatase (AP) substrate kit (Vector Labs, Burlingame, CA). Numbers of plaques were counted per well and the neutralization titers were calculated by adding a trend line to the neutralization curves and using the following formula to calculate 50% endpoints: antilog of [(50+y-intercept)/slope].

Mice RSV challenge study

Four- to 6-week-old female BALB/c mice (BALB/cAnNCr strain code #555) from Charles River Labs (n=5 per group) were intraperitoneally (i.p.) injected with 20 pg/mouse of RSV G specific monoclonal antibodies, RSV G antibody (131-2G), and phosphate buffered saline (PBS, naive control). Mice were intranasally (i.n.) infected with 1 xlO⁶ PFU/ ml of RSV A2 (rRSV-A2-L19-FFL) or RSV Bl (RSV-B1-FFL) under isoflurane anesthesia to determine the efficacy of protection and histopathological effects as previously described (Fuentes et al., Sci Rep 7:42428, 2017). Mice were sacrificed by CO2 asphyxiation 5 days post-RSV challenge (the day with peak viral load), and blood and lungs were collected. For determination of the viral load and cytokine profile, the right lobe of the lung was collected.

In vivo imaging of RSV-infected mice

Whole body live imaging of infected mice was performed using IVIS imaging system as previously described (Fuentes et al., Vaccine 35:694-702, 2017). In brief, mice were anesthetized in an oxygen-rich induction chamber with 2% isoflurane and administered 20 pl of REDIJECT™ D-Luciferin bioluminescent substrate (Perkin Elmer) intranasally. After a 5-min interval, mice were placed in the IVIS 200 Imaging systems (Xenocorp) equipped with the Living Image software (version 4.3.1.). Bioluminescence signals were recorded for 2 min for whole body and for 1 min for lungs and nasal cavities, respectively. Images were analyzed with the Livingimage® 4.5 software (PerkinElmer) according to manufacturer’s instructions.

Lung viral titers by RSV immuno-plaque assay

Plaque formation units indicating RSV replication were visualized and quantified by immune-plaque assay with the Palivizumab antibody. Lung tissues were collected 5 days post challenge and individually measured lung RSV lung viral titer. Individual lungs (unperfused) were weighed and homogenized on ice in 1 mL DMEM, 2% FBS using an Omni tissue homogenizer. The clear supernatant was obtained by centrifugation at 3,795 xg for 10 min for a total of 2 centrifugations. Lung viral plaque-forming units (PFU) were determined by immune-plaque assay in Hep-2 cells. Media controls and lung homogenates mixed with RSV were incubated on HEp-2 cells for 1 h 37°C, 5% CO2. After 4% formalin fixation, the plaques were detected with anti-F MAb (palivizumab) and then HRP conjugated anti-human IgG (Fc) antibodies were used. Stained and developed individual plaques were using Pierce® DAB substrate kit (Invitrogen).

Lung histopathology and inflammation scoring

The left lung was harvested from each individual mouse at 5 days post challenge and immediately fixed with 10% neutral buffered formalin. Lung samples were embedded in paraffin in the dorsoventral position. Subsequently, sections of tissue blocks were obtained and stained with hematoxylin and eosin (H&E) and analyzed under light microscopy (Lee et al.. Hum Vaccin Immunother 13:2594-2605). For histopathological analysis the tissue slides were examined and scored blindly by a certified veterinary pathologist, including the following categories: epithelial alterations in alveolitis, bronchiolitis, perivascular, and interstitial space (Lee et al., Hum Vaccin Immunother 13:2594-2605). Inflammation and focal aggregates of infiltrating epithelial alveolar cells in the airways, blood vessel and interstitial space were blindly examined, and measured using a semiquantitative scale (0 to 3) (0 = absent; normal), 1 (mild inflammation; <20% of lung affected), 2 (moderate inflammation; 20-40% of lung affected), and 3 = severe; 40-60% lung affected) by light microscope as previously described (Lee et al., EMBO Mol Med 14:el3847, 2022). The scores were subsequently converted to a combined histopathology scale of 0-12.

Statistical Analysis

Statistical analyses were performed using GRAPHPAD PRISM™ version 8 (Graph Pad software Inc, San Diego, CA). Data were analyzed for significance using the student t-test, one-way ANOVA with Tukey’s test for multiple comparisons, or a two-way ANOVA with Bonferroni posttests. The difference was considered statistically significant when the P value was less than 0.05. Correlations were calculated with a Spearman two- tailed test. P values less than 0.05 were considered significant with a 95% confidence interval.

EXAMPLE 2: Generation and identification of five classes of RSV G-specific monoclonal antibodies

Six-week-old female C57BL/6 mice were immunized intramuscularly twice with recombinant nonglycosylated G proteins produced in E. coli, from either RSV-A2 strain termed REG-A (n=6), or RSV-B1 strain termed REG-B (n=6) (Fuentes et al., J Virol 89:8193-8205, 2015), at 28-days interval. Mouse spleens were isolated at 7-days following the second vaccination and used to generate hybridomas, followed by single cell cloning. Clones were screened against glycosylated forms of RSV-G protein produced in mammalian cells from either RSV-A2 (RMG-A2) or RSV-B1 (RMG-B1) by ELISA (Fuentes et al., J Virol 89:8193-8205, 2015; Fuentes et al., Sci Rep 7:42428, 2017). The clones that showed strong anti-RSV G antibody binding to any of the RSV G proteins were further expanded into large flasks and used for antibody purification using Protein A chromatogr phy . All MAbs were subjected to a multi-tier epitope mapping and specificity analysis using ELISA or SPR technologies as summarized in FIGs. 7A-7B. Cross-reactivity of MAbs against G proteins of RSV A2 and B 1 strains were determined by ELISA with recombinant glycosylated forms of G protein produced in mammalian cells of RSV-A2 (RMG-A2) or RSV-B1 (RMG-B1) (FIGs. 1A-1E). Fine epitope mapping was performed using G-derived peptides from RSV-A2 strain previously identified using GFPDL analysis of post-RSV infection infant sera (FIGs. 1A-1E) (Fuente et al., PLoS Pathog 12:el005554, 2016).

Five classes (G0-G5) of MAbs were identified (FIGs. 7A-7B). Class GO included conformational- dependent antibodies that bound to the intact glycosylated G proteins but not to any of the individual RSV A2 derived G peptides (SPR binding <10 RU). MAb 12F12 bound RMG-A2 only, while MAbs 68C7, 69C1, and 75F10 bound RMG-B1 only in ELISA. The binding to a CCD-deleted non-glycosylated REG-A2 protein (REG- A2 delCCD) was also measured using SPR. MAb 12F12 bound REG-A2 delCCD, while the three RSV-B1 specific GO MAbs did not bind REG-A2 delCCD. These MAbs may target sites that are less conserved between A2 and BI G proteins (FIGs. 1A-1E). One GO Mab (77D2) showed strong cross-reactivity against both RMG- A2 and RMG-B1 proteins in ELISA. While both 12F12 and 77D2 showed similar reactivity to RMG-A2 in ELISA, in SPR, 12F12 showed much higher binding to CCD deleted unglycosylated REG-A2 protein (REG-A2 delCCD) than 77D2, suggesting differences in their epitope footprints.

Class G1 MAbs specifically targeted the CCD region (aa 172-186), similar to the previously described MAb 13L2G (FIGs. 7A-7B). All the G1 MAbs demonstrated stronger binding to RMG-A2 than RMG-B1 protein in ELISA and no binding to the CCD deleted RSV G protein in SPR. The G1 MAbs reacted to CCD peptide (aa residues 172-186) in SPR. All G1 MAbs demonstrated cross-reactivity between RSV-A2 and RSV- B1 G proteins, especially MAb 40D8 and MAb 7H9.

Class G2 included two MAbs (7C6 and 7G6) that reacted with RMG-A2, but neither to RMG-B1 in ELISA and nor to CCD-deleted REG-A2 protein in SPR. These class G2 MAbs bind primarily to N-terminal peptide (aa residues 61-90) of RSV-G.

Class G3 MAb 48E2 targets a discontinuous epitope consisting of two peptides (residues 129-152 and 169-207) that flank the CCD motif and form the stem of the CCD loop. This MAb is cross-reactive against both RMG-A2 and RMG-B1 in ELISA and binds to CCD-deleted REG-A2 protein in SPR (FIGs. 7A-7B).

Class G4 MAb 72E6 binds much stronger to RMG-B1 than to RMG-A2. It also targets a discontinuous epitope flanking the CCD motif consisting of the peptides upstream and downstream of the CCD loop, but less strong than the cross-reactive MAb 48E2 (FIGs. 7A-7B). This weak binding may reflect amino acid differences between A2 and Bl in these regions (FIGs. 1A-1E).

Class G5 MAbs 7H11 and 23B4 bind strongly to RMG-A2 and to a lesser degree with RMG-B1. These G5 MAbs bind CCD-dclctcd REG-A2 protein (REG-A2 delCCD) and to the peptide encompassing residues 169-297 (downstream of CCD) in SPR (FIGs. 7A-7B). These data demonstrated that vaccination of mice with non-glycosylated G proteins from RSV-A2 and RSV-B1 elicited MAbs that strongly bound to the glycosylated G proteins derived from the RSV A2 and RSV Bl strains. Further mapping using REG-A2 delCCD protein and peptides spanning the RSV A2 G protein in SPR, identified 5 classes of antibodies. In addition to targeting the CCD or epitopes upstream or downstream of the CCD (at the stem of CCD loop), Class GO MAbs that bound only glycosylated intact RSV G protein from either subtype that did not bind linear RSV-G peptides were identified, and an antibody binding to a site in N- terminal region. Similar to MAb 131-2G, all the isolated MAbs did not neutralize RSV-A2 or RSV-B1 in vitro (FIGs. 1A-1E).

The reactivity of 7H11 and 43A11 is similar. In addition, the 68C7 epitope is similar to 73C1 , since it binds to amino acids 169-207 and displays same reactivity to RSV -G protein from A2 and Bl strains.

EXAMPLE 3: Protective efficacy of MAbs against RSV A2 and RSV Bl in mice challenge model: impact of prophylactic MAbs treatment

To determine the prophylactic protective efficacy of the MAbs, 4-6-week-old female BALB/c mice (5 mice per group) were intraperitoneally (i.p.) injected with 20 pg/mouse of RSV G specific MAbs, or MAb 131- 2G, or with PBS (negative control) (FIG. 2A). The MAbs that were used for pre-treatment prior to RSV-A2 and RSV-B1 challenge were selected based on their epitope mapping, representing classes G0-G5 (FIGs. 7A-7B). The protective efficacy of these MAbs targeting different sites in RSV-G protein was determined by challenging mice with RSV-A2 line 19F expressing firefly luciferase [RSV-A2-L19-FFL] or RSV Bl expressing firefly luciferase (RSV-B1-FFL) to track RSV infection in the mice model using live imaging, as previously described (Fuentes et al., Vaccine 35:694-702, 2017). One day after RSV infection, mice were intranasally (i.n.) infected with IxlO⁶ PFU of RSV-A2-L19-FFL or RSV-B1-FFL as previously described (Fuentes et al., Sci Rep 7:42428, 2017). RSV dissemination in the nasal cavity and lungs were inferred using fluorescence measurements obtained via whole body live imaging. Mice were sacrificed 5 days post-RSV challenge (the day of peak viral load). RSV infectious viral titers were measured by plaque forming units (PFU) in the lungs. Additionally, the lungs were used for histopathological evaluations (FIGs. 2A-2B).

Infectious replicating RSV titers in lungs were determined by immune-plaque assay in Hep-2 cells. Both RSV-A2 and RSV-B1 replicated in lungs with peak titers >10⁴ PFU/gram tissue on day 5 post-viral challenge. MAb 131-2G blocked the infectious virus titers in the lungs of animals infected with either RSV-A2 or RSV-B1 (FIGs. 3A-3B). Surprisingly, all anti-G MAbs significantly reduced infectious viral loads by more than 2 logfold on day 5 post-RSV challenge (FIGs. 3A-3B).

For histopathological analysis, the lung sections on day 5 post-RSV challenge were examined and scored by a certified veterinary pathologist, blinded to the treatment groups, including the following categories: epithelial alterations in alveolitis, bronchiolitis, perivascular, and interstitial space (Lee et al., Hum Vaccin Immunother 13:2594-2605, 2017). Inflammation and focal aggregates of infiltrating cells were examined and measured using a semiquantitative scale (0 to 3) (0 = absent; normal), 1 (mild inflammation; <20% of lung affected), 2 (moderate inflammation; 20-40% of lung affected), and 3 = severe; 40-60% lung affected) by light microscope. The lung histopathology scores for the four attributes were then combined to a scale of 0 to 12.

The lung pathology scores on day 5 following RSV-A2 challenge varied for different MAb-treated animals but were significantly lower than the PBS-treated control animals (FIGs. 3C-3D), which was similar to MAb 131-2G treated animals, except for G1 MAbs 7H9 and 2B11, and G3 MAb 48E2-treated animals (FIG. 3C). Interestingly, class GO MAb 77D2, G1 MAb 40D8, 1D9 and 36E10 as well as G2 MAb 7G6 treated animals showed lower pathology than 131-2G treated animals following RSV-A2 challenge, although these differences did not reach statistical significance (FIG. 3C).

All mice that received prophylactic treatment of MAbs prior to RSV-B1 challenge demonstrated reduced lung pathology scores, similar to MAb 131-2G treated animals compared with lung pathology observed in the PBS control treated animals (FIG. 3D). Lungs of mice treated with few MAbs including Gl-MAb 40D8 were similar to those observed for uninfected control animals following RSV-B1 challenge.

Together, this data shows that the various classes of MAbs, targeting different regions of RSV-A2 and RSV-B1 G proteins given one day prior to challenge, reduced lung pathology following either RSV-A2 or RSV- B1 challenge in the mouse model.

EXAMPLE 4: Anti-G MAbs protect from RSV dissemination in lungs: Live imaging of viral spread in the lungs of RSV infected animals

RSV can spread within the host either via infection of target cells by the viral inoculum or the released RSV particles, but also more efficiently via direct cell-to-cell transmission (Cifuentes-Munoz et aL, PLoS Pathog 14:el007015, 2018). To understand the dissemination of RSV in untreated and MAb-treated animals, whole body live imaging of infected mice was performed using IVIS imaging system as previously described (Fuentes et al., Vaccine 35:694-702, 2017; Lee et al., EMBO Mol Med 14:el3847, 2022). None of the MAbs (including MAb 131 -2G) reduced virus transmission in the nasal cavity as measured by flux (photons/sec) units using live imaging of infected mice (FIGs. 4A-4B). On day 5, the lung fluxes varied among the MAb- treated animals. Interestingly, the positive control MAb 131-2G reduced lung fluxes after RSV-B1 (~5-fold) more efficiently than after RSV-A2 infection (~2-fold) and was not statistically different from the PBS (untreated) control animals (FIGs. 4C-4D). Importantly, in RSV-A2 challenged animals, several MAbs reduced day 5 lungfluxes more efficiently than 131-G2, including class GO MAb 77D2, G1 MAbs 40D8, 1D9, 12G11, 22B11 and 3E10, G3 MAb 48E2, as well as G5 MAb 7H11 in RSV-A2 infected animals (FIG. 4C).

For RSV-B1, 4 of the 7 MAbs controlled viral spread in lungs compared with the PBS control (FIG. 4D). Importantly, none of the MAbs-trcatcd animals demonstrated enhanced viral loads compared with the PBS- treated animals by lung-flux measurements. Interestingly, the cross-reactive antibodies from three different classes: GO MAb 77D2, G1 MAb 40D8 and class G5 Mab 7H11, demonstrated significant reduction in lung fluxes against both RSV-A2 and RSV-B1.

Correlations were calculated with a Spearman two-tailed test to determine the relationship between lung pathology and RSV spread in lungs (Flux units) or the lung infectious viral titers (PFU). A statistically significant correlation was observed between the lung pathology scores and lung flux measurement on day 5 for individual mice across all groups for RSV-A2 (p=0.0317) (FIG. 5A) and RSV-B1 (p= 0.0058) (FIG. 5B), but not with lung infectious viral titers (FIGs. 5C-5D).

Together, this data demonstrates that several anti-G MAbs targeting multiple sites, including conformation-dependent class GO MAb 77D2, CCD-specific class G1 MAb 40D8, and carboxy terminus of CCD class G5 MAb 7H11, showed cross-reactive protection from lung pathology and RSV dissemination following challenge with either RSV-A or B subtypes (FIG. 6).

Thus, a panel of RSV G-targeting MAbs that were mapped to different sites in RSV G in addition to the CCD motif were generated and evaluated for their effectiveness in reducing viral dissemination in the lungs and protection from lung pathology. The previously described protective anti-G MAb 131-2G were used as a benchmark (Boyoglu-Barnum et al., Virology 483:117-125, 2015; Chirkova et al., J Gen Virol 96:2543-2556, 2015; Choi et al., Viral Immunol 25:193-203, 2012). Epitope mapping and relative prophylactic effectiveness in reducing lung infectious RSV titers, RSV spread in lungs and protection from lung pathology following either RSV-A2 and RSV-B1 infection is schematically summarized in FIG. 6. Any discrepancy between the in vitro neutralization and the in vivo results for anti-G antibodies could be due to the absence of Fc-receptor interactions and effector cells in the in vitro system.

Similar to MAb 131-2G, the new MAbs did not neutralize RSV in vitro. However, in vivo these anti-G MAbs reduced virus dissemination to the lungs either equally or better than 131-2G following challenge with RSV-A2 or RSV-B1 (FIG. 6). Importantly, among the more effective MAbs, MAb 77D2 (GO class), MAb 40D8 (G1 class), and MAb 7H11 (G5 class) are cross-reactive against both RSV-A2 and RSV-B1 (FIGs. 6-7). A correlation between lung pathology and RSV dissemination (lung flux), but not with infectious viral titer in the lungs, was noted. Without being bound by theory, the lung pathology following RSV infection could be a result of viral dissemination primarily by cell-to-cell spread and inflammatory response in the lungs. These anti-G MAbs were more efficient in restricting the viral dissemination in lungs as measured by live imaging of RSV challenged mice, suggesting a role of these antibodies in blocking cell-to-cell spread of RSV. Without being bound by theory, the mechanisms of protection from viral dissemination may involve Fc mediated functions including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and antibody-dependent complement deposition (ADCD), similar- to some anti-F antibodies elicited by infection or vaccination (Bartsch ct al., Cell 185:4873-4886, 2022).

Experiments are further performed in human nasal organoids (HNOs). In infection studies, the disclosed monoclonal antibodies inhibit RSV/A replication in the HNOs. Thus, the disclosed anti-G cross-reactive MAbs add to the arsenal of MAbs for the inhibition of RS V, and are of use for prevention of disease.

In view of the many possible aspects to which the principles of our invention may be applied, it should be recognized that illustrated aspects are only examples of the invention and should not be considered a limitation on the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. An isolated monoclonal antibody or antigen binding fragment thereof, comprising a heavy chain variable (VH) region and a light chain variable region (Vi.) comprising a heavy chain complementarity determining region (HCDR)l, a HCDR2, and a HCDR3, and a light chain complementarity determining region (LCDR)l, a LCDR2, and a LCDR3 of the VH and VL set forth as any one of: a) SEQ ID NOs: 81 and 85, respectively (77D2); b) SEQ ID NOs: 41 and 45, respectively (68C7); c) SEQ ID NOs: 73 and 77, respectively (73C1); d) SEQ ID NOs: 49 and 53, respectively (40D8); e) SEQ ID NOs: 9 and 13, respectively (1 D9); f) SEQ ID NOs: 33 and 37, respectively (12G11); g) SEQ ID NOs: 1 and 5, respectively (36E10); h) SEQ ID NOs: 25 and 29, respectively (7H11); i) SEQ ID NOs: 57 and 61, respectively (43A11); j) SEQ ID NOs: 17 and 21, respectively (48E2); k) SEQ ID NOs: 65 and 69, respectively (7H9); l) SEQ ID NOs: 89 and 93, respectively (22B11); m) SEQ ID NOs: 97 and 101, respectively (75F10); n) SEQ ID NOs: 105 and 109, respectively (7G6); o) SEQ ID NOs: 113 and 117, respectively (72E6); or p) SEQ ID NOs: 121 and 125, respectively (23B4), wherein the monoclonal antibody or antigen binding fragment thereof specifically binds to an attachment (G) protein of Respiratory syncytial virus (RSV).

2. The isolated monoclonal antibody or antigen binding fragment of claim 1, wherein the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 are set forth as: a) SEQ ID NOs: 82, 83, 84, 86, 87, and 88; b) SEQ ID NOs: 42, 43, 44, 46, 47, and 48; c) SEQ ID NOs: 74, 75, 76, 78, 79, and 80; d) SEQ ID NOs: 50, 51, 52, 54, 55, and 56; e) SEQ ID NOs: 10, 11, 12, 14, 15, and 16; f) SEQ ID NOs: 34, 35, 36, 38, 39, and 40; g) SEQ ID NOs: 2, 3, 4, 6, 7, and 8; h) SEQ ID NOs: 26, 27, 28, 30, 31, and 32; i) SEQ ID NOs: 58, 59, 60, 62, 63, and 64; j) SEQ ID NOs: 18, 19, 20, 22, 23, and 24; k) SEQ ID NOs: 66, 67, 68, 70, 71, and 72; l) SEQ ID NOs: 90, 91, 92, 94, 95, and 96; m) SEQ ID NOs: 98, 99, 100, 102, 103, and 104; n) SEQ ID NOs: 106, 107, 108, 110, 111, and 112; o) SEQ ID NOs: 114, 115, 116 (DEY), 118, 119, and 120; or p) SEQ ID NOs: 122, 123, 124 (EGY), 126, 127, and 128, respectively.

3. The isolated monoclonal antibody or antigen binding fragment of claim 1 or claim 2, wherein the VH and the V comprise the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 and the amino acid sequences of the framework regions of the VH and the VL are at least 90% identical to those of: a) SEQ ID NOs: 81 and 85; b) SEQ ID NOs: 49 and 53; c) SEQ ID NOs: 73 and 77; d) SEQ ID NOs: 41 and 45; e) SEQ ID NOs: 9 and 13; f) SEQ ID NOs: 33 and 37; g) SEQ ID NOs: 1 and 5; h) SEQ ID NOs: 25 and 29; i) SEQ ID NOs: 57 and 61; j) SEQ ID NOs: 17 and 21; k) SEQ ID NOs: 65 and 69; l) SEQ ID NOs: 89 and 93; m) SEQ ID NOs: 97 and 101; n) SEQ ID NOs: 105 and 109; o) SEQ ID NOs: 113 and 117; or p) SEQ ID NOs: 121 and 125, respectively.

4. The isolated monoclonal antibody or antigen binding fragment of any one of claims 1-3, wherein the VH and the VL comprise amino acid sequences set forth as: a) SEQ ID NOs: 81 and 85; b) SEQ ID NOs: 49 and 53; c) SEQ ID NOs: 73 and 77; d) SEQ ID NOs: 41 and 45; e) SEQ ID NOs: 9 and 13; f) SEQ ID NOs: 33 and 37; g) SEQ ID NOs: 1 and 5; h) SEQ ID NOs: 25 and 29; i) SEQ ID NOs: 57 and 61; j) SEQ ID NOs: 17 and 21; k) SEQ ID NOs: 65 and 69; l) SEQ ID NOs: 89 and 93; m) SEQ ID NOs: 97 and 101; n) SEQ ID NOs: 105 and 109; o) SEQ ID NOs: 113 and 117; or p) SEQ ID NOs: 121 and 125, respectively.

5. The isolated monoclonal antibody or antigen binding fragment of claims 1-4, wherein the antibody comprises a human constant domain.

6. The isolated monoclonal antibody or antigen binding fragment of any one of claims 1-5, wherein the antibody is a human antibody.

7. The isolated monoclonal antibody of any one of claims 1-6, wherein the antibody is an IgG.

8. The isolated monoclonal antibody of any one of claims 1-7, comprising a recombinant constant domain comprising a modification that increases the half-life of the antibody.

9. The isolated monoclonal antibody of claim 8, wherein the modification increases binding to the neonatal Fc receptor.

10. The isolated monoclonal antibody of claim 9, wherein the recombinant constant domain is an IgGl constant domain comprising M428L and N434S mutations.

11. The antigen binding fragment of the antibody of any one of claims 1-6.

12. The antigen binding fragment of claim 11, wherein the antigen binding fragment is a Fv, Fab, F(ab')2, scFV or a scFVz fragment.

13. The antibody or antigen binding fragment of any one of claims 1-12, conjugated to an effector molecule or a detectable marker.

14. A mulispecific antibody comprising the antibody or antigen binding fragment of any one of claims 1-13.

15. An isolated nucleic acid molecule encoding the antibody or antigen binding fragment of any one of claims 1-13, or the multipecific antibody of claim 14.

16. The isolated nucleic acid molecule of claim 15, wherein the nucleic acid molecule is RNA.

17. The nucleic acid molecule of claim 15 or claim 16, operably linked to a promoter.

18. A vector comprising the nucleic acid molecule of any of claims 15-17.

19. A host cell comprising the nucleic acid molecule of any one of claims 15-17 or vector of claim 18.

20. A composition comprising an effective amount of the monoclonal antibody or antigen binding fragment of any one of claims 1-13, the multispecific antibody of claim 14, the nucleic acid molecule of any one of claims 15-17, or the vector of claim 18, and a pharmaceutically acceptable carrier.

21. A method of producing a monoclonal antibody or antigen binding fragment that specifically binds to respiratory syncytial virus (RSV), comprising: expressing one or more nucleic acid molecules encoding the monoclonal antibody or antigen binding fragment of any one of claims 1-13 in a host cell; and purifying the monoclonal antibody or antigen binding fragment.

22. A method of detecting the presence of RSV in a biological sample from a subject, comprising: contacting the biological sample with an effective amount of the monoclonal antibody or antigen binding fragment of any one of claims 1-13 under conditions sufficient to form an immune complex; and detecting the presence of the immune complex in the biological sample, wherein the presence of the immune complex in the biological sample indicates the presence of the RSV in the sample.

23. The method of claim 22, wherein detecting the presence of the immune complex in the biological sample indicates that the subject has disease caused by an RSV infection.

24. A method of reducing disease caused by an RSV infection in a subject, comprising administering an effective amount of the monoclonal antibody or antigen binding fragment of any one of claims 1-13, the multispecific antibody of claim 14, the nucleic acid molecule of any one of claims 15-17, the vector of claim 18, or the composition of claim 19 to the subject, wherein the subject has disease caused by an RSV infection.

25. The method of claim 24, comprising administering an effective amount of more than one monoclonal antibody or antigen binding fragment of any one of claims 1-13.

26. The method of claim 24 or claim 25, wherein the method treats the RSV infection in the subject.

27. Use of the monoclonal antibody or antigen binding fragment of any one of claims 1-13, the multispecific antibody of claim 14, the nucleic acid molecule of any one of claims 15-17, the vector of claim 18, or the composition of claim 20 to reduce disease caused by an RSV infection in a subject or to detect the presence of RSV in a biological sample.